Green and Sustainable Preparation of Flower Like ZnO Nanostructures via Soft Bio-template Approach for the Enhancement of Biomedical Applications

Investigation onthe biomedical applications of ower like Zinc Oxide (ZnO) nanostructures (NSs)synthesized by using aqueous extract of Oryza punctata (red rice) is reported for the rst time. For the sustainable preparation of ZnO NSs, the precursors zinc nitrate and the rice extract act as the bio-template material. The powder X-ray diffraction analysis demonstrates the crystallographic structure and purity of the bio-prepared ZnO NSs. Fourier transform infrared spectral studies exhibits the existence of several functional groups in the rice extract and synthesized ZnO NSs. The FT-Raman spectroscopy analysis used to detect the presence of the defects in the synthesized material. The UV absorption onset shows the peaks at 270 nm & 370 nm for rice extract and ZnO NSs, respectively. The optimal energy bandgap value for the ZnO NSs was found to be 3.29 eV. The zinc oxygen vacancies, zinc interstitial and surface defects were determined by photoluminescence spectroscopy. The dynamic light scattering analysis reveals the material stability and average particle size distribution of the prepared ZnO NSs. The ower like morphology of the ZnO NSs was conrmed by eld emission scanning electron microscopy analysis. ZnO NSs were tested for cytotoxicity activity against MCF–7 cell line and antibacterial properties using two gram positive (Staphylococcus Aureus, Bacillus Subtilis) and two gram negative bacteria (Salmonella Paratyphi, Escherichia Coli). Signicant antioxidant activity was studied by the bio-prepared ZnO NSs against scavenging of DPPH (di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium) free radicals. The ower like ZnO NSs showed a signicant anti-arthritic activity which exhibited a maximum inhibition of protein denaturation (78.94±1.62 %) and membrane protective activity (81.12±1.25 %) at a dose of 0.5mg/mL concentration.

[26] reported DNA molecules are used to control the ZnO morphological structures by adjusting the pH values. Han et al. [27] have fabricated zinc oxide Nanoparticles (NPs) from natural silk material for photoluminescent property analysis. Nouroozi et al. [28] have developed brush-like ZnO nanorods via bio template of albumen (Egg white) in the sol-gel method. Ramimoghadam et al. [29,22] have fabricated different morphologies like ake, rose, rod, ower and 3-D star like structures of ZnO by hydrothermal method, where palm olein and rice acts as a bio-template for synthesis of various ZnO NSs. Dong et al. [30] have reported egg-shell membrane acts as the bio-template for fabricating hierarchical ZnO brous through a solution soakage approach followed by a thermal treatment. Tomczak et al., [31] have prepared peptide assisted growth of ower-like ZnO structures. Hence, an effort has been made to synthesis ower like ZnO NSs using Oryza punctata (red rice) rice extract and succeeded. Because rice extract acts as a soft bio-template material which plays a crucial role for synthesize of various functional NMs [23]. Oryza punctata is one of the most troublesome weeds of cultivated rice in the southern United States [32]. The family and species of the Oryza punctataarePoaceae ⁄ Gramineae -grass family and Oryza punctata Kotzchy ex Steud.-Species [32]. The carbohydrate component is majorly present in Oryza punctata, which is most fascinating for agriculture bio resources that can be employed as non-metallic bioprecursor to develop the NMs [22]. Polymeric chains form in the carbohydrate component via glucose (starch) units, which are separated in linear amylase and amylopectin [33,23]. These features are crucial structural factors in the development of novel functional NMs [33].Besides, Oryza punctata also contains bioactive compounds comprising phenolic antioxidants that have the capability of reducing the risk of diseases such as, coronary heart disease, preventing platelet aggregation, reducing the risk of cancer and inhibiting oxidative damage of lipids and low-density lipoproteins. Proanthocyanidins accumulation in rice bran gives the rice red pigment. Phytoconstituents such as peptides and phenolic compounds exhibit antioxidant and anticancer properties [34]. Ferulic acid and protocatechuic acid are the abundant phenolic compounds present in Oryzapunctata. Finocchiaro et al. [35] conveyed that proanthocyanidins of Oryzapunctata show potent antioxidant capacity.
Especially, biomedical applications are achieved by green synthesized ZnO NSs depend on their particle size, morphology, speci c surface area and powder concentration, etc [36]. In this connection, ZnO NSs like a sponge, spherical, pyramid, hexagonal and rod shapes are fabricated from different leaves extracts of Cassia stula, Tecomacastanifolia, Azadirachtaindica (L.), Cordiamyxa and Costuspictus for effective antibacterial activity [36][37][38][39][40]. Also, Balraj et al., [41] synthesized spherical shaped MgO nanomaterial from Streptomyces sp. and used to test MCF-7 cancer cell with various concentrations. Lingaruju et al., [42] developed the ZnO NPs from Euphobiaheterophylla (L.) leaves and reported the potential anticancer activity against lung (A549) and hepatocarcinoma (HepG2) cells. Shobha et al., [43] reported the cytotoxicity effect using green synthesized ZnO NPs from Ricinuscommunis seeds onMDA-MB-231 cancer cells. Ngoepe et al., [44] prepared biogenic ZnO nanomaterial for lung cancer activity. Sukri et al., [45] reported that Punicagranatum fruit peels extract was utilized to synthesis the spherical and hexagonal shaped ZnO NSs, showed more effective on Colorectal cancer cell (HCT116) and normal colon cells (CCD112) activity than the fruit extracts. As well as, Rajkumar et al. has developed ZnO NPs using Andrographis paniculata leaves extract for anti-oxidants, anti-diabetic and anti-in ammatory activity to reduce the sugar level and to in ammations [46]. Agarwal et al. [47] have fabricated spherical like ZnO NPs via Kalanchoe pinnata leaf extract for excellent anti-in ammatory activity. Thatoi et al. [48] reported comparative studies on biomedical applications like antibacterial, antioxidant, antidiabetic and antiin ammatory activities. All the biological activities have been tested against green synthesized Ag-NPs and ZnO-NPs under photo-condition using the aqueous leaves extracts of Heritiera fomes and Sonneratia apetala. As a result, the goal of our research is to develop ower-like ZnO NSs using the bio-template approach in order to improve the biological applications.

Materials
The Oryzapunctatais found in Tamil Nadu, India's southernmost state. Merck India Ltd., provided all the chemicals required for the experiment. For the synthesis, Double Distilled (DD) water is employed as the solvent.

Synthesis of ower like ZnO NSs using Oryzapunctata extract
The Oryzapunctata rice was taken and weighed20 gm. The rice was thoroughly washed with tap water for 2 to 3 times. After that, 100 ml of DD water was added to the washed rice taken in the round bottom ask. Subsequently, the impurities were puri ed through Whatman No. 1 lter paper after being heated at 60°C for 1 hour. For the further step, the ltered extract was utilised as a capping and reducing agent.
In 50 ml of DD water, 0.1 M zinc acetate was dissolved and 5 ml of red rice extract was added dropwise to the precursor solution while stirring constantly for 2 hours. The solution's colour changed from clear white to light red and a precipitate was formed. The resulting solution was centrifuged for 15 minutes at 16,000 rpm. Finally, the obtained product was dried in a hot air oven for 12 hours at 80°C and dried powders were employed for further characterization. Figure 1 shows a schematic representation of the synthesis of ZnO NSs.

Growth mechanism of ower like ZnO NSs from the rice extract
The carbohydrate component is majorly present in the red rice. The carbohydrate consists of polysaccharides which play multiple roles in the synthesis of oxide-based nanosized materials such as coating or capping, functionalizing, stabilizing, poring or coordinating agent [22]. Figure 2(a) shows the function of starch in the synthesis of ZnO. Starch is a carbohydrate component made up of concentric rings in which amylase and amylopectin, both polymers of α-glucose units [23]. The polymeric structure of amylase consists of a linear and helical-shaped carbonaceous matrix, containing multiple polyols or hydroxyl groups, which form a protective layer that prevents agglomeration and acts as a shield for metal ions that perform a structure-directing role [49].The amylopectin chains expose an important number of hydroxyl groups, giving a strong hydrophilic character to starch granules and also involved both inter or intramolecular supramolecular association, which can coordinate transition metal ions and maintaining the NPs highly aggregated [49]. The small number of amylose molecules can form complexes with Zn 2+ ions because of their high number of coordinating functional groups [50]. The majority of the Zn 2+ ions may be closely associated with the starch molecules, so the initial crystal growth and nucleation might preferentially occur within the regions of both high starch and Zn 2+ ions concentration leading to the formation of NPs [50]. They aggregate in the next step and by this process the small petals like structure will form. Then, the small petals like particles aggregate and form ower like ZnO NSs which is shown in Fig. 2 The antibacterial activity of ower like ZnO NSs against the selected bacterial strains was carried out by disc diffusion method. The agar plates were prepared and 0.1% of different inoculum (B. subtilis, E. coli,S. paratyphi and S. aureus) suspensions were uniformly swabbed on agar plates. Various concentration of ower like ZnO NSs (30, 40, 50 and 60 µl/ml) were loaded on sterile disc (6 mm) and then the disks were loaded in surface of the culture plates. The disk loaded plates and control plates (without any disk) were incubated for 24 h. Streptomycin was used as a positive control. At the end of the experiment, the zone of inhibition was analyzed in millimeter.

DPPH radical scavenging assay
The antioxidant activity of ower like ZnO NSs was measured using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. Brie y, 50 µl of ZnO NSs (12.5-1000 µg/mL) was added to the 100 µl of DPPH solution in 96 well plates. The reaction mixture solution was mixed properly and kept for 1 h dark incubation. The colour reduction was determined by absorbance value (517 nm) using UV-Visible spectrophotometer. Ascorbic acid was employed as positive control to determine the resultant activity. The Radical Scavenging Activity (RSA) was determined by following equation:

In vitro cytotoxicity analysis
The cytotoxicity effect of ZnO NSs was determined by MTT (3-[4,5-dimethylthiazol-2-yl]2,5diphenyltetrazolium bromide) based colorimetric assay against human MCF-7 cell lines (breast cancer cell line). The MCF-7 cancer cells were collected from National Centre for Cell Science (NCCS-Pune, INDIA, 411007). The cells were cultivated in Dulbecco's Eagle's Media with 10% of Fetal Bovine Serum (FBS) supplementation. The cells were maintained at room temperature (37°C) with standard atmosphere (5% of CO 2 , 95% of aeration and 100% relative humidity) and the culture medium was changed two times per week.
The medium containing the selected concentrations of ower like ZnO NSs (6.25, 12.5, 25, 50 and 100 µg/ml)were added to the MCF-7 cells (96 well plates) and incubated at 37°C in a CO 2 incubator. After 24 h incubation, 15µl of MTT dye solution (5mg/ml MTT in phosphate buffered saline) was added to the MCF-7 cells (each well) and maintained at 37°C. After 4h incubation, 100 µl of Dimethyl sulfoxide (DMSO)was added to the each well and the culture plates were read at 570 nm. The % of cell inhibition was calculated and the cells were subjected to morphological analysis.

Protein denaturation inhibition activity
Bovine Serum Albumin (BSA) (5% w/v of 2.4 mL aqueous solution) and various concentrations (100-500 µg/mL) of ZnO NSs were added in reaction container. The pH of the solutions were adjusted to 6.3 using HCl (1 N) and incubated for 20 mins at 37°C. After incubation, the sample solutions were maintained at 70 C for 10 mins and after the samples taken to room temperature, 2.5 ml of phosphate buffer (pH 6.3) was added to each tube. The absorbance was measured using a spectrophotometer at 660 nm. Diclofenac was used as a reference standard [51]. The percentage of protein denaturation inhibition can be calculated by the following equation  (1) Where 'β' denotes full-width half maximum of the peak and 'λ' represent the X-ray wavelength. The average crystalline size was found to be 25.98 nm. The dislocation density (δ) (or) sample defects was evaluated from the average crystalline size using the given equation, The prepared material's dislocation density was found to be 14.8x10 − 4 (nm) −2 . The strain was developed as a result of the crystal defect and distortion in the synthesized material. The Williamson-Hall (W-H) technique was used to compute the strain (ε). ε = (βcos(θ))/(4) ………… (3) According to Hooke's law, stress (σ) can be determined from strain (ε) and there is a linear proportionality relationship between stress (σ) and strain (ε) within the elastic limit. σ = Cε………… (4) Where, 'C' signi es bulk Young's modulus (1.46 ± 10 10 N/m 2 ). The strain (ε) and stress (σ) of the ower like ZnO NSs were found to be0.00134% and 196 MPa, respectively [52][53] 3.2 Fourier Transform Infrared Spectroscopy (FT-IR) The liquid samples were subjected to FT-IR analysis using a Perkin-Elmer spectrometer in the range of 400-4000 cm − 1 . The FTIR spectra of Oryza punctata rice extract and the synthesized ZnO NSs are shown in Fig. 4 (a & b).From the Figure, the peaks at 3549 cm − 1 and 3456 cm − 1 reveal the -OH stretching vibration occurs in the Oryza punctata rice extract due to the presence of water (H 2 O), phenolic OH, alcoholic OH or carboxylic OH groups [22,51]. The small peak at 2922 cm − 1 corresponds to the stretching vibration of aliphatic CH groups in the rice extract [22].The peaks at 1575 cm − 1 indicate the -C = C-stretching vibration, which may be due to amide and OH − of the carboxyl group of amino acid and proteins present in the extract [54].The peak at 1418 cm − 1 may attributed to the angular deformation of the C-H bending vibration in the starch molecule [22]. The band at 1018 cm − 1 assigned to the stretching vibration of the C-O-C group in the hydro glucose ring of the starch molecule [22].The small stretch at 414 cm − 1 region denoted the bending vibration of Zn-O [55].

FT-Raman Spectroscopy
FT-Raman spectroscopy is used to examine the vibrational, stretching and rotational properties of the molecules present in the synthesized material. Figure 4(  . The high exciton binding energy of ZnO (60mV) promotes recombination of free excitons between the conduction and valance bands, which generates UV NBE emission [59,60].The peak at 453 nm is ascribed to blue emission which creates due to zinc interstitial (Zn i ) defect [59]. The broad DLE band (or) green emission

Dynamic Light Scattering (DLS) analysis
The stability of the synthesized NMs and the particle size (ranging from 5 nm to 5 µm) was analyzed by the DLS experiment in a liquid state using the instrument of Zeta sizer nano-series (Malvern). The pH of the dilute ZnO solution was measured as 6.9. Normally, biosynthesized NPs demonstrated the particles are monodispersed due to the polydispersity index value of 0.2 obtained from the colloidal solution [66]. From the Fig. 6 (a), the synthesized material's potential stability was around − 32.9 mV, which indicate the material's negative charge potential value may be attributable to the phytoconstituents (starch and carbohydrates) present in the rice extract and also con rms the presence of gross electro-static force with the synthesized ZnO NSs [51]. So, the ZnO NSs indicates potentially stable material. The particle size distribution of ZnO NSs is revealed in Fig. 6(b). From the particle size distribution, the size of the ZnO NSs was calculated as 224 nm.  [22] and Amutha et al., [23] have reported on the structural formation of branched pattern using soft bio-templates and starch to demonstrate that the semi-crystalline granules are made from concentric rings (amylose and amylopectin) in which the basic components are aligned perpendicularly to the growth rings and the granule surface. In the ower like ZnO NSs, each pedal size was achieved around 100 to 200 nm. Figure 8(d) illustrates the EDAX spectrum of ZnO NSs, which exclusively indicates the presence of Zn and O elements without any impurities. The corresponding weight and atomic percentage of zinc (Zn) and oxygen (O) elements are given in the EDAX spectrum.

Field Emission
3.8 Antibacterial activity Figure 8 (a-d) depicts the antimicrobial property of ower-like ZnO NSs, which was tested using zone inhibition methods on both gram positive (Staphylococcus aureus, Bacillus subtilis) and gram negative (Salmonella paratyphi, Escherichia coli) bacteria. Bacterial inhibition zones are in millimetre (mm).By increasing the concentration of ZnO NSs, the growth rate of all the considered bacteria is reduced with the maximum inhibition of growth achieved at 60 g/ml. The highest inhibition zone was attained in Bacillus subtilis (28 mm) when compared to other bacterias such as Escherichia coli (21 mm), Salmonella paratyphi (18 mm) and staphylococcusaureus (16 mm). The Minimum Inhibitory Concentration (MIC) states that the minimum concentration of NPs required to impeded the growth of testing microorganisms. According to the statement, Salmonella paratyphi (10 mm) had a higher MIC value in 30 g/ml than Staphylococcus aureus (9 mm), Escherichia coli (8 mm) and Bacillus subtilis (7 mm), as presented in Table 1

Antioxidant activity
In the standard metabolic process, levels of antioxidants and free radicals are equalized. The overproduction of free radicals results in oxidative damage, leading to a variety of chronic diseases, such as diabetes, cancer and in ammation. In the biological system, antioxidant plays a signi cant part in scavenging toxic free radicals and supports in preventing damage of tissues and cells including DNA, proteins and lipids caused by free radicals. Natural antioxidants are in high demand due to their potential for disease prevention and health control [72].The antioxidant activities of green synthesized ZnO NSs are assessed using DPPH, which is a simple and fast approach. The colour of DPPH turns from purple to yellow and gets converted into 1,1-diphenyl-2-picrylhydrazine with a decrease in absorbance at 517 nm after reduction by ZnO NS's indicates the scavenging potentials of the NPs. The slow color change of DPPH solution from purple to pale yellow in the presence of ZnO NSs is due to shifting of electron density present at oxygen atom to the odd electron present at nitrogen atom in DPPH [73]. Antioxidant activity of ower like ZnO NSs was found ( Figure.9) to increase with the increase in concentration in a dosedependent manner from 12.5 µg/mlto 1000 µg/ml. IC 50 of ZnO NSs is 50.22 µg/ml and the IC 50 value of standard ascorbic acid is 42.18 µg/ml. Oryza punctata rice is known to consist of a signi cant amount of phenolic acids and proanthocyanidins, both reveal valuable biological activities incorporated with good antioxidant activity [74]. Proanthocyanidins belong to condensed tannins and are polymers and oligomers of avan-3-ols. It shows good antioxidant and free radical scavenging activity because of the presence of various phenolic hydroxyl especially an ortho-dihydroxyl group. The phenolic ring confers antioxidant property by stabilizing and delocalizing unpaired electrons [75,76].

Invitro cytotoxicity
The evaluation of cytotoxicity is an essential part of toxicology assessment because it explains the cellular response to a toxicant. Figure 10(d) depicts the variation in cell activity of MCF-7 cells exposed to 6. 25, 12.5, 25, 50 and 100 g/ml concentrations by MTT assay. Figure revealed the gradual increase in percentage of cell inhibition ability by varying the concentration of ZnO NSs from6.25 to 100 µg/mL. The concentration required to kill 50% of cells (IC 50 ) was 75.18 g/ml. The results demonstrate that, reduction in size of the viable cells happened when increase the concentration of ZnO NSs synthesized from Oryza punctata rice extract. Microscopic images of the impact of ZnO NSs on cancer MCF-7 cell line shown in Fig. 10 (b & c) con rms the decrease in number of viable cells compared to that of the control (Fig. 10 (a)). The reduction in no of cells may be due to the Reactive Oxygen Species (ROS) generation exceeds the anti-oxidative defensive capability of the cell and hence decrease in cell viability occurs due to oxidative damage of the cell components. Gunaratneet al., observed to copherols and tocotrienols in rice bran exhibits anticancer activity [77]. Premanathan et al., reported the basic mechanism for cytotoxicity of ZnO NSs towards cancer cells might be by stimulating the production of ROS, which are responsible for the induction of apoptosis [78].

Antiarthritic activity of ZnO NSs
Denaturation of proteins is one of the characteristics that in uence joint damage in rheumatoid arthritis and consequently promote the overproduction of autoantigens. Production of autoantigen/antibodies is associated with type-III hypersensitivity reaction, which is particularly related to arthritis, glomerulonephritis and erythematosus. The alteration of covalent, electrostatic, hydrophobic and cleavage of disul de bonds are probably involved in the protein denaturation mechanism [80]. Non-Steroidal Anti-In ammatory Drugs (NSAIDs) like aspirin, diclofenac, ibuprofen and naproxen provide a protective effect against protein denaturation [79]. The present investigation on the inhibitory effect of ZnO NSs was evaluated against protein denaturation (serum albumin). The different dose of test samples (0.1, 0.2, 0.3, 0.4 and 0.5 µg/ml) provided signi cant protection against denaturation of protein.
The increased absorbance of the test sample indicated the stabilization of protein and the values were compared with standard NSAID (Diclofenac sodium). The ower like ZnO NSs showed inhibitory activity of 78.94 ± 1.62 % at 0.5mg/mL concentration and the standard diclofenac sodium exhibited inhibition of 88.56 ± 1.41 % at a concentration of 0.5 mg/mL (Fig. 11).
The RBC membrane is similar to the membrane of lysosome and the mechanism of stabilization of lysosomal membrane is an important in the in ammation pathway by preventing the release of activated neutrophil, bactericidal enzymes and proteases, which causes further tissue in ammation and damage.
NSAIDs are commonly used to suppress the lysosomal enzyme release or stabilizing the lysosomal membrane [81]. In this study, the ower like ZnO NSs showed the signi cant RBC membrane protection when compared to standard diclofenac (NSAIDs). The ZnO NSs exhibited the 81.12 ± 1.25 % membrane stabilization at the 0.5mg/mL concentration and diclofenac showed an inhibition of 89.24 ± 0.94 % at a concentration of 0.5 mg/mL (Fig. 12).

Conclusion
Flower-like ZnO NSs were fabricated using bio template method for a wide range of biological activities.
The synthesized material had a ower-like structure and was evenly distributed, as evidenced by the FESEM analysis. Excellent antimicrobial activity was achieved at higher concentrations of ZnO NSs due to production of ROS in the cell wall membrane that is more useful for raising antimicrobial activity against Staphylococcus aureus, Bacillus subtilis (gram-positive)and Salmonella paratyphi, Escherichia coli (gram-negative) bacteria. The ower-like ZnO NSs considerably scavenged the radicals in a dosedependent manner (12.5 to 1000 µg/ml) and the IC 50 value of ZnO NSs is 50.22 µg/ml was observed due to free radicals. The percentage of cell inhibition increases with increasing concentration of ZnO NSs against MCF-7 breast cancer cell line. The green synthesized ZnO NSs showed signi cant inhibition of protein denaturation and membrane denaturation activity and that suggests to be used as an antiin ammatory as well as anti arthritic agent. Hence the present study concluded that the green synthesized ower-like ZnO NSs from Oryza punctata might be used for various biological and therapeutical applications.          DPPH radical scavenging activity of ower like ZnO NSs using ascorbic acid as a standard Protein denaturation inhibition activity of ower like ZnO NSs synthesized from Oryza Punctata Figure 12