Eco-Friendly Synthesis of Cerium Oxide Nanoparticles from Lycium cooperi

1. Introduction

Cerium oxide (CeO₂) is a n-type semiconductor material with a band gap of approximately 3.3 eV [1]. Interestingly, CeO₂-NPs have garnered significant attention due to the improved optical, electrical, catalytic and thermal properties, characterized with chemical stability compared to the CeO₂ non-nanostructured precursor [2]. Moreover, CeO₂-NPs are considered a highly promising material for various applications, including optoelectronic devices [3], energy conversion and storage [4], catalysis [5], tissue engineering [6], biotechnology [7], nanomedicine and antioxidants in biological systems [8]. In this regard, CeO₂-NPs have been synthesized using different chemical strategies such as sol-gel, colloidal synthesis, hydrothermal, and precipitation methods [9,10]. These synthetic approaches allow precise control of nanoparticle size, morphology, and chemical composition. However, an important disadvantage of the chemical method often involve toxic solvents and significant pollutant substances. Thus, considering those detrimental issues it has been collectively applied green precipitation mechanisms using plants extracts as a facile, economic technique and commonly used for CeO₂-NPs. On the other hand, plant extracts contain secondary metabolites (e.g. flavonoids, catechins, coumarins and more) that act as chelating, reducing, and stabilizing agents to obtain controlled metallic nanoparticles [11,12,13]. In addition, many of these secondary metabolites exhibit antioxidant, anti-inflammatory, and antimicrobial activity, with significant potential biomedical applications.

Considering a novel greener strategy, the genus Lycium, which belongs to the Solanaceae family with 80 species in different regions worldwide [14,15] can be selected as a promissory choice for the synthesis of CeO₂-NPs. In recent years, several studies have explored the chemical composition and pharmacology of this genus, leading to the identification and isolation of over 200 compounds. These compounds include flavonoids, alkaloids, polysaccharides, steroids, terpenoids, carotenoids, amides, and essential oils [14]. The diversity of phytochemicals suggested the Lycium genus as a highly promising for the green synthesis of nanoparticles, which has been scarcely explored in this application. It has recently been reported that CeO₂-NPs were synthesized using the green precipitation method, employing various plant extracts such as Origanum majorana L. leaf extract [16], Acorus calamus [17], Calotropis procera flower extract [18], Azadirachta indica [19], Artabotrys hexapetalus leaf extract [20], Abelmoschus esculentus [21], Moringa oleifera leaf extract [22] and Rheum turkestanicum extract [23]. Nonetheless, these methods required extended synthesis periods, the use of ammonium and sodium hydroxides as precipitants, and showed large physicochemical variations, highlighting the importance of the green precursor agent.

The main objective of this work was to synthesize controlled, well-dispersed, homogenous spherical CeO₂-NPs from secondary metabolites of L. cooperi aqueous extract, taking advantage of the chelating and reducing properties [11-13]. The characterization of the morphological, optical, and structural properties were measured. To the best of our knowledge, no information about the use of this plant for the green synthesis of nanoparticles was found.

2. Experimental Details

2.1. Morphological Characteristic of L. cooperi Plant

Figure 1 (a) displays the L. cooperi plant specimen harvested under controlled conditions in a greenhouse at the Instituto de Ingeniería, Universidad Autónoma de Baja California in Mexicali, Baja California, Mexico. Figure 1 (b) shows the characteristics of the leaves and flowers. According to the voucher specimen from the databases of the University of California, Berkeley (Figure 1. (c)), the taxonomic identification is in accordance to the L. cooperi [24]. Table 1 presents the main morphological characteristics of the plant, along with information about its habitat and geographical distribution [24].

2.2. Preparation of L. cooperi Extract

The L. cooperi leaves were harvested to obtain an aqueous extract. Initially, the leaves were washed with abundant deionized water (DI) and then left to air-dry at room temperature for one day. Then, 10 g of dry leaves were pulverized and mixed with 100 mL of DI. The mixture was then heated to 60°C for 30 min with constant stirring. Finally, the L. cooperi aqueous extract was filtered using (filter paper, Whatman N°4, 55 mm) and stored at 4 °C for later use. Figure 2 shows the preparation process of the L. cooperi aqueous extract.

2.3. Phytochemical Characterization of L. cooperi Extract

Phytochemical tests were conducted to identify the secondary metabolites in the aqueous extract of L. cooperi as follows:

• Alkaloids

A few drops of Hager’s reagent (picric acid) were added to 2 mL of extract. The formation of a yellow color crystalline precipitate indicates the presence of alkaloids.

• Flavonoids

To evaluate flavonoids, we prepared two test tubes containing 2 mL of L. cooperi and 2 mL of DI. Then, for comparison of the samples, it was added 2 mL of NaOH (0.1 M). A change in the color solution to yellow indicates the presence of flavonoids.

• Saponins

Vigorously shake 5 mL of extract and kept static for 10 min. The presence of a stable froth indicates that saponins are in the extract.

• Tannins

A few drops of FeCl₃ are added to 2 mL of L. cooperi extract. A green condensed formation indicates positive.

• Terpenoids

To 5 mL aqueous extract are mixed with 2 mL of chloroform and 1 mL of concentrated H₂SO₄. If positive, a red-brown color ring appears in the lower chloroform layer phase.

• Cardiac glycosides

The aqueous extract (5 mL) is mixed in 2 mL of ferric chloride-glacial acetic acid solution and 1 mL of H₂SO₄ concentrated. The appearance of a brown ring at the interface indicates the presence of glycosides.

• Carbohydrates

To the aqueous extract (5 mL) it was added 2 mL of acetic acid and 2 mL of chloroform. Consequently, three drops of concentrated H₂SO₄ is added to the mixture. The presence of an orange precipitate indicates positive to carbohydrates.

The functional groups of the secondary metabolites and certain chromophore groups within the aqueous extract of L. cooperi were characterized by FT-IR (PerkinElmer Frontier FT-IR; 4000 – 400 cm^-1) and UV-Vis (Shimadzu 260; 200 – 800 nm) analyses.

2.4. Green Synthesis of CeO₂-NPs

A precursor solution of 0.1 M Ce³⁺ was prepared by dissolving 2.17 g of cerium nitrate hexahydrate (Ce(NO₃)₃ • 6H₂O) in 50 mL of L. cooperi aqueous extract. The mixture was heated to 80°C for 1 h with constant stirring to produce cerium hydroxide (Ce(OH)₃) precipitates. Ce(OH)₃ precipitate was centrifuged at 4000 rpm, then vacuum filtered, washed at environmental conditions with DI, absolute ethanol, and dried overnight at 60°C. Finally, Ce(OH)₃ was calcined at 400 °C in a muffle furnace for two hours in the air to obtain CeO₂-NPs. Figure 3 illustrates the synthesis process of CeO₂-NPs using the green precipitation method.

2.5. Characterization of CeO₂-NPs

The morphology, particle size, and chemical composition were determined by SEM (Tescan LYRA 3 XMH) equipped with an EDS (Quantax Bruker X-Flash 6160) and HR-TEM (JEOL 2010). The SEM micrograph was acquired using an accelerating voltage of 10 kV and a magnification of 40 kX. The mounting conditions for CeO₂-NPs was dispersing 1 mg of CeO₂-NPs in 5 mL of absolute ethanol through ultrasonication for 15 minutes. Subsequently, a 10 μL aliquot was extracted, and the dispersion was deposited onto a TEM grid or carbon tape (SEM) for further study. The TEM image was captured using an accelerating voltage of 200 kV. The structural and crystallographic information of CeO₂-NPs were obtained by TEM and XRD (Panalytical Empyrean) with Cu Kα radiation source (λ = 0.15406 nm) and a real-time multipass detector X´Celerator, with scanned over the 2-theta range 20 – 90 at room temperature. FT-IR spectra were recorded in the 400 – 4000 cm⁻¹ range (Frontier Perkin Elmer) in the attenuated total reflection mode. The optical properties of the CeO₂-NPs were characterized by UV-Vis from 200 – 800 nm (Shimadzu 260).

3. Results and Discussion

3.1. Phytochemical Analysis of L. cooperi Aqueous Extract

In Table 2, we presented the results of the phytochemical tests. Initially, the Hager, ammonia, Keller-Kellani, and Benedict tests showed positive results, indicating the presence of alkaloids, flavonoids, cardiac glycosides, and carbohydrates, respectively. On the other hand, the analyses for saponins, tannins, and terpenoids were negative. The findings demonstrate that the aqueous extract of L. cooperi contains the metabolites flavonoids and carbohydrates, which are chemically highlighted due to the numerous hydroxyl groups (-OH). These metabolites are known for their ability to act as chelating and reducing agents in the formation of nanoparticles in aqueous solutions [25], eliminating the need to add acids or bases. These metabolites can reduce Ce³⁺ ions to Ce⁰, initiating the nucleation process for Ce(OH)₃ formation, which is then calcined to obtain oxidized CeO₂-NPs.

Figure 4 (a) illustrates the FT-IR spectrum of L. cooperi extract. The 3340 cm^-1 and 1420 cm^-1 peaks correspond to the O-H bond stretching and bending, and 2928 cm^-1 and 2850 cm^-1 peaks correspond to –CH₂ bond stretching [26]. Moreover, the peak at 1564 cm^-1 shows the stretching behavior of the aromatic rings C-C bond, the band at 1090 cm^-1 is attributed to the C-O stretching, and a shoulder band at 1626 cm^-1 for stretching vibration of the carbon-oxygen double bond [27]. Finally, a peak is observed at 598 cm^-1 suggesting the presence of halide groups in the extract. The families of secondary metabolites identified by the phytochemical reactions in the L. cooperi extract correspond to the functional groups reported in the molecular structure by FT-IR.

The absorption band observed at 225 nm in the UV-Vis spectrum of the L. cooperi extract (Figure 4. (b)) is attributed to the electronic transitions of π → π* orbitals, which occur in the structure of the chromophore groups present in the sample.

FT-IR and UV-Vis analysis confirm the presence of green-derived metabolites corresponding to a mixture of flavonoids, cardiac carbohydrates, alkaloids, and polymeric carbohydrates identified through phytochemical tests.

3.2. Physicochemical Analysis of CeO₂-NPs

The SEM micrographs show the agglomeration of CeO₂-NPs, highlighting a homogenous spherical morphology and showing an average size of 86±10 nm (Figure 5. (a)). On the other hand, the chemical composition of the CeO₂-NPs by means of EDS illustrated the presence of Ce and O, suggesting the formation of high-pure CeO₂-NPs (Figure 5. (b)).

Figure 6 (a) displays the TEM micrographs of the CeO₂-NPs showing a spherical morphology and size distribution of 6.9 ± 1.5 nm (inset Fig. 6. (a)). Compared to the results obtained by SEM (Fig. 5. (a)), this size reduction is attributed to the process of dispersion and sample preparation by ultrasound. Furthermore, the high-resolution image (Fig. 6. (b)) confirms the crystalline structure of CeO₂. We detected an interplanar distance of 0.31 nm on the (111) plane, which is in accordance with previous works [28]. Additionally, the electron diffraction image confirmed the presence of the cubic phase structure of the CeO₂, as expected.

In Figure 7 the XRD patterns of CeO₂-NPs exhibit peaks values (2θ) of 28.35°, 32.85°, 47.39°, 56.09°, 58.79°, 69.09°, 76.37°, 78.96°, and 88.15°, corresponding to (111), (200), (220), (311), (222), (400), (331), (420) and (422) planes, respectively. These peaks are agree with the typical signals of CeO₂-NPs showing a cubic crystalline structure (ICSD 98-006-1595) as demonstrated in previous studies [17,18,19,20,21,22]. The crystallite size (D) was calculated by the Scherrer's equation:

$$D={\displaystyle \frac{0.9\lambda }{\beta \cos \theta }}$$

(1)

Where λ is the wavelength, β is the full width at half maximum (FWHM), and θ is the Bragg's angle expressed in radians. The calculated crystal size was ~6.5 nm.

Additionally, the interplanar distance ($d$) for the plane (111) was determined by Bragg's equation:

$$d={\displaystyle \frac{n\lambda }{2\sin \theta }}$$

(2)

Where n is the order of diffraction. The results indicated that the interplanar distance was 0.31 nm for the plane (111), consistent with the HR-TEM findings, and supported by previous works of green-strategies and thermal CeO₂-NPs works [28].

The FT-IR spectrum (Figure 8) exhibited a peak at 405 cm⁻¹, which corresponds to the Ce–O stretching vibrational mode [19]. Low intensity peaks are displayed at 1315, 1502 and 1630 cm^-1 respectively, which are attributed to traces of organic matter from the phytocompounds of the extract. The peak at 3350 cm^-1 indicated the presence of O–H groups; nonetheless, it was a low intense formed peak [17,22].

The optical bandgap of the CeO₂-NPs was determined by the Tauc plot method (Figure 9) from the UV-Vis absorption spectrum (inset Figure 9). The Tauc expression relates the optical band gap, E_g, and absorption coefficient, α, by the following expression:

αhν = B [(hν − E_g)]ⁿ

(3)

Where hν is the photon energy, B is the proportionality constant, and n is a number that depends on the nature of the electronic transitions responsible for the absorption. Eg was determined by fitting the linear zone of (αhν)² vs hν dependence, and extrapolating to the energy. The optical band gap obtained for CeO₂-NPs was ~3.29 eV. This value corresponds to the band gap energy reported in the literature for CeO₂-NPs [1,18].

Table 3 compares the results reported in the literature with our work findings regarding the size and optical properties, as well as the reaction time of the CeO₂-NPs obtained through green synthesis using various plant extracts. Our method results in shorter synthesis times compared to those reported in the literature [16,17,18,19,20,21,22,23].

4. Conclusions

CeO₂ nanoparticles were synthesized by the green precipitation method from an aqueous extract of Lycium cooperi. The SEM and EDS results indicate the formation of spherical CeO₂-NPs, with average size of ~85 nm and an elemental composition of Ce and O atoms. However, the TEM results indicate a ~7 nm average size; this reduction in the CeO₂-NPs size is attributed to the ultrasonic dispersion process. Thus far, this extract has shown great potential as a green precursor for controlling the synthesis of spherical-shaped CeO₂-NPs.

The XRD and HR-TEM analyses revealed the formation of cubic structured CeO₂-NPs crystal, and the FT-IR spectrum confirms the presence of the stretching Ce-O bond. Moreover, the calculated CeO₂-NPs bandgap was ~3.29 eV, expressed from the UV-Vis absorbance spectra and Tauc plot method.

This study has demonstrated an affordable and eco-friendly method for obtaining CeO₂-NPs. Notably, it is reported for the first time the application of L. cooperi extract as a green-derived strategy for controlling the size, morphology, and distribution of cerium oxide nanoparticles. The environmentally friendly synthesis method and the characteristics of the resulting CeO₂-NPs can open a new route for extensive biomedical and industrial applications.