Bioconjugate Synthesis of NiFe2O4 Using Juglans Regia Leaves Extract: Phytochemical Analysis, Optical Activity, Removal of Ciprofloxacin and Congo Red From Water


 In recent years, the biogenic synthesis of nanoparticle received enormous attention due to simple and eco-friendly path. Here in Jr.NiFe2O4 nanoparticles (NPs) were synthesized first time using the leave extract of Juglans regia via a straightforward process. The physio and phytochemical analysis of plant confirm the presence of macromolecules which function as bio-reductant and stabilize the nanoparticles. The Jr.NiFe2O4 NPs were characterized by UV-visible, FTIR spectroscopy, PXRD pattern, SEM and TGA/ DTA analysis. The nanoparticles proved to be optically active having a value of indirect bandgap of energy in the range of 1.53 eV. Besides, the ciprofloxacin (CIP) and Congo red (CR) degradation ability of Jr.NiFe2O4 NPs were also discussed. It is seen that the highest degradation rate was acquired for CIP using pH = 3, at 254 nm, while 85 % of removal rate was analysed for CR. The kinetic studies in case of CR removal followed pseudo-first-order. Overall, these data recommend an innovative inspiring application of a plant-mediated synthesis of Jr.NiFe2O4 NPs.


Introduction
Water plays a key role in everyday life but the unconstrained release of organic pollutants in water produces an extremely dangerous effect on human beings as well as on aquatic life. It has been seen that the conventional way of wastewater treatment leads to the production of wastewater-borne microorganisms to higher concentrations of Fluoroquinolones (FQ) 1

. A minimum inhibition
concentration of the most famous and powerful second-generation FQ, ciprofloxacin (CIP) has been continuously found in wastewater and induce bacterial resistance and many side effects like neurological disorder and ruptured tendons. According to many studies ciprofloxacin (CIP) can be degraded or removed from the wastewater by adsorption applications and advanced oxidation processes (AOPs) including sonification, ozonation, photolysis and heterogeneous photocatalysis 2 .
On the other hand, highly developed industrialization and unrestrained discharge of organic dyes is the major cause of water contamination 3 . One of the most utilized dyestuffs is azo dyes having -N=Nlinkage bonded to aromatic rings. The azo dyes such as Congo red (CR) dye is extensively used in the fabric industry 4,5 . 5 . Several approaches are adopted for degradation of CR dye like adsorption, coagulation, ion exchange, chemical oxidation and biodegradation etc [6][7][8] . However heterogeneous photolysis technique is considered the most propitious approach to destabilize organic pollutant from water. Initiation of photocatalysis decontamination of dyes favours the expense efficacious strategy in the use of sustainable energy system 4,9 . Nowadays, nanomaterials of various forms, shape and morphologies, e.g., carbonaceous nanomaterials, dendrimers, carbon nanotubes, zeolites, nanofibers and metal-containing nanoparticles have been widely employed for wastewater treatment 10 . However, the recyclability of these materials is a fundamental problem due to their nano size 10 . This problem is overwhelmed using magnetic nanoparticles (MNPs) materials which can be easily recycled by the application of magnetic filtration.
The variety of magnetic nanomaterial composites like Fe@SiO 2 , Fe 3 O 4 @C, Fe 3 O 4 @TiO 2 , PEO (poly(ethylene oxide)Fe 3 O 4 @PPO (poly(propylene oxide)), Fe 3 O 4 @PNIPAM (poly(Nisopropylacrylamide)),Fe 3 O 4 @PDA (polydopamine), Fe 3 O 4 @CNTs (multiwalled carbon nanotubes), Fe 3 O 4 @MIPs (molecularly imprinted polymer-encapsulated particles), iron oxide-oxyhydroxide/rGO, Fe@CS (carbon spheres), etc. have been used for wastewater treatment 11 . The spinal ferrites and its derivatives are famous for the remediation of various water pollutants 12 . Recently magnetic poly (styrene-2-acrylamido-2-methyl propanesulfonic acid) and magnetic (1,2,4,5-benzenetetracarboxylic acid) nanomaterials have been proved very effective for the removal of pharmaceuticals viz. atenolol, diclofenac sodium and ceftriaxone sodium and Congo red dye from water respectively 13,14 . The surface modification of magnetic nanoparticles is a quite simple and cost-effective approach which fall in the category of desired applications. The fabricated magnetic nanoparticles formed as an inorganic-organic blend by connecting organic species on the inorganic sideshow mechanical stability due to inorganic core and flexibility in solution due to organic modifications. By taking the advantages of magnetic nanoparticles, we synthesized NiFe 2 O 4 by using the leaf extract of Juglans regia. The green strategy has been adopted to contrive metal oxide-containing spinel ferrites by using numerous plants, for instance, rambutan, Malachra capitata, Mirabilis jalapa, lemon eucalyptus, Hibiscus rosa-sinensis black nightshade neem [15][16][17][18][19][20][21][22] . According to our literature survey, there is no report of Juglans regia leaves extract mediated synthesis of nickel ferrite nanoparticles (Jr.NiFe 2 O 4 NPs) and it's potential to remove the ciprofloxacin and Congo red dye from water, which is the subject of this paper. Furthermore, the physio and phytochemical analysis of Juglans regia leaves and the extract is also discussed. Sigma-Aldrich). The dye used for photolysis was Congo red (C 32 H 22 N 6 Na 2 O 6 S 2 , Sigma-Aldrich); All chemicals were used as received.

Physiochemical Analysis of Juglans regia leaves
The physicochemical analysis was carried out by following the guidelines and protocols (in triplicates) approved by the WHO 23 .

Macroscopic analysis
In the preliminary step, an organoleptic investigation was performed using the sense of organ to outline the nature and basic parameters of the flower extract.

Determination of moisture content (loss on drying)
10 grams of the leaves were taken in moisture dish and initially air-dried followed by oven-drying at 105-110°C for 20 minutes to remove water contents completely. The weight loss (mg/g) was determined by total moisture content.

Estimation of total ash
Around 2 grams of leaves were taken in the crucible and extend in layers onto the crucible. The material burnt at 500 -600 °C. The white residue of the leaves was due to the non-existent carbon.
The percentage of ash content was calculated by the following.

Estimation of acid-insoluble ash
The ash contents obtained from crucible were blended with 2M HCl (25 mL) and covered with a watch glass. The mixture was boiled for 5 minutes and the acid-insoluble contents were obtained by filtering the reaction mixture through ashless filter paper. After that washed the filter paper with hot water, dried, ignited and weighed.

Estimation of water-soluble ash
The ash contents were taken in a crucible and 25 mL water was added to it and covered with a watch glass. The reaction mixture was boiled for 5 minutes and the water-insoluble contents were obtained on ashless filter paper, ignited at elevated temperature. The weight of water-soluble ash was calculated by subtracting the weight of water-insoluble ash from the weight of total ash. The following formula was used to calculate the percentage of water-soluble ash.

Estimation of water-soluble extractive
In a round-bottomed flask, 5 g of the finely powdered leaves were dissolved in water (100 mL) and shook on an electrical shaker for 6 hours. After 12-hour maceration the reaction mixture was filtered.
The filtrate was dried to calculate the weight of the water-soluble extract. The percentage of watersoluble extractive was determined by the following formula.

. Preparation of leaves extract
The Juglans regia leaves were initially ground to fine powder followed by hot continuous extraction in a Soxhlet extractor, with various known solvents (nonpolar to polar). Before extracting with the next solvent, leaves powder was dried at less than 50°C in a warm air oven. All the fractions were mixed, concentrated in a water bath, and stored in the refrigerator for qualitative analysis and synthesis of ferrites nanoparticles.

Characterization of phytochemicals
Plants are considered as biosynthetic laboratory having a variety of organic compounds which the medicinal value of that plant. The qualitative identification of these organic compounds in Juglans regia leaves extract was conducted according to the conventionally practised procedure.

Test of tannins
1 mL of extract was further diluted with 20 mL of water and boiled in a vial and then filtered. In the filtrate 0.1% FeCl 3 solution was added and the appearance of brownish-green colouration showed the presence of tannins.

Test of saponin
In 2 mL of the extract, 20 mL of water was mixed with water and boiled for 15 minutes under continuous stirring. The layer of foam formed, showed the presence of saponin.

Test of flavonoids
In a reaction vial, 2 mL of extract, NH 4 OH and concentrated H 2 SO 4 (5 mL each) were mixed and placed undisturbed. The appearance of yellow colouration authenticated the presence of flavonoids.

Test of steroids
To 2 mL of extract, H 2 SO 4 and acetic anhydride (2 mL each) were mixed in a test tube and reaction mixture. No change in colour from violet to bluish green showed the absence of steroids in the extract.

Test of terpenoids
The terpenoid content was determined by mixing 5 mL of the extract with chloroform (2 mL) and concentrated H 2 SO 4 (3 mL). The reaction mixture was continuously stirred but no reddish-brown colour is seen which showed the absence of terpenoids or terpenes.

Test of triterpenoids
In a test tube, acetic anhydride, the extract, chloroform (1 mL each) and concentrated H 2 SO 4 (2 mL) were thoroughly mixed. The solution did not turn reddish violet which showed the absence of triterpenoids in the extract.

Test of alkaloids
One mL of extract was mixed with a few drops of Mayer's reagent. The formation of white precipitates showed the presence of alkaloids.

Test of polyphenols
The mixture of extract (1 mL) and ethanol (4 mL) was initially boiled followed by the addition of 2-3 drops of ferric cyanide solution. The appearance of a bluish-green colour confirmed the presence of polyphenol.

Test of anthraquinones
In a reaction vial, of 5 mL extract, dilute H 2 SO 4 , benzene and dilute NH 4 OH(1 mL each) were mixed and the emergence of rose-pink colouration confirmed the presence of anthraquinones.

Test of glycosides
To the 1 mL of the extracts, 1 mL of conc. sulphuric acid was added and allowed to stand for 2 min. a reddish colour precipitate showed the presence of glycosides.

Test of coumarins
To 3 mL of extract, 2 mL of 10% NaOH was added. No yellow colouration confirmed the absence of coumarins. The removal of CIP from the water was conducted at different pH by using diverse UV lamps working at varying wavelengths, aimed to predict the wavelength at which maximum degradation of CIP could obtain. It was checked that 1 hour of reaction time was enough to degrade CIP to its maximum, but the reaction was performed for 70 minutes to confirm that no more observable CIP concentration was degraded in extra 10 minutes. It was seen that the increased degradation rate was acquired for CIP using pH = 3, at 254 nm than longer 365 nm wavelength. Same conditions were applied for all reactions with UV visible spectrometer. Rate constant was also calculated from kinetic facts at varying pH by using a kinetic equation which was employed for batch reactions. attributes of Juglans regia leave extract were expressed in terms of percentage including moisture content (2.03%); total ash content (4.98%); acid-insoluble ash (0.86%), water-soluble ash (6.2%); alcohol-soluble extractives (8.1%); and water-soluble extractives (9.54%).
These results of phytochemical analyses supported the water-soluble extractive method owing to its easy procedure and cost-effectiveness. The qualitative results of the phytochemical analysis revealed the presence of high concentrative components including triterpenoids (18.87%); tannins (18.28%); glycoside (16.98%); steroids (16.56%), and (14.36%). Whereas the saponins (5.88%) and alkaloids (9.07%) were present in low concentrations. The terpenoids, coumarins, triterpenoids and steroids were absent. The results signify the major contribution of Juglans regia leaves extract in the formation of Jr.NiFe 2 O 4 NPs.

UV-Visible Spectroscopy
The UV-visible graph of nickel ferrite samples S1 (Jr.NiFe 2 O 4 ) and S2 to S6 annealed at different temperature 600-1200°C are depicted in Fig. 1. Absorption spectra of small nanoparticles less than 20nm are in the range of 323-350nm wavelength usually with a single sharp band. As the size of nanoparticles increases redshift seen due to light scattering process. The shape, environment and composition of the prepared nanoparticles affect the scattering of light. The sample S6 annealed at 1200 degree showed two absorption bands at 372nm and 484nm because of larger size nanoparticles.
Higher dispersion was observed as the annealing temperature increased from 600-1200°C that is consistent with the size increasing upon calcination 24 . small band appeared at 2861 cm -1 is ascribed to the C-C bond or C-O bond due to organic impurities 25,26 . As annealing temperature has increased the impurities was burned out and the peak was  Two more peaks were seen at diffraction pattern of (104) and (024) which were indexed to β-Fe 2 O 3 .
The other peaks were expected because of the presence of an excess of oxygen during the annealing process. In detail, two peaks at 2θ 24.12 and 49.36 are referred to β-Fe2O3. The additional peaks revealed the development of a minor phase beside the major phase of nickel ferrite in sample S3 and S4 that was annealed at 800°C and 900°C respectively. The minor phase is considered as an intermediate phase that usually appears during the formation of nickel ferrite. But as the annealing temperature was raised to 1200°C the minor phase diffraction peaks disappeared as the nickel ferrite phase or major phase was fully developed. Crystallite size determined manually using Debye-Scherrer's formula(Eq. 10). The results are cross-checked by using area plot in Eva software.  The synthesis of nanoparticles was due to the interaction between capping biomolecules and nickel ferrite through an electrostatic bond or hydrogen bond.  Where "h" is the Planck's constant, "ν" is the frequency of the photon, "α" is the absorption coefficient, "Eg" is the energy of bandgap, "A" is the constant and "n" is the nature of the transition. (3) A plot of (hνF(R∞)) 1/2 verses hν is attained by using the Kubelka-Munk function. The curve of (hν-(hνF(R∞)) 1/2 ) on the horizontal axis hν and a vertical axis (hνF(R∞)) 1/2 is obtained. Here, eV is the unit of hν and associated with λ ashν= 1239.7/λ. The curve that is the value of (hν-(hνF(R∞)) 1/2 ) and the respective tangent, related to the procedures of steps (3) and (4), are depicted for each sample in Fig. 5a  The TG curve (Fig. 6) Fig. 7a,b.
Linear line with an incline of -k was obtained because of the plot of the natural log of final concentration verse time which showed that the reaction was first order shown in Fig. 8a  3.9. Removal of Congo red (CR) dye Evaluation of photocatalytic experiment for the removal of CR was conducted via UV-visible spectroscopy shown in Fig. 9,10. It was seen that the intensity of the absorption band of CR at 498 nm was decreased gradually as exposed to sunlight for 75 minutes. 85 % of removal rate was analysed for CR which showed remarkable photocatalytic activity of Jr.NiFe 2 O 4 NPs.

Fig. 9 Absorption Spectra of CR dye at different time intervals
The phenomenon of photocatalysis involved the transition of electrons from a ground state to excited state when light intensity is absorbed by the catalyst, leading to the production of valence band hole (h + VB ) and conduction band electron (e -CB ). Light-induced electrons (e -CB ) covert the molecular oxygen into superoxide radical (O2˙) and hydroxyl radicals are generated by reacting water molecules with a highly oxidizing hole (h + VB ). Valence band hole (h + VB ) has greater strength to draw electrons of CR dyestuff which lead to the degradation of contaminant. Therefore, it can be concluded that Hole (h + VB ) and radicals generated during the phenomenon are the main species to detoxify the organic dye 31 .

Fig. 10 Degradation rate of CR under visible light irradiation
Kinetic studies were also conducted for estimating the rate of reaction for photocatalytic activity.
Straight-line showed that degradation experiment followed pseudo-first-order kinetics as shown in            Kinetic plot for degradation of CR, a plot of -ln(A t /A o ) vs time

Fig. 11b
Kinetic plot of degradation of CR, a plot of A t /A o vs time