Biosynthesis, Characterization, Adsorption and Antimicrobial studies of Manganese oxide Nanoparticles Using Punica Granatum Extract

topical concentration and bactericidal effects 7 . In this study, we sought to synthesize


Introduction
Nanotechnology is a burgeoning area of study in medicine.It is the responsibility of nanoparticles, an intermediary between micro materials and atomic structures, to improve the physical qualities such as surface area and volume ratio 1 .The development of novel medications has used the therapeutic characteristics that herbal plants and their derivatives contain 2 .MnO2 has attracted the attention of numerous researchers due to its impact and electromagnetic properties 3 .MnO2 has been synthesized using various techniques, including self-reacting microemulsion, deposition, and solid reaction 4 .However, using natural compounds to decrease and stabilize Mn metal into nanoparticles is more environmentally friendly, less expensive, and more straightforward than the preceding procedures 5 .Escherichia coli, Klebsiella pneumonia, and Pseudomonas aeruginosa are pathogenic microorganisms that can harm individuals with weakened natural defenses and result in severe systemic disease 6 .Due to their nanoscale size, NPs can penetrate biofilms and bacterial cell walls and have a cytotoxic effect.They can also increase the effectiveness of current antibiotics by preventing their detection and providing a method of targeted delivery to microorganisms to maximize their topical concentration and bactericidal effects 7 .In this study, we sought to synthesize MnO2 NPs using Punica Granatum extract as the reducing and

Materials and Methods
Obtaining samples Punica granatum was gathered and marked from a nearby source, and we used hydrated manganese sulfate.

Preparation of Punica Granatum extract and MnO2 NPs
Deionized water has been used to wash the fresh pomegranate peel and eliminate dust.To create homogeneous powders, the dry pomegranate peel is carefully blended in a mixer.After that, 20 g were ground up and combined with 200 ml of deionized water.The mixture was heated for 30 minutes at 60 °C while being stirred.After filtering, the solution was placed in the refrigerator.From the preparation of MnO2, NPs were created using the green synthesis method.As a result, 0.1M, 50 ml of MnSO4.H2O, and 100 ml of pomegranate peel extract were added slowly (one drop per second) and stirred for 30 minutes.The solution was then given 40 ml of 1 N NaOH.The pH level rose to 10-12.The result was a dark black crystal precipitate cleaned with deionized water (all steps were done with centrifuge and then decantation).They were dried for 4 hours at 120 °C before being dried again for 4 hours at 250 °C.Manganese oxide nanoparticles were produced as a black powder.

Adsorption study
To create a stock solution, 10g of CoCl2.6H2O were dissolved in one litre of distilled water to produce 10000 ppm.The stock concentration for NiCl2.4H2O and CuCl2.2H2O was 5000 ppm because the stock was made by dissolving 5g in 1 litre of distilled water.

Adsorption of metal ions on the surface of MnO2
NPs was performed by adding 0.1 g of the adsorbent nanoparticle to 50 ml of a 1000 ppm metal ion solution in a shaker water bath at 26 ºC and shaking at 150 revolutions per minute (rpm).The adsorbent was then separated from the solution at specific times by centrifuging.A visible spectrophotometer measured the remaining clear solution to determine the remaining concentration after adsorption using the calibration curve

Biological Activity Study
Using the disc diffusion method in a nutrient medium (jellos medium) type Muller Hinton agar, the antimicrobial activity of the synthetic MnO2 NPs in concentrations of about (25, 50, and 75) mg/L, was tested against two reference bacterial strains (G+) Staphylococcus aurous, and (G-), Escherichia coli, and the fungus Candida albicans.Likewise, the antifungal activity of a nutrient medium based on potato dextrose was measured using the same method.

FT-IR analysis
The FTIR of MnO2 shows in Fig. 1, bands at 590 and 532 cm -1 in the FTIR spectra of MnO2 are attributed to the Mn-O stretching mode, proving the presence of the Mn-O bond in the MnO2 structure 8,9 .

UV-Visible analysis
The UV-Vis absorption spectrum of the biosynthesis of MnO2 NPs is shown in Fig. 2. The absorption peak in this spectrum at 348.0 nm was due to the transition holes process between Mn and O.

EDX analysis
The EDX spectrum of MnO2 Nps shows the expected peaks for manganese and oxygen.Having  SEM and TEM analysis SEM and TEM were used to determine the Morphology and shapes of nanomaterials.Low amounts of rods in nanostructured, unconsolidated forms of MnO2 NPs can be seen in SEM and TEM measurements of Figs. 5 and 6.In the TEM picture, the MnO2 nanoparticles emerged as UNconsolidated structures at the nanoscale.It should also be highlighted that the samples exhibit high pore content, which distinguishes them in adsorption applications.The shape of Mno2 nanoparticles was found to be packed together in the TEM image.Due to the accuracy of the measurement, the sample's shape cannot be determined with absolute certainty, but it appears to contain measurements of the sample's spherical internal structure that are zero-dimensional (all of its dimensions are nanoscale), which is highly preferred in surface chemistry for nanomaterial's 10 .

AFM analysis
AFM surface analysis must be thoroughly examined due to numerous factors, such as deformations or image artifacts resulting from a tip and contamination, which may produce misleading results.The decision to operate in contact or without contact is one of the critical factors.The contact mode, or degree of surface contact, between the sample and its tip, damages MnO2 NPs severely.The tip is placed very close to the sample but not in contact with it; hence the only mode necessary for this task is the non-contact one.In terms of optical behaviour, Figs.7 and 8 shows the development of three-dimensional spherical clusters of MnO2 Nps following metallization.Due to the environmentally friendly synthesis of the nanomaterials, the sample's surface has pores, is highly rough, and tends to have an amorphous shape 11,12 .The size of the prepared oxide nanoparticles ranges between 15.00 to 50.00 nm, as shown by the Height Accumulation Distribution Report of MnO2 NPs.This confirms that the manganese oxide made using pomegranate peel extract is a nano oxide 12 .

Adsorption Study
For each ion, the adsorption time profile was shown in a comparison of the adsorption behaviour of the prepared MnO2 nanoparticles.The fact that Co (II) exhibits continuous adsorption growth suggests that the process is far from equilibrium and that this is not a straightforward type of adsorption.Instead, it is a precipitation process in which metal oxide nanoparticles act as crystallization nuclei to cause the crystallization of the cobalt chloride salt.The plateau of equilibrium is more distinct for Ni (II) and Cu (II), particularly for Ni (II) Fig. 9.The MnO2 surface is the largest in an alternate form.This arrangement may be caused by convergences between the atomic radius of the element and that of adsorbate metal ions, which make them easily incorporate with the metal oxide's lattice active sites [13][14][15] .The adsorption rate of Ni(II) is the highest in the time scale and conditions of our experiment at all surfaces, whereas Co(II) and Cu(II) ions are close in magnitude, as shown by the above figures.The adsorption process' rate is influenced by (i) charge, (ii) size, and (iii) electronic interactions.Since all ions have the same charge, the first factor (charge) cannot be the leading cause of this difference.Size influences the diffusion process in both the bulk of the solution and the adsorbent mass 16 .According to this theory, Co (II) ought to have the highest adsorption rate, followed by Ni (II) and then Cu (II).However, the observed decrease in the Co (II) adsorption rate and the unrestricted linear growth of the adsorbed portion suggest that there is still another process occurring along with adsorption, which is the Co (II) oxidation by metal oxide 17 ; the adsorption percentage of ions on the surface of manganese oxide of mixed Co, Ni, and Cu were 32.79 %, 75.00%, and 30.20%.

Antimicrobial Study
The antibacterial activity of the synthesized MnO2 nanoparticles was tested using the agar well diffusion method against the bacteria Escherichia coli, Staphylococcus aureus, and the Candida fungus in different concentrations of 25, 50, and 75 mg/L 18,19 and compared with Amoxicillin and Metronidazole as a drug, DMSO solvent medium served as the controls as antibiotics.The antimicrobial activities of the MnO2 nanoparticles were evaluated by examining the inhibition zone of growth against the used pathogens and adjusting the concentration of the nanoparticles.Table 2, Fig. 10 show the inhibition zone of growth in (mm) of MnO2 NPs against the bacterial pathogens, two Bactria, and one fungus [20][21][22][23][24] .

Conclusion
MnO2 NPs with a crystal size of 30.94nm were synthesized by a biosynthetic method using Punica Granatum extract and MnSO4.H2O salt as starting materials.The product was diagnosed and confirmed to be a nanocomposite by several techniques, including EDX, AFM, SEM, TEM, EDX, UV-vis and IR.It was found that manganese oxide has a thin cluster morphology in its total form.They exhibit antimicrobial activity that significantly slows down bacterial species Escherichia coli and Staphylococcus aureus growth.As well as antifungals.The adsorption of three metal ions, Co, Ni, and Cu, was also studied at the same time with the removal from water by MnO2 NPs, i.e. they are percent effective at 32.79 %, 75.00%, and 30.20% in removing salts and heavy elements that are considered water pollutants.

Figure 9 .
Figure 9. Adsorption time evolution of the metal ions on the MnO2 surfaces.

Figure 10 .
Figure 10.The Inhibition zone of growth.