https://orcid.org/0000-0002-7632-5261
Abstract
In recent decades, nanotechnology has garnered significant attention for its diverse applications. Zinc Oxide (ZnO) nanoparticles (NPs) biosynthesized using plant extracts as both reducing and capping agents offer versatile solutions to various biological challenges. This study aimed to advance ZnO nanoparticle synthesis using a low-toxicity, cost-effective phytochemical method employing Eucalyptus globulus leaf extracts. Optimization of key factors (time, temperature, plant extract volume, and reagent concentration) was conducted to achieve high yield, stability, and controlled size. Optimal conditions were determined as 4hours, 60°C, 1:1 ratio, and 1mM concentration. Characterization of the synthesized ZnO NPs was performed through UV-visible spectroscopy, Scanning Electron Microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR), and Energy Dispersive X-ray Diffraction (EDAX). UV—visible analysis revealed a characteristic absorbance peak at 370nm, confirming ZnO NP formation. SEM affirmed the spherical ZnO NPs with a particle size of 25–151nm. EDAX demonstrated high purity, highlighting Zinc (Zn) and Oxygen (O) atoms. FTIR spectroscopy identified key phytochemical bands, elucidating the nanoparticle’s ability to reduce Zn ions, including polyphenols (3739.97cm-1), surface hydroxyl groups (3419.79cm-1), and C-OH stretching (1575.84cm-1). As a result of this research, nanoparticle synthesis can be made eco-friendly and economically viable, making it useful for environmental and industrial purposes.
1. Introduction
The prefix “nano” is referred to as a Greek prefix meaning “dwarf” or something very small and depicts one thousand millionths of a meter (10−9m). Structures and molecules on nanometer scales ranging from 1 to 100nm are studied in nanoscience, and nanotechnology is used to implement them in practical applications like devices (Bayda et al., Citation2019). Despite their small size, nanoparticles possess enhanced reactivity, thermal conductivity, and chemical stability because of their larger surface-to-volume ratio (Khan et al., Citation2019). It results in a change in their biological and physical properties (Verma et al., Citation2019). Nanoparticles are of many different types, classified based on their morphology, size, physical, and chemical properties. They are ceramic, metallic, metallic oxide, and carbon-based nanoparticles (Ealia & Saravanakumar, Citation2017; Suresh et al., Citation2020).
Among all metal oxide-based nanoparticles, zinc oxide nanoparticles are the most preferred. Zinc oxide is a semiconductor having high catalytic activity and a large surface area. It has antimicrobial and antitumor activities (Gancheva et al., Citation2016). It is extensively used in cosmetic lotions as it maintains UV-blocking and absorbing capabilities (Borysiewicz, Citation2019). Biocompatibility, low cytotoxicity, and cost-effectiveness have made zinc oxide nanoparticles (ZnO NPs) a promising option for many industries, including optics, electrics, packaged foods, and medicine (Alyamani et al., Citation2021).
Zinc oxide nanoparticle synthesis can be done by using different methods. The synthetic methods are classified generally into two approaches: The top-down approach and the bottom-up approach (Paul et al., Citation2023). Top-down Approach is a destructive approach in which molecules of larger size are converted to smaller-sized units by decomposition and then used to form suitable nanoparticles. Physical methods of vapor deposition (PVD), and grinding/milling are examples of this method. The bottom-up approach is the reverse action of the top-down approach because simpler substances are used for nanoparticle synthesis in it. Techniques such as reduction and sedimentation, green synthesis, biochemical synthesis, spinning, and sol-gel are included in the bottom-up approach (Jadoun et al., Citation2021).
Plants are used in green technology to synthesize nanoparticles. Green synthesis produces high catalytic activity and is free of toxic substances, so it has no chance of being hazardous. Phytochemicals produced and secreted from the plant act as a reducing and stabilizing agent. (Paul et al., Citation2023)
Synthesis of green nanoparticles has attracted extensive interest worldwide because of their biocompatibility and huge potential for utilization as catalysts, antimicrobial agents, energy harvesting, cancer/gene therapy, and sensing (Rana et al., Citation2020). It is chosen over physical and chemical approaches because it saves time and energy. Different parts of plants like roots, leaves, flowers, stems, and seeds are used for the synthesis of nanoparticles by using the green method and all of these parts are rich in phytochemicals (Ramesh et al., Citation2015).
Plant source is preferred for the synthesis of nanoparticles because it results in large-scale and stable production (Qu et al., Citation2011). In bioreduction metal oxides and ions are reduced to metal nanoparticles with zero valencies with the help of phytochemicals like polysaccharides, alkaloids, vitamins, amino acids, and terpenoids (Mahmood Ansari et al., Citation2021). Several factors affect the synthesis, applications, and characteristics of nanoparticles. They directly affect the size, shape, and distribution. Highly optimized conditions result in nanoparticles of accurate size and shape having greater stability. These also help in large-scale production in much less time including the pH of the solution, temperature, concentration of extract, and volume (Gouws & Hamman, Citation2020; Patra & Baek, Citation2015).
The demand for plant-derived products has increased in recent years for therapeutic use. Aromatic herbs are being used worldwide for primary health care (Gouws & Hamman, Citation2020). These traditional resources are used by almost 80% population of developing countries. The biosynthesis of nanoparticles using Eucalyptus plant extract is not seen every so often in the literature (Ying et al., Citation2022).
Over the past 10years, nanoparticles have become increasingly popular as anti-inflammatory agents. When dosage and size are optimized, it exhibits unique physical qualities such as low toxicity and precise targeting, ranging within the size range of 1–100nm. Due to its distinct optical, therapeutic, and electrical capabilities, ZnO nanoparticles have received more attention than other metal and metal oxide nanoparticles (Kim et al., Citation2011).
The eucalyptus tree is a very diverse genus of flowering trees in the family Myrtle, Myrtaceae. It is native to Australia but also found in South East countries (Jo et al., Citation2022). Leaves of Eucalyptus are very useful because of their diverse health-promoting medical activities. It is used against respiratory and cold infections. The leaves of Eucalyptus are the main source of eucalyptus oil in the world. Essential oils derived from its leaves and buds have antiseptic and antibacterial properties (Sabo & Knezevic, Citation2019). In traditional medicine, leaves are immersed or decocted or used externally in baths, lotions, or enemas to combat asthma, bronchitis, tonsillitis, colds, urinary problems and bleeding. Inhale boiled dry leaf vapor to fight asthma, cough, flu, wheezing, and diphtheria, or inhale fine leaf powder. The leaf soup of Eucalyptus is used to cure malaria. Eucalyptus leaves are also used against stomach aches. The gum resins obtained from Eucalyptus trees are used for curing diarrhea (Siripireddy & Mandal, Citation2017).
Using Eucalyptus globulus Labill as an environmentally friendly reducing and capping agent during the production of zinc oxide (ZnO) nanoparticles is the main goal of this study. The overall objective of the research is to increase the production and quality of ZnO nanoparticles by optimizing the synthesis process and concentrating on important reaction parameters including temperature, pH, and reaction time. Moreover, the study attempts to provide a thorough characterization of the synthesized ZnO nanoparticles. This includes using analytical methods like UV-Vis spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and X-ray diffraction (XRD). By using these techniques, the optical, morphological, and structural properties of the ZnO nanoparticles can be methodically investigated, resulting in important discoveries for the field.
2. Rationale
The use of Eucalyptus leaves, as a reducing agent is eco-friendly and reduces the environmental impact associated with traditional chemical synthesis methods. Eucalyptus-derived nanoparticles are likely to be biocompatible, making them suitable for various biomedical applications. This study advances the field of nanotechnology by offering a green alternative for ZnO nanoparticle synthesis, which can have diverse applications in electronics, sensors, catalysis, and more. Promoting the use of naturally sourced reducing agents aligns with sustainable and cost-effective practices in nanomaterial production, especially in developing regions. This research aims to bridge gaps in green nanotechnology and provide a sustainable alternative for nanoparticle synthesis.
3. Materials and methods
The green synthesis, optimization, and characterization of ZnO nanoparticles was done in the Applied Environmental Biology Research Laboratory, Department of Botany, University of Punjab, Lahore. Eucalyptus globulus Labill. leaf extract and Zinc sulfate (ZnSO4) were used to synthesize ZnO nanoparticles by the green synthesis method at optimized conditions. Characterization of the synthesized nanoparticles was done using scanning electron microscopy, UV-visible spectroscopy, and particle size analysis. All of the reagents used in the study were of laboratory grade. All the reagents including ZnSO4 used in the study were procured from Merck Co., Germany, and Sigma-Aldrich Chemical Co., USA.
3.1. Preparation of leaf extract
Fresh leaves of E. globulus were collected from the district of Sheikhupura. After that, they were brought to the laboratory for further processing. The leaves were washed using distilled water to remove all dust particles from them or any other impurities that can cause problems in the synthesis of nanoparticles. Washed leaves were placed at room temperature for drying. After the leaves were completely dried, they were ground to powder form using a willy mill and stored at 25 °C. This was done with intense care to avoid any kind of contamination. The powder was free from all impurities in it. For the preparation of leaf extract, about 20gm of leaf powder was added to 100ml of deionized water and kept boiling at 80° for about one hour. After an hour, a light black colored solution was observed. It settled down at room temperature. The precipitate formed was then filtered. The size of the Whatman filter was 20μm and the supernatant obtained from this was stored at 4° for further use.
3.2. Preparation of reagent
For the preparation of the reagent, 0.01647g of ZnSO4 was dissolved in 100ml deionized water to prepare a 1mM solution. This solution was prepared by stirring method. To prepare 2mM, 3mM, 4mM, and 5mM solutions, 0.03294g, 0.04941g, 0.06588g, and 0.08235g of ZnSO4 were used respectively.
3.3. Optimization of procedure for nanoparticle synthesis
Some operating parameters such as time, temperature, the volume of extract, and conc. of the solution were optimized during the synthesis of ZnO nanoparticles.
4. Optimization of time
For optimizing the time of reaction to obtain the maximum number of nanoparticles, different time intervals were used. 10ml of the extract was mixed with 10ml of the reagent in a vial and the reaction time was maintained at 0min, 4h, 8h, 12h, 16h, 20h, and 24h and room temperature, 40°, 60° and 80° respectively. A color change was observed after different time intervals according to the formation of nanoparticles. After each interval, the mixture was poured into Eppendorf’s and its centrifugation was done at 13,000 rpm (4°/15min). The supernatant was thrown away and the pellet was washed two times repeating the centrifugation process. UV-visible spectroscopy confirmed the formation of the nanoparticles when subjected to it. The time interval at which the maximum nanoparticles were formed, was considered an optimized time interval.
5. Optimization of temperature
The temperature of the reaction was optimized using different temperature ranges (40°, 60°, and 80°). For that purpose, 10ml extract and 10ml reagent were mixed in vials and kept at different temperature ranges for the time interval selected. Samples were centrifuged after that at 13000rpm/4° for 15minutes in Hettich Centrifuge Universal 320. The supernatant was discarded and the obtained pellet was washed two times and stored for further analysis. The optimized temperature range was selected by UV-visible analysis.
6. Optimization of extract volume ratio
In this process, the extract volume ratio was optimized using different concentrations of extract in the overall solution. The volume of reagent (1mM, 2mM, 3mM, 4mM) and plant extract was maintained at 1:1, 1:2, 1:3, 1:4, and 1:5 respectively. Extract and reagent were mixed in these ratios and were kept for the selected time and temperature. The mixture was centrifuged at 13,000 rpm/4° for 15minutes. The supernatant was discarded and the pellet was washed. The pellet was stored for further analysis. The volume at which the maximum nanoparticles were formed, was selected as the optimized volume.
7. Optimization of reagent concentration
The concentration of reagent was optimized using different ranges. Reagent concentration was maintained at 1mM, 2mM, 3mM, 4mM, and 5mM. The reagents for these concentrations were mixed with the optimized volume of plant extract for the selected time and temperature. The sample was centrifuged after that (13,000 rpm/4° 15minutes) and the supernatant was discarded. The pellet was washed and stored at 4° for further analysis. Optimized solution concentration was selected by UV-visible analysis.
7.1. Synthesis of ZnO nanoparticles
The reaction mixture was prepared by mixing the leaf extract and reagent in the optimized ratio and the concentration was placed at optimized conditions. The color change confirmed the formation of ZnO nanoparticles. After that, its centrifugation was done at 13,000 rpm (4°/15mins). The supernatant was thrown away and the pellet was washed two times. The resulting pellet was subjected to UV-visible spectroscopy that confirmed the formation of nanoparticles.
7.2. Characterization of ZnO nanoparticles
The final ZnO nanoparticles formed were characterized using different techniques, i.e. UV-visible spectrophotometry, SEM, and Particle size analysis.
8. UV-visible spectroscopy
The formation of Zinc Oxide nanoparticles was confirmed by UV-visible spectroscopic analysis. Sample absorption was checked from the 300–800nm range of wavelength in Model METASH Spectrophotometer UV/VIS spectrophotometer. It confirmed the scattering and absorption of light passing through the sample product. For UV-visible analysis, 1ml distilled water was added to the pellet formed. It was vortexed and poured into a glass vial. 3ml distilled water was added to dilute it. Absorption was found at 300–800nm wavelength and the peak was noted.
9. Scanning electron microscopy (SEM)
The surface structure of the synthesized ZnO nanoparticles was identified using scanning electron microscopy. It identified the size and structure of the nanoparticle. Nanoparticles were prepared at optimized conditions and centrifuged to get their purified form. These purified nanoparticles were dried for SEM analysis. The sample drop was taken on a carbon-coated grid to prepare the sample film. The extra solution was wiped off. This film was allowed to dry and observed on the SEM grid.
10. Scanning electron microscopy (SEM) & energy dispersive X-ray analysis (EDX)
Scanning electron microscopy was done to know the morphology of the synthesized ZnO nanoparticles. The size and shape were confirmed by SEM. A high beam of electrons technique was used to scan the surface morphology of ZnO nanoparticles synthesized at optimum conditions. The high-energy electrons interact with the atoms in the sample. For this purpose, ZnO nanoparticles were prepared from Eucalyptus globulus Labill. Leaf extract at optimized conditions (4h/60°/1:1/1mM). The clear cloud formation and color change confirmed the formation of nanoparticles. The resulting sample was centrifuged at 13000rpm for 15minutes at 4°. The supernatant was discarded and the pellet was washed 3 times. The washed pellet was completely dried in a hot air oven at 65° for 2h. After the pellet was completely dried, it was crushed to powder form and placed on a carbon-coated grid. It was then observed under the scanning electron microscope.
Energy dispersive X-ray analysis (EDX) was done to check the purity of ZnO nanoparticles. It gave the elemental composition of the synthesized ZnO nanoparticles. Moreover, it also confirmed the atomic weights of atoms present in the nanoparticle. EDX spectrum of ZnO NPs presented in Figure .
11. Fourier Transform Infrared spectroscopy (FTIR)
This analysis was done to know about phytochemicals, that were present in the plant material. These phytochemicals were responsible for Zinc ions reduction and the stabilization of ZnO NPs. FTIR determined the functional groups of the ZnO nanoparticles. The infrared spectroscopy method was used to obtain the spectrum. The description of functional groups present on the ZnO NPs was studied at the spectrum range of 4000–450cm−1 in this technique. The sample was prepared by dissolving ZnO NPs, prepared at optimized conditions, uniformly in a dry medium of KBr. A transparent disc was formed by compressing the sample. The sample was dried and subjected to FTIR analysis.
12. Results
The present study illustrated the formation of ZnO NPs from Eucalyptus globulus Labill. Leaves extract is used as a reducing agent. The formation was confirmed by UV-visible analysis. Optimization of different parameters was done during the process of synthesis. The resulting nanoparticles were characterized using various techniques.
12.1. Optimization of parameters
For synthesizing ZnO NPs, four parameters were optimized. They include time, temperature, solution concentration, and extract volume ratio. The -by-two factorial method was used for optimization. Optimized conditions were selected by varying all the above-mentioned parameters. Those with the best results were considered optimized conditions.
12.2. Effect of time
The reaction time plays a key role in nanoparticle synthesis. Reaction time caused aggregation and depression in the formed nanoparticles. The nanoparticles formed were confirmed by UV-visible analysis.
12.2.1. At room temperature
Figure showed room temperature, the maximum results were obtained after 24hours of reaction time. The peak shifted between 370-390nm. After 12 and 16hours of reaction time, minimum results were obtained but without any peak. Other time intervals showed a flat line on the spectrophotometer. 24hours of reaction time was selected as the optimum period at room temperature, however, the nanoparticles formed were not even stable at room temperature.a
Figure 1. Ultraviolet-visible spectra for the production of zinc oxide nanoparticles by leaf extract of Eucalyptus globulus Labill. At room temperature for various periods of incubation.
12.3. At 40oC
At 40° temperature, 8hours of reaction time gave maximum results and an absorbance peak at 370nm. It showed a clear color change. 4 and 12hours of reaction time showed results but lesser than those at 8hours. They showed an absorbance peak below the one formed for 8hours. The nanoparticles formed were in a dispersed state. 0min, 16h, 20h, and 24h showed flat graphs in UV spectrophotometric analysis that depict no nanoparticle formation. 8hours of incubation period was selected as the optimum temperature at 40° as it showed maximum production of nanoparticles. Ultraviolet-visible spectra for the production of zinc oxide nanoparticles by leaf extract of Eucalyptus globulus Labill. at 40∘C for various periods of incubation shows in Figure .
Figure 2. Ultraviolet-visible spectra for the production of zinc oxide nanoparticles by leaf extract of Eucalyptus globulus Labill. At 40°C for various periods of incubation.
12.4. At 60°
At 60° temperature, the reaction time of 4hours showed the maximum production of nanoparticles, confirmed by UV-vis analysis. Figure . shows the ultraviolet-visible spectra for the production of zinc oxide nanoparticles by leaf extract of Eucalyptus globulus Labill. at 60∘C for various periods of incubation. A high peak of absorbance between 370-390nm was observed. An obvious color change from yellowish-green to brownish was observed. 8 and 12hours of incubation time also showed absorbance but lesser than that of 4hours of incubation time. 0minutes, 16, 20, and 24hours showed minimum results with no peaks. Stable nanoparticles were formed after 4hours of reaction time at 60°. Therefore, it was selected as an optimum period.
Figure 3. Ultraviolet-visible spectra for the production of zinc oxide nanoparticles by leaf extract of Eucalyptus globulus Labill. At 60°C for various periods of incubation.
12.5. At 80°
At 80° temperature, 4hours of incubation time showed maximum results along with an absorbance peak at 375nm. UV-vis analysis showed the maximum absorbance at 4h. A visible color change was observed during this period for 80°. Figure . shows the ultraviolet-visible spectra for the production of zinc oxide nanoparticles by leaf extract of Eucalyptus globulus Labill. at 80∘C for various periods of incubation. The other period at this temperature doesn’t show any clear color change. While all other periods showed an almost straight line. No clear color change was observed at 0min, 8h, 12h, 16h, 20h, and 24h.
Figure 4. Ultraviolet-visible spectra for the production of zinc oxide nanoparticles by leaf extract of Eucalyptus globulus Labill. At 80°C for various periods of incubation.
The reaction temperature plays an important role in nanoparticle synthesis. It affects the quality of nanoparticles. The mixture of extract and reagent was placed at RT, 40°, 60°, and 80° for 24h, 8h, 4h and 4h respectively. Maximum results were obtained at 60° for 4h. 40° and 80° also showed absorbance but lesser than that of 60°. RT showed minimum absorbance. The nanoparticles formed at 60°/4h were stable. They showed clear color changes and a clear cloud formation. Therefore, the optimum temperature required for the synthesis of nanoparticles was selected to be 60°C. Ultraviolet-visible spectra for the production of zinc oxide nanoparticles by leaf extract of Eucalyptus globulus Labill. at 60∘C for 4h of incubation shown in Figure .
Figure 5. Ultraviolet-visible spectra for the production of zinc oxide nanoparticles by leaf extract of Eucalyptus globulus Labill. At 60°C for 4h of incubation.
12.6. Effect of reagent concentration
Reagent concentration affects the formation of nanoparticles. Optimized reagent concentration was found by using different concentrations of reagent for nanoparticle synthesis. The concentration that gave a larger number of stable nanoparticles of fine quality was selected as the optimized concentration.
12.6.1. At 1mM concentration
1mM zinc sulfate was mixed with (1:01, 1:02, 1:03, 1:04, and 1:05) 1ml, 2ml, 3ml, 4ml, and 5ml plant extract in different vials. These vials were incubated for the optimized time and temperature of 4h/60°. Maximum absorbance was observed in the nanoparticles formed in the 1:01 mixture. The highest Absorbance peak at 382nm wavelength was observed. The nanoparticles formed in this vial were stable and a clear color change was observed from yellowish green to brownish green. Similarly, 1mM concentration also gave results at 1:2, 1:3, 1:4, and 1:5 but they showed lesser absorbance. Ultraviolet-visible spectra for the production of zinc oxide nanoparticles by leaf extract of Eucalyptus globulus Labill. With 1mM conc. of zinc sulfate shown in Figure .
Figure 6. Ultraviolet-visible spectra for the production of zinc oxide nanoparticles by leaf extract of Eucalyptus globulus Labill. With 1mM conc. Of zinc sulfate.
12.6.2. At 2mM concentration
Figure . showed the reaction mixtures with 1mM reagent concentration and five different extract volumes before placing them for incubation at 60∘C for 4h. 2mM zinc sulphate was mixed with 1ml, 2ml, 3ml, 4ml, and 5ml of plant extract and incubated for 4h/60°. Maximum results were obtained at 1:02 while minimum results were obtained at 1:04 with 2mM concentration or zinc sulfate. The nanoparticles formed with 2mM conc. showed absorbance but lesser than that observed with 1mM concentration. Very little color change was observed.
Figure 7. Ultraviolet-visible spectra for the production of zinc oxide nanoparticles by leaf extract of Eucalyptus globulus Labill. With 2mM conc. Of zinc sulfate.
12.6.3. At 3mM concentration
3mM conc. Zinc sulfate was mixed with plant extract in 5 different ratios, 1:01, 1:02, 1:03, 1:04, and 1:05. A low peak of absorbance was observed. Maximum results were observed in 1:01 and minimum in 1:04. The nanoparticles formed were aggregated. They were not even stable. 1:01 and 1:03 showed peaks while the others showed flat lines on the spectrum obtained by UV visible spectrophotometer. Reaction mixtures with 1mM reagent concentration and five different extract volumes after placing them for incubation at 60∘C for 4h shown in Figure .
Figure 8. Ultraviolet-visible spectra for the production of zinc oxide nanoparticles by leaf extract of Eucalyptus globulus Labill. With 3mM conc. Of zinc sulfate.
12.6.4. At 4mM concentration
4mM concentration of zinc sulfate was added to the plant extracts of 1ml, 2ml, 3ml, 4ml, and 5ml, the vials were incubated for the optimized time and temperature of 4h/60°. 4mM concentration of zinc sulfate was less effective compared to 3mM. Spectra showed peaks even lower than that of 3mM. A very slight color change was observed. Ultraviolet-visible spectra for the production of zinc oxide nanoparticles by leaf extract of Eucalyptus globulus Labill. With 4mM conc. of zinc sulphate shown in Figure .
Figure 9. Ultraviolet-visible spectra for the production of zinc oxide nanoparticles by leaf extract of Eucalyptus globulus Labill. With 4mM conc. Of zinc sulphate.
12.6.5. At 5mM concentration
Figure . shows the ultraviolet-visible spectra for the production of zinc oxide nanoparticles by leaf extract of Eucalyptus globulus Labill. With 5mM conc. of zinc sulphate. 5mM concentration of zinc sulfate was added to the plant extract in 5 proportions of 1:01, 1:02, 1:03, 1:04, and 1:05. The vials were placed at 60° for 4hours. No color change was observed. The resulting nanoparticles were subjected to a UV-visible spectrophotometer. Almost flat graphs were observed. Other than that, 1:01 and 1:02 showed little peaks between 370 to 390nm. Finally, it was concluded that maximum nanoparticles were formed with a 1mM concentration of zinc sulfate. 1mM conc. of zinc sulfate was proved to be much more reactive and a clear cloud formation of nanoparticles was observed. The higher concentration causes agglomeration or nanoparticles.
Figure 10. Ultraviolet-visible spectra for the production of zinc oxide nanoparticles by leaf extract of Eucalyptus globulus Labill. With 5mM conc. Of zinc sulphate.
12.7. Effect of extract volume
The volume of plant extract plays a vital role in nanoparticle formation. The phytochemicals present in the plant extract react with the reagent to form nanoparticles. If they are increased, they start reacting with each other and decrease the formation of nanoparticles. Five different volumes of plant extract were used to get the optimized volume. 1:1, 1:2, 1:3, 1:4, and 1:5. Equal ratio (1:1) of both extract and reagent was proved to be the best one because it gave maximum results. 1:2, 1:3, 1:4, and 1:5 also formed peaks. However, these peaks were lower than those formed at 1:1. The optimized volume ratio selected was 1:1 in which 5ml of reagent was mixed with 5ml of plant extract. Ultraviolet-visible spectra for the production of zinc oxide nanoparticles by leaf extract of Eucalyptus globulus Labill. With 1:1 volume of extract and 1mM conc. of zinc sulphate shown in Figure .
Figure 11. Ultraviolet-visible spectra for the production of zinc oxide nanoparticles by leaf extract of Eucalyptus globulus Labill. With 1:1 volume of extract and 1mM conc. Of zinc sulphate.
12.7.1. Characterization
The ZnO nanoparticles synthesized at optimum conditions were characterized by using three techniques, UV-visible analysis, Scanning Electron Microscopy, and energy-dispersive X-ray Analysis (EDX)
12.8. UV-visible analysis
The Zinc Oxide nanoparticles synthesized using Eucalyptus globulus leaf extract at optimum conditions of 4h, 60°, 1mM conc, and 1:1 volume of leaf extract were characterized using UV visible spectrophotometer. An absorbance peak was observed at 370nm. It showed the maximum production of nanoparticles. Ultraviolet-visible spectra for the production of zinc oxide nanoparticles by leaf extract of Eucalyptus globulus Labill. with 1:1 volume of extract and 1mM conc. of zinc sulphate for 4h at 60∘C was shown in Figure .
Figure 12. Ultraviolet-visible spectra for the production of zinc oxide nanoparticles by leaf extract of Eucalyptus globulus Labill. With 1:1 volume of extract and 1mM conc. Of zinc sulphate for 4h at 60°.
12.9. Scanning Electron Microscopy (SEM) and EDX
Figure . showed the SEM images of ZnO nanoparticles synthesized from Eucalyptus globulus Labill. Leaf extract. The morphology of ZnO nanoparticles synthesized at optimized conditions was scanned by using a Scanning Electron Microscope. SEM images detected the size of the synthesized nanoparticles, ranging from 25nm to 151nm. The shape of the nanoparticles was observed to be irregular and was in dispersed form. EDS analysis confirmed the presence and elemental composition of Zn and oxygen present in the synthesized nanoparticles which indicates the purity of the nanoparticle. The spectrum showed three peaks which were identified as Zn, O, and C. It is believed that the carbon may be due to the presence of carbohydrates. The peaks were formed because of the plasmon resonance of ZnO NPs.
Figure 13. SEM images of ZnO nanoparticles synthesized from Eucalyptus globulus Labill. Leaf extract.
Figure 14. EDX spectrum of ZnO NPs.
FTIR was done to know the phytochemicals present in nanoparticles formed, at optimized conditions that were responsible for the reduction of Zn ions and stabilization of nanoparticles. Bands were formed at 3739.97cm−1, 3419.79cm−1, 2889.37cm−1, 2310.44cm−1, 1622.13cm−1, 1575,84cm−1, 1409.90cm−1, 1202.00cm−1, 1213.23cm−1, 1115.78cm−1, 1066.64cm−1,985.02cm−1, 769,80cm−1, 675.09cm−1 and 597.93cm−1, 3739.97cm−1 was due to the OH stretching of polyphenols. 3419.79cm−1 corresponds to a surface hydroxyl group. The band at 1575.84cm−1 was due to C-OH stretching. 769.80cm−1 was due to the C-Cl stretching of alkyl halides.
13. Discussions
Green synthesis is an environment-friendly method for the synthesis of ZnO nanoparticles. ZnO nanoparticles are nontoxic and bio-safe. They are widely used in medicine and cosmetics. They also show antimicrobial activity. The green synthesis of ZnO nanoparticles has become essential because of its vast applications (Subramaniam et al., Citation2019).
ZnO nanoparticles were synthesized in the present experiment by using Eucalyptus globulus Label. leaf extract. The plant leaves were dried and crushed to powder form and 20gm of that powder was added to 100ml of distilled water. The mixture was boiled and filtered. The filtrate was mixed with ZnSO4 reagent. Various factors that affected the synthesis were optimized to get the highest production of nanoparticles. The color change confirmed the formation of nanoparticles. Time, temperature, extract volume, and reagent concentration were optimized for getting highly stable, smaller in size, and better ZnO nanoparticles. The best results were obtained after 4h at 60° with 1:1 volume and 1mM conc. of ZnSO4. After the reaction time, the resulting solution with clear color change was centrifuged at 4°/15min and 13000rpm. The pellet formed was subjected to UV-vis analysis that confirmed the ZnO nanoparticles formation.
Siripireddy et al (Sabo & Knezevic, Citation2019). synthesized ZnO nanoparticles using Eucalyptus globulus leaf extract. He prepared the extract by adding 20gm of leaf powder into 100ml of distilled water and boiling it on a hot plate (Sabo & Knezevic, Citation2019). In another experiment, Narayana carried out Nelumbo nucifera leaves mediated synthesis of ZnO nanoparticles. He prepared the extract using 20gm of leaf powder in 100ml distilled water. After that, he added 20ml of that extract into a 20ml reagent to get better nanoparticles. He placed the mixture at 60° and got stable and better nanoparticles. It is a constituent of the present experiment (Siripireddy & Mandal, Citation2017).
In the present experiment, 4 factors that affect nanoparticle synthesis were optimized. These factors were time, temperature, plant extract volume, and reagent concentration (Shown in plate ). The sample was placed for 7 different time intervals (0min, 4h, 8h, 12h, 16h, 20h, and 24h). the best results were obtained at 4h. It gave the maximum peak of absorbance. In another research, nanoparticles were synthesized zinc oxide nanoparticles using green technology. They placed the reaction mixture in an incubator for 4h and got the best results (Subramaniam et al., Citation2019).
Plate 1.Reaction mixture at seven different time intervals before incubation.
Zinc nanoparticles have aroused the interest of the scientific community due to their many uses in plasmonics, cosmetics, catalysts in the biomedical field, pure water technologies, data storage, optoelectronics, sensors, and textiles (Faisal et al., Citation2020; Jan et al., Citation2020; Poonguzhali et al., Citation2022). Zinc nanoparticles have excellent bio-optical and physicochemical properties, making them among the most widely used nanoscale materials for wound healing, dental applications, catheter modification, cancer therapy, drug delivery, and anti-viral, anti-inflammatory, anti-angiogenesis, and antimicrobial agents (Chabattula et al., Citation2021; Faisal et al., Citation2021).
Siripireddy et al (Sabo & Knezevic, Citation2019). synthesized ZnO nanoparticles using Eucalyptus globulus leaf extract. He prepared the extract by adding 20gm of leaf powder into 100ml of distilled water and boiling it on a hot plate (Siripireddy & Mandal, Citation2017). In another experiment, Narayana carried out Nelumbo nucifera leaves mediated synthesis of ZnO nanoparticles. Sample prepared the extract using 20g of leaf powder in 100ml distilled water. After that, prepared extract and added 20ml of that extract into a 20ml reagent to get better nanoparticles. He placed the mixture at 60° and got stable and better nanoparticles (Narayana et al., Citation2020). It a is constituent of the present experiment.
In the present experiment, 4 factors including time, temperature, plant extract volume, and reagent concentration that affect nanoparticle synthesis were optimized (Shown in plate ). The sample was placed for 7 different time intervals (0min, 4h, 8h, 12h, 16h, 20h, and 24h). The best results were obtained at 4h. It gave the maximum peak of absorbance. In another research, nanoparticles were synthesized zinc oxide nanoparticles using green technology. They placed the reaction mixture in an incubator for 4h and got the best results (Ogunyemi et al., Citation2019).
Plate 2. Reaction mixtures after placing them at Room Temperature for seven different time intervals.
Plate 3. Reaction mixtures after placing them at 40°C for seven different time intervals.
\Plate 4. Reaction mixtures after placing them at 60°C for seven different time intervals.
Plate 5. Reaction mixtures after placing them at 80°C for seven different time intervals.1.2.
Plate 6. Reaction mixtures after placing them for selected time intervals at different temperatures.
Plate 7. Reaction mixtures with 1mM reagent concentration and five different extractvolumes before placing them for incubation at 60°C for 4h.
Plate 8. Reaction mixtures with 1mM reagent concentration and five different extract volumes after placing them for incubation at 60°C for 4h.
Plate 9. Reaction mixtures with 2mM reagent concentrations and five different extract volumes after placing them for incubation at 60°C for 4h.
Plate 10. Reaction mixtures with 3mM reagent concentrations and five different extract volumes after placing them for incubation at 60°C for 4h.
Plate 11. Reaction mixtures with 4mM reagent concentrations and five different extract volumes after placing them for incubation at 60°C for 4h.
Plate 12. Reaction mixtures with 5mM reagent concentrations and five different extract volumes after placing them for incubation at 60°C for 4h.
Plate 13. Reaction mixtures after placing them at 60°C/4h with 1mM reagent concentration and different plant extract volumes.
By optimizing factors such as reaction time, temperature, reagent concentration, and extract volume ratio, researchers can maximize the yield of nanoparticles. This not only reduces material wastage but also ensures that a sufficient quantity of nanoparticles is produced for comprehensive analysis (Ananthi et al., Citation2023). The synthesis process is well-controlled, and it becomes easier to replicate the experiment multiple times, increasing the reliability of the study’s findings. Proper optimization leads to the formation of nanoparticles with desired characteristics, such as size, shape, and stability. This is crucial for applications in various fields, including nanotechnology, medicine, and materials science (Iqtedar et al., Citation2020).
In another experiment done by Song & Yang, ZnO nanoparticles were fabricated using Piper betel leaf extract by heating the reaction mixture at 90° for 4h. The resulting nanoparticles were stable and gave the highest peak of absorbance (Song et al., Citation2010).
For optimizing the temperature, 4 different temperature ranges were used (RT, 40°, 60° and 80°). 60° gave the best results as the highest peak of absorbance and clear color change was observed at this temperature range. Alamdari et al (Alamdari et al., Citation2020a). synthesized ZnO NPs from Sambucus ebulus leaf extract. He placed the reaction mixture for 3h at 70° for the green synthesis of ZnO nanoparticles (Alamdari et al., Citation2020a). Ossai et al. fabricated ZnO nanoparticles by green synthesis. They stirred the reaction mixture at 60° and got a clear cloud of ZnO NPs (Ossai et al., Citation2020). Selim et al. performed the green synthesis of ZnO NPs, placing the reaction mixture in a hot air oven at 60° overnight. A clear cloud formation was observed (Selim et al., Citation2020).
The plant extract volume was optimized by checking the results of five different volume ratios (1:1, 1:2, 1:3, 1:4, and 1:5). The best results and absorption peaks were obtained at equal volume ratios. The optimized volume was considered 1:1. In an experiment done by Raffie et al (Rafiee et al., Citation2018), who synthesized ZnO nanoparticles using Eucalyptus melidora leaf extract. Raffie et al. used a 1:1 volume of leaf extract as the optimized volume and got the best results (Rafiee et al., Citation2018).
Reagent concentration was optimized by using five different concentrations, i.e., 1mM, 2mM, 3mM, 4mM, and 5mM. The highest peak and best results were obtained with 1mM concentration of reagent. Ahmad et al. conducted ZnO NPs synthesis using Eucalyptus globulus leaf extract. He used 1mM conc. of zinc nitrate to prepare the reagent that gave the best results (Ahmad et al., Citation2020). In an experiment conducted by Krishnaraj et al., they optimized the conditions for the green synthesis of ZnO nanoparticles. They used three different reagent concentrations (1mM, 2mM, and 3mM). 1mM conc. showed the rapid synthesis of NPs (Krishnaraj et al., Citation2012).
ZnO-NPs were characterized using a variety of analytical techniques, including UV-visible spectroscopy, FTIR, SEM, EDX, and XRD. According to UV-visible spectroscopy, the sample absorbed energy at 430nm, which is a typical peak value for ZnO-NPs. The results were validated by X-ray spectroscopy (Talam et al., Citation2012). Aside from that, an absorption peak at 430nm with no other peak demonstrated the nanoparticles’ exceptional purity. Many investigations have revealed a significant absorption peak of ZnO-NPs below 450nm wavelength, which was related to the sample’s redshift at 500 and 700 degrees Celsius. They have also shown that in materials transitions when an electron obtains energy, it transitions from a lower to a higher energy level (Alamdari et al., Citation2020b).
The vibrations of alkanes, phenol, alcohols, aromatics, alkenes, alkyl halides, and aliphatic amines were revealed by FTIR analysis of zinc nanoparticles. Song et al. also reported similar results (Song et al., Citation2011). Furthermore, –C=O–, C—O–C, and C—O stretching vibrations were shown to generate maxima in carboxylic acid, polysaccharide, and amino acid, respectively (Stanciu et al., Citation2019). The same results were found. The created ZnO-NPs had particle sizes in the range of 45.8nm, as estimated by Nano Measurer and ImageJ analysis, as confirmed by SEM micrographs. The nanoparticle size was higher in this work which might be attributed to changes in synthesis settings such as temperature, incubation period, plant extract type, and handling applications (Osuntokun et al., Citation2019).
EDX analysis showed pure ZnO-NPs phases and a strong peak in the EDX spectrum, showing that the test sample contained pure Zinc. The EDX spectra of ZnO-NPs were obtained using a simple precipitation process using zinc as the starting material. Pure ZnO-NPs with substantial peaks have been successfully synthesized, according to the EDX spectrum. Additional peaks in the spectrum, however, were detected, suggesting that plant biomolecules were involved in nanoparticle synthesis. Throughout their examination, they found the same EDX pattern of ZnO-NPs with great purity. Parra & Haque, employed EDX analysis to assess the purity of ZnO-NPs and discovered pure zinc in the spectrum, as well as other peaks, suggesting that the sample was pure. XRD analysis was used to assess the size and crystallinity of the biosynthesized zinc nanoparticles. The XRD spectrum demonstrated the planar alignment and crystalline structure of ZnO-NPs. Numerous XRD reflection planes at 2 Theta and angles such as 30.73, 33.4, 35.27, 46.53, 55.2, 62.03, and 67.4 degrees indicate the fcc crystal structure, as attested by JCPDS Card No. 36–1451. The average crystal size, according to Scherer’s equation is 45.32nm. The XRD reflection planes show an fcc crystal shape with an average crystal size of 48.81nm, which matches the International Center of Diffraction Data card (JCPDS-36-1451) and supports the crystalline hexagonal structure synthesis (Parra & Haque, Citation2014; Vidya et al., Citation2013).
14. Conclusion
In conclusion, this study aimed to synthesize zinc oxide (ZnO) nanoparticles using Eucalyptus globulus Labill. The research successfully optimized various synthesis parameters, including reaction time, temperature, reagent concentration, and extract volume ratio, to enhance the yield and quality of ZnO nanoparticles. The use of Eucalyptus leaves as a reducing agent offers an eco-friendly alternative to traditional chemical synthesis methods, contributing to reduced environmental impact and biocompatible nanoparticle production.
The results showed that the optimal conditions for ZnO nanoparticle synthesis were as follows: a reaction time of 4hours, a temperature of 60°C, a reagent concentration of 1mM, and an equal volume ratio of plant extract and reagent (1:1). These conditions resulted in the formation of stable ZnO nanoparticles with a clear color change and an absorbance peak at 370-390nm.
Characterization of the synthesized ZnO nanoparticles was carried out using UV-visible spectrophotometry, scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDX), and Fourier Transform Infrared Spectroscopy (FTIR). The UV-visible analysis confirmed the formation of ZnO nanoparticles, while SEM images revealed the size and irregular shape of the nanoparticles, ranging from 25nm to 151nm. EDX analysis confirmed the purity of the nanoparticles, indicating the presence of Zn and oxygen. FTIR provided insights into the phytochemicals responsible for reducing Zn ions and stabilizing the nanoparticles.
Overall, this research contributes to the field of green nanotechnology and provides a sustainable alternative for nanoparticle synthesis, which can find applications in electronics, sensors, catalysis, and various biomedical areas. The use of Eucalyptus as a reducing agent aligns with environmentally friendly and cost-effective practices, making it a valuable addition to the field of nanomaterial production. Further research in this direction may open up new possibilities for green and eco-friendly synthesis methods.
Conflict of interest
The authors declare that they have no competing interests.
Authors’ contributions
All authors contributed equally to this work. All authors have read and approved the final version manuscript.
Data availability statement
No data were used to support the findings of the study.
Disclosure statement
No potential conflict of interest was reported by the author(s).
References
- Ahmad, H., Venugopal, K., Rajagopal, K., De Britto, S., Nandini, B., Pushpalatha, H. G., Konappa, N., Udayashankar, A. C., Geetha, N., & Jogaiah, S. (2020). Green synthesis and characterization of zinc oxide nanoparticles using Eucalyptus globules and their fungicidal ability against pathogenic fungi of apple orchards. Biomolecules, 10(3), 425. https://doi.org/10.3390/biom10030425
- Alamdari, S., Sasani Ghamsari, M., Lee, C., Han, W., Park, H. H., Tafreshi, M. J., Afarideh, H., & Ara, M. H. M. (2020a). Preparation and Characterization of Zinc Oxide 52 Nanoparticles Using Leaf Extract of Sambucus ebulus. Applied Sciences, 10(10), 3620. https://doi.org/10.3390/app10103620
- Alamdari, S., Sasani Ghamsari, M., Lee, C., Han, W., Park, H. H., Tafreshi, M. J., Afarideh, H., & Ara, M. H. M. (2020b). Preparation and characterization of zinc oxide nanoparticles using leaf extract of Sambucus ebulus. Applied Sciences, 10(10), 3620. https://doi.org/10.3390/app10103620
- Alyamani, A. A., Albukhaty, S., Aloufi, S., AlMalki, F. A., Al-Karagoly, H., & Sulaiman, G. M. (2021). Green fabrication of zinc oxide nanoparticles using phlomis leaf extract: characterization and in vitro evaluation of cytotoxicity and antibacterial properties. Molecules, 26(20), 6140. https://doi.org/10.3390/molecules26206140
- Ananthi, S., Kavitha, M., Balamurugan, A., Kumar, E. R., Magesh, G., Abd El-Rehim, A. F., Rahale, C. S., Suryakanth, J., Sharmila Rahale, C., & Srinivas, C. (2023). Synthesis, analysis and characterization of camellia sinensis mediated synthesis of NiO nanoparticles for ethanol gas sensor applications. Sensors and Actuators B: Chemical, 387, 133742. https://doi.org/10.1016/j.snb.2023.133742
- Bayda, S., Adeel, M., Tuccinardi, T., Cordani, M., & Rizzolio, F. (2019). The history of nanoscience and nanotechnology: From chemical–physical applications to nanomedicine. Molecules, 25(1), 112. https://doi.org/10.3390/molecules25010112
- Borysiewicz, M. A. (2019). ZnO as a functional material, a review. Crystals, 9(10), 505. https://doi.org/10.3390/cryst9100505
- Chabattula, S. C., Gupta, P. K., Tripathi, S. K., Gahtori, R., Padhi, P., Mahapatra, S., Biswal, B. K., Singh, S. K., Dua, K., Ruokolainen, J., Mishra, Y. K., Jha, N. K., Bishi, D. K., & Kesari, K. K. (2021). Anticancer therapeutic efficacy of biogenic am-ZnO nanoparticles on 2D and 3D tumor models. Materials Today Chemistry, 22, 100618. https://doi.org/10.1016/j.mtchem.2021.100618
- Ealia, S. A. M., & Saravanakumar, M. P. (2017, November). A review on the classification, characterisation, synthesis of nanoparticles and their application. Proceedings of the IOP conference series: materials science and engineering, Ukrain (Vol. 263, p. 032019). IOP Publishing.
- Faisal, S., Jan, H., Shah, S. A., Shah, S., Khan, A., Akbar, M. T., Rizwan, M., Jan, F., Wajidullah, Akhtar, N., & Syed, S. (2021). Green synthesis of zinc oxide (ZnO) nanoparticles using aqueous fruit extracts of Myristica fragrans: Their characterizations and biological and environmental applications. ACS Omega, 6(14), 9709–23. https://doi.org/10.1021/acsomega.1c00310
- Faisal, S., Khan, M. A., Jan, H., Shah, S. A., Rizwan, S., Shah, M., Akbar, M. T., Akbar, M. T. (2020). Edible mushroom (flammulina velutipes) as biosource for silver nanoparticles: From synthesis to diverse biomedical and environmental applications. Nanotechnology, 32(6), 065101. https://doi.org/10.1088/1361-6528/abc2eb
- Gancheva, M., Markova-Velichkova, M., Atanasova, G., Kovacheva, D., Uzunov, I., & Cukeva, R. (2016). Design and photocatalytic activity of nanosized zinc oxides. Applied Surface Science, 368, 258–266. https://doi.org/10.1016/j.apsusc.2016.01.211
- Gouws, C., & Hamman, J. H. (2020). What are the dangers of drug interactions with herbal medicines? Expert Opinion on Drug Metabolism & Toxicology, 16(3), 165–167. https://doi.org/10.1080/17425255.2020.1733969
- Iqtedar, M., Riaz, H., Kaleem, A., Abdullah, R., Aihetasham, A., Naz, S., & Sharif, S. (2020). Biosynthesis, optimization and characterization of ZnO nanoparticles using Bacillus cereus MN181367 and their antimicrobial activity against multidrug resistant bacteria. Revista Mexicana de Ingeniería Química, 19(Sup. 1), 253–266. https://doi.org/10.24275/rmiq/Bio1605
- Jadoun, S., Arif, R., Jangid, N. K., & Meena, R. K. (2021). Green synthesis of nanoparticles using plant extracts: A review. Environmental Chemistry Letters, 19(1), 355–374. https://doi.org/10.1007/s10311-020-01074-x
- Jan, H., Khan, M. A., Usman, H., Shah, M., Ansir, R., Faisal, S., Ullah, N., & Rahman, L. (2020). The aquilegia pubiflora (Himalayan columbine) mediated synthesis of nanoceria for diverse biomedical applications. RSC Advances, 10(33), 19219–19231. https://doi.org/10.1039/D0RA01971B
- Jo, I., Bellingham, P. J., McCarthy, J. K., Easdale, T. A., Padamsee, M., Wiser, S. K., & Richardson, S. J. (2022). Ecological importance of the Myrtaceae in New Zealand’s natural forests. Journal of Vegetation Science, 33(1), e13106. https://doi.org/10.1111/jvs.13106
- Khan, I., Saeed, K., & Khan, I. (2019). Nanoparticles: Properties, applications and toxicities. Arabian Journal Chemistry, 12(7), 908–931. https://doi.org/10.1016/j.arabjc.2017.05.011
- Kim, S. H., Lee, H. S., Ryu, D. S., Choi, S. J., & Lee, D. S. (2011). Antibacterial activity of silver-nanoparticles against staphylococcus aureus and Escherichia coli. Microbiology and Biotechnology Letters, 39(1), 77–85.
- Krishnaraj, C., Ramachandran, R., Mohan, K., & Kalaichelvan, P. T. (2012). Optimization for rapid synthesis of silver nanoparticles and its effect on phytopathogenic fungi. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 93, 95–99. https://doi.org/10.1016/j.saa.2012.03.002
- Mahmood Ansari, S., Saquib, Q., De Matteis, V., Awad Alwathnani, H., Ali Alharbi, S., Ali Al-Khedhairy, A., & Mendoza-Diaz, G. (2021). Marine macroalgae display bioreductant efficacy for fabricating metallic nanoparticles: intra/extracellular mechanism and potential biomedical applications. Bioinorganic Chemistry and Applications, 2021, 1–26. https://doi.org/10.1155/2021/5985377
- Narayana, A., Bhat, S. A., Fathima, A., Lokesh, S. V., Surya, S. G., & Yelamaggad, C. V. (2020). Green and low-cost synthesis of zinc oxide nanoparticles and their application in transistor-based carbon monoxide sensing. RSC Advances, 10(23), 13532–13542. https://doi.org/10.1039/D0RA00478B
- Ogunyemi, S. O., Abdallah, Y., Zhang, M., Fouad, H., Hong, X., Ibrahim, E., Masum, M. M. I., Hossain, A., Mo, J., & Li, B. (2019). Green synthesis of zinc oxide nanoparticles using different plant extracts and their antibacterial activity against xanthomonas oryzae pv. oryzae. Artificial Cells, Nanomedicine, and Biotechnology, 47(1), 341–352. https://doi.org/10.1080/21691401.2018.1557671
- Ossai, A. N., Ezike, S. C., & Dikko, A. B. (2020). Bio-synthesis of zinc oxide nanoparticles from bitter leaf (vernonia amygdalina) extract for dye-sensitized solar cell fabrication. Journal of Materials and Environmental Science, 11(3), 421–428.
- Osuntokun, J., Onwudiwe, D. C., & Ebenso, E. E. (2019). Green synthesis of ZnO nanoparticles using aqueous brassica oleracea L. var. italica and the photocatalytic activity. Green Chemistry Letters and Reviews, 12(4), 444–457. https://doi.org/10.1080/17518253.2019.1687761
- Parra, M. R., & Haque, F. Z. (2014). Aqueous chemical route synthesis and the effect of calcination temperature on the structural and optical properties of ZnO nanoparticles. Journal of Materials Research and Technology, 3(4), 363–369. https://doi.org/10.1016/j.jmrt.2014.07.001
- Patra, J. K., & Baek, K. H. (2015). Green nanobiotechnology: Factors affecting synthesis and characterization techniques. Journal of Nanomaterials, 2014, 219–219. https://doi.org/10.1155/2014/417305
- Paul, C. A., Kumar, E. R., Suryakanth, J., & Abd El-Rehim, A. F. (2023). Structural, microstructural, vibrational, and thermal investigations of NiO nanoparticles for biomedical applications. Ceramics International, 49(16), 27230–27246. https://doi.org/10.1016/j.ceramint.2023.05.273
- Poonguzhali, R. V., Kumar, E. R., Arunadevi, N., Srinivas, C., Khalifa, M. E., Abu-Melha, S., & El-Metwaly, N. M. (2022). Natural citric acid assisted synthesis of CuO nanoparticles: Evaluation of structural, optical, morphological properties and colloidal stability for gas sensor applications. Ceramics International, 48(18), 26287–26293. https://doi.org/10.1016/j.ceramint.2022.05.311
- Qu, J., Yuan, X., Wang, X., & Shao, P. (2011). Zinc accumulation and synthesis of ZnO nanoparticles using physalis alkekengi L. Environmental Pollution, 159(7), 1783–1788. https://doi.org/10.1016/j.envpol.2011.04.016
- Rafiee, B., Ghani, S., Sadeghi, D., & Ahsani, M. (2018). Green synthesis of Zinc Oxide Nanoparticles Using Eucalyptus Mellidora Leaf Extract and Evaluation of its Antimicrobial Effects. Journal of Babol University of Medical Sciences, 20(10), 28–35.
- Ramesh, M., Anbuvannan, M., & Viruthagiri, G. (2015). Green synthesis of ZnO nanoparticles using solanum nigrum leaf extract and their antibacterial activity. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 136, 864–870. https://doi.org/10.1016/j.saa.2014.09.105
- Rana, A., Yadav, K., & Jagadevan, S. (2020). A comprehensive review on green synthesis of nature-inspired metal nanoparticles: Mechanism, application and toxicity. Journal of Cleaner Production, 272, 122880. https://doi.org/10.1016/j.jclepro.2020.122880
- Sabo, V. A., & Knezevic, P. (2019). Antimicrobial activity of Eucalyptus camaldulensis Dehn. plant extracts and essential oils: A review. Industrial Crops and Products, 132, 413–429. https://doi.org/10.1016/j.indcrop.2019.02.051
- Selim, Y. A., Azb, M. A., Ragab, I., & Abd El-Azim, M. H. (2020). Green synthesis of zinc oxide nanoparticles using aqueous extract of deverra tortuosa and their cytotoxic activities. Scientific Reports, 10(1), 1–9. https://doi.org/10.1038/s41598-020-60541-1
- Siripireddy, B., & Mandal, B. K. (2017). Facile green synthesis of zinc oxide nanoparticles by Eucalyptus globulus and their photocatalytic and antioxidant activity. Advanced Powder Technology, 28(3), 785–797. https://doi.org/10.1016/j.apt.2016.11.026
- Song, Z., Kelf, T. A., Sanchez, W. H., Roberts, M. S., Rička, J., Frenz, M., & Zvyagin, A. V. (2011). Characterization of optical properties of ZnO nanoparticles for quantitative imaging of transdermal transport. Biomedical Optics Express, 2(12), 3321–3333. https://doi.org/10.1364/BOE.2.003321
- Song, J. Y., Kwon, E. Y., & Kim, B. S. (2010). Biological synthesis of platinum nanoparticles using diopyros kaki leaf extract. Bioprocess and Biosystems Engineering, 33(1), 159. https://doi.org/10.1007/s00449-009-0373-2
- Stanciu, G. D., Luca, A., Rusu, R. N., Bild, V., Beschea Chiriac, S. I., Solcan, C., Bild, W., & Ababei, D. C. (2019). Alzheimer’s disease pharmacotherapy in relation to cholinergic system involvement. Biomolecules, 10(1), 40. https://doi.org/10.3390/biom10010040
- Subramaniam, V. D., Prasad, S. V., Banerjee, A., Gopinath, M., Murugesan, R., Marotta, F., Sun, X. F., & Pathak, S. (2019). Health hazards of nanoparticles: Understanding the toxicity mechanism of nanosized ZnO in cosmetic products. Drug and Chemical Toxicology, 42(1), 84–93. https://doi.org/10.1080/01480545.2018.1491987
- Suresh, K. C., Surendhiran, S., Manoj Kumar, P., Ranjth Kumar, E., Khadar, Y. S., & Balamurugan, A. (2020). Green synthesis of SnO 2 nanoparticles using delonix elata leaf extract: Evaluation of its structural, optical, morphological and photocatalytic properties. SN Applied Sciences, 2(10), 1–13. https://doi.org/10.1007/s42452-020-03534-z
- Talam, S., Karumuri, S. R., & Gunnam, N. (2012). Synthesis, characterization, and spectroscopic properties of ZnO nanoparticles. International Scholarly Research Notices, 2012, 1–6. https://doi.org/10.5402/2012/372505
- Verma, A., Gautam, S. P., Bansal, K. K., Prabhakar, N., & Rosenholm, J. M. (2019). Green nanotechnology: Advancement in phytoformulation research. Medicines, 6(1), 39. https://doi.org/10.3390/medicines6010039
- Vidya, C., Hiremath, S., Chandraprabha, M. N., Antonyraj, M. L., Gopal, I. V., Jain, A., & Bansal, K. (2013). Green synthesis of ZnO nanoparticles by calotropis gigantea. Int J Curr Eng Technol, 1(1), 118–120.
- Ying, S., Guan, Z., Ofoegbu, P. C., Clubb, P., Rico, C., He, F., & Hong, J. (2022). Green synthesis of nanoparticles: Current developments and limitations. Environmental Technology & Innovation, 26, 102336. https://doi.org/10.1016/j.eti.2022.102336