Biogenic Synthesis of Magnetite Nanoparticles using the Roots of Mirabilis Jalapa for Efficient Removal of Eluent, Anti-Bacterial, and Antioxidant: A Sustainable Approach.

Magnetite nanoparticles hold quite immense applications in the fields of drug delivery, environmental remedies, and magnetic resonance imaging as they are biocompatible and can stabilize and reduce. An eco-friendly approach that is viable and sustainable for synthesizing magnetite nanoparticles with controlled properties has new avenues for their applications in various fields. Magnetite nanoparticles are often used in the removal of organic eluents as they can adsorb heavy metals which binds on the surface and cleans the polluted area. Magnetite nanoparticles synthesized from the roots of Mirabilis Jalapa have a variety of uses in the treatment of lesion infections. As antioxidants, they reduce the oxidative stress caused by free radicals. Morphology, size, and form of Magnetite nanoparticles are determined by UV-visible spectroscopy, SEM, EDX, TEM, TGA, XRD, and FTIR, respectively.


Introduction
Nanotechnology has a large assortment of unique applications in the fields of chemical, physical, optical, electrical, mechanical, magnetically, and catalytic properties due to their demand in the present world of science and technology [1][2][3] .The goal of nanotechnology is to prepare better, simpler, more efficient, eco-friendly nanoparticles [4][5][6] .The nanoparticles form as a cluster with nano-sized having a range of 100 nm 7 .The bio-genic synthesized nanoparticle has become trending as it possesses many biological applications towards anti-cancer, antidiabetic, anti-microbial, anti-hyperglycaemic, anti-oxidants, and cardioprotective effect due to its abundant in nature and eco-friendly availability [8][9] .
Metallic nanoparticles like magnetite illuminate towards catalytic, biosensors, ferrofluids, separation, and purification of bio-molecules due to their non-toxic and bio-compatibility for researchers as they exhibit various oxidation states, remove inorganic and organic heavy metal pollutant that is present in water [10][11][12] .Various methods are employed for the synthesis of Fe3O2NPs such as physical and chemical approaches, co-precipitation approach, hydrothermal, spray laser pyrolysis, etc [13][14] .To determine the morphology of synthesized nanoparticles that may be in the form of nanorods, nanotubes, nanowires, nanocage nanoflowers, etc. these methods may lead to toxicity, are hazardous to the environment, and may cause adverse effects on mother nature.Hence it has become important for researchers to synthesize nanoparticles with high bio-compatibility and harness them to synthesize NPs that are low-cost, renewable, benign reactions, sustainable, and non-toxic [14][15] .Iron oxide nanoparticles transformed the world as a supplement to combat disease and solve environmental issues.Different parts of plant material like fruit, leaves, flowers, roots, seeds, stem, and bark were used to synthesize Iron oxide NPs.This study is concerned with the potential application of biogenic synthesis of magnetite nanoparticles that are harnessed in the utility of natural resources acting as stabilizing, capping, and reducing agents [16][17] .These harness techniques can be innovatively used in various applications.In this study, Fe3O2NPs are used as an antibacterial agent in the treatment of harmful infection caused by lesions, as an antioxidant it reduces oxidative stress that arises due to odd unpaired electrons, and as a catalyst, it removes eluent that damages the environment.Fe3O2 NPs were prepared using the roots of Mirabilis Jalapa, which belongs to the Nyctaginaceae family the marvel of Peru a perennial herbaceous bushy plant that is also called the 4'oclock plant [18][19] .In some areas where the climatic conditions are warm, the roots can grow to more than 18-20 kg.The Mirabilis Jalapa roots are made into a paste and used as a medicine in Ayurveda, Siddha, and in some traditional methods for curing diseases like arthritis, joint pains, piles, and inflammation [21][22][23][24] .
Herewith, we prepared magnetite nanoparticles from the roots of Mirabilis Jalapa.The prepared nanoparticles were characterized UV-visible spectroscopy, Scanning Electron microscopy (SEM), Energy dispersive X-ray spectroscopy (EDX), Transmission Electron Microscopy (TEM), Thermogravimetric analysis (TGA), X-ray diffraction (XRD) and Infrared spectroscopy techniques (FT-IR).Further, the prepared nanoparticles catalytic, antioxidant and antibacterial properties are investigated.

Materials
Analytical reagents of Iron II sulphate heptahydrate (FeSO4.7H2O),Iron III Chloride hexa hydrate (FeCl3.6H2O),NH3, and Malachite green dye were purchased from Merch, and Milli-Q-water was used for the analysis.

Preparation of plant extract
From Visakhapatnam's neighborhood, a fresh Mirabilis Jalapa plant was procured.The Mirabilis Jalapa root was cut free from the plant and properly cleaned under a running tap then with 2D water and immersed for 30 minutes to eliminate dust.The collected roots were peeled, sliced into fine pieces, and dried for 10 to 15 days in the shade.After drying, the root was ground into a fine powder.The powder was used for additional testing after being kept in an airtight container and used for extract preparation (Figure 1&2).

Synthesis of Iron oxide nanoparticles
The magnetite NPs have been synthesized using the co-precipitation method with roots of MJ extract that act as a capping and reducing agent (Figure 2).In this typical procedure, a 1:1 ratio of FeSO4•7H2O about 2.78 g, and 0.55 g of FeCl3•6H2O were prepared and 10 ml of each solution was added to 80 ml of Milli-Q water.This solution was then subjected to heated for 50 min at 80 °C stirring constantly on a hot plate.5 mL of 5 % aqueous root extract of Mirabilis jalapa was added to the reaction mixture. 2 mL of 5 % ammonia was added to the reaction and the pH was maintained.A brownishblack precipitate was observed and the reaction was stopped and filtered 25 .The obtained, Fe3O2NPs were allowed to centrifugation and washed thrice with Milli-Q water to free from impurities then dried in an oven at 50 0 c, and used for further study.To enhance the yield optimisation conditions were employed.

Optimization conditions for the synthesis of magnetite NPs
Optimization studies were done to avoid agglomerisation and to enhance the yield of Fe3O2NPs by changing different concentrations of FeSO4, and FeCl3, root extract, pH, temperature, and reaction time.The conditions for the synthesis of Magnetite NPs are identified by the appearance of a characteristic band from 390 nm to 405 nm in the UV-visible spectrophotometer.

Optimizing with FeSO4 and FeCl3 concentration
Different ratios of FeSO4 and FeCl3 solutions were prepared and used to enhance the production of magnetite NPs.The Concentrations were taken in 1:1 ratio, 2:1 ratio, 2:3 ratio, and 1:2 ratios of FeSO4 and FeCl3.1:2 ratios of ferrous sulphate and ferric chloride were favorable in enhancing the yield and the UV-Visible spectral analysis as shown in Fig.

Optimizing with root extract concentration
Various percentages of MJ root extract (1%,2%,4%.5%)were prepared to synthesize magnetite nanoparticles.It was observed that as the percentage of the extract increased the color of the reaction also turned darker, the SPR band of maximum absorbance was observed at 404 nm which is shown in Fig. (4) when the reaction was performed using 5% root extract.

Optimization of pH:
P H studies were done to synthesize Magnetite NPs to enhance yield. 10 ml of 5%v root extract, a 1:2 ratio of FeSO4 and FeCl3 solutions were added, and different pH conditions were maintained (5,7,8,9) by adding NH3 and HCl.pH-9 was observed to be quite favorable with maximum absorbance at 406 nm shown in Fig. ( 5)

Optimization of reaction times
To obtain stable NPs the duration of the reaction mixture is been increased, and various reaction times were carried out for 80 min, 90 min, and 120 min at pH-9, 5% root extract with 1:2 ratios of FeSO4 and FeCl3 was used to increase the yields of magnetite NPs at 70 0 C. The color of the reaction mixture turned yellowish orange to blackish brown and the absorbance was recorded which is shown in Fig. (6).

FT-IR studies
The functional groups in synthesized iron oxide nanoparticles were done with FT-IR analysis using the instrument Prestige 21 Shimadzu.These spectroscopy studies enable the identification of various Phyto-chemical constituents / bio-molecules that are capable of reduction and stabilization [26][27] and are shown in Fig ( 8).
Peaks were identified at 1090 cm -1 that corresponds to strong C-O stretching with primary alcohols, 1610 cm -1 for strong C=C stretching indicates the presence of α, β -unsaturated ketones, 1710 cm -1 for strong C = O stretching with conjugated acids, and 3546 cm -1 for strong broad O-H stretching shows the presence of alcohols.

SEM Studies
The Scanning electron microscope (SEM) enables us to determine the surface morphology, size, and shape of Iron Oxide NPs.The morphological dimensions were assessed by using SEM (ZEISS GEMINI SEM).The SEM images are shown in Fig. (9a-c).The SAED image of prepared nanoparticles is shown in Fig ( 9d

EDX Studies
The chemical composition of the resulting nanoparticles is made possible with the use of EDX analysis.Iron and oxygen show a strong signal on the EDX, and this information makes it possible for the presence of both iron and oxygen in the form of iron oxide nanoparticles with maximum peak intensity at 0.6 e.v for iron with 65.3% and 0.5 ev for oxygen at 32.9 % 28 is displayed in Fig. (10).

TEM studies
TEM examination of iron oxide nanoparticles was carried out using the FEI Tecnai G2 20 S-Twin.The SAED pattern known as selected area electron diffraction and image were used to study the TEM images of iron oxide nanoparticles that were made utilizing environmentally friendly methods using the roots of Mirabilis Jalapa.The produced nanoparticles had been discovered to be crystalline.Iron oxide nanoparticles were found to have a 45 nm particle size.

TGA studies
TGA study was carried out on synthesized magnetite nanoparticles to incorporate the surface density, identify of nanoparticles composition, determine the effect of the additive present, oxidative assessment and to know the thermal stability as well as purity of the sample.In this process, the temperature of the furnace under which the sample is kept whose weight is measured with the help of an analytical balance provided in it.The loss of weight was observed when the instrument was run from 40-1000 0 c temperature.It was observed that about 18% of total mass was lost by the magnetite NPs and the following graph is shown in Fig 13.

Adsorption Studies
Due to the increase in globalization, there is a tremendous spread in trade and industries that enormously increases the pollution of the environment causing serious damage to Mother Nature.The eluents from industries and factories get mixed with water bodies, in the soil and get contaminated.This not only affects the water bodies but also affects the aquatic living system.Dye is the major substance that has to be deprived.Nanoparticles can be used to control these eluents and help the environment.Biogenic synthesized magnetite nanoparticles are proven to be the best pollution deprivers as they are non-toxic and eco-friendly.In this study magnetite nanoparticles that are synthesized using Mirabilis Jalapa root extract are used as pollution deprivers to remove Malachite Green dye, a cationic dye.
Adsorption isotherm analyses were done to identify the maximum adsorption capacity of synthesized magnetite NPs.This method enables in evaluation of the performance of adsorbent in the removal of Malachite Green dye that gets contaminated by the water bodies.Various methods of adsorption techniques were performed in the removal of M.G dye such as Langmuir, Freundlich, and Temkin isotherm models [29][30][31] .Langmuir adsorption assumes monolayer adsorption on homogeneous surfaces without interaction of adsorbate molecules and also provides insight into the nature of adsorption on the surface of magnetite NPs this also helps in depriving MG dye of getting polluted into the environment.A saturated state is formed at which further adsorption does not take place as well and there is no further interaction between the molecules that are adsorbed on neighboring sites.The Freundlich isotherm allows for multi-layer adsorption and is nonuniform energies.This enables us to heterogeneity.The Temkin isotherm involves the interaction between adsorbate over the surface of the adsorptive in a thermodynamical manner.The adsorption isotherms (Fig. 14) parameters R 2 values were calculated from the above techniques are mentioned in Table (1).These results in the regression coefficient values of the Langmuir model (R 2 = 0.99458), the Freundlich model (R 2 = 0.98518), and the Temkin model (R 2 =0.94872) were been evaluated.Adsorption techniques are better fitted to Langmuir isotherm than Freundlich and Temkin isotherms on the surface of Fe 2O3 is a monolayer.The monolayer adsorption capacity (qmax) of Fe2O3 NPs was theoretically calculated and found to be (34.0136)mg/g.Fe2O3 NPs have comparative adsorption efficiency towards organic eluents.In this study, the adsorption mechanism was done using the kinetics of pseudo-first and second-order reactions (Fig. 15) for obtained experimental data, and the parameters and constants of Fe2O3 NPs are shown in Table 2.

Anti-bacterial studies
Lesions carry bacteria damaging the healthy tissues and increasing the infection as they host the immune system.Lesions can transfer from one organ to many organs as some harmful pathogenic microorganisms present in lesions and delay the healing process causing severe pain.To minimize the infections caused by lesions, antibiotics are prescribed to decrease the infection and proper care must be taken [32][33][34][35] .Antibiotics work on infection and kill the growth of bacteria in lesions they do not heal virus or fungus infections.In a few cases, they may need additional interventions such as antibiotics and sometimes surgical debridement.Though antibiotics are necessary for curing diseases improper usage may disrupt the immune system of our body.Biogenic synthesized magnetic nanoparticles target bacteria to reduce contamination and enhance the healing process in the lesion.They can stimulate blood circulation, tissue production, and healing ability.In the present study staphylococcus and E. coli were isolated from the infected lesions and an anti-bacterial assay was performed using disk diffusion / Kerby-Bauer method.The sterile agar growth medium was poured into a petri dish.The infected lesions were collected with a sterile swab and spread on an agar plate.
Streaking was done on the entire surface followed by incubation for 48 hours at 37 0 c, the bacteria developed on the agar plate and were identified by the gram staining process.The isolates of staphylococcus aureus and E. coli images are shown in Fig. (16).Agar medium was prepared and known concentrations of Fe2O3 NPs were prepared with a suitable solvent and added to the agar medium which was punched with the help of a sterile borer.Ciprofloxacin was used as a reference antibiotic and was allowed for incubation for 24 hours at 37 0 c.A Himedia zone scale was used to measure the inhibition zone.Which were shown in the below Table (3) and the anti-bacterial activity of staphylococcus aureus and E. coli with ciprofloxacin antibiotic on Fe2O3 NPs is shown in Fig. (17).

Anti-oxidant studies
Antioxidant activity can be performed in the laboratory to measure the antioxidant capacity of the desired sample.A substance that stops oxidative damage caused by reactive substances called free radicals and reactive oxygen species.Antioxidants are used in food sciences, medicine, and cosmetics, they can neutralize the free radicals.As food preservers antioxidants can retard the action of oxygen on food, they are more reactive towards oxygen than the food.In this study, DPPH is used in assessing the potential of Fe2O3 NPs as it is a rapid simpler, and stable free radical that reacts with antioxidants and turns to violet, and slowly its color becomes pale violet when kept in the dark for half an hour 36 .The degree of color is related to its antioxidant activity.The greater the loss in colour of DPPH greater the antioxidant activity.The antioxidant potential of Fe2O3 NPs is measured using a UV-visible spectrophotometer at 517 nm.For the control, Ascorbic acid was used that reduce and neutralize highly reactive oxygen.The percentage inhibition was done using the formula.the antioxidant efficacy is shown in Fig. Mean ± SD was calculated and shown in the table (4)

Conclusions
The Magnetite nanoparticles have been synthesized from the roots of Mirabilis Jalapa.The prepared Magnetite nanoparticles characterized using UV-visible spectroscopy, Scanning Electron microscopy, Energy dispersive X-ray spectroscopy, Transmission Electron Microscopy, Thermogravimetric analysis, X-ray diffraction and Infrared spectroscopy techniques were used.The prepared particles are acted as a catalyst in depriving eluent, an antioxidant that removes oxidative stress, and as an anti-bacterial agent to cure infective lesions.

( 18 )
% inhibition= {(AC-AS)/AC*100} AC = Absorbance of Ascorbic acid as a control AS = Absorbance of the magnetite nanoparticles after performing an antioxidative assay It was observed that the absorbance of D.P.P.H reduced with a sign of color change from violet to pale yellow color with the increase in the concentration of Fe2O3 NPs.

Table ( 3
): Zone of inhibition of Fe2O3 NPs and zone of inhibition.

Table ( 4
): The antioxidant efficacy using different concentrations of synthesized magnetite NPs against DPPH.