Green formulation, chemical characterization, and antioxidant, cytotoxicity, and anti-human cervical cancer effects of vanadium nanoparticles: A pre-clinical study

2.1 Material

Bovine serum, Dulbecco's Modified Eagle Medium (DMEM), antimycotic antibiotic solution, 2,2-diphenyl-1- pikrilhydrazil (DPPH), dimethyl sulfoxide (DMSO), decamplmaneh fetal, 4- (Dimethylamino) benzaldehyde, hydrolyzate, Ehrlich solution, and borax-sulfuric acid mixture, Dulbazolic mixture Modified Eagle Medium (DMED), all were afforded from the US Sigma-Aldrich company.

2.2 Preparation and synthesis of vanadium nanoparticles containing Calendula officinalis

To obtain the aqueous extract of the plant, first the plant parts was rinsed by tap water and then dried in shadow. Next, 100 gr of the dried branches of the Calendula officinalis leaves were poured in a container containing 1000 ml boiled water, and the container lid was tightly closed for 4 h. Then, the container^’s content was filtered, and the remaining liquid was placed on a bain-marie to evaporate. Finally, a tar-like material was obtained, which was powdered by a freeze dryer.

The biosynthesis of VNPs@C.officinalis was carried out according to the previous study (Prasad et al., 2018). A 25 ml of aqueous extract solution (10 mg/mL) was added to 25 ml of sodium metavenadatein at the concentration of 0.02 M (Deionized water was used for all steps of this section). The mixture was stirred for 90 min at room temperature. The formed yellow-orange precipitate was triplet washed with water and centrifuged at 12000 rpm for 15 min subsequently. The residue was dried and kept in a vial for the chemical characterization and evaluation of its biological activity.

2.3 Chemical characterization techniques

To characterize and measure of different parameters of the biosynthesized VNPs@C.officinalis, various techniques, including UV–Vis. and FT-IR spectroscopy, XRD, SEM, and EDS techniques, were applied. The UV–Vis. spectra were obtained using Cray 50 UV–Vis. spectrophotometer (200–800 nm); The FT-IR spectra were recorded using a Shimadzu FT-IR 8400 in the range of 400–4000 cm⁻¹(KBr disc); MIRA3TESCAN-XMU was used to report the FE-SEM Images and EDS result. The XRD pattern of VNPs was recorded in the 2θ range of20–80° by a GNR EXPLORER instrument (voltage of 40 kV, a current of 30 mA, and Cu-Kα radiation 1.5406 Å).

2.4 Anti-human cervical cancer properties of vanadium nanoparticles containing Calendula officinalis

The MTT assay is a procedure of colorimetric based on reducing and breaking of yellow tetrazolium crystals by the enzyme succinate dehydrogenase to form insoluble purple crystals. In this method, unlike other methods, the steps of washing and collecting cells, which often cause the loss of a number of cells and increase the work error, have been eliminated and all test steps from the beginning of cell culture to reading the results with a photometer are performed on a microplate, so the repeatability, accuracy and sensitivity of the test are high (Mao et al., 2016; Namvar et al., 2014; Sankar et al., 2014; Tahvilian et al., 2019; You et al., 2012). If the test is performed on cells attached to the plate, an appropriate number of cells (about 2,000 cells) must first be cultured in each of the wells. Then we select the control and test wells and add the appropriate amount of mitogen or drug to the test wells and place the plate in the incubator for the required time so that the desired substance affects the cells (Mao et al., 2016; Namvar et al., 2014; Sankar et al., 2014; Tahvilian et al., 2019; You et al., 2012). At the end of the incubation time, discard the supernatant and add 200 μl of culture medium containing half an mg/ml of MTT solution to each well and put it again in a carbon dioxide incubator for 2 to 4 h at 37 °C. During incubation, MTT is regenerated by one of the enzymes of the mitochondrial respiratory cycle i.e., succinate dehydrogenase. The regeneration and breakage of this ring produce purple-blue crystals of formazan that are easily detectable under a microscope (Mao et al., 2016; Namvar et al., 2014; Sankar et al., 2014; Tahvilian et al., 2019; You et al., 2012). At the end, the optical absorption of the resulting solution can be read at 570 nm and the cells number can be calculated using a standard curve. For each cell line, there is a linear relationship between the number of cells and the light absorption of the final solution. Therefore, to examine each cell type, a standard curve related to the same cell line must be drawn and used (Mao et al., 2016; Namvar et al., 2014; Sankar et al., 2014; Tahvilian et al., 2019; You et al., 2012).

In this research, we used the following cell lines to evaluating anti-human cervical cancer and cytotoxicity effects of vanadium nanoparticles using an MTT method.

1. Human cervical cancer cell lines:

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     C-33 A [c-33a].

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     SiHa.

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     Ca Ski.

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     DoTc2 4510.

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     HT-3.

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     LM-MEL-41

2. Normal cell line:

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     HUVEC.

15 ml of RPMI 1640 medium containing 10% FSC (10 mg/ml penicillin and 100 mg/ml streptomycin) in a culture flask, placed in a CO₂ incubator for 2 h to equilibrate the medium. Under safe conditions (using insulated gloves and goggles) the frozen cell vial was removed from the nitrogen storage tank. To avoid the possibility of explosion of the vial (due to the possible entry of liquid nitrogen into the vial), loosen the lid, after disinfecting the outer surface of the vial with 70% alcohol, under the hood to remove nitrogen gas. Close the vial lid again and immediately melt it in a pan at 37° C. The melting process should be completed in about 1 min and the cells should be avoided from overheating. The medium was added dropwise to the vial and then its contents were taken out and centrifuged with the medium in 15 cc sterile test tubes. After centrifugation, the supernatant was removed and the cells were suspended again in the medium and transferred to a pre-prepared flask containing the medium and FBS and incubated (Arunachalam et al., 2013).

Cell line used in RPMI 1640 medium containing penicillin (100 IU/ML), streptomycin (100 IU/ML), glutamine (2 mmol) and 10% fetal bovine serum (FBS). They were incubated at 37 °C and in an atmosphere containing 0.5 CO₂. Cells began to grow in 75 cm² T-flasks in 15 ml medium with an initial number of 1–2 × 10⁶ cells. After three days and covering the flask bed with the cell, the adhesive layer to the bottom of the flask was separated enzymatically using trypsin-verson and transferred to a sterile test tube for 10 min at 1200 rpm. The cells were then suspended in a fresh culture medium with the help of a Pasteur pipette and the suspension was poured into 100-well plate flat wells (for cell culture) using an 8-channel sampler of 100 μl. One column of wells was kept cell-free and as a plank containing only culture medium. In another column, it was considered to contain culture medium and healthy cells and in other columns, it was considered to contain culture medium and cell line cells. One of these columns, which contained culture medium and cells and did not contain vanadium nanoparticles, was considered as a control (Arunachalam et al., 2013).

The plates were incubated in the incubator for 24 h to return the cells to normal from the stress of trypsinization. After this time, suitable dilutions of the prepared vanadium nanoparticles (0–1000 µl/ml) and 100 μl of each dilution were added columnar to the plate wells (Thus, the final concentration of the studied compound in the wells was halved. Therefore, the concentrations were prepared twice as much to reach the final concentration after being added to the well). The cells were incubated for 37 h at 37 °C and 5% CO2 in the atmosphere. After 72 h, 20 μl of MTT solution (5 mg/ml) was added to each well. The plates were incubated for 3 to 4 h and then the residue was removed and 100 μl of DMSO was added to each well to dissolve the resulting formazan. After 10 min, using shaking the plates, the optical absorption of Formazan at 570 nm was read using a plate reader. Wells containing cells without vanadium nanoparticles were considered as control and the optical density of wells without cells and only culture medium were considered as blank. The percentage of cell viability was calculated using the following formula (Arunachalam et al., 2013): $$\mathit{Cell}viability(\%)=\frac{\mathit{Samble}A}{\mathit{Control}A}\times 100$$

The closer is the obtained value to the IC50 of vanadium nanoparticles, the stronger is the cell viability activity of the material. The graph of the IC50 of the vanadium nanoparticles was produced by drawing the percent inhibition curve versus the vanadium nanoparticles concentration. First, three stock samples with variable concentrations (0–1000 µg/mL) of vanadium nanoparticles were prepared. Then, a serial dilution was prepared from each sample, and IC50 of the above samples was measured separately, following which their mean was calculated (Arunachalam et al., 2013).

2.5 Antioxidant activities of vanadium nanoparticles containing Calendula officinalis

Analysis of antioxidant capacity by the DPPH radical method is a well-known test for measuring the antioxidant power of various compounds. The basis of this method is based on the reduction of free radical DPPH by antioxidants in the absence of other free radicals in the environment. A compound is generally compared to a known antioxidant compound such as Butylated Hydroxytoluene (BHT). Analysis of antioxidant capacity by the DPPH method is a test that has received much attention in the fields of food, pharmaceuticals and biotechnology and is used to develop and introduce new antioxidants. The basis of this method is based on the reduction of free radical DPPH by antioxidants in the absence of other free radicals in the environment, which results in color in the environment whose intensity can be measured by spectroscopy. DPPH is a stable free radical that has an unpaired electron on one of the nitrogen bridge atoms. Radical inhibition of DPPH is the basis for assessing antioxidant capacity (Shaneza ‘Aman et al., 2018): $$\mathit{DPPH}°\left(\mathit{purple}\right)+H--A\to DPPHH\left(\mathit{yellow}\right)+A°$$

DPPH is a stable radical whose methanolic solution has a purple color that shows the highest light absorption at 519–595 nm. The basis of this method is that the DPPH radical acts as an electron acceptor of a donor molecule such as an antioxidant, thus converting DPPH to DPPH2. In this case, the purple color of the environment turns yellow, so the absorption intensity decreases to 595 nm. Antioxidant properties can be determined by measuring the decrease in adsorption intensity by spectroscopy (Shaneza ‘Aman et al., 2018).

In the present study to measure the antioxidant properties of the vanadium nanoparticles, 2 ml of DPPH (100 μM) dissolved in methanol with 2 ml of the vanadium nanoparticles at the concentrations of μg/ml. The resulting mixture was placed at 25 °C for 30 min. Then the samples absorbance was measured at 520 nm by spectrophotometer (Spectramax Gemini XS; Molecular Devices, Sunnyvale, CA) and the amount of antioxidant effect was determined by the below formula (Shaneza ‘Aman et al., 2018): $$\%Inhibition=[(A_{\mathit{blank}}-A_{\mathit{blank}})/A_{\mathit{blank}}]\times 100$$

The blank sample consisted of a mixture of 2 ml of vanadium nanoparticles and 2 ml of methanol and a sample containing 2 ml of DPPH and 2 ml of vanadium nanoparticles with the concentrations used was considered as the negative control. BHT was also used as a positive control. Calculating the 50% inhibition error (IC50) is an excellent way to compare drug activity that dose in which 50% of the final activity of the drug occurs is the criterion for measurement and comparison. In this test, the amount of IC50 for different repetitions of the test was also calculated and is compared with IC50 of BHT molecule, which is an indicator of antioxidant activity. The closer IC50 is to BHT, the stronger the antioxidant activity. In the following experiments, the inhibition concentration of 50% of the vanadium nanoparticles was calculated by plotting the inhibition percentage curve against the vanadium nanoparticles concentration. In the next step, a serial dilution was prepared from each sample and 50% inhibition concentration of three separate samples was measured and its mean was calculated. All experiments were performed three times (Shaneza ‘Aman et al., 2018).

2.6 Statistical analysis

The data were gathered and given into the “SPSS-24” a computer software program and was analyzed by “one-way ANOVA”, and then the Duncan posthoc test (p ≤ 0.01).

3.1 Characterization of vanadium nanoparticles

3.1.1

3.1.1 FT‐IR analysis

The method of FT-IR spectroscopy is used for detecting unknown substances, determining the quality or uniformity of the sample, determining the amount of ingredients in a mixture, identify mixtures of organic and inorganic compounds provided they are both solid or liquid, thin layer analysis, analysis of adhesives, coatings and adhesive enhancers or binders, identification of polymers and polymer mixtures, analysis of solvents, cleaners and detergents unknown, percentage of decomposition or non-polymerization of polymers and paints due to heat, UV or other factors, determination of the degree of crystallization in polymers, and analysis of resins, composite materials and metal nanoparticles (Kang et al., 2014; Karthik et al., 2020).

In FT-IR spectra of metal oxides, the vibration band for metal–oxygen bond usually appears in 400 to 700 cm⁻¹. Fig. 1 presents the FT-IR spectra of VNPs and the plant extract. As it can be seen, the both FT-IR spectra are closed together that confirm the green synthesis of VNPs using C. officinalis extract. The peaks at 459, 541, and 740 cm⁻¹ attributes to the bending vibration of V-O or V-O-V. These peaks for vanadium oxide nanoparticles have been reported previously with a small difference in the wavenumber (Kang et al., 2014; Karthik et al., 2020). FT-IR technique is a reliable method to evaluate the plant secondary metabolites as the capping and reducing agents of sodium metavenadatein precursor to VNPs. The presence of different-IR bands correlates to the presence of various functional groups in C. officinalis extract. For example, peaks in 3417 and 2925 cm⁻¹ related to O-H and aliphatic C-H stretching; the peaks at a range of 1471 to 1662 cm⁻¹ correspond to C = C and C = O stretching, and peak at 1096 cm⁻¹ could be ascribed to ‐C‐O stretching. These peaks can be considered for the presence of various compounds in the plant extract such as phenolic, flavonoid, saponins, quinones, terpenoids which have been reported previously (de Oliveira Carvalho et al., 2018; Długosz et al., 2018; Verma et al., 2018). In biosynthetic of metallic nanoparticles, the secondary metabolite of plant extracts, as reducing, stabilizing, and dispersing agents usually bind to NPs over their functional groups of hydroxyl and carbonyl (Ghidan et al., 2016).

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Fig. 1

FT-IR spectrum of biosynthesized VNPs@C.officinalis and C. officinalis extract.

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3.1.2

3.1.2 XRD analysis

X-ray diffraction is applied to investigate the crystalline materials structures. The X-ray region in the electromagnetic spectrum is in the range between the beam and the ultraviolet. Using this spectral region, information can be obtained about the structure, material, and quantification of the elements. Therefore, x-ray diffraction methods have many applications in decomposition chemistry. For a pure substance, the X-ray diffraction pattern is the same as for a fingerprint. So far, the diffraction pattern of more than 75,000 inorganic and organic compounds has been collected (Karthik et al., 2020). Using this database and with the help of the search and matching method, the composition of each material can be determined. X-ray diffraction is used in characterizing the crystal structure of materials, including measuring the average distances between layers and atomic series, determining the position of a single crystal or grain and the composition of atoms …, examining the crystal structure of unknown materials, determining structural characteristics including lattice parameter, size and shape (Karthik et al., 2020). In the XRD method, the size of the crystals can be determined in certain conditions using the Scherer equation. It is also used to detect crystal phases and their position and to measure thickness, thin films and multilayers. Although X-ray diffraction has many applications, its most important application is in estimating the size of crystals in crystal structures (Karthik et al., 2020).

Fig. 2 shows the XRD pattern of VNPs@C.officinalis. The result, same to FT-IR spectra, approves the synthesis of vanadium oxide. Eight peaks at 2θ values of 25.27, 27.95, 37.63, 44.94, 49.49, 53.56, 66.71, and 76.71 are indexed as (2 0 2), (1 0 3), (2 0 2), (4 1 0), (2 0 5), (4 0 4), (4 0 6), and (2 0 2) planes. These peaks are matched as well as to those of standard database JCPDS no: 85–2422 for vanadium oxide. The peaks at different degrees are also reported previously (Karthik et al., 2020). The average crystal size of VNPs@C.officinalis was calculated using X-ray diffraction according to the Scherrer equation $$D=\frac{k\lambda }{\beta cos\theta }$$

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Fig. 2

XRD Pattern of VNPs@C.officinalis.

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The vanadium oxide nanoparticles had an average crystal size of 28.83 nm.

3.1.3

3.1.3 SEM and TEM analysis

Today, SEM is used not only in materials science, chemistry and physics, but also in many fields such as medical and biological sciences. The high resolution of SEM makes it one of the most-powerful and comprehensive tools for examining and analyzing a wide range of microstructure characteristics of samples at the nanometer to micrometer scale. Using the above technique, we can determine how the particles are placed from each other, their morphology and size (Ahmeda et al., 2020; Baghayeri et al., 2018).

SEM and TEM images of VNPs@C.officinalis are shown in Fig. 3 (a-c). The both techniques depict a spherical morphology for the VNPs. This morphology for biosynthesized VNPs has been reported previously (Aliyu et al., 2017; Karthik et al., 2020). The figure also confirms the uniformity, well dispersed, and homogeneous of the VNPs. Similar to the other metallic nanoparticles, which have synthesized using green chemistry approaches, a propensity to aggregate is observed for VNPs@C.officinalis. This property has been reported by others for VNPs, Zn NPs, SnNPs, AgNPs, and TiNPs (Ahmeda et al., 2020; Baghayeri et al., 2018; Karthik et al., 2020; Mahdavi et al., 2020; Mahdavi et al., 2019; Seydi et al., 2019; Sintubin et al., 2009). The average size of diameter for VNPs was 38.14 nm by SEM images and according to TEM image most of nanoparticles were less than 100 nm. In our review of literature, different sizes have been reported for the biosynthesized V₂O₅ NPs. According to the previous studies, VNPs has been synthesized in various value for the particle size from 10 to 100 nm (Aliyu et al., 2017; de Oliveira Carvalho et al., 2018; Deepika et al., 2020; Talavera et al., 2013).

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Fig. 3

a, b: SEM Images; c: TEM image of VNPs@C.officinalis.

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3.1.4

3.1.4 EDS analysis

EDS is a method that uses X-ray energy to analyze the structure and chemical composition of samples on a small scale. Using the EDS analysis method, qualitative and quantitative analysis can be performed on a wide range of metallurgical, biological, mineral and ceramic samples. Using the obtained information, it is possible to investigate the quantity and quality of specific phases and areas with homogeneous chemical composition. In other words, this method can be used for microanalysis (Deepika et al., 2020). In EDS analysis, by measuring the energy of the emitted rays, we can identify the type of element under study, which is a method of qualitative analysis. By measuring the intensity of X-rays, the concentration of sample elements can be determined, making it possible to quantitatively analyze unknown samples. The higher the observed ipak for the element in the graph, the higher the concentration of that element in the sample. Also, as the energy of the electron beam increases and the atomic weight of the elements decreases, more depth can be obtained from the sample information (Deepika et al., 2020).

The EDS analysis of the synthetic NPs, as a semi-quantitative technique to recognize the elements, is shown in Fig. 4. The diagram presents the elemental composition profile of the VNPs@C.officinalis. The presence of vanadium was confirmed by the signals below of 1Kev (for VLα), around 5 Kev(for VKα), and after5 Kev (for VKβ). These signals are as well as match to those of biosynthesized VNPs that was reported previously (Deepika et al., 2020). The single before 0.5 Kev belongs to oxygen. This signal can be attributed to the oxygen in vanadium oxide nanoparticles and to the organic molecules present in C. officinalis extract that linked to the surface of VNPs@C.officinalis. The presence of carbon on the surface of VNPs@C.officinalis was approved by around 0.3 Kev.

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Fig. 4

EDS analysis of VNPs@C.officinalis.

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3.2 Therapeutic properties of vanadium nanoparticles

Free radicals are molecules that do not have a complete electron shell, which increases the chemical reaction relative to others. Free radicals are formed if you are exposed to tobacco smoke and radiation. In humans, the most important free radical is oxygen. When an oxygen molecule (O₂) is exposed to radiation, it removes an electron from the other molecules, destroying DNA and other molecules (Abdoli et al., 2020; Jalalvand et al., 2019). Some of these changes cause disease. Problems such as heart problems, muscle failure, diabetes and cancer are all caused by these free radicals. Antioxidants act like a broom against free radicals, destroying free radicals and regenerating damaged cells. Laboratory evidence has shown that antioxidants can prevent cancer (Abdoli et al., 2020; Jalalvand et al., 2019).

Studies have shown that the antioxidant features of vanadium nanoparticles green synthesized by pharmaceutical herbs are more significant than other metal nanoparticles. So far considerable antioxidant activities of metallic nanoparticles green-synthesized by several pharmaceutics herbs like Gundeliatournefortii L., Allium noeanum Reut. ex Regel, Falcaria vulgaris, Thymus vulgaris, and Camellia sinensis have been confirmed.Vanadium nanoparticles green-synthesized by pharmaceutics herbs show noticeable antioxidant activities against free radicals formation in the living system. Vanadium nanoparticles green-synthesized-formulated have important redox activities and have a noticeable role in free radicals dismantle (Abdoli et al., 2020; Jalalvand et al., 2019).

In this study, we assessed the antioxidant properties of Calendula officinalis leaf aqueous extract green-synthesized vanadium nanoparticles by using the DPPH test. The assay is used to evaluate the free radical scavenging activities of different antioxidant materials. This approach depends on the reduction of free radical DPPH by antioxidants in the absence of other free radicals, this action generates a color that absorption intensity can be evaluated by spectroscopy. The highest light absorption of DPPH radical at 515–520 nm in which methanolic solution is purple and acts as an electron absorber of a donor molecule like an antioxidant, thus changing DPPH to DPPH2. Consequently, the purple color of the solution becomes yellow, therefore the absorption intensity decreases at 515–520 nm (Shaneza ‘Aman et al., 2018).

We can find out the importance of antioxidant activities by evaluating the absorption intensity reduction through spectroscopy. The scavenging capacity of Calendula officinalis leaf aqueous extract green-synthesized vanadium nanoparticles and BHT at different concentrations expressed as percentage inhibition has been indicated in Fig. 5.

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Fig. 5

The antioxidant properties of vanadium nanoparticles (A) and BHT (B) against DPPH. The numbers indicate the percent of free radical (DPPH) inhibition in the concentrations of 0–1000 μg/mL of vanadium nanoparticles and BHT.

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Oxidation from reactive oxygen species can cause cell membrane disintegration, damage to membrane proteins, and DNA mutation that result is the onset or exacerbation of many diseases such as cancer, liver damage, and cardiovascular disease. Although the body has a defense system, constant exposure to chemicals and contaminants can lead to an increase in the number of free radicals outside the body's defense capacity and irreversible oxidative damage (Shaneza ‘Aman et al., 2018). Therefore, antioxidants with the property of removing free radicals play an important role in the prevention or treatment of oxidation-related diseases or free radicals. Extensive molecular cell research on cancer cells has developed a targeted approach to the biochemical prevention of cancers that the goal is to stop or return cells to their pre-cancerous state without any toxic doses through nutrients and drugs (Abdoli et al., 2020; Jalalvand et al., 2019). Numerous studies have been performed on using natural compounds as anti-cancer agents in relation to appropriate antioxidant activity (Shaneza ‘Aman et al., 2018). It seems the high anti-cervical malignancy properties of the recent vanadium nanoparticles are related to its antioxidant compounds in the Calendula officinalis leaf aqueous extract. Calendula officinalis contain triterpenoid esters and the carotenoids flavoxanthin and auroxanthin (antioxidants and the source of the yellow-orange coloration) (Jalalvand et al., 2019). The leaves contain other carotenoids, mostly lutein (80%), zeaxanthin (5%), and beta-carotene. Plant extracts are also widely used by cosmetics, presumably due to the presence of compounds such as saponins, resins, and essential oils (Jalalvand et al., 2019).

In the antioxidant test, the IC50 of vanadium nanoparticles and BHT were 144 and 201 µg/mL, respectively (Table 1).

In the cellular part of the study (Toxicological part), the treated cells with different concentrations of the present vanadium nanoparticles were assessed by MTT assay for 48 h about the cytotoxicity properties on normal (HUVEC) and cervical malignancy cell lines i.e. C-33 A [c-33a], SiHa, Ca Ski, DoTc2 4510, HT-3, and LM-MEL-41. The absorbance rate was evaluated at 570 nm, representing viability on normal cell line (HUVEC) even up to 1000 μg/mL for vanadium nanoparticles (Fig. 6).

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Fig. 6

The cell viability of vanadium nanoparticles against normal (HUVEC) cell line.

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The viability of malignant cervical cell line reduced dose-dependently in the presence of vanadium nanoparticles. The IC50 of vanadium nanoparticles were 237, 259, 226, 409, 335, and 192 µg/mL against C-33 A [c-33a], SiHa, Ca Ski, DoTc2 4510, HT-3, and LM-MEL-41 cell lines, respectively (Fig. 7 and Table 2).

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Fig. 7

The anti-human cervical cancer properties (Cell viability (%)) of vanadium nanoparticles (Concentrations of 0–1000 µg/mL) against common human cervical cancer cell lines i.e. C-33 A [c-33a] (A), SiHa (B), Ca Ski (C), DoTc2 4510 (D), HT-3 (E), and LM-MEL-41 (F).

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The numbers indicate the percent of cell viability in the concentrations of 0–1000 μg/mL of vanadium nanoparticles against several cervical cancer cell lines.

Metallic nanoparticles have various parameters such as shape, size, texture, etc. Sizes have very significant properties in the therapeutic effects of nanoparticles, some studies reported that small metallic nanoparticles have better entrance into cells and have significant anti-cancer activities. It has been assessed that particle size lower than 50 nm showed an excellent remedial feature in the corresponding cancer cell lines (Mao et al., 2016; Namvar et al., 2014; Sankar et al., 2014; Tahvilian et al., 2019; You et al., 2012). As you see in Figure of FE-SEM of our study, the average size of vanadium nanoparticles synthesized by Calendula officinalis leaf aqueous extract is less than 50 nm. Metallic nanoparticles have been utilized to treat various cancers including Lewis lung carcinoma, human glioma, human lung cancer, uterus cancer, lung epithelial cancer, colon cancer, and mammary carcinoma (Abdoli et al., 2020; Jalalvand et al., 2019).

Maybe important anti-human cervical cancer potentials of vanadium nanoparticles synthesized by Calendula officinalis leaf aqueous extract against malignant cervical cell lines i.e., C-33 A [c-33a], SiHa, Ca Ski, DoTc2 4510, HT-3, and LM-MEL-41due to their antioxidant activities. Corresponding reports have indicated the antioxidant materials like pharmaceutics plants and metallic nanoparticles especially vanadium nanoparticles reduce the volume of tumors by scavenging free radicals (Katata-Seru et al., 2018). Generally, the presence of free radicals in the body causes countless chain reactions in the body that gives rise to the production of more free radicals, also causes a variety of mutations in the structure of DNA and RNA, and cause an uncontrolled increase in malignant cell proliferation and finally damage the body (Beheshtkhoo et al., 2018; Sangami and Manu, 2017). The high concentration of free radicals present in all kinds of cancer, free radicals causes tumorigenesis and angiogenesis (Beheshtkhoo et al., 2018; Katata-Seru et al., 2018; Sangami and Manu, 2017).

Many studies worldwide have reported that vanadium nanoparticles synthesized by pharmaceutics plants have a significant role in eliminating free radicals and are involved in preventing the growth of malignant cells (Oganesvan et al., 1991; Radini et al., 2018).