Low-cost, simple and eco-friendly biosynthesis of CeO 2 -NPs using extract of Pelargonium hortorum for the photocatalytic and antioxidant applications

In this paper, firstly the phytochemical composition in the aqueous extract of Pelargonium , growing wildly in the center of Iran, was studied. Folin–Ciocalteu and Aluminum chloride methods and the GC/MS technique were used to determine the total phenolic, flavonoid contents, and volatile constituents in the extract, respectively. The amounts of the total phenolic and flavonoid contents in the extract were found to be 136.5 mg GA/g and 63.9 mg RU/g, respectively. Twenty-one compounds, representing 15 (about 80.0%) of the total volatile constituents were identified. The main components of the volatile constituent were α-pinene (25.28%) and fenchyl acetate (20.63%). Secondly, a fast, simple, and eco-friendly biosynthesis of cerium oxide nanoparticles (CeO 2 -NPs) were investigated using the extract of Pelargonium as a natural reducing and stabilizing agent. Several advance techniques such as Ultraviolet-Visible (UV–Vis) spectroscopy, X-ray diffraction (XRD), Scanning electron microcopy (FESEM), Dynamic light scattering (DLS), and Brunaure Emmett-Teller (BET) were used for the analysis and characterization of the synthesized nanoparticles. The results of the characterization analysis showed that the prepared nanoparticles were spherical in shape, with an average size of 28 nm and zeta potential of -25.8 mV, and had 33.84 m 2 /g BET surface area, 9.31 nm mane diameter pore, and a total pore volume of 0.078 cm 3 /g. Finally, the practical applications including the antioxidant and photocatalyst activity of the biosynthesized CeO 2 NPs were evaluated. An analysis of antioxidant and catalytic activities showed that the biosynthesized CeO 2 -NPs have a good ability of scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical with the IC 50 value of 42.7 μg/mL and also good reduction ability of hexavalent chromium {Cr(VI)} ions with a reduction efficiency of


Introduction
Cerium oxide nanoparticle (CeO2-NPs) is one of the important metal oxides that could be used in optical, medical, and catalytical applications for its unique physicochemical properties including biocompatibility, high stability, and special surface chemistry [1][2][3]. CeO2-NPs are usually synthesized via chemical methods, which mostly employ toxic reducing agents, posing various threats to the environment. In addition, the CeO2 prepared with these methods is toxic and unstable, reducing its efficiency [4,5]. Accordingly, in recent decades, a safe, non-toxic, eco-friendly, and low cost approach, known as biological (green) synthesis, is used by researchers for production of metal oxide nanoparticales, including CeO2 [6][7][8]. This approach employs various biological resources such as plants, microbes, or any other biological derivative (primary and secondary metabolites) believed to lead to reduction and stabilization.
Studies show that these compounds are polyphenols, flavonoids, tannic acid, terpenoids, carbohydrates, amines, enzymes, etc., which function as natural antioxidants [1,9]. In general, the high antioxidant activity of the plant or microorganism results in an extract or any other biological derivative with high reducing capacity, which in turn increases the efficiency of the synthesized nanoparticle [9].
Numerous published studies are available on biological synthesis of CeO2-NPs using plant extracts or other biological derivatives for multipurpose applications. In these studies, green synthesized CeO2-NPs have been mostly examined for biomedical purposes such as antimicrobial, anticancer, anti-larvicidal, and antioxidant therapies and photocatalytic degradation of organic compound applications [2][3][4][5][6][7][8][9][10][11]. However, there are few studies dealing with an antioxidant and the photocatalytic role of CeO2-NPs synthesized by the green method on heavy metal ions of wastewater treatment. For example, Xiao et al. reported an excellent photocatalytic activity for CeO2/BiOIO3 heterojunction with oxygen vacancies and Ce 4+ /Ce 3+ redox and its application in removal of the Hg 2+ ion [12]. Balakumar et al. (2019) used an ultrasound-assisted method to improve the structure of CeO2@polyprrole core-shell nanosphere and its photocatalytic reduction of Cr(VI) [13]. Kashinath et al reported microwave mediated synthesis and characterization of CeO2-GO hybrid composite for removal of chromium ions and their antibacterial efficiency [14]. Saravanakumar et al. reported construction of novel Pd/CeO2/g-C3N4 nanocomposites as efficient and visible-light photocatalysts for hexavalent chromium detoxification [15]. In another study, multi-functional properties of ternary CeO2/SnO2/rGO nanocomposites, including visible light driven photocatalyst and heavy metal (Cd 2+ and Pb 2+ ) removal were reported by Priyadharsanet al. [16].
The aim of this research is biological synthesis of CeO2-NPs using aqueous extracts of Pelargonium hortorum, a growing plant in Iran, as a natural reducing and stabilizing agent.
Pelargonium belongs to the Geraniaceae family with about 300 species, mostly known as an ornamental plant; however, it is also used in the manufacture of herbal medicines for its unique medicinal properties [17]. The literature includes studies using several flavonoids in extracts extracted from geranium, including quercetin 3-O-glucoside, quercetin 3-O-pentose,   isorhamnetin aglycone, kaempferol 3-O-, kaempferol 3-O-glucoside, kaempferol 3,7-di-Oglucoside, rhamnoside-glucoside, quercetin 3-O-pentoside-glucoside, myrisetin 3-O-glucoside-rhamnoside, and quercetin 3-O-rhamnoside-glucoside [17,18]. These compounds (polyphenolic compounds including flavonoids) act as natural reducing agents in extracts and it is predicted that these extracts could act as a potent green and non-toxic reluctant in synthesis of nanoparticles [9,18]. There is paucity of research on the use of geranium extract for synthesis of metal oxides. Most of the studies mentioning the ability of geranium extract as a reducing agent reported synthesis of noble metals nanoparticles such as silver and gold and their application in medicine [18][19][20][21][22]. The review of the literature carried out in this study indicated that so far there is no phytochemical study of Pelargonium hortorum and its use for biosynthesis of pure CeO2-NPs for antioxidant and photocatalytic applications, and especially in the photocatalytic reduction of hexavalent chromium, Cr(VI), ions.
Chromium ion has been classified as an important water pollutant, which mostly appears in form of Cr(VI) and Cr(III) states in aquatic environments. Cr(VI) is widely found in wastewater originating from many industries dependent on chromium; thus, concentration of Cr(VI) in the effluent of these industries is higher than the allowable limit extractable (0.1 mg/L) in surface waters [23][24][25]. According to the literature, ions of Cr(VI) are more toxic than Cr(III) ions, thus, more dangerous to the environment and health of living organisms; therefore, reduction of Cr(VI) to Cr(III) ions could be a suitable strategy for reducing the risks of Cr(VI) ions and easy separation of this ion from water. Given solubility of Cr(VI) ions in water, it is difficult to remove them from water whereas Cr(III) ions can be easily precipitated by alkalizing the solution and separated in solid sludge form [ 26,27].
To the best of my knowledge, this study is the first report on chemical composition of the extract of Pelargonium hortorum, grown in central Iran, looking for a simple and eco-friendly biosynthesis of pure cerium oxide (CeO2-NPs) using this extract as a green bioreducing reagent. In this regard, the phytochemical composition including total phenolic, flavonoid, and volatile constituents in the extract of Pelargonium hortorum was determined by Folin-Ciocalteu and aluminum chloride methods and GC/MS technique, respectively. In addition, the microstructure, optical properties, and some other properties of the biosynthesized CeO2-NPs were characterized by using various advanced techniques including Ultraviolet-Visible (UV-Vis) spectroscopy, X-ray diffraction (XRD), Dynamic light scattering (DLS), and Brunaure Emmett-Teller (BET). Moreover, the performance of the biosynthesized nanoparticles was used in 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging and photocatalytic reduction of Cr(VI) ions as the model metal ion.

Materials
The Pelargonium hortorum plants used for the study were collected from the center of Iran (Arak, Markazi province) at the beginning of Aug 2020 and were used after they were approved by the herbarium expert of the Botanical Garden of Arak University. The materials applied in this study were purchased from Merck and Aldrich chemical companies with high purity and used as such, without further purification. For example, Cerium nitrate purity ≥99.0% was purchased from Aldrich. Potassium dichromate (>99.9%), 1,5-diphenylcarbohydrazide (DPC) purity>99.0%, for metal indication, n-hexane (purity >99%), anhydrous sodium sulfate (>98.5%), hydrochloric acid purity >36%, sodium hydroxide (>97%), methanol (purity >99%), and ethanol with purity of >99.9%, were all Merck products. Folin-Ciocalteu reagent was purchased from Sigma-Aldrich Corporation. Gallic acid (GA) with purity of >98%, sodium carbonate (purity >99%), rutin (RU) with purity of >97%, and aluminum chloride (purity >98.9%) were also purchased from Merck. Double distilled water with conductivity of less than 0.2 μS/cm was used in this study for preparation of all of the experiment solutions.

Preparation of the Pelargonium hortorum extract
For this purpose, first some parts of fresh leaves and flowers of Pelargonium hortorum were selected and washed. Then 10 g of this material were carefully weighed and transferred to a 250 mL beker containing 100 mL of double distilled water. The prepared mixture was heated below the boiling temperature for 1 hour. Finally, the prepared solution was cooled and filtered and then stored in a dark bottle at 4 ° C until use in the next steps.

Determination of the phytochemical composition
Total phenolic and flavonoid contents of the Pelargonium hortorum aqueous extract after addition of Folin-Ciocalteu and aluminum chloride as the coloring agents were determined by a UV-Vis spectrophotometer at λmax = 765 nm and λmax = 415 nm, respectively [28,29]. Briefly, 0.5 mL of each diluted extract was mixed with 0.5 mL of each reagent (Folin-Ciocalteu reagent in presence of sodium carbonate or 5% aluminum chloride methanolic solution). After 30 min, the absorbance rates were read at 765 nm and 415 nm, respectively, for determining the total phenolic and favonoid contents. To determine the total phenolic and flavonoid contents by standard calibration curves of gallic acid (GA) and rutin (RU) was used and the results were expressed as milligrams of GA and RU equivalent per gram of each extract (mg GA/g and mg RU/g) [28,29].
The volatile compounds in the Pelargonium hortorum aqueous extracts were isolated using Syringe to Syringe dispersive liquid-phase microextraction (SS-DLPME) method [30]. Nhexane was applied as the extraction solvent. Briefly, 2 mL of aqueous extract were put in a Syringe and then mixed with 2 mL n-hexane solvent. After agitation for 10 min, 2 mL of the organic solvent was separated by decantation, dried with anhydrous sodium sulfate, and then

Biosynthesis of CeO2-NP
The biological synthesis of CeO2-NP was performed according to the common methods reported in the literature [6][7][8] yet with some modifications and optimization in the synthesis effectiveness variable, including the cerium salt concentration, extract volume, temperature, and time. Optimizations in the process of synthesizing CeO2-NP was based on achieving the highest absorption spectra intensity within 300-330 nm (due to charge transfer from O2P to Ce4f orbitals) [31], which was measured and recorded by a UV-Vis spectrophotometer. Under optimum condition, 20 mL of aqueous extract of Pelargonium hortorum, as the reducing agent, was added to 80 mL of 0.1 M cerium solution (2.91 g of Ce(NO3)2.6H2O salt were dissolved in 100 mL double distilled water). Then the solution was mixed by magnetic stirring for 2h at 80°C and the water was removed until a whitish-brown gel was obtained, which indicated the biosynthesis of cerium nanoparticale compounds [32]. The obtained gel was washed several times with double distilled water and ethanol and then was air dried in an oven at 70 °C for 1 h. Finally, the dried biosynthesized produced was calcined at 400 C for 2 h. Ka radiation X-ray source was used. To analyze the distribution size and zeta potential of CeO2-NPs, a DLS analyzer (HORIBA SZ-100) was applied. Field emission scanning electron microscopy (FESEM, TESCAN, and Mira III) was employed to determine the morphology of CeO2-NPs. The EBT N2-adsorption (BELSORP, Mini II) was carried out to determine the surface properties of the biosynthesized CeO2-NPs.

Antioxidant activity
The free radical scavenging activity of the Pelargonium hortorum aqueous extract and the biosynthesized CeO2-NPs was determined by the percentage of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging efficiency [33]. Briefly, 2 mL of either of the Pelargonium hortorum aqueous extract or CeO2-NPs solution in different concentrations (5-100 μg/mL in methanol) was added to 3 mL of methanolic DPPH solution, kept for 30 min in dark, and then centrifuged. After that, the absorbance of the supernatant at 517 nm was determined using a UV-Vis spectrophotometer. The absorbance at 517 nm of methanolic DPPH solution without aqueous extract or CeO2-NPs was also analyzed for control. Percentages of radical scavenging where A0 is the DPPH control absorbance and AS is the DPPH absorbance with aqueous extract or CeO2-NPs.

Photocatalytic activity
The photocatalytic reduction of the hexavalent chromium ions from aqueous solutions was studied to investigate the photocatalytic activity of the biosynthesized CeO2-NPs. 10 [32]. In addition, creation of a strong absorption spectra at a maximum wavelength of 325 nm due to surface plasmon resulting to the charge transfer from O2P to Ce4f orbitals was another sign of the production of CeO2-NPs (Fig 1), which is highly consistent with the reported results [6,31,32]. Also, the optimization of the conditions for the production of the largest amount of CeO2-NPs with dimensions less than 50 nm was performed by measuring the intensity of the absorption spectra and displacement at the maximum wavelength position by a UV-Vis spectrophotometer. The conditions that lead to recording the highest absorption and displacement spectra of blue shift due to the production of more nanoparticles and their smaller size, respectively, were selected as the optimal conditions. Accordingly, 80 mL of cerium salt of 0.1 M, 20 mL of the extract, temperature 80 °C, and time of 2 h were selected as the optimal conditions for biosynthesis of CeO2-NPs. Furthermore, the direct optical band gap energy as a property in the biosynthesized CeO2-NPs was estimated as 3.81 eV by the Tauc equation (Eg = 1239.8/λ) [38], where Eg and λ are the band gap energy (eV) and wavelength (nm) of the absorption edge in the UV-Vis spectrum, respectively. It should be noted that when the amount of the extract was increased to more than 20 ml, the size of the particle produced was larger (it showed a red shift) and the band gap decreased, a trend that has been reported in some previous studies as well [6]. " Fig. 1" The FT-IR spectroscopy was carried out to identify the functional groups of the biosynthesized CeO2-NPs. Fig. 2 shows the FT-IR spectrum of the biosynthesized CeO2-NPs where the broad reported results in the previous studies [6,40]. The crystalline size of the biosynthesized CeO2-NPs was calculated using Scherrer's formula (D=0.89λ/βcosθ) [41] and was accordingly found to be 29 nm for the highest peak at 2θ=25.22°. " Fig. 3" Moreover, a DLS analysis was applied to determine the zeta potential charge (ZPC) and estimate the hydrodynamic particle size distribution of the biosynthesized CeO2-NPs, as shown in Fig. 4. It was found that the net surface charge of the biosynthesized CeO2-NPs was -25.8 mV, which shows its good colloidal stability [6]. Also, the DLS histogram (inset in Fig.4) indicates that the biosynthesized CeO2-NPs has a narrow pattern with an average hydrodynamic diameter of about 34 nm.
Furthermore, FE-SEM image was used to identify the morphology and estimate the particle size of the biosynthesized CeO2-NPs (see Fig. 5). This figure shows a uniform distribution of the biosynthesized CeO2-NPs and a spherical shape as well as a mean particle size of 33 nm, which is in good agreement with the estimated results from the XRD and DLS techniques. In addition, the EDX analysis (illustrated in Fig. 5) was used to study the chemical elements in the biosynthesized CeO2-NPs. The EDX results showed the major peaks correspond to the Ce and O element. Such correspondence was not detected for other elements, which confirms the purity of the biosynthesized CeO2 -NPs.

"Figs.4 & 5"
Also, the specific surface area, pore volume, and pore radius of the biosynthesized CeO2-NPs were characterized by N2 adsorption-desorption porosimetry using a BET analyzer. Fig 6 shows the N2 adsorption-desorption isotherm of the biosynthesized CeO2-NPs. This isotherm exhibits a type IV isotherm with an H2 hysteresis loop in the p/p0 range of 0.35-0.98, which indicates formation of a mesoporous host in all the CeO2-NP structures [42]. The BET-specific surface areas of the biosynthesized CeO2-NPs are 33.84 m 2 /g with mean pore diameters of 9.31 nm and total pore volume of 0.078 cm 3 /g. The inset figure shows the pore size distribution of CeO2-NPs. The main peak was located in the range of about 2.4 nm, which confirms the mesoporous nanoparticles classification [42]. " Fig. 6"

Antioxidant activity
The antioxidant potential of the Pelargonium hortorum extract was evaluated by the scavenging potential of stable methanolic DPPH free radicals (Fig. 7). In the presence of the extract, the color of the DPPH solution changed from deep violet to light yellowish with an increase in the contact time [33]. The percentage of the scavenging of DPPH increased with an increase in the extract amount from 5 to 100 μg/mL and reached about 85.4% at about 100 μg/mL. The results suggest that the phenolic components (total phenol and flavonoid) contributed significantly to the antioxidant capacity of the extract. The antioxidant activity of the biosynthesized CeO2-NPs was also evaluated using the DPPH method. The maximum antioxidant activities (69.8%) were exhibited when 100 μg/mL of the CeO2-NPs was used.
Since 100% of the free radical could not be scavenged by a certain substance [43], the antioxidant potential was evaluated by the IC50 value (the scavenging concentration needed to scavenge 50% of the DPPH free radical) of the substance. The results show that the IC50 of extract and CeO2-NPs were found to be 29.3 μg/mL and 42.7 μg/mL, respectively.   (6) which, in acidic media indirectly was generated H2O2 molecules leads to rapid reduction of Cr(VI)ions [44]. In this section, the kinetic reduction of the Cr(VI) ions was also investigated and analyzed by − 0 = equation. From the slope of 0 plot versus time (Fig. 8), the rate constant (k) was obtained as 0.079 1/min and the coefficient (R 2 ) was also determined as 0.981 for the process. The rate of the Cr(VI) ions reduction in this process was fitted to the pseudo-first-order kinetic model. Finally, the stability of the biosynthesized CeO2-NPs was evaluated after its regeneration. Briefly, the used CeO2-NPs was washed with hydrochloric acid 0.1 M and then sonicated in boiling double distillate water for 15 min, and then dried in oven at 80 °C for 2 h. The results revealed (Fig. 8) that the Cr(VI) reduction efficiency after a six time treatment process was reduced about 6%. " Fig. 8"

Conclusions
In this work, the phytochemical composition in the aqueous extract of Pelargonium hortorum from Iran, was studied. The amounts of the total phenolic and flavonoid contents in the extract were found to be 136.5 mg GA/g and 63.9 mg RU/g, respectively. Seventeen compounds