Green Synthesis of Encapsulated Copper Nanoparticles Using a Hydroalcoholic Extract of Moringa oleifera Leaves and Assessment of Their Antioxidant and Antimicrobial Activities

The synthesis of metal nanoparticles using plant extracts is a very promising method in green synthesis. The medicinal value of Moringa oleifera leaves and the antimicrobial activity of metallic copper were combined in the present study to synthesize copper nanoparticles having a desirable added-value inorganic material. The use of a hydroalcoholic extract of M. oleifera leaves for the green synthesis of copper nanoparticles is an attractive method as it leads to the production of harmless chemicals and reduces waste. The total phenolic content in the M. oleifera leaves extract was 23.0 ± 0.3 mg gallic acid equivalent/g of dried M. oleifera leaves powder. The M. oleifera leaves extract was treated with a copper sulphate solution. A color change from brown to black indicates the formation of copper nanoparticles. Characterization of the synthesized copper nanoparticles was performed using ultraviolet-visible light (UV-Vis) spectrophotometry, Fourier-transform infrared (FTIR) spectrometry, high-resolution transmission electron microscopy (HRTEM), scanning electron microscopy (SEM), and X-ray diffraction (XRD). The synthesized copper nanoparticles have an amorphous nature and particle size of 35.8-49.2 nm. We demonstrate that the M. oleifera leaves extract and the synthesized copper nanoparticles display considerable antioxidant activity. Moreover, the M. oleifera leaves extract and the synthesized copper nanoparticles exert considerable anti-bacterial activity against Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Enterococcus faecalis (MIC values for the extract: 500, 250, 250, and 250 µg/mL; MIC values for the copper nanoparticles: 500, 500, 500, and 250 µg/mL, respectively). Similarly, the M. oleifera leaves extract and the synthesized copper nanoparticles exert relatively stronger anti-fungal activity against Aspergillus niger, Aspergillus flavus, Candida albicans, and Candida glabrata (MIC values for the extract: 62.5, 62.5, 125, and 250 µg/mL; MIC values for the copper nanoparticles: 125, 125, 62.5, and 31.2 µg/mL, respectively). Our study reveals that the green synthesis of copper nanoparticles using a hydroalcoholic extract of M. oleifera leaves was successful. In addition, the synthesized copper nanoparticles can be potentially employed in the treatment of various microbial infections due to their reported antioxidant, anti-bacterial, and anti-fungal activities.

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 [34]. The concoction of M. oleifera leaves extract contains isomers of caffeoylquinic acid, isomers of feruloylquinic acid, tannins, gallic acid, and several flavonoids like quercetin, kaempferol and their glycoside derivatives [35]. The chemical structures of these compounds are shown in Figure 1. Collectively, these findings suggest that the M. oleifera leaves extract could serve as a nutritional supplement and a possible stabilizing agent for the formed copper nanoparticles. Based on the total phenolic content estimation of the M. oleifera leaves extract before and after the reaction (Table 2), Scheme 1 was 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 250 mg of copper nanoparticles from 0.04 M of copper (II) ion solution. The other reducing and binding chemical entities present in the concoction aid in the formation and stabilization of the synthesized copper nanoparticles by encapsulating the nanomaterial. Comparing the total polyphenol content before and after the synthesis, there was a reduction in total polyphenol content. This substantiates the deduction that some polyphenols are involved in the bio-reduction and subsequent stabilization of the nanoparticles by encapsulation. Table 2. Estimation of total phenolic content before and after the synthesis of copper nanoparticles.

Sample
Total Phenolic Content (mg/g of Dried Leaves) M. oleifera leaves extract (before synthesis) 23.0 ± 0.3 M. oleifera leaves extract (after synthesis) 17.0 ± 0.4 Scheme 1. Schematic representation of the synthesis of copper nanoparticles.

Size and Morphology of the Synthesized Copper Nanoparticles
The high-resolution transmission electron microscopy (HRTEM) image of synthesized copper nanoparticles was captured using a JEOL-TEM 2100 plus electron microscope ( Figure 2). EDS (energy dispersive X-ray spectroscopy) studies indicate only the presence of elements in the sample ( Figure 3).
The synthesized copper nanoparticles were obtained in ethanol to give a colloidal solution. The synthesized copper nanoparticles are amorphous in nature and they agglomerate upon storage. EDS analysis confirmed the presence of the elemental copper nanoparticles with Kα line at 8.04 and Lα line at 0.92 (X-ray energies for copper). The unlabeled peaks correspond to sulphur (Kα line at 2.3) and oxygen (Kα line at 0.52). These two elements can appear from sulphur-containing phytochemicals that had been encapsulated into the nanomaterial. The absence of other elemental peaks reflects the purity of the sample. The scanning electron microscopy (SEM) images ( Figure 4) indicate that the size of the synthesized copper particles is 35.8-49.2 nm. The macromolecules that encapsulated the copper nanoparticles promote the agglomeration of the nanomaterial in layers. So, the smallest grains give the best chance to capture the size of the nanoparticle, as the effect of agglomeration will be negligible.
X-ray diffraction analysis confirms the amorphous nature of the synthesized copper nanoparticles ( Figure 5). This substantiates the electron diffraction pattern observed in the HRTEM images ( Figure 2), suggesting that the synthesized copper nanoparticles are amorphous. Since the nanomaterial was synthesized using a plant leaf extract, the agglomeration occurs due to the encapsulation of nanomaterial by the phytochemicals. These organic moieties form layers of sheets of agglomeration. Using X-ray diffraction (XRD) analysis, we can observe only long-range disorder in the spectrum.

Fourier-Transform Infrared (FTIR) Spectroscopy of the M. oleifera Leaves Extract and the Synthesized Copper Nanoparticles
The FTIR spectra of the M. oleifera leaves extract and the synthesized copper nanoparticles are shown in Figures 6 and 7, respectively. This vibration spectroscopy data can be used to understand the biomolecules involved in the synthesis of copper nanoparticles. The bands around 3400 cm −1 and 1630 cm −1 are broad in the M. oleifera leaves extract ( Figure 6), corresponding to the vibration mode of a hydroxyl group, mostly found in polyphenolic molecules such as tannins, flavonoids, and glycoside derivatives. The signals at 1638 cm −1 and 1068 cm −1 correspond to N-H bending and C-N bond stretching, respectively. The FTIR spectrum of the synthesized copper nanoparticles ( Figure 7) depicts a sharp narrow band at 3432 cm −1 , corresponding to the N-H vibration mode. The FTIR of the extract gave a broad signal at 3400 cm −1 , corresponding to hydrogen-bonded poly-hydroxyl groups of the phytochemicals present in encapsulated copper nanoparticles. The signals at 1631 cm −1 and 1072 cm −1 correspond to N-H bending and C-N bond stretching respectively.
This comparison shows the participation of the hydroxyl group in the synthesis of copper nanoparticles. Moreover, the binding characteristics of amino groups in the M. oleifera leaves extract and their signals in the FTIR spectrum of the synthesized copper nanoparticles confirm the encapsulation of copper nanomaterial during the synthesis (Figures 6 and 7). This could be the reason for agglomeration of the synthesized copper nanoparticles to give an amorphous morphology. The UV-Vis absorption spectrum of the M. oleifera leaves extract is shown in Figure 8, with λ max at 390 nm. The UV-Vis spectrum of the synthesized copper nanoparticles reconstituted in dimethyl sulfoxide (DMSO) solvent is shown in Figure 9, with λ max at 260 nm.  The copper nanoparticles usually absorb from 280 nm to 360 nm, while absorbance is usually detected at 620 nm for the aqueous copper sulfate solution. The detection of λ max at 260 nm and absence of absorbance at 620 nm confirms the reduction of copper ions in the aqueous solution and formation of copper nanoparticles.

Antioxidant Activity of the M. oleifera Leaves Extract and the Synthesized Copper Nanoparticles
Using DPPH assay against ascorbic acid as a standard, the percentage of antioxidant activity of the M. oleifera leaves extract and the synthesized copper nanoparticles was assessed. As shown in Table 3, the M. oleifera leaves extract exerted considerable antioxidant activity, while the synthesized copper nanoparticles displayed a lower 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 Copper Nanoparticles
The potential anti-bacterial activity of the synthesized copper nanoparticles was evaluated against Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Enterococcus faecalis. Streptomycin (10 µg/500 µL) was used 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 copper nanoparticles, respectively. The nutrient broth was also used as a negative control. The minimum inhibitory concentration (MIC) values measured in presence of the M. oleifera leaves extract and the copper nanoparticles are given in Table 5. The growth of the indicated species of bacteria in presence of different concentrations (7.8-1000 µg/mL) of M. oleifera leaves extract and the copper nanoparticles is shown in Table 6. In presence of the M. oleifera leaves extract, the MIC values against Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Enterococcus faecalis were in the range of 250-500 µg/mL ( Table 5). The anti-bacterial activity of the synthesized copper nanoparticles was observed with MIC values (250-500 µg/mL) ( Table 5). The anti-bacterial activity (MIC values) of the synthesized copper nanoparticles was comparable to that observed for the M. oleifera leaves extract (Table 6). A sample of resazurin microtiter assay plates for the M. oleifera leaves extract and the synthesized copper nanoparticles is given in Figure S1.  Table 6. Anti-bacterial activity data (growth (+) and no growth (-)). No.

Anti-Fungal Activity of the M. oleifera Leaves Extract and the Synthesized Copper Nanoparticles
The potential anti-fungal activity of synthesized copper nanoparticles was evaluated against Aspergillus niger, Aspergillus flavus, Candida albicans, and Candida glabrata. Ketoconazole (10 µg/500 µL) was used 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 copper nanoparticles, respectively. The nutrient broth was also used as a negative control. The minimum inhibitory concentration (MIC) values measured for the M. oleifera leaves extract and the copper nanoparticles are given in Table 7. 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 copper nanoparticles is shown in Table 8. The copper nanoparticles displayed more effective anti-fungal activity against Candida albicans and Candida glabrata than the M. oleifera leaves extract (Tables 7 and 8). In presence of the M. oleifera leaves extract, the MIC values against Aspergillus niger, Aspergillus flavus, Candida albicans, and Candida glabrata were found to be 62.5, 62.5, 125, and 250 µg/mL, respectively (Table 7). In presence of the synthesized copper nanoparticles, the MIC values against Aspergillus niger, Aspergillus flavus, Candida albicans, and Candida glabrata were found to be 125, 125, 62.5, and 31.2 µg/mL, respectively (Table 7). A sample of resazurin microtiter assay plates for the M. oleifera leaves extract and the synthesized copper nanoparticles is given in Figure S2. These findings indicate that the copper nanoparticles displayed more effective anti-fungal activity against Candida albicans and Candida glabrata compared to the M. oleifera leaves extract (Tables 7  and 8). Moreover, it is concluded that the enhanced anti-fungal activity of copper nanoparticles against Candida species makes them a better therapeutic choice for direct application as antimicrobial agents.  Table 8. Anti-fungal activity data (growth (+) and no growth (-)).

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 copper nanoparticles and other experimental analyses.

Synthesis of the Copper Nanoparticles
Copper sulfate pentahydrate (1g) 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 copper nanoparticles were collected by centrifugation and repeatedly washed with de-mineralized water. The synthesized copper nanoparticles were dried at 105 • C for 1 h, and subsequently reconstituted in DMSO solvent.

Characterization of the Size and Morphology of the Synthesized Copper Nanoparticles
The completion of synthesis was characterized using a UV-3600 Plus UV-Vis double beam spectrophotometer (Shimadzu, Kyoto, Japan). The formation of copper 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 copper nanoparticles was ascertained by XRD using a XRD-6000 diffractometer (Shimadzu, Kyoto, Japan). The size of the synthesized copper 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.

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. Experimental details of the phytochemical analysis are given in Supplementary Materials. The total phenolic content in the M. oleifera leaves extract was estimated as gallic acid equivalent by Folin-Ciocalteu polyphenol assay [36]. The protein content in the M. oleifera leaves extract was estimated by Lowry's method [37].

DPPH Assay (Antioxidant Activity Percentage-AA%)
The antioxidant activity percentage (AA%) (scavenging activity) of the M. oleifera leaves extract and the synthesized copper nanoparticles was assessed by DPPH free radical scavenging assay. A total of 1 mg of ascorbic acid (standard) was dissolved in 1 mL 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 copper 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 copper 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. To each tube, 1 mL of 0.1 mM DPPH radical solution in ethanol was added. 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: The total antioxidant capacity (TAC) of the M. oleifera leaves extract and the synthesized copper nanoparticles was assessed by phosphomolybdenum assay as ascorbic acid equivalent. A total of 1 mg of ascorbic acid (standard) was dissolved in 1 mL 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 copper 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 copper 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 sulphuric 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, so as 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 copper nanoparticles were calculated and expressed as ppm equivalent of ascorbic acid.

Anti-Bacterial Activity using Resazurin Microtiter Assay
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 [38][39][40]. 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. As a standard protocol, a loop full of bacterial culture was inoculated in 10 mL of the appropriate broth medium for 6 h at 37 • C in a Bio-Oxygen Demand (BOD) incubator. A volume of 100 µL of sample containing 10 mg/mL was pipetted into the first well of the plate. To all other wells, 50 µL of nutrient broth was added, and the tested sample was serially diluted. Subsequently, 10 µL of resazurin solution and 10 µL of bacterial suspension were added to each well. Each plate was wrapped loosely with cling film to prevent dehydration. The plates were incubated at 37 • C for 18-24 h. The color change was then assessed visually. A color change from purple to pink (or colorless) was recorded as positive, indicating cell growth (i.e., (+) means growth and (-) means no growth). The lowest concentration at which color change occurred was considered to be MIC. Streptomycin (10 µg/500 µL) was used 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 copper nanoparticles, respectively. The nutrient broth was also used as a negative control.

Anti-Fungal Activity Using Resazurin Microtiter Assay
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. As a standard protocol, a loop full of fungal culture was inoculated in 10 mL of the appropriate broth medium for 6 h at 37 • C in a Bio-Oxygen Demand (BOD) incubator. A volume of 100 µL of sample containing 10 mg/mL was pipetted into the first well of the plate. To all other wells, 50 µL of nutrient broth was added, and the tested sample was serially diluted. Subsequently, 10 µL of resazurin solution and 10 µL of fungal suspension were added to each well. Each plate was wrapped loosely with cling film to prevent dehydration. The plates were incubated at 37 • C for 18-24 h. The color change was then assessed visually. A color change from purple to pink (or colorless) was recorded as positive, indicating cell growth (i.e., (+) means growth and (-) means no growth). The lowest concentration at which color change occurred was considered to be MIC. Ketoconazole (10 µg/500 µL) was used 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 copper nanoparticles, respectively. The nutrient broth was also used as a negative control.

Conclusions
The green synthesis of copper nanoparticles using a hydroalcoholic extract of M. oleifera leaves was successful. The formation of copper nanoparticles and the role of biomolecules in this synthesis were confirmed by UV-Vis absorption. FTIR spectrometry supports the encapsulation of copper nanoparticles by the phytochemicals present in the M. oleifera leaves. The amorphous nature of the synthesized copper nanoparticles encapsulated by phytochemicals was ascertained by XRD and the electron diffraction pattern from HRTEM images. The particle size of the synthesized copper nanoparticles was measured to be 35.8-49.2 nm using SEM imaging. EDS analysis confirms the presence of copper element in synthesized encapsulated nanoparticles. Our study reveals that the M. oleifera leaves extract and the synthesized copper nanoparticles display considerable antioxidant activity. We also demonstrate that the M. oleifera leaves extract and the synthesized copper nanoparticles exert considerable anti-bacterial activity against Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Enterococcus faecalis, with MIC values in the range of 250-500 µg/mL. Similarly, the M. oleifera leaves extract and the synthesized copper nanoparticles exert relatively stronger anti-fungal activity against Aspergillus niger, Aspergillus flavus, Candida albicans, and Candida glabrata, with MIC values in the range of 62.5-250 µg/mL and 31.2-125 µg/mL, respectively. Indeed, the anti-fungal activity of the synthesized copper nanoparticles is more effective than that of the M. oleifera leaves extract against Candida albicans and Candida glabrata. We also conclude that the anti-bacterial and anti-fungal activities were not deterred in the process of green synthesis of the copper nanoparticles. These findings suggest that the synthesized copper nanoparticles can be a promising therapeutic candidate for the treatment of various bacterial, and particularly, fungal infections such as candidiasis. Due to encapsulation, the synthesized copper nanoparticles can be employed in direct medical application as antimicrobial agents. Yielding non-toxic material and reducing the production of wasteful products is a huge advantage of green synthesis of nanoparticles. Moreover, compared to the incinerated nanoparticles that are synthesized by conventional thermal-chemical methods, the described green synthesis approach leading to the synthesis of nanoparticles encapsulated with phytochemicals/polyphenols is of significant therapeutic value.
Supplementary Materials: The following are available online, Figure S1: Resazurin microtiter assay plates for the M. oleifera leaves extract and the synthesized copper nanoparticles, Figure S2: Resazurin microtiter assay plates for the M. oleifera leaves extract and the synthesized copper nanoparticles, Experimental details: Phytochemical tests.

Conflicts of Interest:
The authors declare no conflicts of interest.