Facile preparation of Fe₂O₃ nanoparticles mediated by Centaurea alba extract and assessment of the anti-atherosclerotic properties

2.1 Materials

The Centaurea alba plant, which is applied in this study as a factor for reducing metal ions and stabilizing nanoparticles, has been prepared. FeCl₃·6H₂O, ethanol and methanol were purchased from Merck.

2.2 Preparation and extraction of aqueous extract

First, it was necessary for the experiments to provide a pure solution of the extracts of the leaves of Centaurea alba plant. To do this, 1.5 g of leaf powder was added to 60 mL of deionized water. Then, the resulting solution is placed on a hot plate at 60° C for 2.5 h to dissolve plant agents and metabolites well in the solvent. The resulting solution was poured into the Falcon tubes, and centrifuged at 5000 rpm for 10 min to separate the plant residue from the extract. Then, the resulting extract was moved into test tubes, and placed in the refrigerator to keep it fresh and healthy.

2.3 Green synthesis and chemical characterization of iron nanoparticles

Various factors were involved in the synthesis of Iron nanoparticles (FeNPs). These factors include temperature, time, and concentration of iron and volume ratio of iron solution to extract solution. Different experiments were designed and tested to achieve the optimal conditions for each of these factors. During the complete duration of the experiment, the concentration of the extract was kept constant. In a typical procedure, 2 mL of the prepared extract solution was diluted in 30 mL of deionized water. Then, 20 mL of FeCl₃·6H₂O solution (0.001 M) was added to the extract solution with a volume ratio of 1 to 1.5 mL and the mixture was placed on a hot plate at 60 °C while being mixed. After 30 min of the reaction time, the color of the reaction solution changes completely and the synthesized nanoparticles collect as sediment at the bottom of the reaction vessel.

Different spectroscopic and imaging techniques were used to evaluate and identify the FeNPs. At the first step, UV–vis spectrum (Cary UV–Vis 4000, Agilent) was used to identify iron nanoparticles. Typically, iron nanoparticles have two absorption spectra in the range of 200–300 nm. The FTIR spectrum (PERKIN-ELMER, RXI) was used to identify nanoparticles and plant metabolites in plant extracts. Electron microscope techniques such as SEM (LEO 1430VP, UK) and TEM (H9500, HITACHI) were also used to examine the shape and size of nanoparticles.

2.4 Antioxidant activities of FeNPs

Degree of decolorization of the 2 and 2-diphenyl-1-picryl-hydrzyl purple solution corresponds to the ability of the hydrogen atoms or electrons to give off different compounds and nanoparticles in the test solution, where DPPH was used a stable radical compound. Thus, 100 μl of various dilutions of nanoparticles in methanol was added to 10 mL of 0.005% DPPH solution in methanol. The samples were analyzed against blank at 518 nm after incubating it for an hour at room temperature. The formulae mentioned below depict the DPPH inhibition percentage (Abdoli et al., 2020): $$\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\left(\%\right)=\frac{\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{A}.}{\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{A}.}x100$$

In this formula, “Control A” shows the negative control of light absorption that lacks nanoparticles, and “Sample A” expresses the amount of light absorption of different concentrations of nanoparticles (Arunachalam, 2003).

2.5 Cytotoxicity potentials of FeNPs

MTT assay serves as a quantitative technique for the determination of live cell population. It is well known that live cells could do oxidative metabolism, in case of MTT assay, the oxidative process leads to the breaking down of MTT dye and results in producing a dye ranging from yellow to blue (Pashaie et al., 2017).

In this research, we used the HUVEC cell line (Human normal cell line) to evaluate the cytotoxicity effects of FeNPs using an MTT method.

To prevent the fungal growth, the cells were maintained in a DMEM medium with 1% penicillin/streptomycin antibiotic and 10% bovine embryos.Temperature of 37 ° C and 5% CO₂ with 95% moisture are considered ideal for cell growth, which was provided by the NÜVE incubator (EC160 model).When the cell reaches 70% of cell growth, they were separated from the flask by using trypsin-ethyldiaminetetraacetic acid and has been centrifuged for 1-6 min at 1700 rpm.Cell precipitate was prepared in suspension in 1 ml of culture medium. The cell suspension was scrutinized to determine its viability by mixing them with equal proportion of tryptan blue and the cells were counted with a neobar slide under light microscope. After confirming the infectivity of the cells, cells with 90% viability was proceeded for further process (Pashaie et al., 2017).

Tetrazolium salt calorimetric method (MTT) as exploited to investigate the effect of nanoparticles on proliferation.For this test, 10⁴ cells were added to each 96-well plate well. The cells were incubated for 24 h and concentration of 1-1000 µg/mL was treated on cancer and normal cells for 24, 48 and 72 h.

After these times, 20 μl of MTT solution and 200 μl of base culture medium were added to each well. The plate was then placed at 37°C in dark CO₂ incubated for about 4 hours in the dark.After the incubation, each well was filled with 100 microliters of DMSO, where the optical readings were taken at 492 nm and 630 nm using the ELISA. The cell viability can be evaluated by the below expression (Pashaie et al., 2017): $$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}(\%)=\frac{\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{A}.}{\mathit{ControlA}.}x100$$

2.6 Anti-atherosclerotic potentials of FeNPs

2.7 Statistical analysis

To compare the results, in addition to the formula mentioned above, which was calculated as an average of 5 repetitions of experiments. The results were analyzed using SPSS software version 22 and the statistical differences between the treatments were examined by t-test and P ≤ 0.01 was considered significant.

3.1 Chemical characterization of FeNPs

3.1.1

3.1.1 Nanoparticle analysis using UV–Vis spectroscopy

The effect of reaction temperature, time of the reaction, iron concentration and amount of extract solution on the synthesis of FeNPs were investigated using UV–Vis spectroscopy. The results are presented in Fig. 1. In each experiment one of the factors was evaluated while the other factors were kept constant. According to the results, the optimum condition for preparation of FeNPs was 0.001 M FeCl₃ solution, 1.5 vol ratio of extract solution to Iron solution, 60 °C and 30 min reaction time.

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Fig. 1

UV–Vis analysis of FeNPs.

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3.1.2

3.1.2 Analysis of FeNPs using FT-IR spectroscopy

FT-IR technique is another qualitative method in characterization of nanoparticles. The presences of the bands in the specific wavenumber regions shows useful information about the metallic nanoparticles. For example, if the nanoparticles are formed as metal oxide, the peaks at 400 to 700 cm⁻¹ belong to metal oxygen bond. The presences of the peaks at the other region are attributed to the different bonds for organic compounds present in the plant extract that bind to the NPs. The FT-IR spectra of AgNPs is shown in Fig. 2. The peaks at wavenumbers of 437, 530, and 5581 cm⁻¹ belong to Ag-O bond. Literatures has stated the peaks for green synthetic AgNPs with a little difference in wavenumber (Karpagavinayagam and Vedhi, 2019; Yazdani, 2019; Ting and Yien, and Jia En Chin, 2020; Fazlzadeh, 2017), Moreover, the peaks at 3490 and 2979 cm⁻¹ (O-H and aliphatic C-H stretching), 1480 to 1751 cm⁻¹ (C = C and C = O stretching), (1031 cm⁻¹ ‐C‐O stretching) belong to the various bonds of organic compounds in A. maurorum extract that exist as the plant secondary metabolites. These compounds can be comprised different class of compounds such as phenolic, flavonoid, triterpenes, which were reported previously (El-Moslamy, 2017; Karpagavinayagam and Vedhi, 2019; Yazdani, 2019; Ting and Yien, and Jia En Chin, 2020; Fazlzadeh, 2017; Katata-Seru, 2018).

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Fig. 2

FT-IR Spectrum of FeNPs synthesized using Centaurea alba extract.

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3.1.3

3.1.3 Analysis of nanoparticles using FESEM and TEM images

To study the morphological features and the size of the obtained FeNPs, FESEM was exploited. Fig. 3 shows the FESEM image of FeNPs. It could be observed from the figure that the ontained NPs exhibits spherical morphology, where their size range from 10-52 nm.

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Fig. 3

FESEM image of FeNPs.

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Fig. 4 shows the TEM image of FeNPs. The nanoparticles are surrounded by a thick layer of plant organic molecules, which reduces the image resolution of the nanoparticles. These observations are very similar to the observations in other research (Alam, 2019; Devatha et al., 2018; Önal, 2017; Bibi, 2019; Veisi et al., 2019).

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Fig. 4

TEM image of FeNPs.

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3.2 Cytotoxicity, antioxidant, and anti-atherosclerotic activities of FeNPs

Examining the rate of survival, mortality and proliferation under various conditions has become crutial for the determination of cytotoxic effects.Cytotoxicity tests are carried out to assess the effects of various compounds in the cell. Being a non-radioactive colorimetric system, MTT assay greatly contributed to biocompatibility studies of various materials. In MTT analysis according to ISO 10993–5 international standard, different equipments are tested for cytotoxicity, if they do not have toxic effects, they will obtain the necessary standards and licenses and enter the buying and selling market. For testing the cell viability, MTT assay has been considered to be ideal. The primary aim and role of the MTT assay is to assess the toxic effect of compounds, drugs or other supplements. It has also been mentioned as a method used for the determination of cell proliferation or counting in numerous literatures (Tahvilian et al., 2019; You et al., 2012; Mao, 2016; Beheshtkhoo et al., 2018). By affecting the intracellular organs, MTT analysis provides the difference between live and dead cells.In this assay, the cultured cells are treated with desired compounds to evaluate its toxicity. The cell viability was determined for each concentration of the substance. Even though the method is primarily used for water-soluble compounds, it has been recently used for components soluble in organic solvents as well. Hormones, cytokines,growth factors and mitogens greatly influence the behavior and rate of cell proliferation.Moreover, some drugs, usually those which are cytotoxic, results in necrosis or apoptosis of cells or even decrease the cell proliferation rate (You et al., 2012; Mao, 2016; Beheshtkhoo et al., 2018; Radini et al., 2018; Sangami and Manu, 2017). Typically, MTT assay is based on the mitochondrial activity, whose activity is usually stable in living cells. Correspondingly, change in activity of cells is linked to the mitochondrial activity.Work error is usually prevented in MTT assay, as the washing and shrinking steps are eliminated (Katata-Seru et al., 2018; Sankar et al., 2014; Namvar et al., 2014).

In this study, the cells (HUVEC) were treated with FeNPs in different concentrations and the resultant toxicity has been assessed by MTT assay for 48 h.

The absorbance rate was evaluated at 570 nm, which represented viability on normal cell line (HUVEC) even up to 1000 μg/mL for FeNPs (Fig. 5).

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Fig. 5

The cytotoxicity effects of iron nanoparticles against normal (HUVEC) cell line.

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Antioxidant property of the green synthesized FeNPs has been evaluated by DPPH test as a common free.Free radicals are highly reactive unstable molecules or atoms with a free electron, which is formed as a result of a bond breakage (Abdoli et al., 2020; Sujayev et al., 2020; Hummers and Offeman, 1958; Kooti et al., 2017; Shaneza, 2018). The main important FR in humans is O₂. Oxygen molecules in the biological systems are egenrally exposed to various harsh environmental conditions such as stress, radiation, smoke etc, which leads to the production of free radicals and eventually destroys other molecules, compounds and DNA. Antioxidants are generally used to fight those free radicals from damaging (Abdoli et al., 2020). Antioxidants are substance that eliminate the threat of free radicals by inhibiting their production or transforming them into less reactive forms.In our immune system, as a part of inflammatory response, huge number of free radicals are produced by phagocytes, macrophages and neutrophils to fight against pathogens (Abdoli et al., 2020; Sujayev et al., 2020; Hummers and Offeman, 1958). Here, the presence of antioxidants are considered inevitable to prevent any harmful effects against the immune cells (Sujayev et al., 2020; Kooti et al., 2017). Natural antioxidants are found in legumes, fruits and vegetables, which are classified as dietary antioxidants and potentially reduce disease (Sujayev et al., 2020; Hummers and Offeman, 1958; Kooti et al., 2017; Shaneza, 2018). Even though synthetic antioxidants are such as BHT are being produced, they also possess comes up with carcinogenic and hepatotoxic effects. This lead to the reversal of attention to natural resources (Abdoli et al., 2020; Sujayev et al., 2020; Hummers and Offeman, 1958; Kooti et al., 2017; Shaneza, 2018; Arunachalam, 2003). Fig. 6 represents the scavenging capacity of green synthesized FeNPs and BHT at different concentrations.

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Fig. 6

The antioxidant activities of iron nanoparticles and BHT against DPPH.

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In the antioxidant test, the IC50 of FeNPs and BHT against DPPH free radicals were 287 and 191 µg/mL, respectively (Fig. 6).

Feeding rats with high cholesterol supplemented diet resulted in a significant increase in plasma cholesterol, LDL and HDL levels (Fig. 7). The LDL and cholesterol level (P ≤0.01) reduced after administration of FeNPs and STN in test groups. Moreover, treatment with STN (10 mg kg⁻¹) significantly reduced TC level (Fig. 7), (P ≤ 0.01).

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Fig. 7

Effects of FeNPs on plasma lipid profile concentration ((mg dL ^-1)) after six months in control and experimental animals (n = 6). HCD: High cholesterol diet, HCD + L: Low dose (100 µg kg-1) of FeNPs, HCD + M: Medium dose (200 µg kg-1) of FeNPs, HCD + H: High dose (400 µg kg-1) of FeNPs, and HCD + STN: Atorvastatin (10 mg kg-1) group.

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In HCD group, the blood triglyceride levels increased significantly compared to control rats (P ≤ 0.01) (Fig. 7). However, the treatment group exhibited decrease in blood glucose level.

The media and intima thickness of the thoracic aortas from different groups were depicted in Fig. 8. It could be observed from the Fig. 8 that the intimal surface of aorta is smooth, whereas it is found to be intact in the control group. In HCD group, both the intimal and medial layers of aorta showed abnormalities.Furthermore, an increase in diameter in aortic wall was observed upon chronic HCD consumption. The treated group exhibited no significant changes in the histological appearance of aorta, which was very similar to STN and control group. However, the HCD group exhibited medial thickness and vascular fibrosis in the coronary artery wall, which was characterized by appearance of high density blue colour fiber. In 400 µg kg^-1 treated group, vascular fibrosis and blue density were lowered (Fig. 8).

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Fig. 8

Measurements of intima and media layers thickness (µm) of thoracic aortas from rats treated with or without FeNPs (n = 6). HCD: High cholesterol diet, HCD + L: Low dose (100 µg kg-1) of FeNPs, HCD + M: Medium dose (200 µg kg-1) of FeNPs, HCD + H: High dose (400 µg kg-1) of FeNPs, and HCD + STN: Atorvastatin (10 mg kg-1) group.

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Lesions were observed in the medial and intimal layers of HCD group in the microscopic examination of aortic tissue.Atherosclerotic plaques initially develop as intimal precursor lesions at sites which are predisposed to lesion formation (Bentzon et al., 2014). Based on the microscopical pathology, atherosclerotic lesions were categorized into 6 class. Type I and II were classified as fatty streak precursor lesions. Type III has been classified as transition state lesions, which leads to type IV and Type V has the features of fibrosis and calcification, which is known as the complicated plague. Type VI is found with fissure, surface thrombi with or without plaque rupture , surface erosions and plaque hematomas (Stary et al., 1995; Stary, 2000). Rats have been known as cholesterol resistant laboratory animals (Park et al., 2005). To overcome this tolerance, along with HCD, we inhibit the secretion of thyroid hormones by PTU. Since thyroid hormone directly promotes cholesterol metabolism by liver, PTU induces high blood levels of cholesterol (Wang et al., 2014).