Neem (Azadirachta indica) leaf extract mediated synthesis of zinc oxide nanoparticles (ZnO NPs) and their antibacterial activity

Abstract

In this study, we prepared zinc oxide nanoparticles using a quick, safe, and cost-effective method by reducing Zn(NO₃)₂·6H₂O solution with Neem (Azadirachta indica) leaf extract. Qualitative phytochemical screening and FT-IR spectroscopy measurements were employed to validate the presence of active biomolecules such as Flavonoids, phenols, alkaloids, terpenes and tannic compounds. FT-IR, UV–Vis, and XRD spectroscopic techniques were utilized to fully analyze the biosynthesized nanoparticles. The spectrum of UV–Visible spectroscopy indicated UV–Vis spectrum of 321 nm. FTIR spectra showed the absorption peak for the stretching vibration of Zn–O at 544 cm⁻¹. The results obtained supported the formation of ZnO NPs employing A. indica leaf extract as a reducing and stabilizing agent. X-ray diffraction spectrum analysis was also used to investigate the crystal structure. The particle size of ZnO NPs was calculated using the Scherrer’s equation and the result was found to be 19.16 nm. Furthermore, the antibacterial potential of zinc oxide nanoparticles against two clinical strains of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) bacteria was examined by paper disc diffusion method. The result showed a significant inhibition zone of 18 mm against E. coli and an inhibition zone of 15 mm against S. aureus.

Graphical Abstract

Introduction

The Greek word “dwarf,” which refers to particles with sizes between one and one hundred nanometers, is the source of the term “Nano” [1]. Recent reports show that nanotechnology deals with the distribution and morphology of materials having sizes less than 100 nm, and it depends on the nature of Nanoparticles, which are atomic or molecular aggregates [2]. Nanoparticles exhibit distinct physical, chemical, optical, and biological characteristics due to their quantum size effect and large surface area to volume ratio [3, 4]. This feature of nanoparticles enables them to be more reactive than the bulk material as atoms on the surface tend to be more active than those at the center. Changing the size of particles from micrometers to nanometers will bring changes in their properties, such as electrical conductivity, hardness, active surface area, chemical reactivity, and biological activity. This is because a high-surface-area particle has more reaction sites than a low-surface-area particle, which leads to enhanced chemical reactivity [5, 6].

Among the various nanomaterials, metal oxide nanoparticles have drawn a lot of interest from the scientific community due to their tunable size and numerous special qualities, including high chemical stability, high electrochemical coupling coefficient, wide range of radiation absorption, high photostability, etc. [7, 8]. Within the large family of metal oxide NPs, ZnO NPs have attracted considerable attention due to their unique properties, such as their biocompatibility, wide and direct band gap (3.3 eV), and large excitation binding energy (60 meV) [9]. ZnO NPs are widely used in electrochemical sensors [10], photovoltaics as a transparent conducting oxide electrode [11], wastewater treatment, antibacterial, and drug carriers [12, 13], medicine [13], degrading environmental pollutants [14] and photocatalysis [15]. Treatment of rice with Zn based bio fertilizers such as biosynthesized ZnO NPs is also an economical and ecofriendly route for agriculturists to control Zn deficiency in crops to obtain high yield [16–18].

Zinc oxide nanoparticles (ZnO NPs) are significant in antibacterial applications because of their potent ability to kill a broad spectrum of bacteria, even those resistant to conventional antibiotics. The chemical mechanism of action involves a series of steps, beginning with the release of ions and the subsequent generation of reactive oxygen species (ROS) by NPs when interacting with bacterial cell membranes. Zinc oxide can change its physicochemical characteristics under the influence of the environment, and unfortunately depending on its concentration, ZnO can be hazardous to the life of living organisms and ecosystem safety. Therefore, special attention should paid to the control of the release of toxic zinc ions from the solution, especially in vivo studies [19–22]

ZnO NPs are synthesized by several physical and chemical methods such as the hydrothermal, solvothermal, microemulsion, microwave, and thermal methods. Although these physical/chemical approaches are efficient in producing nanoparticles of various sizes and morphologies, they employ expensive equipment, toxic chemicals, non-biodegradable stabilizing agents, potentially hazardous organic solvents [23–25]. To minimize these problems, a safe, cost effective, less hazardous and environmentally friendly synthesis procedure has been developed, namely the biological or green method using plant extract with a low concentration of the chemicals.

ZnO NPs synthesis using plant extracts offers significant environmental benefits compared to traditional chemical. This method eliminates the use of harsh chemicals, reduces toxic waste generation, and utilizes renewable plant-based materials, making the process significantly more eco-friendly and sustainable [26]. As a result, the interest in the synthesis of nanoparticles has shifted towards “green chemistry” and bio-processor approaches. Green chemistry is the design of chemical products and processes to minimize or to eliminate the use and generation of hazardous substances and was developed in principle to guide chemists in their search towards greenness [27, 28]. Green synthetic procedures have gained rising popularity as they can develop clean technology that proactively affects the design of nanomaterials to provide a benign atmosphere for promoting environmental sustainability [9].

Green methodologies facilitate the reduction of dissolved metal ions to zero valence state and eventually lead to the formation of nanoparticles. Components found in plants can function as stabilizing, chelating, reducing, and capping agents when producing nanoparticles [15]. Literature review reports showed that ZnO NPs have been synthesized from various plant extracts such as Parthenium Hysterophorus [29], Garcinia Mangostana [30], Aloe vera [31, 32], Sarcopoterium Spinosum (L.) Spach [33], Murraya Koenigii [34], Albizia Lebbeck Stem bark [35], etc.

Although using green chemistry-based synthesis has advantages over traditional methods for the environment, there are still some unanswered questions regarding the consistency of particle size and shape, reproducibility of the synthesis process, and understanding of the mechanisms involved in producing the nanoparticles via green entities [36]. Therefore, more investigation is required to examine and understand the current green synthesis-dependent processes. Thus, this study investigates the use of Azadirachta indica leaf extract as a capping and reducing agent in the synthesis of ZnO NPs.

Azadirachta indica plant is an evergreen tree that belongs to the Meliaceae family and is found commonly in India, Africa and America [37–39]. Azadirachta indica plant is considered to be the richest sources of drugs for traditional medicine, modern medicine, food supplements, folk medicine, pharmaceutical intermediates and chemical entities for synthetic drugs [36, 38]. The Chemical components incorporate many biologically energetic compounds that can be extracted from A. indica like alkaloids, flavonoids, saponins, Tannins, Catechines, Gallic acid, Limonoids, phenolic compounds, carotenoids, steroids and ketones. [38, 40]. Figure 1 shows some of the chemical components of A. indica plant.

Fig. 1.

Types of extracts and major compounds found in Azadirachta indica plant

Materials and methods

Materials

All the chemicals used in this study were of analytical reagent grade, commercially available and used without further purification. Zinc nitrate hexahydrate (Zn(NO₃)₂·6H₂O, 98.5%) from Loba Chemie Pvt. Ltd, India, sodium hydroxide (NaOH, 98%) from Blulux Laboratories (P) Ltd; Gentamicin from Abcekadtek (P) Ltd, and other chemicals were purchased from different companies. Double distilled water was used throughout the experiment.

Neem (Azadirachta indica) leaf extracts preparation

Azadirachta indica leaf extract was used to prepare ZnO NPs on the basis of cost-effectiveness, its medicinal value and ease of availability. Thus, relatively fresh and healthy A. indica leaves were collected from the local area of Bahir Dar city, Ethiopia. The surface of the leaves were rinsed thoroughly first with running tap water followed by distilled water to remove debris and other contaminated organic contents. Thereafter the leaves were cut into small pieces and then homogenized using mortar and pestle. The aqueous leaves extract was prepared by placing 5 g of washed and fine cut leaves in 250 mL flask along with 50 mL of distilled water. The mixture was then heated at 60 °C for 10 min.

Then, the extract is cooled to room temperature and filtered through Whatman No.1 filter paper to remove particulate matter and to get clear solution. The filtrate was collected and then stored in a refrigerator at 4 °C for further use in the synthesis of ZnO NPs.

Qualitative phytochemical test of neem (Azadirachta indica) leaf extract

Different functional groups from A. indica leaf extract were identified using a qualitative phytochemicals test based on standard methods [41–43]. These functional groups are responsible for the reduction of Zn²⁺ to ZnO NPs and capping and stabilization of the synthesized ZnO NPs. Figure 3 shows the results of qualitative phytochemical screening tests of the leaf extract. As shown in the figure, the phytochemical tests confirm the presence of secondary metabolites such as Alkaloids, Flavonoids, Glycosides, Tannins, Terpenoids and Phenols.

Fig. 3.

Qualitative Phytochemical screening of Azadirachta indica leaf extract for A alkaloids, B Flavonoids, C glycosides, and D tannins, E Terpenoids and F Phenols were examined

Synthesis of zinc oxide nanoparticles

Zinc oxide nanoparticles were synthesized by mixing 2 mL aqueous solution of A. indica leaf extract with 25 mL of 4 mM aqueous Zn(NO₃)₂·6H₂O solution according to the method reported by different literatures with some modifications [2, 7]. The reaction was conducted for 3 h until a pale yellow color was observed (Fig. 2E). The solution heated at 60 °C for 1 h turned from pale yellow to deep yellow colored paste confirming the formation of ZnO NPs [11].

Fig. 2.

Process of synthesis of zinc oxide nanoparticles (A) Fresh Azadirachta indica leaf (B) washed and chopping Azadirachta indica leaf (C) filtering the mixture of Azadirachta indica leaf extract (D) aqueous solution of Zinc nitrate hexahydrate (E) mixture of Azadirachta indica leaf extract and Zinc nitrate hexahydrate solution (F) heating of ZnO NPs colloidal solution (G) ZnO NPs powder

The mixture shown in Fig. 2E was placed in a beaker (Fig. 2F) and heated (calcinated) in an oven at a temperature of 400 °C for 2 h. This calcination process is in agreement with other research works [2, 7]. After subsequent steps of washing, centrifugation, and calcination, white powder of ZnO NPs was obtained (Fig. 2G). The powder was used for further characterization, specifically for XRD analysis. Scheme 1 shows possible reaction mechanism for the formation of white powder ZnO NPs. In the reaction mechanism, aromatic hydroxyl groups present in the phytochemicals are attached to the Zn²⁺ ions from Zn(NO₃)₂.6H₂O to form a stable complex system [11, 12].

Scheme 1.

Possible synthesis mechanism of ZnO NPs using plant extracts

Characterization of zinc oxide nanoparticles

To characterize the synthesized ZnO NPs, we used visual observation and different analytical instruments such as UV–Vis spectroscopy, XRD analysis, FTIR spectroscopy. An Agilent Cary 60 UV–Vis spectrophotometer was used to perform UV–Visible absorption spectral analysis of the synthesized ZnO NPs. The samples were scanned in UV–Visible spectrum with a wavelength range of 200 to 800 nm. A drop of the plant leaf extract and colloid solution of ZnO NPs was combined with KBr powder to create a paste for FT-IR spectroscopic analysis. The paste was then placed into an 8 nm resolution FT-IR (IRAffinity-1S, Shimadzu, Japan) spectrophotometer and scanned in a wave number range of 400–4000 cm⁻¹. The crystalline nature of the zinc oxide nanoparticle was determined by subjecting ZnO NPs powder to X-ray diffraction (XRD, PANalytical X’Pert PRO) at a voltage of 40 kV and a current of 30 mA with Cu K radiation. Estimation of crystalline size (D) of the prepared ZnO NPs was calculated by Debye–Scherrer formula.

Antibacterial activity of ZnO NPs using disc diffusion method

Antibacterial activity of ZnO NP was investigated using the disc-diffusion method against human pathogens such as S. aureus and E. coli. Preparation of the bacteria stock was done by inoculating each inoculation loop with pure culture of S. aureus and E. coli nutrient broth solution. All the equipment and growing media were sterilized by autoclaving at a temperature of 115 °C for 30 min. An overnight culture of inoculum was spread over the Mueller Hinton Agar (MHA) plates by a non-toxic cotton swab on an applicator stick which was dipped into the standardized suspension of bacteria. Subsequently, the filter paper disc of 6 mm in diameter was soaked using sterile forceps in a 30 µl of ZnO NP colloidal solution and in a 30 µl solution of a positive control drug, Gentamicin. Each disc was gently pressed down with sterile forceps to confirm complete contact with the Mueller Hinton Agar (MHA) plate’s surface. The plates were examined for evidence of zones of inhibition, which appear as a clear area around the discs. The maximal zone of inhibition against each species of microbe was measured using a digital electronic caliper and seen after the plates were incubated for 24 h at 37 °C.

Results and discussion

Phytochemical analysis of Azadirachta indica leaf

Phytochemicals are bioactive substances derived from plants. Because the plants that produce them may not have much need for them, they are thought of as secondary metabolites. All plant parts, including the leaves, stem, roots, flowers, fruits, seeds, etc., naturally synthesize them [44]. In this work, the preliminary qualitative phytochemical examination of A. indica leaf extract showed as the presence of alkaloids, flavonoids, glycosides, Tannins, Terpenoids and phenoles. Figure 3 illustrates the distinctive color changes of phytochemicals during the chemical tests using standard procedures [45, 46]. These phytochemicals might help to reduce zinc nitrate and regulate the size of the synthesized ZnO NPs. The bioactive molecules of the plant extracts are essential for capping and stabilizing the synthesized nanoparticles [2, 47].

UV–visible spectra analysis

During the green synthesis of ZnO NPs, the colorless Zn (NO₃)₂∙6H₂O solution started changing its color to pall yellow as soon as the leaf extract of A. indica was added to it (Fig. 2E). This color change to pall yellow is an initial confirmation for the formation of ZnO NPs [7]. The optical characteristics of ZnO NPs revealed a definite maximum absorbance peak at 321 nm (Fig. 4). The result agrees with the range of λ_max values of ZnO NPs reported by the previous researchers [6]. Therefore, the visual observation and UV–Vis spectrum value are taken as confirmatory for the formation of ZnO NPs.

Fig. 4.

The dependence of the absorption coefficient on the wavelength of the incident photons through solution of Azadirachta indica leaf extract (Red), Zinc nitrate hexahydrate (Green) and the synthesized ZnO NPs (Blue)

The UV–Vis spectrum shows broad intense absorption from about 400 nm to lower wavelengths, which is typically associated with a charge-transfer process from the valence band to conduction band [48]. The optical transmission in the visible region is high (> 90%), but an absorption edge at about 321 nm wavelength near the UV region could be observed for the synthesised ZnO NP. The optical band gap of a direct semiconductor can be calculated by analyzing the absorption edge and applying the Tauc model [48–50]

$$\alpha =\frac{A}{\mathit{hv}}{\left(hv-E_g\right)}^{\frac{1}{2}}$$ 1

Taking the square of the above equation gives:

$${\left(\alpha hv\right)}^2=A\left(hv-E_g\right)$$ 2

where α is the absorption coefficient, hυ is the photon energy, A is a constant, and E_g is the optical band gap.

The experimental points fit with (αhν)² versus hυ curve only if the direct electronic transitions are responsible for the photon absorption inside the nanoparticles. An intercept of the straight line, as shown in Fig. 5, yields an optical energy gap of 3.35 eV. This value is in good agreement with the reported band gap of ZnO [48, 49, 51].

Fig. 5.

(αhν)² versus hν plot of the synthesized ZnO NPs

The size and structure of the nanoparticles can be controlled by varying a number of variables such as the concentration of A. indica leaf extract, concentration of precursor solution, reaction time, and pH of the solution. In this study, we tried to check the effect of these variables for the synthesis of ZnO NPs.

Effect of zinc nitrate hexahydrate (Zn(NO₃)₂·6H₂O) concentration

Figure 6 shows the UV–Visible spectrum measured at various Zn(NO₃)₂·6H₂O concentrations with a constant amount of A. indica leaf extract. The concentrations of Zn(NO₃)₂·6H₂O solution used for the experiment were 25 mL of 0.25, 0.5, 1, 2, 4, and 6 mM. As the concentration of Zn(NO₃)₂·6H₂O solution increased, the intensity of the spectra was also increased. At higher concentrations of Zn(NO₃)₂·6H₂O solution, such as 6 mM, the spectrum becomes noisy. It is noteworthy that agglomeration happened on the summits at higher concentration. This suggests that bigger ZnO NPs were formed when the concentration of zinc ions in the solution surpassed the optimum quantity. The UV–Vis spectrum of ZnO NPs obtained using 4 mM of Zn(NO₃)₂·6H₂O is sharp, intense, and smooth. Thus, we used 25 mL of 4 mM Zn(NO₃)₂·6H₂O solution as an optimum value for this work.

Fig. 6.

UV–Vis spectra of ZnO NPs from 2 mL of 6% (m/v) Azadirachta indica leaf extract and 25 mL of a 0.25, b 0.5, c 1, d 2, e 4, f 6 mM Zn(NO3)2·6H2O

Adjustment of pH for ZnO NP production

The effect of pH on the synthesis of ZnO NPs was investigated by adjusting its value using 0.1 M HCl and 0.1 M NaOH. The UV-Visible spectra of ZnO NPs produced at pH values of 1, 3, 5, 7, 9, 11,and 13 are shown in Figure 7. At pH values of 1, 3, 5, and 7, there is no distinct absorbance peak for the ZnO NP. But there is a high intensity absorbance peak at pH 9. This indicates that a large number of ZnO NPs formed with smaller diameters [52]. At pH 11 and 13, the particles become unstable and agglomerated so that the peak intensity decreases and finally disappears [53, 54]. This result confirmed the vital role played by pH in controlling the shape and size of the ZnO NPs. The optimum pH value chosen for this work was pH 9.

Fig. 7.

UV–Vis spectra of ZnO NPs produced using 2 ml of 6% (m/v) Azadirachta indica leaves extract and 25 ml of 4 mM zinc nitrate at various pH values: a 1, b 3, c 5, d 7, e 9, f 11, and g 13

Effect of reaction time on the synthesis of ZnO nanoparticle

To assess the stability of the synthesized ZnO nanoparticles, UV–Visible spectroscopic measurements were used at different time intervals. The reaction time intervals used were 2, 24, and 48 h. As shown in Fig. 8, the maximum UV–Vis absorbance of ZnO NPs is exactly the same in intensity, broadness, and λ_max value for both reaction times. The result confirms the stability of the synthesized ZnO NPs.

Fig. 8.

UV–Vis spectra of ZnO NPs showing the effect of variation of time

The study of FT-IR spectra

Azadirachta indica leaf extract has an intense FTIR spectrum (Fig. 9A) centered at 3465 cm⁻¹, which can be ascribed to hydrogen-bonded O–H stretching vibrations of phenol, alcohol, carboxylic groups, etc. [26, 55]. Absorption spectrum at 1638 cm⁻¹ is attributed to the vibrations of C=O and N–H group of proteins and enzymes [26, 56]. The absorption spectrum at 690 cm⁻¹ has been assigned to C–H bending in alkynes [11]. There are also absorption bands for the A. indica leaf extract corresponding to the vibrations of different functional groups. An absorption band was also seen at 3435 cm⁻¹ for the synthesized ZnO NP (Fig. 9B), which was linked to the –OH stretching vibrations of alcohol or phenolic compounds [7]. The intensity of the ZnO NPs spectrum decreased in comparison to the spectra of A. indica leaf extract. This result confirms that phytochemicals such as alcohols, Flavonoids, and carboxylic groups are involved in the synthesis of ZnO NPs [7]. An absorption band observed in the region between 400 and 600 cm⁻¹ confirmed the presence of metal–oxygen [M–O] bond stretching vibration [26, 57]. Thus, the formation of zinc oxide nanoparticles was confirmed by the spectrum of the stretching vibration of Zn–O observed at 544 cm⁻¹ [57]. The existence of N–H and O–H bonds in the FTIR spectrum indicated that proteins, phenolics, and flavonoid compounds in A. indica leaf extract are responsible for the bio reduction of Zn.²⁺ ions and stabilizing process of ZnO NPs [13, 26]

Fig. 9.

FT- IR spectra of: A Azadirachta indica leaf extract and B ZnO NPs

XRD analysis

The XRD diffraction peak measurement was carried out with Cu Kα radiation (k = 0.15406 nm), and 2θ ranges from 10° to 80°. The diffraction peaks (Fig. 10) obtained for the synthesized ZnO NPs are; 31.74°, 34.38°, 36.30°, 47.46°, 56.54°, 62.88°, 67.96°, and 77.21°. The peaks correspond to (100), (002), (101), (102), (110), (103), (112), and (202), lattice planes respectively. All the peaks were assigned using the JCPDS 00-036-1451 [2, 3] and proposed the existence of the hexagonal Wurtzite structure of ZnO NPs. The results obtained are in good agreement with previous experimental findings [2, 3, 51]. The crystallite size (D) of ZnO NPs was calculated from the full-width at half-maximum from XRD patterns using the Debye Scherer equation [7]:

$$D=\frac{K\lambda }{\beta cos\theta }$$ 3

where D is the crystallite size, λ = 0.15406 nm, which is the wavelength of the X-ray for Cu target Kα radiation, β is the peak width at half maximum of an XRD, K = 0.89, which is the Scherer’s constant and θ is the Bragg diffraction angle.

Fig. 10.

X-ray diffraction patterns of the synthesized ZnO NPs

Thus, the average crystalline size of the synthesized ZnO NPs calculated using the Debye–Scherrer formula was found to be equal to 19.16 nm.

ZnO NPs analyzed by XRD will typically be high-quality if the XRD pattern displays sharp and well-defined crystalline peaks with minimal background noise. The % crystalline of the ZnO NPs was determined using the relation:

$$\%\text{crystallinity}=\frac{Areaofcrystallinepeaks}{Areaofallpeaks\left(Crystalline+Amorphouse\right)}\times 100$$ 4

Based on Eq. 4, the % crystallinity of the synthesized ZnO NPs is calculated to be 86.19%. This value indicates a good crystalline level of the synthesized ZnO NPs. A large value of % crystalline indicates a high degree of crystalline order.

Antibacterial activity of synthesized ZnO NPs

The antibacterial activity of ZnO NPs examined against E. coli (gram −ve) and S. aureus (gram +ve) using the agar well-diffusion method is shown in Figure 11. The protection level against the two bacterial strains was determined on the basis of zone of inhibition (mm). The maximum zone of inhibition of ZnO NPs against E. coli (gram −ve) was 18 mm. The electrostatic interaction between the negatively charged bacterial cell wall and the positively charged Zn²⁺ ions may be the cause of this outcome, which leads to bacterial cell wall rupture and eventual cell death [58].

Fig. 11.

Antibacterial activity of ZnO NPs (A) 1,2,3 forZnO NPs and 4 for Gentamicine against E. coli (B), 1 and 2 for ZnO NPs and 3 for Gentamicine against S. aureus

Possible mechanisms that may be used to explain the antibacterial activity of ZnO NPs are (1) the release of reactive oxygen species (ROS), which causes oxidative stress and damage to DNA and eventually leads to cell death (2) the dissolution of ZnO NPs into zinc ions (Zn²⁺), which interact with the cell membrane of bacteria, destroying the cellular integrity and causing bacterial cell death; and (3) direct electrostatic interactions between ZnO NPs and bacterial cell membranes that alters the permeability of the plasma membrane by disrupting its structure [58, 59]. According to the different literatures, it can be concluded that the inhibition of bacterial growth demonstrated by ZnO NPs displayed that they possessed strong antibacterial efficiency. This excellent antibacterial effect of biosynthesized ZnO NPs is due to the larger surface area of smaller NPs in size, the excessive production of highly reactive oxygen species, including H₂O₂, OH radicals, and singlet oxygen, causes a disturbance in the cellular membrane and leads to bacterial death [59, 60]. It is inferred that green synthesized ZnO NPs are good antibacterial catalysts against resistant bacterial strains. ZnO NPs synthesized via this route showed better result on both gram positive and gram negative strains, as they were stabilized by different secondary metabolites of A. indica leaf extract. Results of the anti-bacterial activity (Fig. 12) showed that E. coli was more susceptible to ZnO NPs than S. aureus which is supported by the works of different researchers [58, 61].

Fig. 12.

Antibacterial activity of ZnO NPs against bacterial strains

Future perspectives

The authors recommend that further research will be done to analyze and comprehend the real biological synthesis-dependent mechanisms and also to explore different indigenous plants in Ethiopia for the synthesis of nanoparticles.

Conclusions

In this work, ZnO NP has been successfully synthesized through a green synthetic pathway with the aid of A. indica leaf extract as reducing, stabilizing as well as capping agent. The zinc oxide nanoparticles were characterized using color change of the nanoparticle solution, UV–Vis absorption spectroscopy, XRD, and FT-IR spectroscopy. ZnO NPs have been found to have a maximum absorbance at a wave length of 321 nm. FTIR spectrum elucidated the phytochemicals present in A. indica leaf extract. The presence of absorption peak at 544 cm⁻¹ indicates the formation of ZnO NPs. The synthesized ZnO NPs were found to have a crystalline nature with a hexagonal structure, as confirmed by powder X-ray diffraction analysis. The average crystallite size of the nanoprticle was calculated using the Debye–Scherrer equation and was found to be 19.16 nm. ZnO NPs synthesized through the above green route have better antibacterial activity against bacterial strains. It was found that the ZnO NP has an inhibition zone of 18 mm for E. coli and 15 nm for S. aureus.

Author contributions

The contribution of Mr Elimneh Tsegahun to this manuscript is data acquisition during his MSc study under the super vision of Dr Muluken Aklilu. He also write the manuscript. The contribution of Dr Muluken Aklilu is supervision of Mr Elimneh Tsegahun while he was doing the reserach to collect data and Editing the manuscript.

Funding

No funding have been received to conduct this research.

Data availability

All the data are included in the manuscript and also aveliable with corresponding author upon request.

Declarations

Ethics approval and consent to participate

Fresh Azadirachta indica leaves were collected from wild forests found around the local area of Bahir Dar city, Ethiopia. The collection of leaves complies with the national guidelines of the Ethiopian biodiversity institute, with no need for further affirmation.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.