One-pot green synthesis of iron oxide nanoparticles from Bauhinia tomentosa: Characterization and application towards synthesis of 1, 3 diolein

The green synthesis of NPs through plant extracts can be a modest, one-pot alternative synthesis to the conventional physical or chemical method. The prime focus of this study is to produce MNPs by the reducing effect of Bauhinia tomentosa leaf extract, and it was immobilized in porcine pancreatic lipase (PPL). Synthesized NPs were characterized by field emission scanning electron microscopy (FESEM), X-ray diffraction (XRD) and Raman spectroscopy, UV–Vis Spectrometry, Thermogravimetry, and Differential Scanning Calorimeter (DSC), Zeta potential test, VSM, BET and Fourier Transform Infrared Spectroscopy (FTIR). The effect of process parameters was studied, about the efficiency of immobilization are enzyme stability, the extent of enzyme reusability, its separation from products, the activity of immobilized enzyme, recovery, and its loss. Finally, the immobilized lipase was used for the synthesis of 1,3-diolein using enzyme-mediated esterification of oleic acid and glycerol. Under optimized condition (reaction temp-55 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^\circ $$\end{document}∘C; molar ratio-2.5:1; pH-7) diolein yield was achieved to be 94%. Therefore, this work was further used for the industrial production of 1,3-diacylglycerol since a perfect enzyme-catalyzed process was observed.

. The degradation of textile waste by catalytic activity was effective using NPs formed by a reduction reaction and also exhibited antibacterial activity 16 . Lakshmi Pravallika et al., 2019 synthesized iron oxide nanoparticles using ethanolic extract of Centella asiatica (CAIONPs) by reducing ferrous and ferric chlorides which were administered to Swiss albino mice with a dosage of 2000 mg/kg body weight. Nil effects of the NPs on various tissues were revealed by histopathological studies, indicating that green synthesized NPs were safe for use in biomedical and drug delivery systems 17 . In a similar study by Izadiyan et al., 2018, iron oxide nanoparticles were synthesized using Juglans regia green husk extract by co-precipitation method of FeCl 3 and FeCl 2 and the cytotoxicity tests were performed on mouse embryonic fibroblast cell lines and human colorectal adenocarcinoma cell lines by MTT assay which had no toxic effect on both normal and cancerous cell lines 12 . Khatami et al., 2019 synthesized super-paramagnetic iron oxide nanoparticles (SPIONs) produced using a zero-calorie stevia extract which acts as both reducing and stabilizing agents. The antioxidant effect studied by DPPH assay indicated the activity of produced NPs in the acceptable range 18 . Table 1 reports the comparative studies of the synthesis of iron oxide nanoparticles from various sources reported in the literature with the present work. Bauhinia tomentosa is a legume species in the Fabaceae family, rich in phytochemicals such as flavonoids, quinones, tannins, etc. act as stabilizing and reducing agents in NPs production. It plays a significant role in the formation, capping, and stabilization of Iron (II) oxide nanoparticles due to the presence of phytochemical and bioactive compounds. The process was demanding due to the presence of polyphenols and antioxidants which shield the NPs from oxidation and aggregation 19 .
In the case of 1,3 diolein, the enzymatic approach was employed due to environmental pleasantness, safety, and mild reaction condition with improved yield. Conventionally, diacylglycerol was used to reduce the accumulation of body fat. The green synthesis of nanoparticles for enzyme immobilization has benefits to instigate the enhancement of the greater surface area, lower diffusion limitation, particle mobility, thermal stability, storage capacity, modulation of catalytic activity, cost-effective, low toxicity, effective preparation, and availability, and high productivity in terms of binding efficiency with enzymes. In the present work, to maximize the diolein yield and to improve the operational stability of the enzyme, a new synthesis was employed 20,21 . This work emphasizes on green route for the synthesis of Fe 2 O 3 (Iron (III) oxide) nanoparticles produced from Bauhinia tomentosa leaf extract and to synthesize 1,3 diolein using immobilized PPL.

Materials and methods
Materials. The porcine pancreatic lipase (PPL) 5 (Type II, 100-500 U/mg protein using olive oil) was purchased from Sigma Aldrich Co. India. For enzyme activity analysis via the olive oil emulsion method, chemicals were obtained from Hi-Media Laboratories: dipotassium hydrogen Phosphate and Potassium dihydrogen phosphate (preparation of pH 7 buffer), gum arabic, pure olive oil, and Sodium hydroxide. Chromatographically pure monoolein and oleic acid were purchased from Sigma-Aldrich (Shanghai-china). Bradford reagent was prepared using Coomassie brilliant blue, ethanol, phosphoric acid (85% pure), and glycerol. Biosynthetic Iron (II) oxide nanoparticles were used as a support for immobilization which was prepared using leaf extract and Diolein synthesis. The enzymatic esterification of oleic acid and glycerol was done with the support of immobilized PPL. The reaction was carried out in a 50 mL flask on a rotary shaker at 200 rpm 25 . To make up the reaction mixture, 1.5 mmol of oleic acid, 0.5 mmol glycerol, 10 mL of t-butanol, and an appropriate amount of immobilized lipase was added (15% (wt%) of the substrate). 4 Å molecular sieves were added into the reaction mixture to remove the water content. 50 µL of the sample was taken out from the reaction mixture and centrifuged to obtain the supernatant and analyzed by HPLC 26 . All the experiments were done in triplicates. Overall process layout for synthesis of iron oxide nanoparticles from Bauhinia tomentosa and 1, 3 diolein production was presented in the Fig. 1.

Results and discussion
Iron (II) oxide nanoparticles were synthesized using Bauhinia tomentosa leaf extract. Transformation in color was observed from an orangish-brown solution to a black precipitate. The nanoparticles were washed with water and acetone thrice and dried at 90 ºC in a hot air oven to achieve black-colored purified nanoparticles. Ferric Chloride solution of 0.01 M concentration gets reduced to Ferric oxide and gets precipitated in the leaf extract. This reaction materializes in the company of oxidizing agents like Vitamin E 28 . Phytochemicals such as flavonoids, quinines, tannins, etc. act as stabilizing agents in nanoparticle production in the presence of a polar solvent, water. Phenols and terpenoids may play a significant role in the formation, capping, and stabilization of Iron (II) oxide nanoparticles 29 . Also, due to Surface Plasmon Resonance, a color change was observed. For measuring adsorption of material onto planar metal or the surface of metal NPs many standard tools are formed based on SPR 30 .

Characterization of Fe 2 O 3 nanoparticles. UV-vis spectrometry. UV-Vis Spectrometry has revealed
the characteristic formation of nanoparticles during color change based on the absorption spectra. A scanning wavelength measurement from 300 to 900 nm was executed to reveal a peak value at 328 nm which indicated the formation of nanoparticles (Fig. 2). A characteristic peak at 328 nm confirmed the formation of Fe 2 O 3 Nanoparticles 31 .
Fourier transform infrared spectroscopy. FTIR is ascribed to functional groups (=C-H, C=O, N-O, C-O, C-N) present in the compound (Fig. 3). FTIR spectroscopic studies confirm the presence of amides, phenols, nitrogen, and aromatic compounds that has a strong binding affinity with Fe and thus play a significant role in reducing and capping ferrous ions 32  Thermogravimetry and differential scanning calorimeter. Mass changes of a sample as a function of temperature in scanning mode are examined by TGA (dynamic TGA) (Fig. 4). The physical and chemical properties of materials, as a function of increasing temperature, can be determined. This decomposition/degradation temperature bear witness to mass changes in the materials. The approximate temperature of Fe 2 O 3 s transition of interest was found to be around 930 • C. Characterization of coatings on NPs by evolved gas analysis can be achieved using TG-DSC techniques. DSC was grounded on the differences in the amount of heat required to increase the temperature of the sample. In combination with TGA, it was applied to study melting point, gas transitions, and exothermic decompositions. The graph depicts that the decomposition melting of the sample starts at around 250 • C and ends at about 700 • C revealing that the sample was Iron (II) oxide 34 . At a temperature of around 180 °C, the TGA curve showed a weight loss of around 3.0446 percent in the study. This weight loss may be attributed to the removal of water molecules removed by nanoparticles from the atmosphere, during which the sample weight is almost stable, indicating the sample's thermal stability.
Zeta potential and field emission-scanning electron microscopy. Size is an important factor to define NPs although considerable debate exists on the size threshold to distinguish NPs from bulk materials. The particles were dispersed in water with a dielectric constant of 78.5, a refractive index of 1.33, and a viscosity of 0.887 cP 35 . A potential of −16 mV was found which was a good manifestation for nanoparticle formation. The potential difference between the EDL (electric double layer) of electrophoretically mobile particles and the layer of dispersant around them at the slipping plane is reflected by the zeta potential (Fig. 5A). It is also termed electrokinetic potential, the potential at the slipping/shear plane of a colloid particle moving under the electric field. Therefore, the particle size distribution and magnitude of electric charge at the particle surface are determined 36 . Also, a zeta sizer was employed to determine the size of the particles. The size distribution was scanned by intensity (Fig. 5B). However, due to differences in dispersion co-efficient and cluster formation, it did not provide accurate results. The FE-SEM image revealed the size of the synthesized nanoparticles (Fig. 6). Thus, eminently meticulous results were provided by FE-SEM. The average size was observed to be around 70 nm which is acceptable.
X-ray diffraction. X-Ray Diffraction (XRD) was performed to understand the crystalline structure of the nanoparticles. The sample consisting of fine grains of crystalline material to be studied was usually in powdered Brunauer-Emmett-Teller (BET) surface area analysis. N 2 adsorption/desorption isotherms at liquid nitrogen temperature were used to determine the precise surface area (Brunauer-Emmett-Teller, BET) pore size and pore volume of the samples. Figure 8 displays the outcomes of the BET analysis 38 . The synthesized iron oxide nanoparticles display TYPE IV adsorption-desorption isotherm. The prepared nanoparticles showed Brunauer-Emmett-Teller (BET) surface area, pore-volume, and diameter were calculated to be 48.8 m 2 /g with 0.096 cm 3 /g and 7.9 nm respectively. From the adsorption-desorption isotherm, it can be noticed that around 62.04 cm 3 /g of nitrogen was adsorbed at maximum relative pressure (P/P 0 ) of 1 39 . The hysteresis pattern shows that the condensation occurred approximately from 0.4 to 0.9 (P/P 0 ) (Fig. 8). These findings suggest that these particles have a large surface area and are nanometer in size. In contrast to the other samples, the iron oxide Np sample had the highest surface area and had a very small particle size along with a strong adsorption property, according to the BET report 40 .
Vibrating sample magnetometer (VSM) analysis. A vibrating sample magnetometer was used to test the magnetic properties of the iron oxide nanoparticles, at room temperature, the hysteresis loops of the bare Fe 3 O 4 and iron coated NPs are shown in Fig. 9 41 . As the magnetic field is withdrawn from both prepared NPs, the magnetization decreases from a plateau state to zero. This action clearly shows superparamagnetic behavior 42    Effect of various reaction parameters. Finding the effect of various parameters that affect the diolein yield based on reaction time, temperature, substrate molar ratio, and reusability of the immobilized enzyme has been pivoted in this study (Fig. 10). An indispensable role is played by the reaction temperature in biocatalysts. Higher temperature results in the deactivation of the enzyme. This work entails five different temperatures (40, 45, 50, 55, 60, and 65 °C) and was ascertained to observe the diolein yield. At 55 • C, diolein yield reaches the highest value of 92.5%. More than that range, the yield and initial reaction rate of diolein get decreased and simultaneously acyl migration will take place which results in triolein formation and diolein yield reaches optimum value after 7 h of reaction time.
To investigate the optimum level of the substrate molar ratio based on the yield of 1,3 diolein, different ranges were taken to experiment (2:1, 2.5:1, 3:1, 3.5:1, and 4:1). The diolein yield will not be tremendously affected by an escalation in the molar ratio of oleic acid to glycerol. But higher concentrations of oleic acid will simultaneously diminish the yield of 1,3 diolein formation. Therefore, based on molar ratios, no significant difference was observed in the diolein yield. And from this work, it was observed that the substrate molar ratio of oleic acid to glycerol (2.5:1) shows the highest yield of diolein as 94%. Cost efficiency is imperatively influenced by the reusability of the immobilized enzyme 44 . The operational stability of immobilized lipase was carried out under optimized conditions. From the results, it was observed that 90% of the original activity was maintained until 10 cycles and in this case, a maximum yield of 1,3 diolein was achieved 45 . Therefore, the catalytic activity of the enzyme was not lost, and also it was proved how effectively the enzyme binds to the matrix. From the above results, it was clearly shown that Fe 2 O 3 nanoparticles were an eminent matrix for lipase (PPL) immobilization. Therefore, the immobilization of enzymes on a solid support such as nanoparticles is more advantageous due to improved stability, enhanced thermal efficiency and pH, increased enzyme loading, and reusability with simple handling and separation making the process feasible with maximal yield. Table 2 represents the detailed comparison studies reported in the literature for the synthesis of 1,3 diolein using lipase catalysis with the present work. It was found that a higher yield of 1,3 diolein was obtained with the lipase immobilization on the iron  www.nature.com/scientificreports/ oxide nanoparticles and also the immobilized enzyme eases the process of recovery and reuse. This reduces the overall production cost of the 1,3 diolein synthesis.

Conclusion
This work highlighted the green synthesis of Fe 2 O 3 nanoparticles from Bauhinia tomentosa leaf extract and it was efficaciously implemented for lipase immobilization. Moreover, it was the pragmatic approach for enhancing the synthesis of 1,3-diolein by the esterification of oleic acid and glycerol. The phenolic compounds present in Bauhinia leaf extract play a vital role in boosting up the stability of Fe 2 O 3 nanoparticles. The distinct characteristics, size, and shape of Fe 2 O 3 nanoparticles were identified using FTIR and SEM analysis. XRD, TGA, and UV-Vis spectroscopic techniques were used to recognize the crystallographic structure, thermal stability, and optical behavior of the green synthesized nanoparticles were studied. Further, due to the high stability, effectiveness, enzyme activity, greater safety, low energy consumption, and high product quality of the immobilized lipase, it was employed for 1,3-diolein synthesis which will gain momentum for various applications. Finally, this greener optimistic work will aid in the large-scale synthesis of 1,3 diolein using the effective binding of immobilized lipase.

Data availability
The datasets used during the current study are available from the corresponding author on reasonable request.