Green Synthesis and Characterization of Zinc Oxide Nanoparticles (ZnO NPs) from Camellia sinensis Leaf Extract and Its Potential of Antibacterial Activity and Acetyl Cholinesterase Inhibitory Activities

Nanoparticles are widely used in the biotechnology and biomedicalfield. Green synthetic methods of nanoparticles are a simple and environmentallybenign process that declines the demerits of conventional chemical andphysical methods. The synthesis of semiconductor and metal nanoparticles is a risingdo research part, due to the possible applications in the progress of new technologies.The present research information the synthesis and characterization of zinc oxide nanoparticles (ZnO NPs) using C. Sinensis leaf extract. The findings of these studies show the green synthesized ZnO NPs are effective, safe, and eco-friendly as they arestable and have abundant flower shapes with maximum particles in size ranging from 100 nm indiameter. The synthesized ZnO NPs have been tested against the pathogenic microorganismsand showed an excellent zone of inhibition. DPPH radical scavenging activity of synthesized ZnO NPs expressed Original Research Article Baskaran and Subash; JPRI, 33(51A): 134-147, 2021; Article no.JPRI.77312 135 DPPH free radicals as a percentage of inhibition andIC50value of 70.37%. Acetylcholine esterase (AChE) inhibitory activity possesses the maximum amount of the synthesized ZnO NPs. Since the results ofthe current work, it was concluded that the synthesized ZnO NPs exhibited significant antibacterial and acetylcholine esterase inhibitory activities. Hence it can beused as a drug with multifunction in treating Alzheimer’s disease (AD).


INTRODUCTION
Nanotechnology contains the use of materials having nanoscale size in the range of 1to100 nm. In use by nanomaterials have acceptable researchers to have a better understanding of natural science [1]. Nanoscience has been creating a new chemical and biological nanostructure, understanding and uncovering their novel properties and organizing these new structures into larger and more complex functional structures and devices [2]. The inorganic nanoparticles (NPs) like gold, silver, copper, TiO2, CuO and ZnO have deep antibacterial activities. Among the inorganic NPs, ZnO NPs are of particular interest, because they can be prepared easily, inexpensive and safe material for human beings and animals. Nanoparticles can be synthesized by various methods like physical (evaporation, condensation and laser ablation), chemical and biological method. Different metal nanoparticles have been synthesized previously using the evaporationcondensation process Deka et al., [3].They are also generally used in the formulation of health care products. ZnO NPs has entered the scientific spotlight for its high catalytic UV filtering properties, wound healing, anti-bacterial, antifungal activity semiconducting properties and photochemical activity [4].
Pharmacology, biotechnology, nanogenerators, biosensors, gas sensors, varistors, solar cells, photo catalysts and photo detectors are capable candidates for a variety of applications ZnO NPs [5].
ZnO NPsbroad applications in pharmacological biological fields. Mainly, metal oxide nanoparticles and ZnO NPs efficiently protects over a broader UV range than any of the molecular UV-absorbers. The non-toxic nature of ZnO NPs makes it fitting in drug research. ZnO NPs are also use as anti-microbials in numerous drugs medicines and formulations [6]. Toxicological impact of nanomaterials are predictable to relate with the materials of biological components to produce important impacts on the properties and behaviour of body, organs, tissues, cells and macromolecules, cells, tissues, organs and body [7]. On the increasesupport of oxidative stress-mediated damages to the lipids, protein and DNA during Alzheimer's disease prompted for the detection of original molecules that play ansignificant role in cholinesterase inhibitors and scavenging the free radical produced during the pathogenesis of the ailment [8].
In this study, the ZnO NPs are synthesized using simple, eco-friendly and cost effective method. The present work is aimed at synthesizing ZnO NPs from leaf extract of C. sinensis. The synthesized ZnONP's were characterized by Ultra Visible spectroscopy, Scanning electron microscopy, X-Ray Diffraction, FTIR, Zeta potential and also acetylcholine esterase inhibitory and antibacterial activities of prepared ZnO NPs and tested against different human pathogenic microorganisms (bacteria).

Plant Sample
C. sinensis leaves were collected from Aruvankadu in the Distirct of The Niligri's, Tamil Nadu, and taxonomic identification of the plant was confirmed by Botanical Survey of India, Coimbatore (Authentication No: BSI/ SRC/5/23/2019/Tech./18). The collected plant leaves were kept in shade 10 days for at room temperature for complete drying. The plant material was pulverized and used for further investigations.

Preparation of the Plant Extract
Aqueous extract: Aqueous extract of C. sinensis was prepared according to the method of Kumar et al. [9]. 10 g of thoroughly dried leaves powder were immersed in 100 ml of double distilled water at 60°C for 15 minutes. The extract was filtered using Whatman filter paper and stored at 4°C for further use.

Synthesis and Optimization Parameters for ZnO NPs
The method proposed by Gnanasangeetha and Thambavani, [10] was used for the synthesis of ZnO NPs in the selected medicinal plant and the detailed procedure was given in Fig. 1.
Zinc oxide nanoparticles (ZnO NPs) were synthesized using zinc acetate dehydrate Zn (CH 3 COO) 2 . 2H 2 O. (modified) briefly, 5ml of leaf extract was taken and 20 ml of 200mM solution of zinc acetate was added. The solution was stirred continuously for 1 hourand then added 2M NaOH solution (pH 12.0). The mixture was incubated at 60˚C with stirring for 2 hours. A white precipitate was centrifuged at 1400 rpm for 5 min and washed twice with sterile distilled water followed by ethanol. Complete conversion to ZnO NPs was then dried to fine powder at 60˚C.

Characterization of ZnO NPs
The changed pH of reaction mixture was recorded using digital pH meter during the synthesis of ZnO NPs. UV-Vis spectral analysis was performed using Shimadzu 1800 spectrophotometer, whereas the morphology of the ZnO NPs was observed by SEM with EDAX, Structure and composition of ZnO NPs were analysed by XRD. Further characterization was accomplished by FTIR 2800 Shimadzu and Zeta potential.

DPPH Radical Scavenging Activity of Synthesized ZnO NPs
The determination of DPPH scavenging activity of the plant extract was done by the method of Kikuzaki and Nakatani [11]. The sample (25µl) and 0.48ml of methanol were added to 0.5ml of methanolic solution of DPPH. The mixture was allowed to react at room temperature for 30 minutes. Methanol alone served as blank and DPPH in methanol, without the plant extracts, served as positive control. After 30minutes of incubation, the discolouration of the purple colour was measured at 518nm. Ascorbic acid was used as a positive control.

Fig. 1. Synthesis of nanoparticles
The radical scavenging activity was calculated as follows Scavenging activity (%) =

Antibacterial Activity of Synthesized ZnO NPs
The antibacterial activity of synthesized ZnO NPs was performed by agar disc diffusion method against four bacterial strains (Gram-negative bacteria: Escherichia coliMG1655and Staphylococcus aureus, MH605510 Grampositive bacteria: Klebsiella oxytoca11492-1 and Bacillus subtilis. MG859252). All the tested strains were reference strains. The bacterial cultures were grown on nutrient agar medium (Himedia, pH 7.4) at 37ºC. The cultures were maintained at 4°C for further use.
The antibacterial activities of synthesized ZnO NPs were screened by agar well diffusion method Tiwari et al. [12]. The antimicrobial compounds present in the plant extract are allowed to diffuse out into the medium and allowed to interact in a plate seeded with the test organisms. The diameter of zone of inhibition can be measured in millimetres.

Broth Microdilution Method
The broth microdilution method was carried out in a 96-well microtiter plate to determine the minimum inhibitory concentration (MIC). The different concentrations of compounds (1, 0.5, 0.25 and 0.125mg/ml) were diluted in Mueller Hinton broth and the final volume was maintained at 100 µl. The final concentration of DMSO was less than 1%.
From the overnight grown bacterial culture 5 µl was added to the test medium to bring the final inoculums size to 1x105 cfu/ml. The agar plates were incubated at 37˚ for 16 hour and the absorbance was read at 600 nm. The lowest concentration of the compound that inhibits the complete growth of the bacterium was determined as MIC NCCLS, 2000.The percent growth inhibition was calculated by comparison with a control using the formula indicated below; % of growth inhibition = control − test control x 100

Thin Layer Chromatography (TLC) with Bioassay Detection for AChE Inhibition
The TLC with bioassay detection for AChE inhibition was modified from the study of Rhee et

Acetyl Cholinesterase Inhibition Activity
The acetyl cholinesterase inhibition activity was measured using the method described by ingkaninan et al. [15]. 3 ml of 50mM Tris-HCl buffer (pH 8.0), 100 µl of sample solution at different concentrations (3 mg/ml, 1.5 mg/ml, 0.75 mg/ml) and 20µl AChE (6 U/mL) solution were mixed and incubated for 15 min at 30ºC, a 50 µl volume of 3 mM 5, 50-dithiobis-(2nitrobenzoic acid) (DTNB) was added to this mixture. The reaction was then initiated by the addition of 50 µl of 15 mM acetylthiocholine iodide (AChl). The hydrolysis of this substrate was monitored at precisely 405 nm wavelength. At this wavelength the formation of yellow 5-thio-2-nitrobenzoate anion was noticed as the result of the reaction of DTNB with thiocholine, released by the enzymatic hydrolysis of acetylthiocholine iodide. The enzymatic activity was calculated as a percentage of the velocities compared to that of the assay using buffer instead of inhibitor (extract), based on the formula: Enzyme Activity (EA) = X 100 Where, E is the activity of the enzyme without test sample and S is the activity of the enzyme with test sample.

2.11Statistical Analysis
The parameters analyzed of the study were subjected to statistical treatment using SigmaStat Statistical package. All measurements were expressed as mean ± standard deviation. Statistical significance was determined by one-way ANOVA. Values of p<0.05 were considered significant.

Color Change
Decrease of zinc is established by color alteration of the reaction mixture from pale yellow to white and shown in Fig. 2 and Table 1.
Visual colour change is the preliminary test for nanoparticles synthesis.For the duration of synthesis, the transform in color of the solution and development of a yellowish-white precipitate was an indication that zinc acetate had been reduced. Similar results was observed by Santhosh kumar et al. [1],who stated that the ZnO NPs was synthesized using freshly prepared leaf extract of Passifloracaerulea.

UV-Vis Spectrum of synthesizedzno NPs
UV-Vis spectroscopy is asignificant tool for the determination of development shape and stability of nanoparticles. The UV-spectrum of the synthesized ZnO NPs is shown in Fig. 3.
The spectrum reveals a broad peak which was obtained at wavelength 318nm which can be assigned to the intrinsic band gap absorption of ZnO NPs due to the electron transition from the valence band to the conduction band.

SEM Analysis of Synthesized Zinc
Oxide Nanoparticles SEM analysis is through to imagine shape and size of the nanoparticle. The surface morphology of the resulting synthesized ZnO NPs is examined in Fig. 4.

Elemental Mapping Analysis of Synthesized ZnO NPs
The EDAX spectrum Fig. 6 shows only the peaks of zinc and oxygen elements which prove synthesized ZnO NPs.
Synthesized ZnO NPs is essentially free from impurities and it is also seen in the limit of EDAX.

XRD Patterns of Synthesized ZnO NPs
The crystalline size and structure properties of synthesized ZnO NPs are revealed using X-ray diffractions analysis. The XRD carried out with Cu -κα radiation (κ -0.154nm) and 2theta range from 20˚ to 80˚. The XRD image of synthesized ZnO NPs is showed in Fig 7. The The calculated crystallite size of the powder is about 160 nm. Jamdagni et al., [23] observed that the XRD spectrum crystal lattice indices and particles size calculation of ZnO NPs diffraction peaks were observed at 2ᶿ. The peaks have been attributed to hexagonal phases of ZnO NPs. The value of particle size was found to be 16.58 nm.

FTIR Spectra of Synthesized ZnO NPsanalysis
The FT-IR spectra of Camellia sinensis leaf extract before reduction was shown in Figure 8.

Zeta Potential Synthesized
Zeta potential was used to conclude the surface potential of the synthesized ZnO NPs Zeta

Synthesized ZnO NPs
Zeta potential was used to conclude the surface potential of the synthesized ZnO NPs Zeta potential is an essential characterization of stability for aqueous ZnO NPs. A minimum of +30 mV Zeta potential is essential for the indication of stable synthesized ZnO NPs. For the showed nanoparticles, zeta values were measured and found to be -11.6 mV with a peak area of 100% intensity. These values present full stabilization of the nanoparticles which may be the main reason in producing particles sizes with a narrow size distribution as shown in Fig  zeta potential of the synthesized zinc oxide nanoparticles was determined in water as dispersant. The high value confirms the repulsion between the particles and thereby increases in stability of the formulation [24].

The Zeta potential of synthesized ZnO NPs
Article no.JPRI.77312

Plantextract B. ZnO NPs
potential is an essential characterization of stability for aqueous ZnO NPs. A minimum of +30 mV Zeta potential is essential for the ZnO NPs. For nanoparticles, zeta values were 11.6 mV with a peak area of 100% intensity. These values present full stabilization of the nanoparticles which may be the main reason in producing particles sizes with ow size distribution as shown in Fig. 9.The zeta potential of the synthesized zinc oxide nanoparticles was determined in water as dispersant. The high value confirms the repulsion between the particles and thereby increases in

DPPH Radical Scavenging Activity Synthesized ZnO NPs
The inhibition of scavenging activity of synthesized ZnO NPs for DPPH was shown in Fig.10. The inhibition of scavenging activity of the synthesized ZnO NPs was increasing in dose dependent manner and IC 50 value of was70.37%.
Lawrence et al., [25] observed that the DPPH activity of Gmelina arborea extract on radical increased with an increasing concentration of the extract. The IC 50 value of the extract was found to be 124.39µg/ml. Similar results was reported by Mathew et al. [26], showed raw curcumin showed more than 80% DPPH free radical scavenging capacity.

Antibacterial Activity of Synthesized ZnO NPs
The antibacterial activity against obtained zones of inhibition growth of the bacteria which varied against test organisms with different concentration ranging from 20 to 80µl and it is represented in Table 2 and Fig. 11.

Radical Scavenging Activity of
The inhibition of scavenging activity of NPs for DPPH was shown in inhibition of scavenging activity of the synthesized ZnO NPs was increasing in dose value of was70.37%.
observed that the DPPH extract on radical ncentration of the value of the extract was found to be 124.39µg/ml. Similar results was reported , showed raw curcumin showed more than 80% DPPH free radical

Synthesized
The antibacterial activity againstZnO NPs obtained zones of inhibition growth of the bacteria which varied against test organisms with different concentration ranging from 20 to 80µl and it is represented in Table 2 and Fig. 11.
The inhibition of zone produced by the sample extract compared with zones produced by ampicillin was used as a control. The results revealed that the ZnO NPswere able to resist against some of the bacterial species. The NPs showed stronger moderate activit concentration against Escherichia coli Staphylococcus aureus Klebsiellaoxytoca (0.8mm) and Bacillus subtilis (0.9mm).
Similar study was reported by Mishra al., [27]where the ZnO NPs inhibit the microbial growth. Stan et al, [28] have also demonstrated that the synthesized Zinc oxide nanoparticles using Petroselinum crispum extract showed the microbial growth inhibition which has enhanced 2-16 times when compared with the plant extract.

Determination of Minimum Inhibitor Concentration (MIC)
MIC (Minimum inhibitory concentration) for certain bacteria using synthesized ZnO NPs gave maximum zone in well diffusion method and the results are obtainable in Table 3.  inhibition of zone produced by the sample extract compared with zones produced by ampicillin was used as a control. The results were able to resist against some of the bacterial species. The ZnO showed stronger moderate activity in 50µl Escherichia coli (1.2mm), (0.8mm), Bacillus subtilis Similar study was reported by Mishra et [27]where the ZnO NPs inhibit the microbial [28] have also demonstrated that the synthesized Zinc oxide nanoparticles extract showed the microbial growth inhibition which has enhanced 16 times when compared with the plant extract.

Determination of Minimum Inhibitory
MIC (Minimum inhibitory concentration) for certain bacteria using synthesized ZnO NPs gave maximum zone in well diffusion method and the results are obtainable in Table 3.  Similar study was reported by Jamdagni et al. [23] and reported that the synthesized ZnO NPs were tested for microbial pathogens and the strains showed the absence of microbial growth was visually checked to define MIC values.

Thin Layer Chromatography (TLC) with Bioassay Detection for AChE Inhibition
The qualitative results of inhibition of enzyme acetyl cholinesterase in Thin Layer Chromatography showed that synthesized ZnO NPs inhibited the enzyme by the appearance of yellow back ground with white spots was visible after about 5 min. Following are the results of the first test, yellow background with white spots for inhibiting sample and for ZnO NPs were visible after 5 min apparently tested against positive enzyme and gives inhibition at concentration of 5 mg ml -1 in Fig. 12.
Kalanchoebra siliensis extract of 2mg ml -1 concentration of TLC with white spot showed inhibitory property of cholinesterase with flavonoid compounds [29]. Similar results were obtained by Feitosa et al., [30] and found that the acetylcholinestrase inhibitors are successfully used to treat the symptoms of Alzheimer's disease.

Acetyl Cholinesterase (AChE) Inhibitory Activity of Synthesized ZnO NPs
The percent inhibition data and IC 50 value and Concentration (0.75, 0.50, 0.25, 1, 1.25, 1.5µg/ml) of synthesized ZnO NPs is presented in Fig. 13. When studied for its possible inhibitory effect in the in vitro analysis, synthesized ZnO NPs showed AChE inhibitory activity in a dose dependent manner with an IC 50 value increasing the extract concentration. Concentration of 1.25 µg/ml in the synthesized ZnO NPs showed the most potent effect in AChE inhibition activity. Rashed et al., [31] evaluated anti activity from the isolated compound of 80% methanolic extract of brevipedunculata arial parts.

CONCLUSION
The green synthesis of nanoparticles used in this research is found to be non-toxic, eco and have less usage of chemicals compared to the chemical and physical methods. The occurrence of phytochemicals in the leaf extract itself helps in the synthesis of metal oxide nanoparticles by inducing oxidation and reduction reactions. The antibacterial activi synthesized ZnO NPs can be used as a potent antibacterial agent against pathogenic microorganisms and acetylcholinesterase The green synthesis of nanoparticles used in this toxic, eco-friendly, e less usage of chemicals compared to the chemical and physical methods. The occurrence of phytochemicals in the leaf extract itself helps in the synthesis of metal oxide nanoparticles by inducing oxidation and reduction reactions. The antibacterial activity of synthesized ZnO NPs can be used as a potent antibacterial agent against pathogenic microorganisms and acetylcholinesterase inhibitory activity of ZnO NPs is proved to be a promising agent of anti-Alzheimer's activity. ZnO NPs are anticipated to have applications in the drug and pharmacology industries.