A systematic investigation on Elaeocarpus sylvestris leaf extract capped CuO nanoparticles as reducing agent and their antioxidant activity

Recently, the role of plant-based nanoparticles as sustainable catalysts has emerged as a new field in material science. As a result of their wide applicability, bio-designed nanocatalysts have dominated the present study. At this juncture, this study gives the rapid and systematic procedure for the formation of an aqueous Elaeocarpus sylvestris plant leaf extract–capped copper oxide nanoparticles (ESCuO NPs). A benign method has been established for the fabrication of ESCuO nanoparticles that which serves as an innocuous, renewable, and mild reducing agent. Vivid spectrochemical and optical experimental investigation supported the formation of the ESCuO particles. Based on TEM investigation, the produced ESCuO NPs had spherical shape and an average size of 53 nm. The phytochemicals were used as a reducing agent internally without the use of harmful chemicals or extremely high temperatures. The derived ESCuO NPs have been employed as a powerful catalyst to reduce 4-nitrophenol. Furthermore, rate constants for different doses were calculated. The antioxidant efficiency of synthesized ESCuO NPs was determined. This study illustrates the way for cutting-edge synthesis of CuO NPs with numerous applications.


Introduction
As a result of the industrial revolution and the quick development of technology, all types of industries often discharge massive amounts of dangerous chemicals into the air, water, and land, wreaking havoc on the ecosystem.Emerging contaminants are being released into water bodies as a result of the industrial revolution.These new toxins pose dangers to ecosystems and human health, necessitating creative treatment strategies.More specifically, 4-nitrophenol is a straightforward organic aromatic molecule that is widely employed in the production of numerous goods needed in current society, including colors, pesticides, and medications [1].However, toxic chemicals like 4-nitrophenol are not only harmful but also anthropogenic and hindering in nature, so its reduction is a crucial challenge.
Additionally, 4-nitrophenol and its by-products are utilized to create pesticides, insecticides, and herbicides that pose a harm to both the environment and people [2].
In this connection, the significant challenge of reduction of 4-nitrophenol has been performed with the application of nanotechnology with their specific characteristic properties recently [3].There is a plethora of effective research on the use of nanoparticles to reduce 4-nitrophenol.Metal nanoparticles have many uses in the biological, electrochemical, and medicinal domains because of their distinctive features, such as substantial surface area relative to volume ratios and significant surface energies related to their bulk molecules [4][5][6][7].There are various types of metal oxide nanoparticles, but copper oxide nanoparticles are of particular interest due to their potential for usage in both science and industry.Due to their biocidal qualities, CuO has drawn attention and may be used effectively in a variety of biomedical applications, including illness treatment, drug delivery, cellular delivery, and biomedical imaging [8].CuO NPs are employed as heterogeneous catalysts in the pharmaceutical industry as drug delivery, antioxidant, anticancer, and therapeutic agents [9].The extremely unique crystal shapes and large surface areas of copper oxide NPs make them extremely excellent antibacterial particles [10].
Like other nanoparticles, CuO NPs can be produced through chemical and physical methods; however, current research has begun to recognize green CuO nanoparticles due to the green revolution in all fields.To prevent hazardous chemicals and an abrasive environment when using chemical methods and a laborious procedure while using physical methods, a green protocol for the production of CuO NPs has been established.As a result, the biological technique for producing CuO NPs is receiving greater attention because it is simple to use, easy to handle, does not require cell culture, and is environmentally benign [11].Thus, using plant extracts for the synthesis of CuO NPs has already been reported for Gloriosa superba [12], Aloe barbadensis [13], Malva sylvestris [14], and Carica papaya [15].
In the current study, Elaeocarpus sylvestris plant leaf extract is used as a green source for producing copper oxide nanoparticles in a sustainable manner.The Elaeocarpus sylvestris plant has uttermost priority both in the spiritual purpose and scientific research.This plant belongs to the Elaeocarpaceae (Rudraksha) family.These are subtropical and longleaved evergreen shrubs and trees.The research on this plant reveals that phytochemicals like alkaloids, terpenoids, and flavonoids are present, as reported by Amit Dadhich [16].The antioxidant activity of this plant was reported by Amalia Indah Prihantini et al. [17].This shows its action towards depression, stress, pain in the nerve, anxiety, palpitation, migraine, asthma, hypertension, and alleviations related to the liver and arthritis traditionally [18][19][20][21][22]. CuO NPs have been synthesized employing plant leaf extract from Elaeocarpus sylvestris for the first time.The current study investigates the environmentally friendly synthesis of CuO NPs and characterizes them using various microscopic and spectroscopic methods and tested its reduction activity of 4-nitrophenol and antioxidant activities.

Reagents
The chemicals utilized in this research were of superior quality, conforming to analytical grade specifications.Cu(NO 3 ) 2 .3H 2 O, NaOH, 4-nitrophenol, NaBH 4 , ethanol, and DPPH (1,1-diphenyl-2-picrylhydrazyl) were obtained from Merck and employed without any refinement.The Elaeocarpus sylvestris tree leaves were piled up near the Lord Shiva temple, Bommuru, Rajahmundry.Double-distilled water was utilized throughout all synthetic procedures.

Green protocol description of ESCuO nanoparticles
The fabrication of nanoparticles takes place through a variety of methods and approaches.A facile green protocol through the coprecipitation method has substantially emerged recently.Natural (plant biomolecule) capping, stabilizing, and reducing agents are being employed to accomplish CuO NPs by avoiding perilous reagents [23,24].Irrespective of all plant particles, leaves are a good extract yielding part through the coprecipitation method.

Elaeocarpus sylvestris leaf extract preparation
The fabrication of ESCuO NPs was executed by using Elaeocarpus sylvestris leaf extract as a green source operator.Elaeocarpus sylvestris tree leaves were washed to remove the dust particles.To dry these leaves, they were kept in the shade for 15 days.Subsequently, a fine green powder of the dried leaves was obtained by using ultra household mixer.In order to create the 1% aqueous leaf extract, 100 ml of distilled water was combined with 1 g of finely powdered leaves.For 20-25 min, the aqueous mixture was heated to 60 °C while being constantly stirred.Then, to obtain a clear solution free of any particles, the brown aqueous leaf extract was filtered through Whatman No. 1 filter paper.The filtrate was then kept at 4 °C.

Elaeocarpus sylvestris-functionalized CuO nanoparticle synthesis
By using an environmentally friendly source, such as an aqueous solution of Elaeocarpus sylvestris leaf extract, the coprecipitation process was used to create Elaeocarpus sylvestris-functionalized CuO nanoparticles.A typical synthesis technique involved dissolving 1.87 g of Cu(NO 3 ) 2 .3H 2 O in 90 mL of distilled water.At room temperature, the solution is a clear blue color after being thoroughly mixed for 20 to 25 min.The reaction mixture volume was then increased to 100 mL by adding 10 mL of aqueous leaf extract, which caused it to turn dark green.Then, it was stirred for 15 min to get sonicated well with the mixture.Later, the temperature was raised to 80 °C and maintained for up to 1 h, and an intense green color was observed.Accordingly, 1 M 10 mL NaOH was added dropwise, and soon after addition, the mixture turned dark brown, which indicated the formation of Elaeocarpus sylvestris-functionalized CuO NPs as per literature [25].
The workup procedure included washing 2-3 times using distilled water and later two times with ethanol.The prepared CuO NPs precipitate was kept in the oven at 40 °C for drying overnight.

Elaeocarpus sylvestris-functionalized CuO nanoparticle instrumentation
The fabrication of nanoparticles in any way requires a firm footing through instrumental (optical, spectral, and microscopic) techniques.Likewise, ESCuO NPs were put forward through diversified characterization and analytical techniques.These helped us to estimate specific properties of synthesized material to be studied in an accurate scalable manner which is rapid and reliable to understand the obtained results.Here, a variety of instruments and analytical methods, including UV-visible spectroscopy as well as FTIR, X-RD, SEM, EDAX, and TEM-SAED instruments, are used to examine the bio-orchestrated CuO NPs.Shimadzu UV-2600 in the range of 200-800 nm was used for determination of absorbance of Elaeocarpus sylvestris leaf extract and synthesized ESCuO NPs.The FTIR spectra of ESCuO NPs were recorded using Bruker FTIR Alpha spectrometer in the range of 400-4000 cm −1 , and standard KBr pellet method is employed.PANalytical X pert pro diffractometer at 0.02°/s scan rate using Cu-k1 radiation ( λ = 1.54061) was used to obtain the powder X-ray diffraction (XRD) patterns.XRD was used to determine the crystallinity of the synthesized ESCuO NPs.FESEM (FE-SEM model JEOL 6390LA/ OXFORD XMX N) at an accelerating voltage of 0.5 to 30 kV with magnification × 300,000 equipped with energy-dispersive X-ray spectroscopy (EDAX) of resolution 136 eV detector area 30 mm 2 is used to investigate the morphology and elemental composition of ESCuO NPs.HRTEM (HR-TEM model JEOL/JEM 2100) at an accelerating voltage of 200 kV with point resolution 0.23 nm and lattice resolution 0.14 nm equipped with selected area electron diffraction patterns (SAED) is used to investigate the size and shape of the synthesised ESCuO NPs.The HRTEM images of various resolutions of the nanoparticles are collected.

Reduction of 4-nitrophenol by ESCuo NPs
Here, normally used procedure has been employed as per literature [26].Commonly, 25 mL of an aqueous solution of 4-nitrophenol was taken in a RB flask for 10 min while stirring to ensure good mixing.Ten milligrams of synthesized NPs made from copper oxide were then added to the reactant at 2.5 mM.Then, to decrease 4-nitrophenol at room temperature, 0.25 M NaBH 4 that had been dissolved in a 25-mL aqueous solution was mixed into the reaction solution.The mixture was a deep yellow color, and it was allowed to mix thoroughly through stirring until it became colorless.The colorless reaction mixture indicated the complete reduction of 4-nitrophenol.Frequently obtained samples of the reaction mixture, then a UV-visible spectrophotometer was utilized to check if the reaction had finished.Additionally, the outcomes were analyzed with and without NaBH 4 .

Antioxidant potentiality
The "DPPH (1,1-diphenyl-2-picrylhydrazyl) scavenging test" is a technique used to investigate the synergistic effect of antioxidant molecules on capped CuO NPs as well as their effectiveness as antioxidants.The ESCuO NPs have been employed for conducting the experiment.It was calculated using the DPPH free radical scavenging potential method and measured according to the procedure described by Dobrucka [27].An aliquot of 3 mL of 0.004% DPPH solution in ethanol and 0.1 mL of ESCuO NPs at various concentrations (100, 200, 300, 400, 500 µg/mL) were mixed, which were shielded from light.The DDPH reagent was made 24 h before and kept out of the light.Each mixture was left for 30 min; using the Shimadzu UV-2600 spectrometer, changes in absorbance intensity were measured at 517 nm.After which DPPH scavenging activity was calculated from using the equation given here, the findings were estimated as an amount of scavenging of a control blank with the absorption value of the investigated materials calculated at 517 nm.

Results and discussion
The hydroxyl and carboxyl groups found in Elaeocarpus sylvestris leaves are the best green sources to act as reducing, stabilizing, and capped operators for CuO NPs, according to the remarkable results achieved by several instrumental approaches.The biosynthesized ESCuO NPs are apt for further catalytic applications as their metabolites constitute amino, hydroxyl, and carboxyl functional groups.

UV-visible spectroscopy
Figure 1 depicts the comparative UV-Vis spectrum of both an aqueous solution of Elaeocarpus sylvestris leaf extract and the synthesized ESCuO NPs.In Fig. 1a, the absorption of the specified signal at 232 nm was observed for the aqueous leaf extract of Elaeocarpus sylvestris.Whereas the absorbance at 232 nm is related to the benzoyl ring system of π-π* or n-π* transitions, these are demonstrating that polyphenols (1)

Radical Scavenging activity(%) =
A control blank − A sample × 100 are some of the constituent phytochemicals in the Elaeocarpus sylvestris leaf extract [28].Figure 1b gives the UV-visible spectrum of ESCuO NPs.Now, the drawn spectrum revealed the absorption peaks at 225 and 262 nm.The peak at 225 nm is attributed to the plant constituents [29], and the peak at 262 indicates the formation of ESCuO nanoparticles [28,30].

FT-IR
The important constituents responsible for the capping, stabilizing, and reducing of synthesized ESCuO nanoparticles are corroborated through FTIR.In this context, the functional group particulars of the plant and of synthesized ESCuO nanoparticles were investigated and depicted in Fig. 2. The FTIR spectrum of the employed biosource is shown in Fig. 2(a).The obtained spectra expressed the key functional groups through its characteristic ranges corresponding to the values of 3323.90, 2107.53,1634.43, and 531.44 cm −1 , respectively.The spectrum shows value at 3323.9 cm −1 confirmed the -OH bond stretching vibration robustly.However, plant literature reveals that there is no compound with N-H functional group.Actually, the employed biosource contains ellagic acid, gallic acid, methyl gallate as major constituents with -OH and -COOH groups [31].Given to the bending vibration of group -OH explicitly is 1634.43cm −1 .Another intense peak at 2106.33 cm −1 is ascertained to C = O in biomolecules.The single intensity peak below 900 is 531.55 cm −1 and is due to the bending vibrations of C-C aromatic ring.Moreover, the FTIR analysis of green-synthesized ESCuO NPs in Fig. 2b exhibited a shift in the transmittance intensity values compared to the spectra of biosource employed.Besides, it confirms the surface functionalization of bio-source of Elaeocarpus sylvestris on the green-synthesized CuO NPs.The FTIR image depicts the peak values at 3795.67, 2801.95,2661.75, 990, and 583 cm −1 , respectively.One of the prominent peaks, called OH stretching, shifted purposefully from 3323.9 cm −1 to 3795.67 cm −1 , indicating that the phenolic -OH of the biosource is tightly bound to the CuO NPs.The stretching frequencies of the C-C and aromatic groups were assigned values of 2801.95 and 2661.75 cm −1 , respectively, while the C-O bond of the plant's components was given a value of 990 cm −1 .It is interesting to note that the Cu-O bond's stretching vibrations are represented by the other band at 583 cm −1 .Therefore, the above results confirmed the typical role of biocomponents of plant such as substituting carboxylic acids and reducing sugars, polyphenols, and proteins which are acted as best in functionalization and stabilization of ESCuO NPs [25,26].
the defined spherical morphology of the orchestrated CuO NPs.The observed pictures showed that the ESCuO NPs had nearly spherical morphologies and were agglomerated and monodispersed.The chemical composition of the Elaeocarpus sylvestris extract is surface linked to this variance in particle shape and size distribution [34,35].In addition to that, the high surface energy of ESCuO nanoparticles caused little aggregation.This character developed because the water medium served as the source of the synthesis process  [36][37][38].Small nuclear particles actively self-aggregate and oriented in a way to form larger spheres explicitly.In addition to this, the SEM micrographs were like shattered pieces (agglomerated particles) on a rough surface.Figure 4e displays the chemical composition of the produced NPs being examined by the EDX method.Only two powerful signals, Cu and O, contribute significantly to the spectrum, with weight percentages of 69.43 and 27.53, respectively.Additionally, the presence of significant concentrations of Cu and O with sharp peaks points to the stability and quality of the synthesized ESCuO NPs.Sodium was another signal that was seen and had a very low intensity (3%).Interestingly, there is an unlabeled peak with less intensity which is ascribed to carbon (0.01%) according to Sone et al. [39].
According to him, the carbon film used to support the CuO samples as well as leaf-based organic compounds that emerged from the aqueous extract could be ascribed for the presence of C found in the powder.Figure 4b depicts the needle-shaped and layered flakes that were visible on the surface.The findings showed that the ESCuO NPs were functionalized by the Elaeocarpus sylvestris leaf extract biomolecules in a significant way.

TEM
Figure 5a-e depicts the micrographs of the synthesized ESCuO NPs.The spherical morphological distribution of orchestrated ESCuO NPs with sizes in various ranges between 9-25 nm is explicitly depicted in Fig. 5.The TEM micrographs illustrate that the particles are scattered, and some appear to be well coated with the leaf extract of Elaeocarpus sylvestris.The different magnifications of TEM images declare that very small particles below 25 nm are observed [36,40].The mean particle size was characterized by "Image J software (National Institute of Health, Bethesda, MD, USA)," and it was 53.99 nm.Correspondingly, the obtained value is in accordance with the crystallite size attained by the Debye-Scherrer equation using XRD.The bright diffraction rings are observed in the "selected area electron diffraction (SAED)" phenomenon of orchestrated ESCuO NPs in Fig. 5f.The areas with white and black dots revealed the hydrophilic and hydrophobic characteristics of CuO nanoparticles, respectively.The well-organized and arranged location of the lattice planes in CuO nanoparticles has been demonstrated by the fringes that appeared [36,41].The polycrystalline nature of the CuO nanoparticles has been found to be consistent with the appearance of white spots and rings in dark nature [36,42].This showed that the CuO NPs are polycrystalline in nature.It is further confirmed that the particles are finely crystallized.The SAED image's diffused ring dot pattern fits the XRD pattern, demonstrating the monoclinic structure of the synthesized ESCuO nanoparticles.

Reduction of 4-nitrophenol using ESCuO NPs
4-Nitrophenol can be reduced by different metal, metal oxide, and bimetallic nanoparticles like Au, Ag, Pt, Pd, and Au-Ag.Eventually, all the aforementioned metals and metal oxides are cost-effective, and some are harmful, but the literature says green-synthesized CuO nanoparticles are also used as reducing agents in organic reactions [43].Nevertheless, CuO NPs are eco-friendly, bioactive, and commercially viable.Here, ESCuO NPs act as blistering agents In the presence of NaBH 4 as a reducing agent, the performance of the as-synthesized ESCuO NPs as a catalyst was evaluated against 4-nitrophenol reduction [14].The conversion of 4-nitrophenol into 4-aminophenol is depicted in Fig. 6.In the absence of NaBH 4 , UV-vis absorbance of p-nitrophenol showed a peak at about 313 nm.However, when NaBH 4 was added to the p-NP solution, the peak at 313 nm vanished, and a new, strong peak at about 400 nm emerged.During the reduction process, a weakly yellowishorange p-nitrophenolate solution formed from 4-nitrophenol transforms into a strong yellowish-orange p-nitrophenolate ion in alkaline conditions [26].The UV-Vis absorbance of the p-nitrophenol and NaBH 4 mixture without a catalyst was the source of the initial, strong spectrum.Following the addition of the catalyst, the peak at 400 nm began to progressively fade while a new peak at about 313 nm began to emerge, signifying the transformation of the  p-nitrophenolate ion into the p-aminophenol, as shown in Fig. 6b.Based on the appearance of the absorbance peak at about 400 nm in the presence of the ESCuO NPs as a catalyst, the amount of time needed to convert 4-nitrophenol completely was calculated [30].When the 10 mg ESCuO NPs as a catalyst was added, the entire conversion of 4-nitrophenol into aminophenol took place in 14 min (Fig. 6c), whereas there was no change when the catalyst was not present.The rate constant (k) of reduction of 4-nitrophenol using different doses of ESCuO NPs was determined (Fig. 6d) (Table 1).The kinetic analysis results showed that the data best fit with pseudo-first-order.The rate constant of the reaction obtained from the slope of a plot Fig. 6d depicts that the rate of catalytic reduction of 4-nitrophenol is the significant for the ESCuO catalyst synthesized using Elaeocarpus sylvestris leaf extract based on the literature.The reason for the higher rate constants when there is a greater concentration of ESCuO nanoparticles is because there is a corresponding increase in the quantity of reactive sites available for the reduction of 4-nitrophenol [26].Figure 6c exhibits 4-nitrophenol reduction by ESCuO NPs and NaBH 4 .Figure 6d depicts the kinetics of reduction followed.

Antioxidant activity
Free radicals are created in biological systems when biomolecules interact with molecular oxygen [44].Numerous studies have looked at the antioxidant activity of many different types of natural and manmade substances [45].The DPPH scavenging assay is thought to be the most used technique for examining a material's antioxidant capacity.The antioxidant activity of ESCuO NPs was investigated as its capacity to reduce the stable nitrogen radical DPPH, which causes gradual decrease in absorbance at 517 nm, with increase in concentration of ESCuO NPs.This further confirms the free radical scavenging activity of ESCuO NPs.Hence, ESCuO NPs were used as radical scavenger and DPPH was used as the radical source.The color of the DPPH solution changed from deep violet to pale yellow in the presence of ESCuO NPs.The antioxidant efficiency CuO nanoparticles and green-synthesized CuO nanoparticles are compared by Naz et al. [46] which confirms that there is definite enhancement in its efficiency due to the capping of plant biomolecules [47].Here, various concentrations of samples were prepared (100, 200, 300, 400, and 500 µg/mL) and placed these samples in a dark condition for half an hour after the addition of DPPH free radical.Then, after, samples were analyzed using spectrophotometer, and percentage of efficiency values shown in the table were calculated using the formula (Eq.1).
These results showed that the antioxidant efficiency of ESCuO NPs is dose-dependent (Table 2).All values represented in the tables are the average ± SD of the results of two separately conducted experiments.Figure 7 shows the free radical scavenging activity of ESCuO NPs, which exhibits up to 61.21 ± 0.03% in 500 µg/mL concentration of ESCuO NPs.

Fig. 1 Fig. 2 FTIR
Fig. 1 UV-visible spectrum of a ES leaf extract and b ESCuO NPs

Fig. 4 a
Fig. 4 a-d SEM pictures of ESCuO NPs; e EDX spectra of ESCuO NPs

Fig. 5 a
Fig. 5 a-e TEM images of ESCuO NPs; f SAED of ESCuO NPs

Fig. 6 a
Fig. 6 a A schematic representation of conversion of para-nitrophenol to para-aminophenol by ESCuO NPs and NaBH 4 .b 4-Nitrophenol reduction with ES leaf extract and NaBH 4 without ESCuO NPs.c 4-Nitrophenol reduction by ESCuO NPs and NaBH 4 .d Kinetics of reduction

Table 1
The kinetic parameters determined during the conversion of para-nitrophenol to para-aminophenol by ESCuO NPs

Table 2
The antioxidant activity of ESCuO NPs Free radical scavenging activity of ESCuO NPsConclusionFinally, a novel, simple, and green method is established in a promising and sustainable way for the production of CuO nanoparticles.To escape from regular chemical methods and their toxicity, an environmentally friendly substitute is the need of the hour.This green protocol with Elaeocarpus sylvestris leaf extract became successful in this way.The fabricated particles are corroborated with respective instrumental analysis.The findings revealed that the formed particles have a spherical shape with a 53-nm size and a face-centered cubic structure.SEM pictures have defined spherical morphology of the orchestrated ESCuO NPs.The EDAX spectrum showed the elemental percentage of Cu and O are 69.43 and 27.53, respectively.ESCuO NPs with sizes in various ranges between 9 and 25 nm is explicitly depicted by TEM instrument.In general, CuO nanoparticles are cheaper than other metal oxides.The ESCuO NPs shown efficient catalytic activity in the conversion of para nitrophenol to para aminophenol.The rate constant (k) of reduction of 4-nitrophenol using different doses of ESCuO NPs was determined, and for 5 mg and 10 mg of ESCuO NPs, rate constants are 0.1477 and 0.1596 and reduction takes place within 24 and 14 min, respectively.Therefore, the ESCuO NPs show efficient catalytic activity in the conversion of para-nitrophenol to para-aminophenol.Furthermore, it proves ESCuO NPs have promising antioxidant potentiality and show the free radical scavenging activity exhibits up to 61.21 ± 0.03% in 500 µg/mL concentration of ESCuO NPs.Therefore, ESCuO NPs act as a good catalyst for elimination of organic pollutants.In future perspective, the study gives the new pathway in a variety of applications, including biomedicine and catalysis.