1 Introduction

In recent time, the green fabrication of nanomaterials has attracted a great deal of interest owing to its environmental friendliness, compatibility with living things, varied biomedical potentials, and effective ability to eliminate waste materials. The literature review determined that there has been lot of reports on the synthesis of nanomaterials using natural products, such as plants, sponges, cyanobacteria, fungi, and algae [1,2,3]. For the production of nanomaterials, plant-derived bioactive compounds and synthetic biomaterials have been used for a variety of biological application [1].

CeO2 is a semiconductor with a large band gap energy (3.19 eV) that is utilized in a variety of applications, including antibacterial activity, sunscreen cream, catalysts, and sensors for solid oxide fuel cells [4]. Typically, physical and chemical processes, such as hydrothermal, flame spray pyrolysis, sonochemical, microwave, sol–gel, and co-precipitation, are normally used to produce CeO2 NPs, which are complicated, costly, time-consuming, and hazardous [5, 6]. The expansion of phytosynthesis of metal and metal oxide NPs using green chemistry embraces great promise. This approach has many benefits, including low cost, large-scale commercial production, and use in pharmaceuticals production. Since soluble Ce3+ salts have antiemetic, bacteriostatic, immunomodulation, antibacterial, and anticancer action, they have been employed for biomedical applications, even though cerium itself has little biological importance in mammalian physiology [7]. A commercial cutaneous burn cream made from cerium nitrate has also been given orphan drug designation by US FDA [8]. In addition to existing naturally, the insoluble oxide of cerium (i.e. CeO2) is also produced as a bulk material and as bioengineered nanoparticles [9]. Moreover, CeO2 NPs have been found to be less hazardous in cultured cells as compared to TiO2 and ZnO NPs [10]. Interestingly, the synthesis and uses of NPs as possible catalytic antioxidants in biology and medicine has been skyrocketed [11,12,13]. Previously, CeO2 NPs were synthesized using natural resources, such as honey [14], egg white [15] and fungal extracellular components [16]. In cell and animal models, CeO2 NPs mimics the antioxidant enzymes and effectively scavenge various reactive oxygen and reactive nitrogen species (ROS and RNS) [17].

Stimulated by the versatile properties of CeO2, herein, the green synthesis of CeO2 NPs using HS is being reported. H. Sabdariffa is a shrub in Malvaceae family that produces calyces-shaped crimson blooms due to the presence of high content of anthocyanins like cyanidin-3-glucoside, delphinidin-3-glucoside, cyanidin-3-sambubioside, and delphinidin-3-sambubioside in flowers. Additionally, the plant contains both organic and phenolic acids, particularly citric acid, hydroxycitric acid, and hibiscus acid, as well as flavonoids like gossipetin, luteolin, and quercetin, along with their corresponding glycosides [18]. Moreover, HS has been documented in the biofabrication of new nanoparticles. For instance, HS flower extract was employed as a reducing and stabilizing facilitator for fabrication of cerium oxide, gold, and silver nanoparticles and showed cytotoxicity potential against U87 glioblastoma cells under hyperglycemic conditions [18, 19]. The outstanding biocompatibility and great stability of magnesium oxide nanoparticles (MgO NPs) in extreme environments provoked researchers to choose Mg in composites [20]. Furthermore, MgO NPs are also very effective in exerting cytotoxicity against various cancer cells [21, 22]. However, to the best of our knowledge, hitherto, there is no report on biofabrication of Mg doped CeO2 NPs and their anticancer, anti-inflammatory and antioxidant activities. Therefore, in the present investigation, HS was utilized for synthesis of Mg doped CeO2 NPs, and its organizational, morphological and biological aspects (anticancer, anti-inflammatory and antioxidant) has been studied in detail.

2 Materials and methods

2.1 Aqueous extraction of HS

Fresh red petals of HS flowers were procured from a local market in Jeddah, Saudi Arabia and were identified by Dr. Haider Abd Algadir, taxonomist, Department of Biology, AlBaha University, Saudi Arabia. The plant was air-dried at room temperature and the samples were preserved in glass jars and flushed with nitrogen. The dried flowers of HS were pulverized into a fine powder in a domestic blender. A typical process involved weighing 10.0 g of clean HS flower petals in a beaker and were added to 400 mL of distilled water. Since non-thermal extraction was the primary goal, a high concentration of anthocyanins was obtained by keeping the aqueous extraction solution at room temperature for 25 h. In order to remove any remaining particles, the resultant red extract solution was vacuum-filtered twice, and extract was then preserved for further studies.

2.2 Synthesis of CeO2 and Mg doped CeO2 nanoparticles

The CeO2 and Mg doped CeO2 nanoparticles were prepared using previously reported methods with suitable modifications [18, 23, 24]. For synthesis of CeO2, 2.0 g of Ce (NO3)3.6H2O was added into 80 mL of red HS flower aqueous extract solution. The solution mixture was assorted consistently for one hour. Then, the resultant solution was transferred to hydrothermal reactor and heated at 180 °C for 24 h. After heating, the solution was filtered and cleaned several times with water and then with ethanol to remove any impurities and kept at 80 °C overnight in oven. For synthesis of Mg doped CeO2, all the aforementioned reaction conditions were followed except Mg (NO3)2.6H2O (5 wt.%) was added into the extract solution. The temperature and time was optimized in the present study to get CeO2 and Mg doped CeO2 nanoparticles with uniform morphology.

2.3 Characterization

The samples were characterized by SEM, EDS, XRD and FTIR. The crystallinity of HAP and Mg doped CeO2 NPs were examined by X-ray diffraction (XRD, Rigaku, Tokyo, Japan). SEM furnished with energy dispersive X-ray (SEM–EDS, JEOL, Tokyo, Japan) was utilized to determine the morphology and microstructure of the prepared materials. The occurrence of functional groups in samples were determined by Fourier transform infrared (FTIR, Thermo Scientific, Waltham, MA, USA) spectrometer at resolution of 4 cm−1 and scanning rate of 0.75 Hz using ATR mode.

2.4 Source and types of cancer cell lines

Three cancer cell lines were used for evaluation of anticancer activity supplied from the American Type Culture Collection (ATCC): HepG2 human liver cancer cell line, MCF-7 human breast cancer cell line, and A-549 lung cancer cell line, while MRC-5 human normal fetal lung fibroblast cell line was used as control.

2.5 Cell line propagation

The cells were raised in RPMI-1640 medium supplemented with 10% inactivated fetal calf serum and 50 µg/ml gentamycin. The cells were maintained at 37 °C in a humidified atmosphere with 5% CO2 and were sub-cultured two to three times a week.

2.6 Cytotoxicity evaluation

Three cancer cell lines (MRC-5, A-549, HepG2) were used for evaluation of anticancer activity. 5 × 104 cells of each cell line in 96-well plate were incubated for 24 h. This followed by the addition of the synthesized nanoparticles in different concentrations and incubation of tested cells for 24 h. The colorimetric assay (MTT assay) was applied and then the optical density was read at 590 nm using the microplate reader to determine the percentage of the viable cells. Based on graphic plots of dose response curves, the IC50 was calculated using GraphPad Prism software [25].

2.7 Antioxidant activity

The antioxidant activity of CeO2 and Mg doped CeO2 NPs was performed at the Regional Center for Mycology and Biotechnology (RCMB) at Al-Azhar University using 2,2-diphenyl-1-picrylhydrazyl radical scavenging (DPPH) [26], hydrogen peroxide scavenging (H2O2) [27] and the total antioxidant capacity (TAC) [28] assays.

2.8 Anti-inflammatory activity

Two methods were employed to examine the anti-inflammatory capacity. The albumin denaturation assay was carried out according to Williams et al. [29] with diclofenac sodium as a reference drug and COX-1 inhibition assay was performed using a COX 1 inhibitor screening assay kit (Catalog number k548, Biovision, USA) as instructed by manufacturer.

3 Results and discussion

To check the crystallographic phase of synthesized samples, XRD was employed. Data in Fig. 1 showed that the XRD spectra of CeO2 and Mg doped CeO2 NPs. All the diffraction peaks shown in Fig. 1a match the crystalline plane (111), (200), (220), and (311), which approves the cubic fluorite phase of CeO2 NPs (JCPDS no. 81-0792) [23, 30]. The XRD arrangements of the synthesized Mg doped CeO2 demonstrated that all characteristic diffraction crests of CeO2 and identical to the crystallographic structure (Fig. 1b) but the peaks were sharply, indicating high crystallinity and comparatively larger crystallite size. This may be attributed to the presence of Mg metal particles. However, the MgO phase was not observed, or any impurity crests were existing in the spectrum, which revealed that that there is no change in phase structure of CeO2 due to Mg doping.

Fig. 1
figure 1

XRD of (a) pure CeO2 and (b) Mg doped CeO2 NPs

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Result in Fig. 2 illustrated that the apparent arrangement and morphological features of pure CeO2 and Mg doped CeO2 NPs. The CeO2 particles are somewhat orbicular in profile, with average size around 100 nm (Fig. 2a, b). The particles were homogeneously distributed throughout the sample. Whereas the XRD spectra of Mg doped CeO2 samples have shown that CeO2 particles are in aggregation, neighboring with Mg particles of varying length (Fig. 2c, d).

Fig. 2
figure 2

SEM of (a, b) pure CeO2 NPs and (c, d) Mg doped CeO2 NPs at different magnifications, respectively

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The EDS spectra of pure CeO2 and Mg doped CeO2 have been conducted to elucidate their chemical composition. The EDS result clearly identifies peaks of cerium (Ce) and oxygen (O) which confirms presence of CeO2 (Fig. 3a). Whereas in case of Mg doped CeO2, additional peak of magnesium (Mg) confirms its presence in Mg doped CeO2 (Fig. 3b).

Fig. 3
figure 3

Elemental compositions of (a) pure CeO2 NPs and (b) Mg doped CeO2 NPs

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The FT-IR spectra of primed CeO2 and Mg doped CeO2 were presented in Fig. 4 which is measured at wave number 400–4000 cm−1range. The broad absorption frequency band observed at 3000–3700 cm−1 is allocated to O–H widening from residual alcohols and water. The crests at 1337 cm−1, 1542 cm−1 and 1645 cm−1 possibly due to the trapped CO2 in air atmosphere (Fig. 4). The absorption band near 854 cm−1 is characteristic of cubic MgO [31]. A strong band allocated to the stretching frequency of Ce–O can be observed around 400 cm−1, indicating establishment of CeO2 [9].

Fig. 4
figure 4

FTIR spectra of (a) Mg doped CeO2 NPs and (b) pure CeO2 NPs

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4 Anticancer activity of pristine CeO2 and Mg doped CeO2 NPs

The anticancer action capacity of the pristine CeO2 and Mg doped CeO2 NPs were performed on three cancer cell lines: HepG-2 (Hepatocellular carcinoma cells), MCF-7(Breast carcinoma cells) and A-549 (Lung carcinoma cells). Vinblastine sulfate was used as the standard anticancer drug. Interestingly, we found that the Mg doped CeO2 NPs boosted cytotoxicity in the HepG-2, MCF-7 and A-549 cell lines. The Mg doped CeO2 NPs exhibited cytotoxicity with IC50 of 109.65 ± 4.13 µg/mL (HepG2), 113.55 ± 3.89 µg/mL (MCF-7) and 79.19 ± 3.07 µg/mL (Fig. 5), while pristine CeO2 NPs displayed IC50 = 95.97 ± 3.85 µg/mL, 126.63 ± 5.71 µg/mL and 102.70 ± 3.97 µg/mL towards HepG2, MCF-7 and A-549 carcinomas, respectively. In case of the standard Vinblastine sulfate drug, the IC50 values for MCF-7, A-549 and HepG2 were found to be 3.58 ± 0.52 µg/mL, 3.03 ± 0.29 µg/mL and 24.48 ± 2.04 µg/mL, respectively.

Fig. 5
figure 5

Anticancer activity of pure CeO2 NPs and Mg doped CeO2 NPs against the three cancer cell lines HepG2 (Liver), MCF-7 (Breast), A-549 (Lung). Data represent the mean values ± standard deviation of three independent experiments performed in triplicate. The IC50 was calculated by regression analysis using GraphPad Prism software. Vinblastine sulfate was used as the reference drug (i.e., positive control)

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Even though, the multiple uses of nanomaterials have led to an exponential rise in the number of nano-textured materials in recent times. Nevertheless, despite their numerous uses, there is limited evidence available regarding their effects on human health and the environment [32]. According to some studies published in literature, unintentional exposure to nanomaterials through inhalation, skin contact, or gastrointestinal absorption can seriously endanger human health [33]. Considering the harmful aspects, the cytotoxicity experiments were carried out on healthy human lung fibroblast (MRC-5) cells to evaluate the safety of the prepared CeO2 and Mg doped CeO2 NPs. As shown in Fig. 5d, on comparing the cytotoxicity of CeO2 and Mg doped CeO2 NPs to the untreated control cells as well as normal cells, results revealed that there were no discernible toxicity or substantial difference in cell viability. Therefore, these nanoparticles did not show negative impacts on healthy lung fibroblast cells, while only exhibited cytotoxic effects on screened cancerous cells. Previous research studies have documented the impact of environmentally friendly manufactured gold nanoparticles on liver and lung cancers, and it is thus anticipated that nanomaterials will mutate cancer diagnosis and therapy [34]. Considering the safety and environmental factors, herein, the prepared eco-friendly Mg doped CeO2 NPs were evaluated, and found to be promising effective candidates to be taken into consideration for the treatment of the aforementioned cancers.

4.1 Antioxidant activity of CeO2 and Mg doped CeO2 NPs

The antioxidant potential of Mg doped CeO2 NPs was investigated using 2,2-diphenyl-1-picrylhydrazyl radical scavenging (DPPH), hydrogen peroxide scavenging activity (H2O2) and total antioxidant capacity (TAC) assays. The obtained results are displayed in Table 1. The nanotextured CeO2 and Mg doped CeO2 NPs dose dependently scavenges DPPH and H2O2 free radicals.

Table 1 The DPPH% and H2O2% scavenging activity of pure CeO2 and Mg doped CeO2 NPs and ascorbic acid as a reference antioxidant agent
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In this study, the antioxidant activity of CeO2 and Mg doped CeO2 NPs was highly dependent on the gradual increase in the concentration of nanoparticles from 0 to 1000 μg/mL, as shown in Table 1. The Mg doped CeO2 NPs depicted DPPH and H2O2 scavenging potential with maximum % inhibition equal to 79.43 ± 1.51 and 72.43 ± 1.51, respectively at 1000 µg/mL. The total antioxidant capacity (TAC) exhibited by Mg doped CeO2 and CeO2 NPs was found to be 76.26 ± 3.1 and 66.73 ± 1.19 mg GAE/g, while ascorbic acid showed 72.68 ± 3.74 mg GAE/g. Furthermore, IC50 exhibited by CeO2 and Mg doped CeO2 NPs in DPPH assay was found to be 369.37 ± 12.13 and 137.78 ± 6.25 respectively. Whereas, in case of H2O2 scavenging assay the IC50 for CeO2 and Mg doped CeO2 NPs was 535.97 ± 14.87 and 340.50 ± 10.26 respectively. It is evident from previously published work that the synthesized CeO2 NPs are gaining a captivating spot as antioxidants in biological and medical field. They are utilized in various treatments related to oxidative-stress/nitrosative stress and various types of malignancies [35, 36], neurodegenerative diseases [37] and chronic inflammation [38]. It was reported that aqueous extracts of HS were more potent antioxidants than extracts prepared using methanol [39]. In this respect, Das et al. [40], demonstrated that CeO2 NPs can remove OH free radicals generated from H2O2 in aqueous solutions, that implicitly suggests that CeO2 NPs have inherent OH radical scavenging capacity which was further supported by Xue et al. investigation that CeO2 NPs competently scavenge OH radicals [41]. Conclusively, the importance of CeO2 NPs in biology and medicine has been fully described by Nelson et al. [17].

4.2 Anti-inflammatory action

Reactive oxygen species is associated with the development of inflammatory diseases via modulation of cytokines such as interlukins, iNOS, TNF-α, therefore the synthesized CeO2 and Mg doped CeO2 NPs were evaluated for anti-inflammatory activity by COX-1 and albumin denaturation inhibitory activities (Fig. 6). The CeO2 and Mg doped CeO2 NPs inhibited COX-1 enzyme, in comparable manner to Indomethacin in a concentration dependent mode. The CeO2 and Mg doped CeO2 NPs both has pronounced potential as COX-1 inhibitors with maximum inhibition of 68.47 ± 1.39% and 87.49 ± 1.23% at 500 μg/mL with IC50 84.58 ± 3.86 μg/mL and 25.98 ± 1.76 μg/mL, respectively, while the standard drug, Indomethacin displayed COX-1 inhibition with IC50 = 0.85 ± 0.03 μg/mL. Likewise, the albumin denaturation is an indicator for inflammatory ailments in which due to external stress, protein undergo denaturation [42]. The CeO2 and Mg doped CeO2 NPs was effective in albumin denaturation inhibition in a concentration dependent manner with maximum inhibition of 72.34 ± 1.82% and 87.49 ± 1.23% at 500 μg/mL with IC50 = 57.87 ± 2.91 μg/mL and 14.85 ± 0.97 μg/mL, respectively. The standard drug Diclofenac demonstrated comparable inhibition to the synthesized NPs. The capability of CeO2 and Mg doped CeO2 NPs to inhibit albumin denaturation further confirms its anti-inflammatory activity. Consequently, inhibition of COX-1 and albumin denaturation clearly suggest that CeO2 and Mg doped CeO2 NPs possess an anti-inflammatory potential (Fig. 6).

Fig. 6
figure 6

Dose response curve of %COX-1 and % albumin denaturation inhibition of pure CeO2 NPs and Mg doped CeO2 NPs. Data represent the mean values ± standard deviation of three independent experiments performed in triplicate. Indomethacin and diclofenac were used as standard drugs

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The inflammation is the body’s defensive response mechanism towards invasive infections [43,44,45]. It is found that the overproduction of ROS is involved in intracellular signaling transduction inflammatory events, which results in chronic inflammation [44]. Plants with anti-inflammatory properties are being used in traditional medicine to treat inflammatory diseases [45]. It has been well recognized that oxidative stress due to generation of free radicals is the primary cause for triggering inflammation and various types of cancers. In this context, the blending of plant based molecules with synthetic biomaterials for development of nanomaterials has been explored for diverse biological activities [1] since the usage of hazardous compounds and their applications in nanomaterials synthesis pose serious problems. Therefore, the design of non-toxic, clean, eco-friendly and biocompatible processes for nanoparticles synthesis has advantages over synthetic ones. Recently, the focus has switched to bioprocessing and usage of green chemistry which uses economical, biocompatible, and environmentally friendly reducing agents to create new nano-textured materials. In this context, the application of cerium nanoparticles in biomedicine and cosmetics have shown pronounced potential [4, 46].

As aforementioned by numerous authors who have discussed the application of nano-scaled CeO2 as antioxidants in biological systems [13], it is clearly suggested that nevertheless, the CeO2 NPs with varied forms and size distributions have so far been produced using physical and chemical techniques. However, the majority of these methods are complicated, involve vacuum conditions, or produce hazardous waste-based precursors. In this aspect, continuous research is being conducted on synthetic processes that are biocompatible and non-toxic CeO2 nanoparticle as part of the growing green synthesis trend [4]. To sum up, the present method has the advantages of using nontoxic reducing agents and simple conventional compounds without any waste by-product. This contribution also describes the fabrication and biological characteristics of green synthesized nanoscale Mg doped CeO2 particles employing HS extract as a powerful reducing agent.

5 Conclusion

In the present study, a green and environmentally friendly method was developed to synthesize CeO2 and Mg doped CeO2 NPs from the H. sabdariffa flower without using any toxic reducing or capping agents. The structure of CeO2 and Mg doped CeO2 NPs were validated using XRD, SEM, EDS and FTIR spectroscopy. The synthesized nanoparticles displayed promising cytotoxicity, antioxidant and anti-inflammatory potentials. Therefore, future studies can focus on the utilization of green synthesized Mg doped CeO2 NPs for preparation of nanodrugs with appropriate consideration of safety procedures. In conclusion, the developed Mg doped CeO2 nanomaterials may be useful in designing efficient and low-cost prodrugs to control the deadliest diseases like cancer.