Antioxidant and Antibacterial Activities of Magnesium Oxide Nanoparticles Prepared using Aqueous Extract of Moringa Oleifera Bark as Green Agents

In this research, Magnesium oxide nanoparticles (MgONPs) was prepared from MgCl2 solution using aqueous extract of M. oleifera bark as green agent. Preparation procedure involved mixing of MgCl2.6H2O solution and the aqueous extract of M. oleifera bark, followed by drop wise addition of NaOH solution. The formation of MgO nanoparticles in this synthesis was confirmed using UVVis absorption. The spherical crystal structure of MgO nanoparticle confirmed XRD analysis. The average particle size of the synthesized MgO nanoparticles measured between 60 100 nm using SEM and TEM images and PSA results. MgONPs synthesized showed good antioxidant activity and antibacterial activity against S. aureus, E. faecalis, E. coli, and S. dysenteriae bacteria.


INTRODUCTION
Green method is a method that uses extracts from plants as agents to form nanoparticles. This method provides a variety of resources in the process of nanoparticle synthesis [1]- [3]. In addition, there are many other advantages in the use of natural materials in the synthesis of nanoparticles. The fact that the use of biodegradable raw materials as a synthesis of nanoparticles has no harmful impact on the environment, leads to a green technology policy, is first and foremost environmentally friendly. Second, the efficiency of synthesis, which promotes further production and further production through the availability of abundant natural raw materials. Thirdly, the nanoparticles produced are biocompatible in comparison with conventionally seized nanoparticles [4].
Moringa Oleifera (M. oleifera) is a multipurpose tropical plant from the Moriceae family which is spread throughout India, Asia, and sub-Saharan Africa [17]. The use of Moringa extract has been widely used in synthesizing metal nanoparticles and metal oxides. Moringa flower extract reportedly can be used to prepare palladium [18], silver [19], and hydroxyapatite [20] nanoparticles. Moringa leaf extract to prepare silver [21], zinc oxide [22], titanium oxide [23] and nickel oxide [24] nanoparticles. Phytochemical analysis of water extracts of M. oleifera bark shows that Moringa wood bark contains compounds such as alkaloids, phenolic acids, terpenoids, and flavonoids [25]. These compounds play a role in the chelation process and are able to reduce metal ions to nanoparticles [26].
The effect of M. oleifera bark water extract for preparation of MgO nanoparticles was examined in this presence research. In this presence study. This research has been confirmed, since the special use of these plant components for nanoparticle preparation is still limited. The MgONPs characteristics were evaluated through UV-Vis absorption spectroscopy, X-Ray (XRD), Electron scanning (SEM). Electron transmission microscopy (TEM) and particle size analyzer (PSA). The results of this analysis were evaluated. The bioactivity of MgONPs was subsequently examined as antioxidant and antibacterial.

Methods 2.2.1. Plant extract
A fresh, M. oleifera sample washed by floating water, dried and then poured into powder and stored at room temperature under direct sunlight. In 100 mL of distilled water, four grams of M. oleifera bark powder was soaked and heated for 20 minutes at 60 ° C. The mixture was then filtered 1 hour with Whatman Filter Paper 1 to isolate the extract from the residue. The mixture was left.

Phytochemicals analysis
The aqueous extract was subjected to phytochemical analysis to detect the presence of carbohydrates, amino acids, glycosides, polyphenols, saponins, steroids, flavonoids, tannins, and alkaloids. Total phenolic was estimated using the Follin-Ciocalteu test [27], and the result was expressed as microgram per milligram (µg/mg) gallic acid equivalent (GAE). Total flavonoid content was determined by the colorimetric AlCl 3 method, using catechin as standard and expressed as microgram per milligram (µg/mg) equivalent of catechin (CE) [28].

Preparation of MgO nanoparticles (MgONPs)
To synthesize nanoparticles, an aqueous extract of M. oleifera barks 50 mL was mixed with MgCl 2 .6H 2 O solution 1 mM 50 mL in a beaker with heating to 90 °C and stirred at 600 rpm. The 1 M NaOH solution was added drop wise until the color of the mixture faded out and precipitate was formed. The mixture was left for 3 hours to maximize the synthesis process. MgONPs  synthesized was centrifuged at 7500 rpm at room temperature and re-dispersed in deionized water and methanol (99%) to remove biological residues. The process was repeated twice, and the solid was dried at 100 °C. The solid was subjected to calcination at 600 °C for 5 hours was used to optimize the formation of oxides.

Characterizations of MgO nanoparticles (MgONPs)
Several techniques were used to describe MgO nanoparticles (MgONPs). The confirmation of MgONPs synthesis was based on the change in the color of the mixture during the reaction and recorded with UV-Visible spectroscopy (Analytic Jena Specord 200 Plus) by scanning the spectrum in the range of 200 -800 nm. The morphology of nanoparticles was studied with scanning electron microscopy (SEM; FEI Inspect-S50). The size and morphology of the MgONPs were investigated by transmission electron microscopy (TEM; Jeol Jem 1400) and the average particle size by the particle size analyzer (PSA; Horiba SZ 100z). The crystal structure of the synthesized MgONPs nanoparticles was confirmed by x-ray diffraction (XRD; PANAnalytical Expert Pro).

Antioxidant activity
Antioxidant activity of Moringa oleifera bark aqueous extract (BEM) and MgONPs was evaluated through (2,2-diphenyl-1-picrylhydrazyl) DPPH radical testing in accordance with the procedure described by Das et al. [29], using ascorbic acid as reference. DPPH 0.1 mM solution is was prepared by dissolving in ethanol. 1 mg of ascorbic acid is was dissolved in 1 mL of methanol. Dilution was carried out to make a standard solution of ascorbic acid with different concentrations (50-500 μg). For each tube containing a standard solution of ascorbic acid (200 μl), 1 mL of 0.1 mM DPPH solution was added and continued with the addition of 800 μl 50 mM Tris-HCl buffer (pH7.4). The final volume is adjusted to 4 mL using ethanol. Stock solutions for BEM and MgONPs were synthesized prepared by dissolving 1 mg of each sample in 1 mL of a suitable solvent (BEM using methanol and MgONPs using DMSO).
Different aliquots of stock solution (50-500 μg) was added to separate tube, and the final volume is adjusted to 2 mL using ethanol. A total of 1 mL of 0.1 mM DPPH solution and 800 μl 50 mM Tris-HCl buffer (pH7.4) was added to each tube. The control was made by mixing 1 mL DPPH 0.1 mM, 800 μL 50 mM Tris-HCl buffer (pH7.4), and 2 mL ethanol. Absorbance was recorded after incubation for 30 minutes at room temperature, measured by UV-Vis spectrophotometer at 517 nm. The percentage of antioxidant activity (% Inhibition) was calculated using the following equation: (1) The mean and standard deviation (SD) were calculated based on triplicate measurements by repeating three times.

Antibacterial activity 2.2.6.1. Microorganism and inoculum preparation
The antibacterial activity of BEM and MgONPs nanoparticles was evaluated against both grampositive (S. aureus and E. faecalis) and gramnegative (E. coli and S. dysenteriae) obtained from the microbiology laboratory of Airlangga University. Bacterial cultures for testing were cultivated on nutrient agar (NA) tilted by selecting a colony from the Mueller-Hinton agar plate (MHA) after 24 hours.
A single bacterial or fungal colony is selected and transferred to the Mueller-Hinton (MHB) broth using a sterilized loop, followed by shaking at 100 rpm at a temperature of 37 °C overnight for a normalized population. The optical density of

Minimum inhibition concentration (MIC) determination
The resazurin microtiter test was used to assess the minimum concentration of inhibition. This approach has been chosen as the easiest and most economical way to simultaneously scan for multiple microorganism isolates and produces satisfactory performance. A 270 mg tablet of resazurin in 40 mL of sterile distilled water was prepared to dissolve the resazurin solution. The test was conducted in aseptic conditions in 96-well plates. The samples were transferred into the plate well with a volume of 100 μL containing 600 mg / mL. Subsequently, the checked sample was applied to all other wells by 50 μL of bacterial suspense and diluted severely. Each well was subsequently supplemented with 10 μL resazurin solution. The plate was covered with film to avoid dehydration and incubated for 24 hours at 37 ° C. The change in color was noticed visually. A change in blue to rose color signaling growth of the cells was considered. In case of a color change, the MIC was registered at the lowest concentration. The mixtures between the sterile distilled water and the DMSO solvent and the nutrient broth were used as adverse controls by Streptomycin (antibacterial) (10 μg / 500 μL).

Phytochemical screening of M. oleifera bark aqueous extract
Different methods are used to qualitatively test phytochemical compounds contained in moringa oleifera (BEM) extracts. The test method used refers to the report Das et al. [29], Qualitative evaluations of several chemical contents are shown in Table 1.  Table 1 shows BEM containing phytochemical compounds such as alkaloids, flavonoids, saponins, and phenolics. These compounds play a role in the chelation process and are able to reduce metal ions to nanoparticles [26].
Total phenolic and flavonoid levels in BEM 21.65 ± 4.25 (µg / mg GAE) and 55.31 ± 3.82 (µg / mg CE) respectively in dry powder, as shown in Figure 1. Extracts of some parts of the plant contain different constituents with distinct functional groups, which can act as reducing or chelating agents in the formation of nanoparticles. Total phenolic and flavonoid levels in bark water extracts are lower than total phenolic and flavonoid levels in water extracts of other M. oleifera plants that have been previously reported. Siddhuraju and Becker [30] reported total phenolic content in leaves is 74.30 ± 9.00 (µg / mg GAE), while Mohammed and Manan [31] reported total phenolic content in ore is 101.79 ± 2.89 (µg / mg GAE). The total flavonoid content in leaf water extract was reported by Okumu et al. [32] amounted to 79.13 ± 13.04 (µg / mg CE).

Characterization 3.2.1. UV-Vis spectroscopy of BEM and MgONPs synthesized
Synthesis of MgO nanoparticles using Moringa aqueous extract followed by a change in color during the synthesis process. The color of the solution changes from clear (MgCl 2 solution) to dark brown when added with Moringa extract. After adding NaOH, the color of the solution changes to brighter, indicating the formation of MgO and Mg (OH) 2 complexes in the solution. Spectrum adsorption of MgO nanoparticles measured in the range of 200-800 nm. Figure 2 shows the UV spectrum with a sharp peak at around 290 nm, which confirms the formation of MgO nanoparticles [28]. Besides, the precursor ion Mg 2+ , MgCl 2 salt does not show a spectrum at the specified wavelength. The existence of a peak of about 280 -290 nm can be attributed to the formation of metal oxide nanoparticles after the addition of plant extracts and NaOH solution [28].

Size and morphology of MgONPs synthesized
In order to correctly assess the atomic position in the lattice structure, a crystalline stage and structure of the synthesized MgO nanoparticulate were studied using X-ray diffraction technologies. The XRD patterns of MgO nanoparticles manufacturing are shown in Figure 3

(2)
Where K is a constant dimension depending on the particular geometry of the target, λ is the wavelength of X-ray radiation, β is the full width at half maximum (FWHM) of the significant peaks in radians, and θ is the Bragg's angle. SEM has been used to carry out morphological tests of MgO nanoparticles synthesized with extract. Figure 3(b) shows an image of the MgO nanoparticles scanning electric microscopy (SEM) showing that the resulting MgO nanos are in the form of spherical particle of 20 to 80 nm. Figure 3 (c) and (d) shows TEM and PSA of MgO nanoparticles prepared. The results of particle size analysis (PSA) show that the MgO with particle size in the range of nanometer with relatively narrow distribution was produced. The samples obtained have the particle size in the range of 60-100 nm. The existence of spheres particles The structure of the synthesized MgO nanoparticles was confirmed by the spherical structure. The average particle size of the synthesized MgO nanoparticles measured between 40 -70 nm. MgO nanoparticles synthesized from leaf aqueous extract have narrower particle size distribution than that of MgO produced using bark aqueous extract, which was attributable to the presence of antioxidants-rich compounds (e.g. flavonoids, phenolic acids, reducing sugars, etc.) [34] [35]. They are biomolecules that play a vital role as outstanding bio-reducing and/or bio-capping agents towards generation of the nanoparticles [36].

Antioxidant
The antioxidant activity of BEM and MgONPs was assessed by DPPH testing using ascorbic acid as a positive control. The free radical capture activity of DPPH BEM and MgONPs is directly related to their concentration. DPPH is a stable compound and accepts hydrogen or electrons from BEM or MgO nanoparticles. This test is often used for the antioxidant activity of compounds present in medicinal plant extracts [37]. The antioxidant activity of MgONPs tends to be higher than that of BEM ( Figure 5)

Antibacterial
The

CONCLUSION
Green synthesis of MgO nanoparticles that were prepared using M. oleifera bark aqueous extract was successful. The formation of MgO nanoparticles in this synthesis was confirmed using UV-Vis absorption. The spherical crystal structure of MgO nanoparticles confirmed XRD analysis. The average particle size of the synthesized MgO nanoparticles measured between 60 -100 nm using SEM and TEM images and PSA results. Our study shows that