The nanotechnology sector stands as one of the most promising technologies for manipulating nanoscale for a wide range of science disciplines, including ceramic materials, cosmetics, food, and pharmaceuticals . Nanoparticles are a promising strategy to improve health and industrial applications due to their exceptional properties, such as small size, high adsorption, high catalytic activity, large surface area, a large number of reactive sites, and chemical stability . Since metal and metal oxide nanoparticles have many valuable properties, including catalytic, optical, electrical, and magnetic properties, they have become very appealing materials in several fields over the past few years . Metal and metal oxide nanoparticles are used in various fields such as solar cells, laser deflectors, photonics, water treatment, biomedicine, transparent, piezoelectric, catalyst and gas sensors [4,5]. Metal oxide nanoparticles, especially ZnO nanoparticles, seem to be particularly effective for antibacterial activities, eco-friendly agrochemicals, photocatalyst degradation, and photocatalysis for environmental remediation . Due to their high stability, ZnO nanoparticles are considered probable next-generation materials as biocidal and fumigating agents. This stability is ascribed to them being more versatile than organic-based decontaminators and anti-microbial agents . As a semiconductor, ZnO has a wideband gap (3.37 eV) and an exciton binding energy of 60 meV, which is an efficient source of excitonic blue radiation. As a result of its inherent ability to absorb UV irradiation, ZnO has been accepted by the food and drug administration (FDA) for use in sunscreens . To synthesize the ZnO nanoparticles, several physical and chemical strategies were used, whereas sol-gel , chemical precipitation , laser ablation , chemical vapor deposition , pyrolysis , hydrothermal , and solvothermal route  have been used. The above methods used to synthesize nanoparticles are complex, costly processes that produce hazardous toxic wastes that are harmful to both humans and the environment , and also limited in biomedical applications because of the toxic chemicals required. To overcome these disadvantages, it is imperative to explore alternative green sources. The process of green synthesis utilizes a variety of natural sources, including plants, bacteria, algae, and fungi . The biosynthesized nanoparticles are nontoxic, biocompatible, and eco-friendly . The natural extract may contain bioactive compounds that may bind to the surface of the nanoparticles, and their density will be dependent on synthesis parameters . Phytochemicals, such as flavones, phenols, amino acids, sugars, carotenes, amides, aldehydes, ketones, etc., present in plants have been widely used for green synthesis. Nanoparticles and biological materials interact to control the surface coatings of the fabricated materials . There have been numerous reports on the synthesis of ZnO nanoparticles with leaf extract such as Solanum nigrum , Cinnamomum verum , Azadirachta indica , Passiflora caerulea , Curry , Moringa oleifers , and their antibacterial activity. Acacia arabica leaf extract is used to biosynthesize ZnO nanoparticles for antimicrobial and antioxidant activity.
The traditional medicinal plant of south India, Acalypha indica (Euphorbiaceae), contains bio-reductants and stabilizers. There are several medicinal applications of these plants, including anti-inflammatory, antifungal, anticancer, and antibacterial effects, which are useful in treating asthma, rheumatism, and pneumonia. The leaf of Acalypha indica shoes a significant number of proteins, carbohydrates, alkaloids, felonies, tannins, phenolics, terpenoids, and amino acids . Nanoparticles of copper oxide , titanium oxide , and silver oxide  have been synthesized using Acalypha indica leaf extract. It has been suggested that phenols, aldoses, and proteins are responsible for the creation of metal oxide nanoparticles. In our study, we synthesize ZnO nanoparticles using the leaf extract of Acalypha indica for the first time. It is easier to use, less expensive, and more eco-friendly than the conventional method. The present study utilized Acalypha indica leaf extract for reducing zinc nitrate to ZnO nanoparticles which were synthesized and characterized using several techniques: UV-Visible spectroscopy (UV-vis), Photoluminescence (PL), Scanning electron microscopy (SEM), Zeta potential (ZE), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Energy-dispersive X-ray spectroscopy (EDX) and Dynamic light scattering (DLS) analysis. Also, nanoparticles produced as photocatalysts are utilized to degrade organic dye and inhibit microorganisms that cause human disease.
MATERIAL AND METHODS
The chemicals such as Zinc acetate dihydrate (Zn (CH3COO)2.2H2O) and all the chemicals and reagents were procured since Merck chemical reagent co distilled this work purchased since the leaves of Acalypha indica plant together form in and around the garden, Nagapattinam, Tamil Nadu, India.
Plant leaf collection
Fresh leaves of plants that is, Acalypha indica free were collected from Nagapattinam. The leaves were identified and authenticated by the Department of Agriculture, Annamalai University in Tamil Nadu. To remove dust particles from the surface of the leaves, they were twice washed with tap water and then repeatedly washed in double-distilled water. The washed leaves were then shade dried for five days. Then, 20 gm of dried leaves were crushed and 50 mL of distilled water was added. After that, a magnetic stirrer was used to stir the mixture, then the mixture was heated for 1 h at 60ºC. When the mixture displayed a yellow color, it was filtered using Whatman filter paper. As a result, nanoparticles of ZnO were prepared from the exact solution.
Biosynthesis of ZnO nanoparticles
The ZnO nanoparticles were biosynthesized by following the Sol-gel method designated by Muthuvel et al. . Briefly, it was prepared by stirring 2 M zinc acerate in 50 mL of deionized water for 30 min at 85ºC. To prepare a NaOH solution, 4 gm NaOH powder was added to 50 mL of distilled water and stirred simultaneously at 85ºC for 30 min. the two solutions were then vigorously stirred together. The 15 mL leaf extract was mixed with the solution drop by drop during this stirring process. After stirring continuously for 2 h using a magnetic stirrer, a white precipitate was obtained. To remove the impurities, the precipitate was filtered and repeatedly washed with distilled water followed by ethanol. After the precipitate was dried at 400ºC for 4 h and the obtained ZnO powder was subjected to further characterization.
Characterization of synthesized nanoparticles
To collect X-ray diffraction data for the formed samples, SHIMADZU-XRD 6000 analytical diffractometers were used. We measure UV absorbance and photoluminescence with a Shimadzu UV-VIS-260 system. The morphology of the synthesized nanoparticles was examined using scanning electron microscopy (Hitachi S-4500 machine). DLS and zeta potential were measured by Malvern Zeta Sizer (ZS 90, USA). Measurements were made with a Bruker tensor 27 FT-IR spectrometer using Fourier transform infrared spectra.
To assess the photocatalyst activity of ZnO nanoparticles under sunlight, MB was selected as the model contaminant for photocatalyst degradation. The stock solution of 50 ppm MB in distilled water was prepared by dissolving of distilled water. Using a UV-vis spectrometer with maximum absorption at 661 nm, the concentration of MB at the initial concentration was determined. Thereafter, 100 mL of stock solution will be poured into a 100 mL beaker, followed by 100 mg of ZnO nanoparticles. After exposing the solution to sunlight for some time, 3 mL, of the solution was withdrawn and the suspended ZnO powder was centrifuged at 6000 rpm for 15 min. after the solution was degraded, the absorbance was measured. This equation was used to estimate the degradation percentage of the photocatalyst ;
Degradation percentage = x 100 (1)
where, Co is the dye concentration before degradation, and Ct is the dye concentration at different times t.
The antibacterial activity of Acalypha indica leaf extract and biosynthesized ZnO nanoparticles were established using the disc diffusion method on four types of bacteria: Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. The samples of 5 and 10 µg/mL concentration were poured onto ach disk and placed on Muller Hinton agar plates. The antibiotic dis Ciprofloxacin was used as the positive control. Incubation was carried out at 35ºC for 24 h. By forming an inhibitory zone around the wells, the antibacterial activity of the samples was determined.
RESULTS AND ANALYSIS
The XRD pattern was used to determine the crystallinity and phase of synthesized ZnO nanoparticles (Fig. 1). The peak positions with 2θ values of 31.7º, 34.9º, 36.4º, 53.8º, 66.4º, 75.5º, and 82.6º can be assigned to planes (100), (022), (101), (102), (110), (103) and (200) which corresponds to hexagonal Wurtzite structure of ZnO nanoparticles (JCPDS card no:5-0664). It is evident from the quality of peaks that the ZnO nanostructures are well-crystalline. The XRD result obtained were very similar to what had previously been recorded . The XRD pattern does not show any additional peaks, demonstrating the high purity of the ZnO nanoparticles This suggests anisotropic growth and a preferred orientation of crystallites, based on the relatively high intensity of the (101) peak. Further analysis of the XRD spectrum is conducted to establish crystal structural parameters such as dislocation density (δ), the crystallite size (D), and microstrain (ε) as described in Eqs 2-4 .
D = (2)
Where, λ is the wavelength of the X-ray source (0.1541 nm), D is the average crystalline size (nm), the angle of Bragg’s diffraction is θ, and the angular peak width at half maximum is given by β in radians along with the (1 1 1) plane. The crystalline size of biosynthesized ZnO nanoparticles has been calculated to be around 16.2 nm, as well as the dislocation density and microstrain, which are about 5.678 and 0.1345, respectively. Table 1 shows the structural parameters values of green synthesized ZnO nanoparticles. The crystalline size of the biosynthesized ZnO nanoparticles from Acalypha indica leaf extract is very small when compared to other leaf extracts (Cinnamomum verum, Azadirachta indica, and Passiflora caerulea) used in synthesizing ZnO nanoparticles [20, 21, 22] and also Acalypha indica leaves extract used biosynthesized iron and silver oxide nanoparticles [30, 27]. The present study has a very small crystallite size of 16 nm, and this will greatly improve antibacterial activity and photocatalysis.
UV-Vis absorption spectroscopy was used to confirm the optical properties of the biosynthesized ZnO nanoparticles. Fig. 2a shows the UV-Vis absorption spectrum of synthesized nanoparticles from 200-800 nm. the figure shows absorption bands at 371 nm that confirm the presence of ZnO nanoparticles in culture filtrate. Therefore, the absorption spectrum of ZnO nanoparticles will show a strong shift towards blue, indicating that their size is less than the exciton Bohr radius . ZnO nanoparticles are characterized by the blue peak, while nanoscale confinement is indicated by a blue shift. Satheshkumar et al. also obtained similar results for the absorption band, similarly , Jamdagni et al., ZnO nanoparticles UV-vis spectrum rage between 330-390 nm . The UV spectrum range of ZnO nanoparticles was also measured at 380 nm by Muthuvel et al. . The band gap energy of ZnO nanoparticles synthesized was determined by UV-vis spectroscopy based on the following Eq ;
(αhν)2 = B (hν - Eg)1/2 (5)
where B is a constant, h is the Planck constant (6.626x 10-34 J Hz-1), hν is photon energy, Eg is the optical band gap energy and α is the absorption coefficient. The band gap energy of synthesized nanoparticles is displayed in Fig 2b. The observed band gap energy of ZnO is 3.34 eV, lower than the band gap of bulk ZnO due to quantum confinement effects . The ZnO nanoparticles synthesized in the present study have a band gap energy in good agreement with that found in Vijayakumar et al. . The present band gap energy is very low for the ZnO nanoparticles synthesized by some chemical methods [12, 14, 15]. The present work demonstrates that ZnO nanoparticles can be obtained by biological means from small band gap values and that this greatly enhances their antibacterial properties.
DLS and Zeta potential analysis
Nanoparticles undergoing Brownian movement are measured by measures of their time-dependent scattering of light, the particle size distribution was determined using dynamic light scattering analysis . In colloidal solution, dynamic light scattering is widely used to
ration the shell thickness of capping agents or stabilizers omnipresent metallic nanoparticles,
as well as the size of the metallic core. Fig. 3a shows the DLS pattern of biosynthesized ZnO
nanoparticles using Acalypha indica leaf extract. The size distribution of synthesized nanoparticles is found to be 46 nm. Furthermore, the negative zeta potential of ZnO nanoparticles, which is found to be -27.47 mV, further confirms the stabilization of synthesized nanoparticles (fig 3b). Particles with zeta potential above +30 mV or below -30 mV are considered stable. With a zeta potential of -27.47 mV, the ZnO nanoparticles synthesized via biosynthesis at 400 ºC are very stable. The high negative value of the zeta potential correlated with the presence of negatively charged groups on the surface of the nanoparticles. This reduction of metal ions and stabilization of nanoparticles could be caused by protein and flavonoids in the leaf extract. There are similar kinds of results reported by Chaudhuri et al. .
The SEM was used to detect the morphology and size of the biosynthesized nanoparticles. The SEM image of synthesized nanoparticles is shown in Fig 4 (a). The figure shows, that the biosynthesized ZnO nanoparticles have a homogeneously spherical morphology. Upon closer inspection, several aggregates of nanoparticles can be seen in the agglomerated lump. Particles are agglomerated, and some individual crystals can be seen in Fig 4(a). In Fig 4(b), the histogram of particle sizes distribution shows that the mean diameter of ZnO nanoparticles is 35 nm. The EDX result shown in Fig 5 further confirms the presence of zinc nanoparticles in oxide form, indicating that the biosynthesis of ZnO nanoparticles provides an effective way to process inorganic matter. Fig. 4 (c-d) shows a surface plot analysis of synthesized nanoparticle images. It has been demonstrated that biosynthesized ZnO nanoparticles have the highest porosity, thus allowing more dye molecules to absorb, which would improve the performance of the photocatalyst.
The FT-IR analysis can be used to identify the possible reducing and stabilizing biomolecules. Wave number range between 400 to 4000 cm-1 was used for FT-IR analysis. Fig. 6 shows the FT-IR spectra of the Acalypha indica leaf extract and biosynthesized ZnO nanoparticles. The FT-IR spectrum of Acalypha indica leaf extract exhibited several peaks at 3417.12, 2948.52, 2487.52, 1409.65, 851.69, and 681.87 cm-1. The peaks at 3417.12 (O-H), 2948.52 (CH2), 2487.52 (stretching mode of C-H), 1409.65 (bending mode), 800-500 (RC=OO) cm-1 are associated with phenols, alkaloids, resins, saponins, tannins, and flavonoids compounds, respectively [26, 27]. As a result of the functional group’s analysis, Acalypha indica leaf extract contained carboxylic and amino groups. It is the responsibility of the groups to bio-transform zinc ions into zinc oxide nanoparticles. In synthesized ZnO nanoparticles, the major absorption bands are 3397.92, 2969.53, 2478.84, 1452.27, 870.44 and 600-400 cm-1. The O-H stretching vibrations are present in flavonoids and phenolics, giving rise to the broad absorption peak, 3397.32 cm-1 . A peak at 296.53 cm-1 is attributed to the C-H stretch of the alkane functional group . An alkyl peak was assigned to the O-H group at, 2478.84 cm-1. The peak at, 1452.27 cm-1 is due to the C=C stretch of the aromatic ring system . Around 870 cm-1, the absorption peak is probably in the O-H functional group . ZnO is considered to have a stretching mode absorption peak between 400 to 600 cm-1. The biosynthesized ZnO nanoparticles exhibit absorption bands between 400 to 600 cm-1 . As a consequence, phytochemicals show a role in the biotransformation of nitrates to oxides. Biosynthesized ZnO nanoparticles showed new characteristic peaks as compared to pure ZnO nanoparticles . 2948.52 and 1409.65 cm-1 were peaks created by flavonoids and phenolics of leaf extract molecules, confirming the presence of phytochemicals on ZnO nanoparticles. Plant phytochemicals reduce the size of nanoparticles during their formation. Mahendra et al. and Vijayakumar et al. also observed similar findings [38, 34].
The study of the PL, a property of biosynthesized ZnO nanoparticles, is interesting because it can give valuable insights into the quality and purity of the material. ZnO nanoparticles are shown in Fig 7 as PL spectra at room temperature. An excitation wavelength of 320 nm was used for the PL measurements. The PL spectra of ZnO nanoparticles at room temperature show four main peaks, 362, 441, 486, and 543 nm. It correlates with the Near band emission of ZnO at 362 nm. The excited electron of a valence band recombines with the holes by radiative recombination . A weak peak at 441 nm was attributed to irradiative excitation annihilation, and a weaker peak at 486 nm to defects in the band gap produced during sample preparation, such as oxygen vacancies . The 543 nm, green band may correspond to a transition between interstitial and vacancy oxygen . This study showed that Acalypha indica leaf extract can synthesize nanoparticles that have intrinsic defects, oxygen vacancy, and surface defects sites that can enhance antimicrobial and photocatalytic activities.
An investigation of the photocatalytic activity of biosynthesized ZnO nanoparticles under sunlight irradiation was carried out using MB dye degraded in an aqueous solution. Fig. 8(a) shows the change in MB absorption spectrum during photocatalytic degradation using ZnO nanoparticles at different radiation times, ranging between 0 to 90 min. After 90 min of irradiation, 96 % of the MB had degraded. It was also visually observed that the dye solution gradually turned from blue to colorless due to the photodegradation of MB. The MB dye shows strong absorption maxima at 661 nm due to the chromophore group, and it gets weaker as the irradiation time increases and becomes invisible within 90 min, indicating that the chromophore in MB molecules is completely removed. Metal oxide nanoparticle size plays a significant part in photocatalyst reduction since a decrease in the size of biosynthesized ZnO nanoparticles improves the adsorption of reactants on impetus surfaces and promotes corrosion. Increasing the surface area of the particles will therefore increase the effectiveness of the catalyst.
Mechanism of photocatalyst activity
The electron in the valence band of the synthesized ZnO nanoparticles absorbs energy as visible light is irradiated and moves to the conduction band (CB), departure a hole in the valence band (VB) (Fig 8c). This leads to the formation of electron-hole pairs, which rise to the surface. Water molecules generate hydroxyl radical (OH¯) when the holes corrode the OH¯ ions, while ambient oxygen is oxidized by the electrons to produce superoxide radius (ºO2¯). The photocatalyst reaction mechanism is depicted below ;
ZnO + hν → ZnO ( + ) (6)
ZnO () + → (7)
ZnO) + → (8)
+ + (or) () + MB dye degradation product (11)
Biosynthesised ZnO nanoparticles are primarily definite by the degradation of color dyes that determine their surface charge, crystallite size, and structure. As a result of photoinduced organic reactions on the catalyst superficial, a photocatalyst system is formed. In the presence of light, electron pores on the surface of the catalyst promote redox reactions, resulting in ºO2¯and OH¯. Due to their powerful photocatalytic properties, these radicals degrade toxins in wastewater.
Based on pseudo-first-order reaction kinetics, we calculated the degradation rate of MB dye in the presence of synthesized ZnO nanoparticles .
ln (Ao/At) = -kt (12)
where t is time (min), in the first order, Pseudo rate constant k, Ao, and At are absorptions of MB dye at time t to zero, respectively. As a function of irradiation duration, in (Ao/At) is 1.04476 min-1 for MB dye. Additionally, the fitting correlation coefficient (R2) is calculated as 0.9952. Ao/At values decrease with time, but MB dye degradation percentages increase with time (Fig 8b). As shown in Table 2, the ZnO nanoparticles synthesized from Acalypha indica leaf extract showed enhanced photocatalytic activity in organic dye than any other leaves extract used to synthesize ZnO nanoparticles.
Figs. 9 shows the antibacterial activity of aqueous leaf extract of Acalypha indica and biosynthesized ZnO nanoparticles were investigated by both Gram-positive (+Ve) and gram-negative (-Ve) bacteria by disk diffusion method. Table 3 shows the diameter of the zone of inhibition (mm). A linear increase in the size of the inhibition zone occurs with an increase in sample concentration, as shown in Table 3. The leaf extracts were minimally active at higher concentrations with zones of inhibition ranging from 15, 12, 11, and 8 mm for Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis. The phytochemicals in Acalypha indica leaf extract may be responsible for the very tiny antibacterial activities present in it. Based on the antibacterial activity results, biosynthesized ZnO nanoparticles were effective against all tested bacteria. At higher concentrations, Bacillus subtilis (21 mm), Staphylococcus aureus (22 mm), Pseudomonas aeruginosa (23 mm) and Escherichia coli (25 mm) were found to be in the zone of inhibition. Gram-negative bacteria are more susceptible to biosynthesized ZnO nanoparticles than gram-positive microbes. There was a difference in the cell wall properties of Gram-positive and Gram-negative bacteria, with Gram positives having an impenetrable outer cell sheath layer, which made them immune to ZnO nanoparticles. In addition, it was proposed that gram-negative bacteria were immune to nanoparticles due to lip polysaccharides on their cell walls. In a report by Mahendra et al. , the antibacterial activities of synthesized nanoparticles were testified to be extra active against gram-negative bacteria than gram-positive microorganisms. ZnO nanoparticles have a variety of mechanisms for causing antibacterial action, the most common of which is the production of ROS and the proclamation of Zn2+, which result in cubicle impairment and death in bacteria . Moreover, because ZnO nanoparticles are cations, they can electrostatically attribute to the negatively charged surface of bacteria, causing them to become damaged. The biosynthesized ZnO nanoparticles exhibited greater antibacterial activities in the current study because of their small size and stability (Table 4). The antibacterial activities of smaller nanoparticles are greater than that of bulk nanoparticles due to their higher surface area and responsiveness. Among the biosynthesized ZnO nanoparticles with an average crystallite size of 3 nm, Muthuvel et al.  observed higher inhibitory activities against Pseudomonas aeruginosa and Escherichia coli microorganisms. In addition, they found ZnO nanoparticles inhibited Escherichia coli growth at a comparatively low concentration of 10 g/mL, which was estimated to be size-dependent .
In conclusion, we have developed a procedure for the synthesis of ZnO nanoparticles with the use of Acalypha indica leaf extract that is environmentally benign and economically, efficient and safe. The XRD results showed that the particles are hexagonal crystalline, and optical properties indicated that the ZnO band gap energy is 3.34 eV. The DLS and ZE analysis showed that the particles were 46 nm in size and had a negative zeta potential of -27.47 eV, suggesting greater stability. FT-IR results revealed that flavonoids and phenolics in Acalypha indica leaf extract might have contributed to the synthesis of nanoparticles. A photodegradation of MB dye was used to evaluate the photocatalytic activity of biosynthesized ZnO NPs. Biosynthesised ZnO nanoparticles demonstrated outstanding photocatalytic performance, as evidenced by 96% degradation of MB dye under natural sunlight. The antibacterial activity of biosynthesized ZnO nanoparticles is probably inhibited by their negatively charged surface against Gram-negative bacteria, particularly Escherichia coli. In the future, bio-synthesized ZnO nanoparticles have the potential for a greater number of applications in sensors, catalysts, and biomedicine.
CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.
DECLARATIONS OF INTEREST