INTRODUCTION:

In advanced material science the field of Nanotechnology is most dynamic. Highly enhanced qualities are shown by Nanoparticles because of their special framework, size and classification.

There are many methods which are used for the formation of ZnO Nanoparticles, which include spray pyrolysis, chemical vapor deposition, gas phase method, hydrothermal synthesis, pulsed laser deposition, micro emulsion, sol gel method and microwave synthesis method. The green synthesis is more powerful than physical and chemical method because it is economical and environment friendly.¹

ZnO is an n-type semiconductor having very extensive and non-stop band gap nearly 3.3 eV at room temperature having dependence on the condition of their synthesis. Zinc-blende and wurtzite are the most common crystalline structures.²

Chemical, physical and biological methods are very simple and easy for the formation of zinc oxide therefore, it is extensively studied among the metal oxides nanoparticles in the previous fifteen years. Molecular beam epitaxy, chemical vapor deposition, thermal evaporation, pulsed laser ablation are successful physical methods of zinc oxide nanoparticles also chemical methods such as spray pyrolysis, hydrothermal, sol-gel and electrodeposition require high vacuum, high temperature, and occasionally unsafe solvents.^3,4

Instead of these formation techniques, the green synthesis method is advantageous because it is eco-friendly and inexpensive showing great purity and capacity in the formation of zinc oxide nanoparticles. Also the extracted compounds from the flowers, seeds, leaves, fruits, pods, roots and microorganisms were utilized for the formation of ZnO nanoparticles.^5,6

Moringaceae is a single genus family having trees and shrubs in it. A tree Moringa oleifera belongs to this family. Tuberculosis, diabetes, fever, ear infections, skin and stomach aches can be treated by using the leaves of this tree. ^7,8

The seeds of Moringa oleifera carry oil nearly 22-40% by weight which have fatty acids for example palmitic acid, oleic acid, Gallic acid, arachidic acid and oleic acid. Ca, K, P, S and Mg are some Mineral elements which are present in the flowers and leaves of Moringa oleifera.⁹

ZnO nanoparticles are synthesized by using the extracts of Moringa leaf utilizing Green method which isvery economical, eco-friendly, and easy to handle. ZnO nanoparticles synthesized from Moringa oleifera show a great activity against gram positive and gram negative bacteria.^10,11.

2. MATERIALS AND METHODS:

2.1 Materials:

All the synthetic compounds utilized in the exploration work are of Analytical grade. Moringa oleifera leaves are collected from flowerbed of Minhaj University Lahore and Zinc Chloride solution is purchased from lab market Anarkali bazar Lahore.

Figure 2.1 Chemicals used in research

2.2 Instruments and Equipments:

Sr. No	Instrument/equipment	Model Name	Company
1.	UV-VIS	2618-013	UH5300
2.	IR	380	Thermonicolet
3.	SEM	VEGA3-TESCAN	TESCAN
4.	Magnetic Stirrer	MS 300	BANTE
5.	Analytical balance	EJ 120	Snowern
6.	Water bath	C116	EISCO

2.3 Preparation of Stock Solution of Plant Extract:

Dry Moringa olefira leaves under the shadow and then grounded very well. After that dilute the 5.0 g of Moringa olefira leaves powder with 100ml of deionized water for some minutes and filter it with whatmann paper 41 and for further analysis prepared 1mM, 2 mM, 3 mM, 4 mM and 5 mM solutions from this stock solution.

Fig. a) Preparation of plant extract, b) Extract of Plant Moringa leaf, (c,d) Filteration of leaves (For removal of dust and other particles).

2.4 Synthesis of ZnO Nanoparticles:

As the precipitationreaction process accelerates, it will synthesize zinc oxide nanoparticles. First separate the extract of Moringa oleifera leaves and mix 3ml of it in 100ml of sodium hydroxide solution. Then add 1ml of this mixture to 200ml beaker with 10ml distilled water and add zinc acetate drop wise into the beaker and stir for 1 hour. Stir for 1 hour at 600rpm at 25°C. Finally, the precipitate is filtered and washed several times with diethyl ether. The precipitate is then dried in a vacuum for 8 hours and heated in a hot air oven at 30⁰C for 2 hours. After that zinc oxide nanoparticles are collected and stored. These stored particles are used for further characterization.

3.0 Characterization of Prepared ZnO NPs:

From the various techniques such as Uv-Vis, XRD, FTIR, and SEM, ZnO NPs characterization is done.

3.1 Sample preparation for FT-IR analysis:

The prepared nanoparticles in solution form were subjected to FT-IR analysis for characterization. Nano solutions were put on sample chamber in FT-IR device against standard solution one by one and analysis is completed. Analysis is done on Cary 630 model of FT-IR made by Agilent Technologies placed in department of chemistry at Quid-e-Azam Campus University of the Punjab Lahore. The existence of moietypresent on the plane of particles is determined by the study of FT-IR. For the FT-IR study sample composition of ZnO NP is 0.ImM/I.0g.

3.2 Sample preparation for UV-Visible spectroscopy analysis:

The prepared four different types of nanoparticles in solution form were subjected to UV-Visible spectroscopic analysis for characterization. Nano solutions were put on sample compartment in UV-Visible instrument against standard solution one by one and thus analysis was completed. Analysis is done on Ultra-3000 model of UV-Visible instrument made by Rittun company placed in department of chemistry at Quid-e-Azam Campus University of the Punjab Lahore. UV-Visible are used to illustrate ZnO NP made from green synthesis. Sample Composition of UV-Visible analysis are 0.1mM/1.0g.

3.3 Sample preparation for SEM analysis:

The prepared nanoparticles in solution form were subjected to SEM analysis for characterization. Nano solutions were first dispersed and then coated on a thin film dried and then analyzed. Analysis is done on Hitatchi SUB8230 regulars model of SEM placed in department of chemistry at Quid-e-Azam Campus University of the Punjab Lahore. Scanning Electron Microscopy (SEM) is a progressive, reasonable method which springs us a clue on external texture, shape of particle and absorbency. Additional porous arrangement extra Will be the worth of fraction elimination.

3.4 Sample Preparation for XRD:

Sample Composition of nanoparticles are 0.1mM/1.0g.

3.5 Antibacterial Activity:

One of the major goal of thatinvestigation is to check the antibacterial of manufacture ZnO nanoparticles of various sizes. For this purpose, two bacteria are selected from two different categories Gram negative bacteria E. coli and Gram-positive bacteria Staphylococcus. Bacterial culture is grown using agar media approach provided with optimum pH, temperature, humidity, nutrients, and time for reproduction in controlled environment according to world-wide accepted protocol at advanced biological lab in the school of biochemistry and biotechnology Quid-e-Azam campus University of the Punjab Lahore.

Antibacterial activity is investigated at pH 8, temperature 35-40^◦C for 12 to 24 hours against E. coli and Staphylococcus bacteria by synthesized ZnO nanoparticles. Major steps are adopted to check the antibacterial activity of synthesized ZnO nanoparticles including selection of bacterial cultures, methodology of bacterial growth, adjustment of the optimum conditions, sterilization of petri dishes, transfer of bacterial culture in agar media plates, application of um litter samples of synthesized ZnO nanoparticles, time allowed to observe antibacterial activity and reporting of results. Moreover, during this whole antibacterial assessment health hazard precautions are acknowledged.

The antibacterial activity of ZnO nanoparticles is successfully tested against E. coli and Staphylococcus by means of Agar-plate media approach in vitro. The samplesare provided same concentration of nanoparticles for 24h, at pH 8 with provided temperature of 35-40^◦C. The magnificent results are obtained at 24 h, and this antibacterial activity is monitored after 3h, 6h, 9h, 12h and 24h using sample of nanoparticles against Gram negative and Gram-positive bacteria.

Sr. No	Staphylococcus Gram (+)	E. coli Gram (-) bacteria
Zone of Inhibition ZOI (mm)
1.	11 mm	8mm

4.0 RESULTS AND DISCUSSION:

4.1 FTIR Spectra Analysis:

The characteristics FTI-IR spectra is obtained for ZnO nanoparticles using range of 3600-500 at scan rate of 40 with resolution of 2 in a good status system. This analysis provided the information about various bonds that are present in the sample of nanoparticles.

Fig 4.1 FTIR Spectra for ZnO NPs (Synthesized by Moringa Leaves).

The Fig 4.1 shows that Various peaks indicate the existence of manymoiety in the sample of nanoparticles. ZnO nanoparticles shows peak at 500 Cm^-1

4.2 UV-Visible Spectra Analysis:

To characterize ZnO nanoparticles UV-Vis spectroscopy is one of the easiest approaches in nanoscience.

Fig 4.2: UV-Visible spectra for ZnO NPs

As ZnO nanoparticles show a stronger adsorption band near 290-315nm in the visible region because of the surface plasmon resonance.

4.3 XRD Spectra for ZnO NPs:

The XRD patterns of the combined ZnO NP show that all peaks of the roots of the ZnO NPs are the same as the standard ZnO NPs data. The height of the layers (Figure 4) of the XRD is very well compared to the formation of a six-sided wurtzite.

The sharp and deep heights show that the samples are very high crystals. The design can be registered by splitting from different planes. No other pollution-related separation peaks are available, which ensures the high purity of the composite products. From XRD data, the peaks are found to be wider; suggests that crystallites are at nanometer size and the width (D) is calculated using the Debye-Scherrer formula 𝐷 = Kλ / βcosθ

When K remains a constant Scherrer, λ is the X-ray wavelength, β is the maximum height of the upper half, and θ is the Bragg angle. The average crystallite size of zinc oxide is measured by XRD data, obtained approximately 48nm.

4.4 SEM Analysis:

After confirmation of XRD results, the sample is pre-presented in the SEM study. The size, shape, and more morphology of the ZnO NPs are shown in the SEM image, as shown in the figure. Detailed classification of the structure shows that the products made are rounded and made of crystal, and their diameter was about 48nm.

Fig. SEM Analysis for ZnO NPs

4.5 Antibacterial Activity:

The data obtained after systematic antibacterial activity; it is found that the synthesized ZnO nanoparticles showed an excellent antimicrobial action. Table 5.1 shows the comparative rates of the zone of inhibition of selected bacteria. In addition, the antimicrobial action of synthesized ZnO is greater on Gram positive bacteria than Gram negative bacteria; it might be due to more multi-drug resistance of E. coli than Staphylococcus bacteria. Synthesized NPs are of 2nm size and has higher microbial activity because Size of NPs is inversely related to antimicrobial activity.

Fig. (a) Gram (+) bacteria, (b) Gram (-) bacteria. The figure shows that Gram + bacteria show higher Zone of inhibition than Gram – bacteria.

The infectious diseases concern to health growth of living organisms including human beings, these are related to medical field and their prevention, treatment and cure is necessary because these can cause long term disability along health regularity cost of the patients, and sometimes could be fatal. Therefore, proper cure of such diseases enhances the probability of resistance of multi-drug microorganisms.

CONCLUSIONS:

In this Paper we have used Moringa oleifera leaf as natural precursor for the production of zinc oxide nanoparticles through a very simple and effective precipitation method. The major benefit of this production method is its simple and cost-effective route. Using this process, we can use Moringa oleifera leaf to produce zinc oxide nanoparticles. SEM and XRD are used to confirm the synthesis of zinc oxide nanoparticles. Zinc oxide nanoparticles are also synthesized for antibacterial activity to be studied against pathogenic diseases.

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Received on 10.01.2022 Modified on 17.03.2022

Asian J. Pharm. Tech. 2022; 12(3):213-217.

DOI: 10.52711/2231-5713.2022.00035