Green synthesis of ZnONps using Horse gram seed aqueous extract and its in vitro evaluation on antioxidant, antidiabetic, anticancer and DNA binding potential

The goal of this study was to access the green production of zinc oxide nanoparticles ( G-ZnO NPs ) using aqueous extract of horse gram seeds ( Macrotyloma Uni ﬂ orum ) . The precursor to the extract ratio ( 2.5:1 ) and pH value ( 8.5 ) , along with the zinc nitrate concentration ( 0.5 M ) , had an impact on the particle size and the green synthesized ZnO nanoparticles. UV Spectroscopy inspection revealed formation of G-ZnO NPs with absorption at 320 nm which is the characteristic absorption of G-ZnO NPs. FTIR, XRD, SEM, EDX and TEM were used to characterize the green synthesized ZnO NPs. The ﬁ ndings demonstrated that the presence of secondary metabolites in the seed extractstabilize and contribute in the production of G-ZnO NPs. A dose-dependent increase in antibacterial activity was seen in evaluation of these NPs against Klebsiella, Staphylococcus aureus , Escherichia coli , Pseudomonas aeruginosa . The antioxidant activity and antidiabetic activity for G-ZnO NPs was also noted to be concentration dependent. The synthesized nanoparticles are found to interact with CT-DNA to produce a hypochromic shift. Further the studies on the G-ZnONPs in MCF-7 cells using the MTT test demonstrated greater cellular inhibition. The results validate that the green synthesized ZnO-NPs


Introduction
In recent years, research has focused increasingly on the interdisciplinary field of nanotechnology.It has an important role across all aspect of science including economy.Every aspect of life that involves the creation, synthesis is manipulation of nanoscale materials [1].The array of different nanoparticles (NPs) has a variety of potential uses in the medical, pharmaceutical, cosmetic, and food industries because of their unique characteristics, including size, shape, and high surface area.The production of NPs has been documented over time using variety of approaches, such as chemical, physical, and biological methods [2].Owing to quick, cost effective, single-step synthesis, use of ecofriendly solvents, and reduced generation of toxic compounds, green synthesis has been the main attraction of the researchers recently and followed widely [3].Studies have proved that the biological materials including natural products found to be the natural reductance with good capping and stabilizing capabilities.
The biological sources for the synthesis of metallic NPs include plant extracts, microorganisms, and enzymes that can reduce the metal ions within the solution into NPs [4].In addition to metal nanoparticles, green synthesis of metallic oxides are also increasingly gaining its importance.They are proved to possess excellent antidiabetic, antioxidant and anticancer properties [5].Further they are also used as (a) sensor (Al 2 O 3 , TiO 2 , CuO, NiO), (b) antibacterial agents (ZnO, CuO, NiO, CeO, Fe 2 O 3 ) and (c) anticancer (TiO 2 , CuO), applications in (d)drug delivery (CuO, ZnO, Fe 2 O 3 ), and as (e) Catalyst (TiO 2 , ZnO) [6].
The ZnO nanoparticles, which belong to metallic oxide NPs, are well known for its multifunctional, less toxic and biocompatibility.It finds use in food industry as an antimicrobial agent [7] and other applications includes as; sunscreens, paints, coatings, optics, and optoelectronics because of their excellent UV protection property.It is also finding its application in environmental chemistry in the treatment of waste water with respect to the removal of heavy metals [8].
Due to its lesser environmental toxicity and contamination, numerous studies have recommended the greener approach for the ecofriendly production of ZnO NPs using naturally occurring biowastes like leaves, flowers, seeds, etc that may be produced at a reasonable cost.The biomass used in the present study is horse gram which is known as Macrotyloma uniflorum botanically [9].Horse gram is an herbaceous plant having numerous therapeutic benefits.Different portions of the plant are used to treat heart disease, asthma, bronchitis, leukoderma, urine discharges, and kidney stones [10].It is an edible seed which contains carbohydrates, phenols, steroids, terpenoids, proteins, and minerals are all present [11].Many research has been conducted to study variable optimization in the production of metallic NPs and to portray the interaction effects between effective factors.Furthermore, from the standpoint of green synthesis, the optimization of independent process parameters is an intriguing topic [12].In comparison to physical or chemical synthesis methods, the utilisation of waste materials eliminates energy requirements, lowers the cost of synthesis, and reduces the need to utilise hazardous chemicals or byproducts, demonstrating the effectiveness of green synthesis [13].Microwave [14,15], sono chemical [16] and thermal decomposition [17] techniques have recently been employed for the quick creation of nanostructures.Thus, to line with the green synthesis of metal oxide nanoparticles in this study attempt have been made to use the biomass horse gram which is underutilized have been exploited for the green synthesis of metallic nanoparticles.
Understanding the potential impacts of green synthesis of NPs, the other aspect of interaction of NPs with DNA and RNA requires research into how NPs interact with nucleic acids.DNA interaction studies suggest that biosynthesized ZnONps can be used for the formation of DNA-Nanoconjugate complex for using them in biomedical applications [18,19].Research report on the interaction of metal NPs with DNA is recently drawing the attention of biologist because of their potential impact on the structural integrity and synthesis of DNA [18].The recent reports on ZnONPs have sparked a lot of attention as a novel, inexpensive, and low-toxic nanomaterial for use in a range of biomedical applications, including anticancer, antibacterial, antioxidant, drug delivery, and bioimaging ones.Chung et al demonstrated a dose-dependent lethal impact of ZnO NPs on human liver carcinoma cancer cells and sliver NPs on HeLa cancer cells.Green production of ZnO NPs utilising an aqueous extract of Deverra tortuosa aerial parts is reported against two cancer cell lines Caco-2 (human colon adenocarcinoma) and A549 (human lung adenocarcinoma).Metal NPs synthesized from various plant sections clearly had distinct lethal effects on different cell types.This is because the potential of NPs to produce cytotoxicity is determined by their form and size, as well as the biomolecules used in their synthesis [20].The environmental-friendly synthesis of ZnO NPs from horse gram and its biological applications such as antioxidant, anti-bacterial, anti-diabetic activities, CT-DNA binding study and Cytotoxic studies were not reported in the literature.Thus, the current study focuses on the synthesis and evaluation of the abovementioned biological applications of ZnO NPs from horse gram.

Preparation of seed aqueous extract
Horse gram seed used for the study was obtained from a farmer in the neighborhood of Tamil Nadu's Vellore District.The dried seeds were powdered using mechanically grinding and sieved through #150 mm mesh and stored in an airtight container.The plant was authenticated by Dr M.U.Sharief, Scientist 'F' & Head of office, Botanical survey of India, Southern Regional Centre, Coimbatore.A voucher specimen was deposited (BSI/ SRC/5/23/2022/Tech/610) for further reference.About 5 g of air-dried horse gram seed (HGS) flour was extracted by ultra-sonic (FAST CLEAN Ultrasonic cleaner) assisted method with 100 ml of distilled water for 2 h at room temperature.Thus, obtained extract was concentrated on a rotary evaporator under reduced pressure and evaporated to dryness.The phytochemical analysis was conducted as per the earlier reported methods [21,22] for the presence of for the presence of flavonoids (lead acetate test), alkaloids (Mayers test), phenols (ferric chloride test), protein (Biuret's test) and amino acid (ninhydrin test), carbohydrates (Benedict's test), and saponins (Froth test).
2.3.Green synthesis of ZnONPs 0.5 M of zinc nitrate solutions were added dropwise in a volume ratio of 5:2 to the horse gram extract while stirring at 80 °C for 60 min using magnetic hotplate stirrer.After completing the process, the precipitate was centrifuged for 30 min.The collected G-ZnO nano particles washed with deionized water followed by ethanol and then dried at 80 °C.The dried sample were calcinated at 400 °C for 1 h as per the earlier reports [23].Following calcination at 400 °C, the mixture of organic solution directly decomposes, releasing G-ZnO nanoparticles [24].

Characterization of G-ZnO NPs
UV-Visible spectrophotometer was used to obtain UV-Visible spectra (JASCO).In order to identify the functional groups, the individual G-ZnONPs and HGSW (blank) were prepared in KBr pellets containing 1% (w/w) of the samples.The instrument was operated in the 400-4000 cm −1 range in frequency (Shimadzu, Japan).To determine the crystalline nature and phase structure of the samples, an X-Ray Diffractometer (Bruker, D8-Advance) was used to perform.An XRD analysis on G-ZnONPs.SEM-EDX, a scanning electron microscope equipped with an EDX (Carl Zeiss), was used to examine the surface morphology and elemental composition of the green synthesized ZnONPs.The particle size was first counted using ImageJ's 'Analyze Particles' method after the images were first made black and white.With EDX area analysis, quantification of individual elements was done.On a HORIBA Scientific and Horiba SZ 100 model, dynamic light scattering study (DLS) and Zeta potential measurements were carried out.10 mg of the sample were sonicated in distilled water for 10 min before to the measurement.A very little amount of the sample was placed on a copper grid that had been coated with carbon before being blown with a hand dryer to eliminate any extra particles.Thus, created thin films of the material was subjected to TEM studies using a JEOL JEM 2100 model.For imaging, the samples were air-dried after being drop-cast on carbon-coated TEM copper grids.

Antioxidant activity
The antioxidant studies on G-ZnONPs were assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) method, reducing power assay, and phospho-molybdenum methods by following the procedures mentioned in the literature with slight modification [25], as discussed below.

DPPH radical scavenging activity
The antioxidant activity of G-ZnONPs was carried out using the reagent DPPH.G-ZnONP was prepared in methanol at various concentrations (40-160 μg ml −1 ) and it was added to 40 μl of 1 mM DPPH solutions and allowed to stand for 30 min at room temperature.The absorbance of the sample was measured at 517 nm.The radical scavenging activity was expressed in terms of percentage inhibition and was calculated using the formula Reducing power assay G-ZnONPs in various concentrations was taken ranging from 40 μg ml −1 to 160 μg ml −1 and was mixed with 0.2 M phosphate buffer and %1 potassium ferricyanide.The absorbance was measured at 700 nm using ascorbic acid as a positive reference standard.

Phospho-molybdenum assay
G-ZnONPs was taken in various concentrations (40-160 μg ml −1 ) and was added to each test tube containing 3 ml of distilled water and 1 ml of molybdate reagent.The sample absorbance was measured at 695 nm.Ascorbic acid was used as a positive reference standard.

Anti-Bacterial activity of G-ZnONPs
The test was measured with four bacterial pathogens, including Pseudomonas, Staphylococcus aureus, Klebsiella and E. coli to assess the bactericidal efficacy of NPs using an agar disc diffusion experiment [26].The bacteria were activated in nutrient broth medium using a shaker incubator (TeifAzmaTeb, Iran) at 37 °C for 24 h.After this time, the optical density of the fresh media was checked using Mcfarland standard No. 0.5, and the bacteria were subsequently grown on Mueller Hinton Agar.On a blank disc, various doses (50, 75 and 100 μg/disc) of the green produced G-ZnO NPs were added before being incubated for 24 h.The standard antibiotic used was ciprofloxacin (10 μg/disc).Based on the diameter of the zone of inhibition (ZOI), a Caliper was used to assess the G-ZnO NPs bactericidal activity [26,27].

In vitro Antidiabetic activity of G-ZnONPs
The α-amylase was dissolved in sodium phosphate buffer (0.5 ml, 0.02 M, pH 6.9) before being added to G-ZnONPs at various concentrations ranging from 20 to 100 g ml −1 .The tubes received 1 ml of a starch solution (1%) before being incubated at 37 °C for an additional 15 min.After that, the reaction was stopped by adding 1 ml of the dinitrosalicylic acid reagent and heating it for 10 min in a pot of boiling water.After further cooling the tubes, the absorbance at 540 nm was measured [28].

2.8.
In vitro DNA binding study UV-vis spectral analysis was used to investigate the interaction between calf thymus DNA (CT-DNA) and G-ZnONPs.The CT-DNA solution was made in 0.1 M Tris-HCl buffer (pH −7.2) with 12 h of stirring.By adjusting the concentration of CT-DNA while maintaining a constant concentration of G-ZnONPs (20 μl), absorption studies were conducted (10-100 μl).After adding various concentrations of CT-DNA, the spectral alterations of G-ZnONPs were observed by observing UV-visible absorption in the region of 300-600 nm [18,19].

Cell culture and cytotoxicity studies
The monolayer cells were detached using trypsin-EDTA to form single cell suspensions, and viable cells were counted using a hemocytometer before being diluted with media containing 5% FBS to achieve a final density of 1×10 5 cells ml −1 .Cell suspension in 100 μl per well was seeded into 96-well plates at a plating density of 10,000 cells/well and incubated to allow for cell adhesion at 37 °C, 5% CO 2 , 95% air, and 100% relative humidity.The cells were treated with repeated concentrations of the test substances after 24 h.They were first dissolved in neat dimethyl sulfoxide (DMSO), and an aliquot of the sample solution was diluted with serum free medium to twice the desired final maximum test concentration [20].
Succinate-dehydrogenase, a mitochondrial enzyme in live cells, cleaves the tetrazolium ring, transforming MTT to an insoluble purple formazan.As a result, the amount of formazan generated is proportional to the number of viable cells.After 48 h, 15 μl of MTT (5 mg ml −1 ) in phosphate buffered saline (PBS) was added to each well and incubated for 4 h at 37 °C.The MTT medium was then removed, and the generated formazan crystals were solubilized in 100 μl of DMSO before being quantified at 570 nm with a microplate reader [29].

Statistical analysis
The significance of the analysis was examined using one way ANOVA test in Graph Pad prism.The study reports the p-value <0.01 was found to be statistically significant and all the data are expressed as mean ± standard error mean.

Phytochemical analysis of the aqueous extract
The aqueous extract of HGS was obtained using ecofriendly greener approach.The extract obtained was filtered and the aliquot was subjected to preliminary phytochemical investigations.The study of the phytochemical analysis confirmed the presence of secondary metabolites present in the aqueous extract of horse gram seed.It is showed in table1.

UV-visible absorption spectral analysis
The reaction between the functional elements of the Horse gram water extract and the zinc precursor is depicted in graphical abstract as a potential mechanism for the synthesis of ZnO utilizing horse gram seed water extract.The terpenoid, steroids and phenolic compounds in the present in the HG aqueous extract function as ligand agents.Nanoparticles are created, stabilized, and shaped through the processes of nucleation and shaping.A valuable investigation to support the bio-reduction of zinc oxide nanoparticles is UV spectroscopy.UV-Vis spectral studies proved that zinc nitrate was bio-reduced into zinc oxide nanoparticles in the presence of horse gram seed aqueous extract.Due to the simultaneous vibration of the electrons of metal nanoparticles in resonance with the light wave, zinc oxide nanoparticles include free electrons, which have the potential to give birth to an SPR (surface Plasmon resonance) absorption band [30].G-ZnONPs easily produced by the extracellular and quick biosynthetic reduction process.According to figure 1(a), zinc oxide has a broad spectrum with an absorption peak at 320 nm.Horse gram seed extract may include amides and acid groups that are crucial for the stabilization and encapsulation of zinc oxide nanoparticles.

SEM and EDX analysis
The shape, size, and purity of the bio-synthesized zinc oxide nanoparticles were examined using SEM and EDX analysis.SEM images of zinc oxide nanoparticles may be seen in figures 2(a)-(c).From the SEM picture shown in figures 2(a)-(c) it is evident that the biosynthesized zinc oxide nanoparticle has the particle size ranging from 30 to 60 nm which is calculated using Image J software.G-ZnO nanoparticles EDX spectrum is displayed in figure 3(d), which shows strong signal for zinc and oxygen.

Zeta potential and particle size analyzer
Using dynamic light scattering (DLS) analysis in an aqueous solution, the hydrodynamic size and polydispersity index (PDI) of the produced Nano-Zinc oxide were determined.Figure 3(c) exhibits the graph of the size distribution of G-ZnONPs.The average size of nano-zinc oxide was determined to be 65 nm, and its low dispersity index (PDI), which is 0.621, confirms the homogeneity and uniform dispersion of G-ZnONPs.The particles in the DLS measurement were slightly larger than the one in the TEM measurements, because the DLS measures the hydrodynamic radius of the particles.Zeta potential measurements can be used to determine the

TEM analysis
The pictures of G-ZnONPs acquired by transmission electron microscopy (TEM) are displayed in figures 3a and b.The creation of spherical nanoparticles is made clear by TEM images and is consistent with the shape of the SPR band in the UV-vis spectra.Gaussian fit graph showed in figure 4. The particle size range from the TEM picture was measured between 14.8 nm, which was consistent with the particle size estimated from the XRD spectrum.

Antibacterial activity
Through the use of the disc diffusion method, the antibacterial activity of the produced zinc oxide nanoparticles was examined against Gram-positive (Klebsiella, Staphylococcus aureus) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacterial species.The zone of inhibition values for the positive control (ciprofloxacin) against all pathogens were assessed and the matching cluster column charts are provided in figures 5(a)-(b).It shows that the produced zinc oxide nanoparticles have demonstrated a significant antimicrobial activity against all four pathogens investigated; nevertheless, Staphylococcus aureus had the greatest antibacterial activity of zone of inhibition values (42 mm).Their different membrane structures account for the differential in antibacterial action (Gram-positive and Gram-negative).Gram-positive bacteria like Klebsiella and Staphylococcus aureus have thick peptidoglycan layers, but Gram-negative bacteria like Escherichia coli and Pseudomonas aeruginosa have weaker peptidoglycan layers that are nonetheless surrounded by a lipid layer on the outside [35][36][37].In the antibacterial activity, zinc oxide nanoparticles first adhere to the bacterial cell membrane's surface before entering the bacteria themselves.Following penetration, they deactivate the microorganism enzymes, which results in the production of hydrogen peroxide.Bacterial cells were therefore dead [29,[38][39][40].

Antioxidant activity of G-ZnONps
The antioxidant activity of G-ZnONPs was evaluated by using the DPPH, Ferric Reducing power determination and phospho molybdenum method, and the results are shown in figure 6.It was observed that in DPPH method, IC50 values of were found to be 5.3 [34].In the FRAP method, IC 50 values of found to be 3.7.The phospho molybdenum assay method again confirms that the antioxidant potential of G-ZnONPs possess good reducing property with IC 50 values of 7.8 [41].

DNA binding studies
The interaction of small molecules with DNA has been clarified using a variety of approaches, including UV-vis spectroscopy, electrochemical methods, and gel electrophoresis.However, compared to other sophisticated procedures, spectroscopic techniques are practical, precise, and extremely sensitive [19].Any drug's binding to DNA can be investigated using the UV-visible spectroscopy method.The present study involves using UVvisible spectroscopy to examine the CT-DNA binding capability of the G-ZnONPs.Through this study, the stability of the G-ZnONPs in Tris-HCl buffer solution was examined.The absorption peak obtained at room temperature after 1 h indicates that the G-ZnONPs were stable in the buffer solution.The G-ZnONPs spectrum showed hypochromic shift during titration with an increasing amount of CT-DNA and confirms the binding effectiveness.According to figure 8(a), the d-d transition gives the G-ZnONPs an absorption peak at 320 nm.From figure, it is clear that there is no change in the absorption maxima of CT-DNA nano-Zinc oxide complexes   In the Wolfe-Shimmer equation, the terms 'ea' and 'eb' stand for the received extinction coefficient of the absorption signal at various concentrations of CT DNA, the extinction coefficient of ZnONPs when fully bound to CT-DNA, and the extinction coefficient of free ZnONPs [48].The plot of [DNA] / (ea-ef) against [DNA] is displayed in figure 8(b).The intrinsic binding constant was determined using the (slope/intercept) of this plot (Kb).Kb for G-ZnONPs-DNA was calculated to be 1.55×10 3 M −1 .

Cytotoxic evaluation
Furthermore, the favorable results of the antimicrobial investigations for G-ZnONPs prompted us to continue investigating the compounds in vitro cytotoxicity in a dose-dependent manner.On the MCF-7, this was studied using an MTT colorimetric test.After a 48-hour incubation period, G-ZnONPs showed a higher decrease in cell feasibility.It is showed in figure 10.The % age of cell inhibition versus complex concentration plot was used to  investigate the ability of chemicals to cause cell death in a dose-dependent way.The higher IC 50 values for micro particles demonstrated that they are not toxic to normal cell lines.The MCF-7 cell lines had IC 50 values of 55.01 μg ml −1 , indicating that they had a larger development inhibitory impact.After a 48 h incubation period, the results clearly demonstrated that G-ZnO nano particles are the most effective against the human breast cancer cell line MCF-7.The overall results of the cytotoxicity of the nano particles are strikingly similar to those seen in the investigation of their antibacterial evaluation.Figure 11 shows a graphical representation of the cytotoxic activity of the three cell lines mentioned above with all of the synthesized nano particles at 48 h. Figure 10 depicts graphs of cell viability versus G-ZnONPs concentration [20].

Conclusion
The bio synthesis of zinc oxide nano particles utilizing an aqueous seed extract of horse gram was shown to be a green method of producing ZnONPs.The FTIR, UV-vis Spectrophotometer, XRD, SEM, EDX, and TEM studies confirms the production of ZnO NPs and were characterized accordingly.The appearance of a surface plasma resonance peak at 320 nm served as confirmation that the G-ZnONPs were bio-synthesized.XRD measurements supported the G-ZnONPs crystalline nature.The G-ZnONPs 100 nm size was revealed by the SEM investigation.Additionally, manufactured G-ZnONPs antibacterial activity has demonstrated that they are effective antimicrobial agents.The G-ZnO NPs shows good antioxidant and antidiabetic activity.MCF-7 cell lines were used to test the cytotoxicity of the synthesised zinc oxide nano particles.This study suggested that the ZnO nano particles were valuable in decreasing cell viability.The interaction between the CT-DNA and ZnONPs revealed that the ZnONPs may bind to the CT-DNA via partial intercalation binding mechanism.Therefore, these interaction investigations imply that the ZnONps that have been generated in this way can be employed to create DNA-Nanoconjugate complexes for usage in bio-medical applications.Thus the green synthesized ZnONPs which contain encapsulated secondary metabolites from horse gram seed extract is found to be potential material with antioxidant, antidiabetic CT-DNA binding properties and cytotoxic studies which can be explored in future for pharmaceutical applications.

Figure 1 (
b) displays the FT-IR spectra of zinc oxide nanoparticles stabilized by horse gram seed extract.For the seed extract, the FT-IR spectra shows major peaks at 3340.01, 2168.24,1633.22,and 1213.71cm −1 .The presence of peak at 3340.01 cm −1 could be due to O-H group in polyphenols.1633.22 cm −1 related to carbonyl group stretching vibrations.The peaks at 1213.71 cm −1 may be described to -C-O-C[31].However, the FT-IR spectrum of the G-ZnO nanoparticles in figure 1(b) reveals the presence of significant peaks at 3421.21, 2852.32,1622.61,1430.22,1010.64, and 526.34 cm −1 , which are connected to vibrations of the -OH stretching mode, CH-stretching of alkanes, C-CH 2 groups.The stretching mode of the zinc and oxygen connection is what causes the bulk zinc oxide to exhibit a high intensity broad band in the IR spectrum at 526.34 cm −1[32,33].

Figure 9 .
Figure 9. Hypothetical representation of interaction of ZnONps with DNA.

Table 1 .
Phytochemical screening of horse gram seed aqueous extract.