Syzygium cumini-mediated Green Synthesis of Magnesium Oxide Nanoparticles and Evaluation of their Antibacterial, Antileishmanial, and Antioxidant Activities

Green protocols for the synthesis of nanoparticles have gained signi�cant attention due to their environmental friendliness, ease, and cost-effectiveness. Objectives: to synthesize magnesium oxide nanoparticles (MgO-NPs) using aqueous leaves extract of Syzygium cumini plant, and to investigate the antimicrobial and antioxidant potential of the synthesized NPs. Methods: The synthesis of MgO-NPs was achieved by mixing a solution of magnesium nitrate + (Mg (NO ) with an aqueous extract obtained from S. cumini leaves to reduce the Mg ions. These 3 2 NPs were characterized by X-ray diffraction (XRD), Fourier-Transform Infrared (FTIR) Spectroscopy analysis, Scanning electron microscopy (SEM), and Energy-dispersive X-ray (EDX) analysis. Results: The transformation in color of the solution from yellow to deep brown along with the UV absorption peak at 294 nm showed the effective synthesis of MgO-NPs. SEM and XRD data revealed cubic-shaped NPs with an average size of 23.73 nm. EDX analysis con�rmed the presence of magnesium and oxygen in the sample at 31.85% and 35.11% weight percentages, respectively. The antibacterial evaluation demonstrated effectiveness against the gram-negative strains Citrobacter koseri and Pseudomonas aeruginosa , with inhibition zones of 28.1 ± 1.25 mm and 27.8 ± 1.25 mm, respectively. MgO-NPs also showed antileishmanial potential against Leishmania tropica promastigotes (68.41 ± 0.05% inhibition at 1000µg/ml). Furthermore, the NPs exhibited antioxidant properties (75.12 ± 4.29% at 1 mg/ml) as determined by the DPPH radical scavenging assay. Conclusions: MgO-NPs synthesized using S. cumini plant leaves extract hold promises as agents for antibacterial, antileishmanial, and antioxidant applications.

gas-phase deposition, and wet precipitation, despite some limitations [4,5,6,7].Advancements in synthesis have made it possible to create eco-friendly and medically useful substances.These substances have properties in various applications [8].Synthesis strategies that employ natural extracts and water are known as green synthesis [9].This method of synthesis is gaining popularity due to its eco-friendliness, simplicity, and non-toxic, especially when using plant extracts.Syzygium cumini (Jamblang, Jamun) is a Myrtaceae tree that has spread naturally from its native Asia to South America, Africa, and parts of the US .The S. cumini plant has potential in various applications

I N T R O D U C T I O N
Nanotechnology involves manipulating materials at the atomic or molecular level, with one dimension in nanometers.Metal nanoparticles (NPs) have recently gained signi cant attention due to their unique features associated with bulk metals.The distinct characteristics of nanoparticles at the nanoscale directly impact their various physical and chemical properties [1,2].Due to their unique properties, nanoparticles have the potential to be used as a new antimicrobial drug to combat the growing threat of infectious diseases and antibiotic resistance [3].Various techniques are available to synthesize MgO-NPs, including sol-gel synthesis, hydrothermal re ux, chemical were purchased from Sigma Aldrich® (MA., USA).All chemicals and reagents used in the experiments were of minimum analytical grade and used without modi cation.Preparation of Syzygium cumini Aqueous Leaf Extract S. cumini leaves were cleaned with tap water and deionized water to remove dirt and particles.The leaves were dried in the shade and crushed into a ne powder using an electric blender (Daigger Scienti c®, USA). 25 g of powder and 250 ml of deionized water were mixed in a water bath for 10 minutes.Then the mixture was incubated in the dark under continuous stirring at 24 ℃, for 12 days.The solution was ltered through Whatman No.1 lter paper, and next, the crude aqueous extract was subjected to water evaporation using a rotary evaporator (RE 100-Pro, Biobase®, China) at 70 ℃.The crude extract was acquired and then placed in an airtight container, where it was stored at a temperature of 5 °C for future utilization.

Green synthesis of MgO NPs
Magnesium oxide nanoparticles were synthesized according to the previously reported protocol with minor modi cations.50 ml (10 mg/mL) of S. cumini aqueous leaves extract was dropped into 50 ml (0.1 M) magnesium nitrate aqueous solution at 50 ℃ for 3 hours on a magnetic hotplate stirrer (MS300HS, Korea).After obtaining a solidliquid dispersion, the mixture was centrifuged at 10,000 rpm for 10 minutes.The excess Mg (NO ) .6HO and 3 2 2 remaining organic molecules were then removed by washing the residue with de-ionized water.To obtain MgO NPs, the residue was dried for 2 h at 70 °C and then calcined

M E T H O D S
for the same time at 300 °C.

Characterization of the Synthesized NPs
The physical and chemical characteristics of MgO NPs were studied using a variety of characterization techniques.For this purpose, UV-vis spectrophotometer (UV-1800 Spectrophotometer, Shimadzu, Japan) in the range of 200-800 was used to con rm the production of NPs, Fourier transformed infrared spectroscopy (FTIR) (L1600300 Spectrum TWO Lita, UK.) was employed within the range of -1 4000-400 cm to identify the capping and stabilizing molecules.Morphological properties of the NPs were evaluated by scanning electron microscopy (SEM) (JSM 5910, JEOL, Japan), and their elemental structure was determined using EDX (INCA 200, Oxford Instruments, UK.).
The thermally annealed samples were analyzed with an Xray diffractometer (JDX.3532,JEOL, Japan) and the corresponding size was determined using the Scherrer equation [15].

Biological Activities Antibacterial Activity
The study tested the antibacterial properties of Magnesium Oxide (MgO) nanoparticles using an agar well diffusion assay [16].The nanoparticles were tested against G r a m -n e g at i ve b a c te r i a l s t r a i n s ( Ps e u d o m o n a s aeruginosa, Providencia stuartii, Proteus vulgaris, Klebsiella pneumoniae, Citrobacter koseri) and Gram-positive (Staphylococcus aureus).Different concentrations of MgO NPs (25, 50, 75, and 100 µg /mL) were tested for their dosedependent activity.The test was conducted in triplicate and the average was taken to determine the antibacterial activity.

Anti-leishmanial Activity
Following the approach, the anti-leishmanial e cacy of MgO nanoparticles was investigated [17].In 96-well microtiter plates supplemented with 10% FBS, 1% (Pen Strep) antibiotic, and 1% HEPES buffer, promastigotes Leishmania tropica was grown and then subjected to various doses of nanoparticles over a period of 48-72 hours at a temperature of 25 °C.Following incubation, the viability of promastigotes was determined using a tetrazolium-dye (MTT) colorimetric approach.Percent inhibitions were derived using the following formula:

Antioxidant Activity
To evaluate the antioxidant capabilities of NPs and ascorbic acid, the DPPH test was utilized [18].A mixture of a 1-millimolar DPPH solution and a 50% methanol solution was prepared, and diverse amounts of NPs and ascorbic acid were introduced.The resulting mixture was incubated for 30 minutes, and the absorbance at 517 nm was subsequently measured.The effectiveness of each spectrophotometer in the range 400-4000 cm as shown in -1 gure 3. The absorption bands appear at 3690 cm , 2217 cm , 1784 cm , 1352 cm , 879 cm , 834 cm , and 522 cm .
-1 -1 The two bands at 3690 cm and 2217 cm were caused by compound was evaluated in triplicate, and the scavenging ability was determined using a formula: Where A0 is the control (DPPH) sample absorbance and At is the test sample absorbance.

Statistical Analysis
We analyzed the data using OriginPro® version 9.8, 2021 or GraphPad Prism® version 7.0 and reported the results as the mean ± standard deviation (SD) of triplicates.

Characterization of the Synthesized MgO Nanoparticles Physical appearance and UV spectroscopy Analysis of MgO-NPs
Figure 1 shows that the aqueous extract of S. cumini leaves turned dark brown when magnesium nitrate solution was added.The UV absorption peak ( gure 2) was measured at 294 nm, which is speci c for MgO-NPs.The band gap energy (4.2eV) and visual evidence suggest that the NPs are small and have a band gap, making them suitable as an antibacterial agent

Energy-Dispersive X-Ray Analysis
The chemical compositions of the MgO-NPs were determined by EDX analysis.

Antileishmanial Activity
Our study on the impact of MgO-NPs on the growth of L. tropica culture is presented in figure 9. Different concentrations of NPs (250-1000 µg/mL) were used and measured the percentage of inhibition over 48 to 72 hours.To assess the e cacy of the NPs, we compared them to Miltefosine (standard antileishmanial drug) and DMSO, a negative control.Our ndings indicated that the inhibition of parasites increased in proportion to the concentration of NPs used.Speci cally, at 1000µg/ml, the NPs inhibited 68.41 ± 0.05%, at 500µg/ml they inhibited 55 ± 0.14%, and at 250µg/ml they inhibited 34.03 ± 0.28%.The IC value was 50 452µg/ml which suggest that these NPs have the potential to serve as an antileishmanial agent.

Antioxidant Activity
Absorbance at 517 nm was used to calculate the radical scavenging activity of DPPH.MgO-NPs showed moderate radical scavenging activity and increased effectiveness with concentration (0.1-1 mg/ml).Ascorbic acid (standard antioxidant) exhibited more e cient radical scavenging capability than the NPs (Figure 10).At 1mg/ml, the antioxidant action of the NPs increased from 8.68 ± 0.61% to 75.12 ± 4.29% with an IC50 value of 1.92mg/ml.alkynes, amino groups, and magnesium oxide.Reduction in peak intensity con rms the involvement of organic molecules in the synthesis.Peaks in the broader and narrower range indicate the stretching of alkynes and respectively [21].The sample has a phase and multiple indexed crystal planes.It's worth noting that no secondary peaks were detected, indicating that the NPs are pure and free from impurities.A published study showed that the synthesized MgO-NPs had a cubic structure and minimal impurities [22].These NPs under SEM had a cubic morphology with a small size along with clusters which are due to synthetic methods and biological incapacitation of phytochemicals.Elemental analysis con rmed the purity of NPs and suggests the formation through metabolites in the plant.Previously, MgO-NPs were spherical with an average diameter of 36.74 nm, and have aggregated into dense, irregular, pointy akes [23].The synthesized substance consistently exhibits antibacterial activity against all strains.NPs may kill bacteria by disrupting the membrane.Increasing the concentration of MgO-NPs enhances their effectiveness.Small size improves their antibacterial properties against both gram-positive and gram-negative bacteria.The increase in surface area results in more active sites on the nanoparticle's surface, displaying signi cant antibacterial properties [24].MgO-NPs showed antileishmanial activity against L. tropica promastigotes by increasing concentration led to more inhibition.MgO-NPs also inhibited promastigote infectivity in a dose-dependent manner [25].Furthermore, our study on the antioxidant potential of NPs found that increasing the concentration of the sample improved their radical scavenging abilities [1].
The use of Syzygium cumini leaf extract is an affordable, eco-friendly, and safe method for producing magnesium oxide nanoparticles (MgO-NPs).The synthesized material underwent comprehensive analysis using various techniques, such as UV-Vis, FTIR, EDX, SEM, and XRD.MgO-NPs demonstrated a remarkable ability to ght against pathogenic microorganisms.Furthermore, the NPs showed potential in inducing parasite cytotoxicity, which could be useful in antileishmanial treatments.The NPs also displayed signi cant antioxidant properties, making them useful as an antioxidant.
of the O-H bond.Band 1784 cm is attributed to the stretching vibration of C=C, whereas band -1 1352 cm results from the stretching vibration of C=C and -1 the bending of the N-H bond.The bands at 879 cm and 834 -1 cm correspond to the bending vibration of the C-H and C-O -1 bonds, respectively, whereas the peak at 522 cm reveals the presence of MgO.38 FBT VOL. 3 Issue.2Jul-Sep 2023 LLP Copyright © 2024.FBT, Published by Lahore Medical Research Center FBT VOL. 4 Issue. 1 Jan-Mar 2024

Figure 1 :
Figure 1: Transformation of yellow color Syzygium cumini extract (B) to dark brown (C) upon the addition of colorless magnesium nitrate solution (A).
Figure.6 demonstrates that the NPs are pure, and the presence of Mg and O ions indicates the successful formation via metabolites contained in the aqueous extract of the S. cumini plant.The quantitative examination revealed that the samples' weighted percentages of Mg and O ions were 31.85 and 35.11 %, while their atomic percentages were 25.58 and 42.85 %, respectively.

Figure 6 :
Figure 6: Energy dispersive X-ray spectroscopy of MgO-NPs using EDX (INCA 200, Oxford Instruments, UK).Biological Applications Antibacterial ActivityThe antibacterial potential of MgO-NPs was investigated by assessing the zone of inhibition against the tested bacterial strains.All bacterial strains were tested with Cipro oxacin as a positive control as depicted in Figure.7.Zone of inhibition was 28.1±1.25 mm (C.koseri), 27.8 ± 1.25 mm (P.aeruginosa), 27.3 ± 1.04 mm (P.vulgaris), 25.1 ± 1.04 mm (P.stuartii), and 24.8 ± 1.25 mm (K.pneumoniae) for gram-negative bacteria and 25.8 ± 0.76 nm for grampositive bacteria.C. koseri (28.1 ± 1.25 mm) was the most susceptible gram-negative strain to the highest concentration.The presence of an inhibitory zone shows that these NPs kill bacteria possibly by disrupting membranes and generating surface oxygen species.Size of i n h i b i to r y z o n e va r i e d by p at h o g e n a n d NPs concentration (Figure8).In this work, increasing the concentration (25-100 µg/ml) of MgO-NPs boosted growth inhibition due to nanoparticle diffusion in agar media.

Figure 9 :
Figure 9: Assessment of Leishmaniasis inhibition by MgO-NPs.(A) represents the inhibition in the negative control using DMSO, while (B, C, D) show the inhibition at different concentrations of MgO-NPs.(E) displays the inhibitory effect of the positive control Miltefosine.Antioxidant ActivityAbsorbance at 517 nm was used to calculate the radical scavenging activity of DPPH.MgO-NPs showed moderate radical scavenging activity and increased effectiveness with concentration (0.1-1 mg/ml).Ascorbic acid (standard antioxidant) exhibited more e cient radical scavenging capability than the NPs (Figure10).At 1mg/ml, the antioxidant action of the NPs increased from 8.68 ± 0.61% to 75.12 ± 4.29% with an IC50 value of 1.92mg/ml.