Effect of volume ratios on the novel Solanum lycopersicum leaf extract-mediated production of ZnO and Co₃O₄ nanoparticles for potential antifungal applications

ABSTRACT

Antimicrobial infectious disease has become a major problem in the world. It has been found as its continual distribution is at extremely frightening rate. The most curable technique is the design and synthesis of effective nano-based drugs as green alternative. In this study, ZnO and Co₃O₄ nanoparticles (NPs) were produced from their precursor salts in the volume ratios of 1:3, 1:1, and 3:1 with Solanum lycopersicum leaf as a capping and reducing agent. Characterizations were performed using TGA/DTA, XRD, SEM, TEM, HRTEM, SAED, UV-Vis, and FTIR techniques. After 450°C and 500°C, ZnO and Co₃O₄ NPs were found to be thermally stable, respectively. For the ZnO and Co₃O₄ NPs in 1:3, 1:1, and 3:1 volume ratios, respectively, their average crystalline sizes were 51, 20, 29 and 26, 21 and 28 nm. The degree of crystallinity is confirmed by SEM in conjunction with TEM-HRTEM, and SAED. Among the numerous volume ratios, Co₃O₄ (1:3) and Co₃O₄ (1:1) showed 100% promised activity against Alternaria solani pathogen.

Antimicrobial infectious disease has become a major serious problem in the modern world. It has been found as its continual distribution is at an extremely frightening rate. The most curable technique for this worldwide issue is the design and synthesis of effective nano-based drugs as green alternative solutions. In this study, ZnO and Co₃O₄ nanoparticles (NPs) were produced from their respective precursor salts in the volume ratios of 1:3, 1:1, and 3:1 with Solanum lycopersicum leaf as a capping and reducing agent. Characterizations were performed using TGA/DTA, XRD, SEM, TEM, HRTEM, SAED, UV-Vis, and FTIR techniques. After 450°C and 500°C, greenly synthesized ZnO and Co₃O₄ NPs were found to be thermally stable, respectively. For the ZnO and Co₃O₄ synthesized in 1:3, 1:1, and 3:1 volume ratios, respectively, their average crystalline sizes were 51, 20, 29 and 26, 21 and 28 nm. The degree of crystallinity is confirmed by SEM examination in conjunction with TEM-HRTEM, and SAED. Among the numerous volume ratios, Co₃O₄ (1:3) and Co₃O₄ (1:1) showed 100% promised activity against Alternaria solani pathogen.

KEYWORDS:

• ZnO NPs
• Co3O4 NPs
• Solanum lycopersicum
• Alternaria solani

Introduction

Global food security associated with environmental sustainability has recently been become a grave global concern. The spread of numerous types of plant caused diseases results in a dramatic decline of crop output and vegetable production from year to year, which is a worldwide issue [1]. The major microorganisms that cause plant diseases include bacteria, viruses, and fungi. The main illness behind the sharp reduction in the production of healthful vegetables and fruits is attributed to various types of fungal pathogenic infections. Comparably, reports showed that ten times as much as bacterial and viral infections, respectively, fungal infections have a mammoth impact on plant yield output. Vegetables and fruit products lose quality due primarily to fungus infections, which leads to poor components and lower storage quality and this inturn causes the for the decline in global food safety. Moreover, such global decline of vegetables and numerous types of fruits leads for the existence of different classes of diseases, especially fatal in developing countries. Due to their great nutritional value, tomatoes are the most consumed fruit worldwide. The high levels of carotene, vitamin C, folate, flavonoids, and ascorbic acid found in tomatoes make them potent antioxidants [2]. However, it is very susceptible to fungal infections caused by diseases and readily putrefies. The tomato fruit is harmed by a number of fungus infections, including Alternaria solani, which is recognized for its dark brown necrotic lesions with concentric rings and influences the product yield. Additionally, the host that ate the fungal-infected tomato is more susceptible to developing illnesses like lung cancer, liver cancer, and respiratory conditions. Because of this, tomatoes have been treated with a variety of antifungal substances, from tomato seedlings to the collection of ripe tomato fruit [3].

However, the fungi developed drug resistance either directly or indirectly harmed the human health system by polluting the environment [4]. In order to manage fungal infections caused by diseases of plants, specifically fruits and vegetables, and to maintain food security, it is therefore necessary to develop and design a very effective and competent new power antifungal drug. Among these, the use and choice of NPs is found to be the most prominent and very cost effective, which may kill fungi that are resistant to many chemical-based fungicidal agents [5]. Previously, reports proved that the antifungal activities of several metal oxide NPs such as TiO₂ [6], ZnO [7], Co₃O₄, and Fe₃O₄, Ag [8] and relatives were verified, that provides a promised activity against potato attack fungal pathogens such as Alternaria solani. Report studies showed that the ability of ZnO and Co₃O₄ NPs to control fungal plant diseases such as Alternaria solani and Fusarium oxysporum through their ability to directly inhibit fungal growth by distorting the growing mycelia and also by eliminating mycotoxins such as fusaric acid. This mechanism enables these metal oxide NPs to easily and effectively control the growth and damage to fungal caused diseases from the different parts of fruits and vegetables.

These NPs are mostly synthesized using a chemical method which is not environmentally friendly, toxic, costly and not safe to handle and then to apply to the targeted applications. In addition, some of those metal oxides were also synthesized using plant extracts as capping and stabilizing agent within a single composition, without any composition variation. However, single composition synthesis of metal oxides from their precursors, salt and leaf extract, might not lead to identifying the impending candidate for the targeted applications. To overcome such a challenge, in the current studies, the potent antifungal Co3O4 and ZnO NPs were synthesized by utilizing (tomato) Solanum lycopersicum leaf extract within numerous volume ratios of the leaf and their corresponding precursor salts.

Metal oxide NPs were previously created using physical techniques like plasma, evaporation-condensation, laser ablation, and sputtering, as well as chemical techniques like hydrothermal, coprecipitation, electrochemistry, thermal decomposition, polyol, microemulsion, and sol-gel. These days, biological methods which are quick, inexpensive, non-toxic, and use less energy most often take the place of these older ones [9]. Plant components like roots, leaves, flowers, peel, seeds, and bark are used in the production of biological NPs, as well as microorganisms like fungi, bacteria, yeast, and viruses. Due to their advantages over chemical and physical synthesis methods, including biocompatibility, non-toxicity, cost-effectiveness, high stability, speed, and environmental friendliness, green synthesis techniques are more approachable and effective [10].

In comparison to other living organisms used for NPs synthesis, plants can be advantageous because the synthesis of NPs by using microorganisms is somewhat difficult due to the involvement of a complex process of maintaining cell cultures, intra-cellular synthesis, requiring longer incubation time in the growth media for reducing metal mediums, and water-soluble phytochemicals do the same in a moment. At the same time, these synthesis techniques require an excellent clean environment and optimization of the numerous parameters during the culturing of the medium is very complex and difficult. The presence of primary metabolites like polysaccharides, proteins, amino acids, and nucleic acids [11]as well as secondary metabolites (phytochemicals) like polyphenols, flavonoids, terpenoids [12,13], alkaloids, tannins, steroids, and glycosides in plant extracts makes plant-mediated synthesis a suitable process for the large-scale production of metal NPs quickly and efficiently [14,15]. Due to this, the potential antifungal activities of ZnO and Co₃O₄ NPs were synthesized using Solanum lycopersicum leaf extract within the three volume ratios of 1:3, 1:1 and 3:1 and to deal with comparative study of volume ratios on the physico-chemical properties of the synthesized NPs and on antifungal applications. However, a lot of previous work has been intensively conducted on the synthesis of doped and un-doped ZnO and Co₃O₄ NPs for various applications. Until now, there has not been any scientific report done on the synthesis of ZnO and Co₃O₄ NPs within different volume ratios from their corresponding precursor salts and Solanum lycopersicum leaf extract as a capping, reducing, and stabilizing agent for antifungal activities. Therefore, the emphasis of the current work is the synthesis of both ZnO and Co₃O₄ NPs within various volume ratios and then to deal with the effect of volume ratio on the structural morphology, crystallinity nature, and light absorption behaviour, as well on thermal stability. Furthermore, the work was also very tight with a comparative study of volume ratios towards the effect on Alternaria solani activity. This attempt is made to investigate the antifungal activity of green bioacid ZnO and Co₃O₄ NPs against the Alternaria solani.

Materials and methods

Chemicals and reagents

An oven, mortar, electronic balance, beaker, pH metre, ceramic crucible, conical flask, measuring cylinder, Whatman filter paper, magnetic stirrer, centrifuge, glass filters, beaker, test tube, plant grinding machine, hot plate, pipets, spatulas, Erlenmeyer flask, volumetric flask, various types of beakers and aluminium foils were used as the equipment. The analytical chemicals such as cobalt acetate hexahydrate (Co(CH₃CO₂)₂.6 H₂O), zinc acetate hexahydrate (Zn(CH₃CO₂)₂.2 H₂O) (Sigma Aldrich), Sodium hydroxide (NaOH) (Sigma Aldrich), ethanol (99.9%) (Lab Tech Chemicals), distilled water, fluconazole, potato dextrose agar and Dimethyl sulphoxide (DMSO Sigma-Aldrich) were used.

Synthesis of ZnO and Co₃O₄ NPs

ZnO and Co₃O₄ NPs were produced using the acetate precursor salts of the relevant metals. The size reduction of bulk salts to the nanoscale was accomplished using the leaf extract of Solanum lycopersicum. Three volume ratios of precursor salt to plant leaf extract were used to create the ZnO NPs: 1:1 (50:50), 1:3 (33:66), and 3:1 (66:33). At room temperature, the plant extract was then added to 0.5 M of Zn(CH₃CO₂)₂.2 H₂O and stirred for 3 h. By adding 0.1 M of NaOH to the already formed suspension dropwise and stirring for 20 min, the pH was adjusted to be 12 in this manner. Each of the suspensions was then stayed at 4 in the refrigerator to improve the formation of precipitate. The precipitate was washed three times with absolute ethanol and distilled water by centrifugation at 2000 rpm for 15 min. ZnO NPs produced biologically were collected on a ceramic crucible dish and dried for 2 h at 80°C in the oven. The oven-dried NPs were then kept for characterization and application after being calcined in a muffled furnace for five hours at 450°C. By adjusting the volume of the precursor salt and plant extract, as indicated in Figure 1, the remaining two ratios of ZnO NPs were formulated in the same manner. The same procedure was followed for Co₃O₄ NPs synthesis from Co(CH₃CO₂)₂.6 H₂O and Solanum lycopersicum leaf extract in volume ratios like 1:1 (50:50), 1:3 (33:66), and 3:1 (66:33) mL, respectively [8,16].

Figure 1. Solanum lycopersicum leaf extract templated synthesis of Co₃O₄ and ZnO NPs.

Characterization techniques

The green formed ZnO and Co₃O₄ NPs were characterized using several characterization techniques, that them to gather immense physico-and surface properties. The thermal stability and the calcination temperature identification of ZnO and Co₃O₄ were confirmed using the TGA-DTA (DTG-60 H, Shimadzu Co., Japan). The formation and crystalline structure of green synthesized ZnO and Co₃O₄ (1:1, 1:3, and 3:1) NPs were determined using an X-ray diffraction pattern (XRD-7000, Shimadzu Co., South Korea). The morphological analysis and particle size calculations of ZnO and Co₃O₄ NPs were investigated using SEM (SEM-EVO 18 model-low vacuum facility-ALTO 1000 Cryo attachment) and TEM (JEOL JEM 2100 HR-TEM) techniques, respectively. Furthermore, SAED and HRTEM micrographs were used to establish the internal planar distance and crystalline structure. The FTIR characterization technique (PerkinElmer 65) was also used to identify the possible functional group present within the leaf extract and also to deal with the formation of Zn-O and Co-O modes of vibrations.

Method of antifungal

By using 10 mg/mL from each of the prepared different volume ratios of ZnO and Co3O4 NPs in the presence of Fluconazole as a positive control, the antifungal activities of the green synthesized ZnO and Co3O4 NPs within volume ratios of 1:3, 1:1, and 3:1 were tested against the Alternaria solani fungi pathogen. The pathogen was isolated and cultured at room temperature in potato dextrose broth. A 230 rpm orbital shaking incubator was then permitted to hold the cultured pathogen. Later, a sterile cotton swab was used to transfer 100 µL from the prepared aliquot culture into the potato dextrose agar on the PDA plate. Each PDA plate was then left to stand for a further 15 min to allow for the process of cultural absorption. Then, using a sterile gel puncher, 5 mm-sized wells were made in the prepared agar. Using a micropipette, the prepared suspensions of ZnO and Co₃O₄ NPs in each of the various volume ratios were poured into the wells on each plate. The wells were then mixed until homogeneity was maintained. The PDA plate containing each of the green nano-drugs and the associated fungus was then cultured for about 20 days at room temperature in an upside-down position, Alternaria solani, followed by measuring the zone of inhibition [17].

Results and discussion

Thermal stability characterization

Thermal stability and calcination temperature identification analysis of synthesized ZnO (1:1) and Co₃O₄ (1:1) NPs was used to characterize the thermal stability of Solanum lycopersicum leaf extract templated ZnO and Co₃O₄ NPs. The obtained TGA-DTA graph of ZnO (1:1) NPs is shown in Figure 2a. The synthesis and centrifugation processes may be attributed to the observed loss of surface-attached water molecules from synthesized ZnO NPs in the temperature range of 25–100°C [18]. Additionally, extra weight loss between 200°C and 350°C was also noticed, which the carbonization process may be responsible for. The use of Solanum lycopersicum leaf extract as a templating agent may have again contributed to this carbonization process because it is both environmentally and economically advantageous. However, it has been noted that weight loss stops around 400°C and continues no further. This intern attributed it to the heat stability of ZnO (1:1) NPs synthesized using a Solanum lycopersicum template. As a result, 450°C was discovered as the calcination temperature for the greenly synthesized ZnO (1:1) NPs, which is found to be too stable.

Figure 2. TGA-DTA graph of Solanum lycopersicum extract mediated synthesized (a) ZnO (1:1) and (b) Co₃O₄ (1:1) NPs.

The thermal stability of Co₃O₄ (1:1) NPs synthesized utilizing the Solanum lycopersicum template was also evaluated and shown in Figure 2b. As can be seen in Figure 2b ,a significant weight loss was noticed between 27°C and 100°C due to the evaporation of solvents that were attached to the surface, such as water and ethanol. Additionally, weight loss remained between 200°C and 450°C, which may be related to the loss of carbon-containing components contained by the use of Solanum lycopersicum leaf extract [19]. No further weight loss was noticed after 450°C, indicating that starting at this temperature, the stability of greenly synthesized Co₃O₄ NPs was attained. As a result, the Solanum lycopersicum-derived Co₃O₄ (1:1) NPs were calcined in a muffle furnace by adding additional preservation (50°C) [20]. This calcination temperature was also used to calcined the other Co₃O₄ NPs volume ratios.

Crystallographic and phase purity characterization

The XRD characterization methodology was used to assess the crystal structure, degree of phase purity, and compute the average crystal size of each of the various volume ratios of green-fabricated ZnO and Co₃O₄ NPs. The characterization was performed at room temperature in the presence of a Cu target for generating Cu Kα radiation with λ = 0.15406 nm, which was recorded in the range of 10–80°. Figure 3a,b display the resulting XRD spectra of Solanum lycopersicum supporting ZnO and Co3O4 with numerous volume ratios. The peaks of ZnO NPs are observed at 31.8°, 34.4°, 36.3°, 47.6°, 56.60°, 62.6°, and 69.1° with corresponding crystal plane values of 100, 002, 101, 102, 110, 103 and 112, respectively with JCPDS card number at 036–1451. Using Debye Scherer’s equation, the calculated average crystalline size of ZnO NPs was determined to be 51, 20, and 29 nm average for the 1:3, 1:1, and 3:1, respectively. The alteration and the change in the calculated average crystalline size of Solanum lycopersicum assisted ZnO NPs were due to the volume ratios of plants and its precursor salts [16]. When compared the ZnO NPs with the other two volume ratios, it has been discovered that the ZnO (1:1) NPs have the smallest average crystalline size. This might be as a result of the optimal aqueous leaf extract of Solanum lycopersicum filling with pores of ZnO [9].

Figure 3. XRD spectra of Solanum lycopersicum leaf extract templated synthesized (a) ZnO and (b) Co₃O₄ NPs.

Similarly, Figure 3b, depicts the XRD spectra of Solanum lycopersicum leaf aqueous extract assisted Co₃O₄ (1:3, 1:1, and 3:1) NPs. The diffraction peaks were observed at 19.2°, 31.8°, 36.96°, 38.6°, 46.9°, 55.7°, 59.8°, and 66.0° corresponding to hkl plane values of 111, 220, 311, 222, 400, 422, 511, and 440, respectively. The diffraction peaks of each of the volume ratios of green synthesized Co₃O₄ NPs were found to fit with the JCPDS card number 042–1467, which confirms the successful fabrication of pure Co₃O₄ NPs. The estimated average crystalline size of Co₃O₄ NPs were found to be 26, 21, and 28 nm for the 1: 3, 1: 1, and 3: 1 volume ratios of its precursor salt to leaf extract, respectively. The larger amount of leaf suspension utilized during the synthesis procedure, which successfully stabilizes the intended NPs, may have allowed for the smallest average crystalline size (21 nm) of Co₃O₄ (1: 1) NPs [21,22].

Optical property characterization

The optical light absorption behaviour of green synthesized ZnO and Co₃O₄ NPs were investigated using UV-Vis (UV-Vis1800-double beam spectrophotometer, Shimadzu, Japan)) spectroscopy. The varying volume ratios of the environmentally friendly synthesized ZnO and Co₃O₄ NPs displayed diverse UV-Vis light absorption behaviours, as shown in Figure 4a,b. A change in the ratio of the plant’s leaf extract and the corresponding precursor salts of ZnO and Co₃O₄ NPs is responsible for the variation in light absorption. Figure 4a depicts the recorded UV-Vis result of ZnO NPs with an absorption wavelength of 361.5, 360.3, and 358.4 nm for the 1:3, 1:1, and 3:1 volume ratios of precursor to leaf extract compositions, respectively. The formation of ZnO NPs is found to be directly confirmed by this study, and it also fits with earlier reports [23]. The calculated bandgap energy for 1:3, 1:1, and 3:1 volume ratios of ZnO NPs were found to be 3.40, 3.46, and 3.45 eV, respectively. The variation in the volume ratios of the plant leaf with the precursor salt of ZnO NPs contributes to the variation in the light absorption within the UV-Vis region and bandgap energy of ZnO NPs. Additionally, the calculated average crystalline size of the XRD (Figure 3) result of ZnO NPs was directly matched with the predicted bandgap energy of each volume ratio of ZnO NPs. Equivalently, the oscillation of electrons from the region of valence to the conduction band, which can occur after exposure to light at a specific wavelength, was responsible for the variation of surface plasmon resonance for each of the different ratios of ZnO NPs. This study supports the idea that leaf extract plays a role in the synthesis of ZnO NPs by changing the corresponding physico-chemical properties of green synthesized ZnO NPs.

Figure 4. UV-Vis spectra of Solanum lycopersicum leaf extract templated (a) ZnO and (b) Co₃O₄ NPs.

Similarly, the green Co₃O₄ NPs UV-Vis absorption spectra are tabulated in Figure 4b and demonstrate maximal light absorption behaviour between 263.3 and 278.5 nm. The discovery of the region’s highest absorption validates the formation of stable Co3O4 NPs, and the outcome is also closely related to the earlier published work [24,25]. The cause of light absorption by Co in Co₃O₄ NPs is the remarkably constant fluctuation of electrons within the conduction band and valence band produced by the binding of an electromagnetic field. The desired Co₃O₄ NPs were produced by reducing cobalt (II) nitrate hexahydrate in the presence of leaf Solanum lycopersicum aqueous extract, which was used extensively as a stabilizing, reducing, and capping agent during the synthesis process. Furthermore, the estimated bandgap energy of Co₃O₄ NPs was found to be 4.7, 4.53, and 4.5 eV for 1:2, 1:1, and 2:1 volume ratios, respectively. Similarly, the change in the volume ratio of the leaf extract to be supplied during the reaction process was linked to the changing of the bandgap energy of each of the distinct compositions of Co₃O₄ NPs. The band gap energy study supported the earlier studies by confirming the generation of stable Co₃O₄ NPs.

Functional group analysis

The formation of Zn-O and Co-O stretching as well as the role of Solanum lycopersicum leaf extract during the synthesis process was investigated via FTIR. Figure 5a,b showed the presence of various functional groups at 3457.7, 2919.4, 2373.2, 1645.3, 1425.4, 1091.8, and 477.6 cm-1 for ZnO NPs and 3435.3, 2927.4, 2373.4, 1638.1, 1387.8, 1068.7, 667.1, and 568.0 cm-1 for Co3O4 NPs. The strongest and broad peak found in both ZnO (3457.7 cm-1) and Co3O4 (3435.3 cm-1) confirmed the presence of O-H stretching mode of vibration for both ZnO and Co3O4 NPs, which might be associated to the O-H stretching of alcohol, ketone, carboxylic acid, phenolic compounds, flavonoids and related bioactive ingredients [26].

Figure 5. FTIR spectra of Solanum lycopersicum leaf extract mediated (a) ZnO and (b) Co₃O₄ NPs.

Furthermore, the slight broad peak observed at about 2919.4 and 2927.4 cm⁻¹ is responsible for the ZnO and Co₃O₄ NPs formation, respectively, which reflects the presence of C-H stretching of hydroxyl compounds. This might be contributed due to the use of the leaf extract of Solanum lycopersicum during the synthesis process of the targeted ZnO and Co₃O₄ NPs. While the low intense peaks exist at around 2373.2 and 2373.4 cm⁻¹ is for ZnO and Co₃O₄ NPs, which depicts the presence of C-O stretching oscillations. Moreover, the peaks located at about 1645.3, 1425.4, 1091.8 cm⁻¹ is for ZnO NPs, while the peak found at about 1638.1, 1387.8, and 1068.7 cm⁻¹ is for Co₃O₄ NPs, which might indicates the presence of C-H and C = C fused C = O stretching vibration of alkane, alkenes, lower molecular weight alcohols ketones and also the surface association of atmospheric CO₂. The narrowest peak is located at about 477.6 cm⁻¹ (Figure 5a), which strongly confirms the Zn-O stretching vibrations. This is a direct confirmation of the formation of metal oxygen bonding between Zn and O. The peaks observed at about 667.1 and 568.0 cm⁻¹ (Figure 5b) could result from the formation of Co (II)-O and Co (III)-O bonds, respectively The current study was found to be directly related with the reports done before [16].

Morphological analysis

For the purpose of surface morphological analysis and also to gain additional supportive information, Solanum lycopersicum leaf extract mediated ZnO and Co₃O₄ NPs was allowed to SEM. Figure 6 (a–c) and (d–f) presents the surface morphology of green synthesized ZnO and Co₃O₄ NPs, which formed within a volume composition of 1:3, 1:1, and 3:1, respectively. The morphological structure of ZnO (1:3) NPs showed a mixture of rode and spherical like surface morphologies, while the remaining volume ratios of ZnO NPs showed nearly spherical. Furthermore, the ZnO (1:3) NPs also showed some aggregation that might be contributed due to the use of excessive volume of the leaf extract as compared to its counterparts. Since, the presence of an excessive amount of leaf extract might lead to the formation of accumulated leaf suspension above the pore size capacity of the ZnO NPs that it can hold in it. Hence, these findings directly fit with the previous reports in the literature too. However, ZnO (1:1) and ZnO (3:1) NPs showed almost homogenized surface morphology in all sections of the surfaces without any aggregation and agglomeration formation, which might be attributed due to the use of optimized extract during the synthesis process [27].

Figure 6. SEM of Solanum lycopersicum leaf extract mediated synthesized (a–c) ZnO and (d–f) Co₃O₄ NPs.

Figure 6d–5f shows the surface structural image of Co₃O₄ NPs synthesized with the volume ratios of 1:3, 1:1, and 3:1, respectively. As can be observed in Figure 6d, the Co₃O₄ (1:3) NPs showed sphere-shaped surface morphology with little aggregation formation, which is a direct confirmation of the use of the extract during the synthesis process. In the same manner, the establishment of aggregation in the case of the 1:3 ratios of the green synthesized Co₃O₄ NPs might be ascribed to the presence of a surplus amount of aqueous Solanum lycopersicum suspension on the surface of Co₃O₄ NPs. This is due to the fact that the presence of excessive amount of the extract would be above the pore-size structure of Co₃O₄ NPs. While the resulting 1:1 and 3:1 volume ratios of Co₃O₄ NPs showed a smooth spherical-shaped morphological structure in the absence of any forms of aggregation and agglomeration. This might be possible due to the existence of a 1:1 fit with the pore-size capability of Co₃O₄ NPs. Similar results were reported in the literature review, which is consistent with the current report [20,28].

TEM-HRTEM and SAED analysis

Using cutting-edge methods like TEM and HRTEM, the thorough morphological characterization and particle size of greenly synthesized ZnO and Co₃O₄ NPs were determined. Additionally, SAED was used to look at the crystallinity and the formation of pure ZnO and Co₃O₄ NPs. All the resulting ZnO NPs volume ratios were found to be spherical structures in its TEM images, as shown in Figure 7a. HRTEM images also showed that ZnO NPs have a spherical morphology. The attainment of small sized green synthesized ZnO and Co₃O₄ NPs was due to the presence of phenolic groups that are found in plant leaves used in the synthesis [29]. This indicated that the bioactive molecules utilized were a good size, reducing and strong capping agent. Being a strong capping agent used for prevention of agglomerates that yield larger NPs as an extended form. The atomic structure of ZnO and Co₃O₄ NPs was obtained from HRTEM images. Following the outer edge, it was possible to establish the location of the lattice in the grain containing the atoms on the plane for both ZnO and Co₃O₄ NPs. To determine the crystalline nature of the biologically synthesized materials, the interatomic distances in the crystallographic planes were calculated [19]. The d-spacing or interplanar distance of ZnO and Co₃O₄ NPs was investigated using Gatan software (GMS3). As shown in Figures 7c and 8b, the d-spacing of ZnO and Co₃O₄ NPs were found to be 0.576 and 0.522 nm, respectively. The interplanar distance was determined by the HRTEM of the Fourier Transform (FT) on the histogram, which indicates the space between the lattice fringe that corresponds to the reciprocal reflections at the specific angles as shown in Figures 7d and 8e.

Figure 7. TEM image (a), HR-TEM (b), selected FFT (c), histogram graph (d), and SAED images (e and f) of green synthesized ZnO NPs.

Figure 8. TEM image (a), HR-TEM image (b), selected HR-TEM image (c), selected FFT histogram graph (d), histogram graph (e)and SEAD images (f) of green synthesized Co₃O₄ NPs.

The crystalline nature of ZnO and Co₃O₄ NPs were revealed using a selected area electron diffraction pattern (SAED) as presented in Figures 7f and 8f, respectively. As shown in Figures 7f and 8f, the monocrystalline and polycrystalline nature of ZnO and Co₃O₄ NPs were investigated, respectively. The plane arrangement shown in SAED indicated good agreement with XRD data. In the SAED image of ZnO NPs, the bright spot is aligned of a single line and parallel to the other, which indicates the monocrystallinity of ZnO NPs, while of the Co₃O₄ NPs, the electron diffraction observed in spherical form around the focusing point, which indicate the presence many crystalline forms [30].

As shown in Figure 9b,d, the average particle size was determined by TEM using ImageJ software for both ZnO and Co₃O₄ NPs, respectively. The average particle size of green synthesized ZnO and Co₃O₄ NPs was found to be 13.61 0.52 and 14.03 0.55 nm, respectively, which shows good agreement with XRD results [31,32]. In addition to this, the particle size distribution was also determined from the TEM images using ImageJ software. From the Histogram graph shown in Figure 9a,c, the particle size distribution was found within the range of 8–30 nm and 8–20 nm for ZnO and Co₃O₄ NPs, respectively. On the other hand, the difference in size distribution of ZnO and Co₃O₄ NPs was most probably due to the presence of polyphenolic molecules. The charge interaction on these compounds develops different reducing abilities [33,34].

Figure 9. The histogram graph showing particles size distribution (a, b) and particle size (c, d) of ZnO and Co₃O₄ NPs, respectively.

Antifungal activities of Co₃O₄ and ZnO NPs

Green synthesized ZnO and Co₃O₄ NPs are outstretched as a good alternative for fungal-caused disease treatment. In this report, the Solanum lycopersicum leaf extract mediated ZnO and Co₃O₄ NPs were applied to the fungal Alternaria Solani, and their potential activities were elucidated. The tests were performed on PDA growth media treated with 100 μL of Co₃O₄ and ZnO NPs [35]. All the three ratios of ZnO and Co₃O₄ NPs were tested at constant concentration and their activities were compared between their ratios and between the two separate NPs. The growth diameter of the fungi was measured by all ratios for both NPs. The growth diameter of ZnO NPs were found to be 1.0, 1.2, and 0.41 mm for 1:1, 1:3, and 3:1 volume ratios, respectively.

As shown in Figure 10, the resulting growth of fungi was observed on the PDA plate treated with ZnO NPs (1:3). However, the growth was highly inhibited on the PDA plate treated with ZnO NPs (3:1). The improved inhibition zone by ZnO NPs (3:1) is due to the high ratio of metal in particles which can interact with the negatively charged cells of fungi. In the case of Co₃O₄ NPs, no fungal growth was observed for the 1:1, and 1:3 volume ratios at 100 μL [36]. However, unlike ZnO NPs, the Co₃O₄ (3:1) NPs do not prevent the growth of fungi at the same concentration, which is due to their large particle size. The larger Co₃O₄ (3:1) NPs cannot easily interact with the peptidoglycan and cross the cell membrane to inhibit fungal growth. The Zn2+ and Co²⁺/³⁺ interact with the cell biomolecules such as polysaccharides and proteins such as chitin, glucan, glucanases, and glycoproteins [37].

Figure 10. The growth zone (mm) of Alternaria solani under ZnO and Co₃O₄ NPs treatment (a) and graph (b).

The green synthesized ZnO and Co₃O₄ NPs can interact with the hydroxyl group found on the glycoproteins. The metal ions of the NPs interact with the hydroxyl of glucan at C₆ of glucose and the amide group of chitin at C₂ of glucose and generate reactive oxygen and nitrogen species. Antifungal drugs such as Fluconazole, azoles, echinocandins, and polyenes are well-known and widely used drugs. These antifungal drugs target Ergosterol and β-(1,3)-D-glucan, which are essential for the survival of these pathogens. In general, the metal ions of biologically synthesized ZnO and Co₃O₄ NPs inhibit the growth of fungi by disintegrating fungal cell walls, damaging the surface of the protein and nucleic acid by generating reactive oxygen species (ROS). As in Figure 10a,b, both NPs showed higher growth inhibition than standard drugs. However, Co₃O₄ NPs showed good fungal growth inhibition for 1:1 and 1:3 volume ratios except 3:1. The better growth inhibition for ZnO NPs (3:1) than Co₃O₄ NPs (3:1) is believed to be due to the shape of the particle, which is clearly shown in Figure 11 and supported by the possible antifungal mechanism [38].

Figure 11. Molecular mechanistic interaction of ZnO and Co₃O₄ NPs in Alternaria Solani cell.

Conclusion

The current findings provide immense scientific direction and promises towards the green alternative synthesis of various types of metal oxide NPs having numerous biomedical applications. In this findings, Solanum lycopersicum leaf aqueous extract was employed as a potential green alternative templating agent for the synthesis of ZnO and Co₃O₄ NPs within the volume ratios of 1:3, 1:1, and 3:1. The physico-chemical and surface structure of the synthesized NPs was verified using advanced characterization techniques such as TGA/DTA, XRD, SEM, TEM, HRTEM, SAED, FTIR and UV-Vis. The green synthesized ZnO and Co₃O₄ NPs were found to be thermally above 450 and 500, respectively. The XRD analysis proves that the green fabricated ZnO and Co₃O₄ NPs were highly crystalline and showed an average crystalline size in the range of 20–51 nm and 21–28 nm, respectively. The SEM-TEM with HRTEM-SAED analysis proves the surface structure of the green fabricated NPs showed a spherical structure with some aggregation and agglomeration formations that happened with increase of the leaf extract. Furthermore, the light absorption behaviour of green achieved by NP’s behaviour was investigated via UV-Vis techniques and the result showed that as the amount of the leaf extract is increased, the light absorption behaviour also altered because of the decrease in conductivity. The presence of various bioactive ingredients within the leaf extract was checked using FTIR analysis and the result showed that the leaf extraction contains different functional groups such as saturated and unsaturated hydrocarbons, ketones, aldehydes, alcohols and carboxylic acids. This analysis also confirms the formation of metal-oxygen stretching too. In the end, the green obtained ZnO and Co₃O₄ NPs was investigated as an effective anti-drug for Alternaria Solani. The antifungal activity of Co₃O₄ NPs was found to be effective as compared to the ZnO NPs, which might be contributed due to the suitable physico-chemcial property of Co₃O₄ NPs and properties also might have resulted from the more homogenized surface morphology. The study can be concluded that as the amount of the extract added is enhanced, the physic-chemical and surface phenomena also vary and so the antifungal activity increases simultaneously.

Acknowledgments

The authors heartly acknowledge Adama Science and Technology University (ASTU) for the financial support.

Disclosure statement

The authors completely declares as they have no any known conflict of interest that could influence the publication of this manuscript.