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BY 4.0 license Open Access Published by De Gruyter Open Access October 27, 2023

Green synthesis, chemical characterization, and antioxidant and anti-colorectal cancer effects of vanadium nanoparticles

  • Yang Nie , Huifang Chen , Junfang Zhu , Bo Li , Haichao Huang , Jianhua Yi EMAIL logo and Rohallah Moradi
From the journal Open Chemistry

Abstract

In this research, we have used a green approach to vanadium nanoparticle (NP) synthesis by the Salvia leriifolia watery extract. The NP characterization was performed by field emission scanning electron microscopy, fourier transform infrared, X-ray diffraction, and energy-dispersive X-ray spectroscopy. The NPs materialized in spherical morphology with an average size of 26.26 nm. The antioxidant activity was assessed using 2-2 diphenyl-1-picrylhydrazil (DPPH) assay, while the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was used to measure anti-colorectal cancer (against HCT-15‎, COLO 320, Caco-2, DLD-1‎, HT-29, and HCT-116 cell lines) and cytotoxicity (against HUVEC cell line) activity of vanadium NPs. Cancer cell line viability decreased dose dependently in the presence of V NPs. The half-maximal inhibitory concentration (IC50) values of V NPs were 149, 125, 173, 83, 131, and 105 µg/mL against Caco-2, COLO 320, DLD-1‎, HCT-15‎, HCT-116‎, and HT-29 cell lines, respectively. In radical scavenging activity, V NPs scavenged DPPH with the IC50 value of 33 µg/mL.

1 Introduction

In recent years, research on herbal medicines to prevent all types of cancer is growing due to the fewer side effects of the chemical drug [1,2,3]. The Salvia genus is an important medicinal plant in different cultures. The genus belongs to the Lamiaceae family, which displays various biological activities such as antioxidant, antimicrobial, anti-diabetic, wound healing, anti-inflammatory, and antitumor [4,5,6,7]. Salvia leriifolia is a plant with several medicinal applications. The plant is used to cure colds, cholera, constipation, liver disorders, and various disorders. The antibacterial, antiulcer, anticonvulsant, and anti-inflammatory activities of the plant have been reported. S. leriifolia is rich in various classes of compounds such as flavonoids, phenolic, and terpenoids [8,9].

Carcinoma is a class of diseases in which cells lose their ability to divide and grow normally and multiply uncontrollably and may spread to the body (metastasis) [10,11,12,13]. The abnormal growth of these cells eventually leads to the formation of large masses (tumors). Tumors are divided into malignant and benign tumors, which are not life-threatening. There is a possibility of cancer at any age, but it increases with age [12,13,14,15]. With the help of technological advances in bioinformatics and molecular techniques, a lot of information has been obtained that will help in the early recognition of cancer, and timely screening for some cancers can help in their early diagnosis [16,17,18,19].

Recently, nanotechnology has become an important and effective technology for science on a global scale. Nanomaterial engineering is a powerful technology that has opened research new ways and development in environmental science, agriculture, cosmetics, material science, bioscience, medicine, food, and information technology. It is applied as a tool to understand how the nano-sized materials change physicochemical properties [20,21,22]. In fact, nanotechnology is a very practical knowledge that includes a wide range of sciences, and developing countries need investment and special attention to this science to improve the situation in the fields of treatment and health, environment, energy, and water. Nanomaterials’ main unit is placed in this range in three-dimensional space [22,23,24,25]. Nanoparticles (NPs) have shown their effects in a broad spectrum against both groups of Gram-negative and Gram-positive bacteria and several cancers. For example, ZnONPs can prevent Staphylococcus aureus, and AgNPs indicate antimicrobial property in a concentration-dependent manner against Pseudomonas aeruginosa and Escherichia coli. However, different types of NPs often have different effects [21,22,23,24]. Their anticancer mechanisms have not been fully determined (9). Anticancer mechanisms of metallic NPs such as vanadium NPs that have been accepted so far are generally described in one of these three models: 1) induction of oxidative stress, 2) release of metal ions, or 3) non-oxidative mechanisms [23,24,25]. There are modern prospects for the recent formulation development based on several types of NPs with various shapes and sizes and outstanding antimicrobial effects [22,23,24,25].

In this study, we focused on the V NP green synthesis using S. leriifolia extract and also evaluated the cytotoxicity properties of the NPs against common human colorectal cancer cell lines, i.e., Caco-2, COLO 320, DLD-1‎, HCT-15‎, HCT-116‎, and HT-29‎.

2 Methods and materials

2.1 Green synthesis of V NPs

A 10 g of the S. leriifolia dried aerial parts was extracted in 200 mL of boiling water. Then, 40 mL of the filtered extract was poured to 40 mL of NaVO3 (0.04 M). The mixture was placed on a stirrer at 35°C for 2 h. The NPs were formed as precipitate in a deep yellow color. Then, this precipitate was centrifuged at a rate of 12,000 rpm for 15 min to separate the NPs. Finally, the NPs were washed with deionized water four times and left in a dark and cold place (4°C) to dry.

2.2 Antioxidant property (DPPH)

In order to check the antioxidant property, 2-2 diphenyl-1-picrylhydrazil (DPPH) test was applied. The color change was measured by spectrophotometry. 1 mL of 1 mM DPPH was added to 1 mL of NPs in different dilutions, shaken well, and kept at 25°C for 0.5 h and in the dark. Then, the absorbance of the mixture at 517 nm wavelength was measured with blank methanol and DPPH solution, and free radical inhibition was calculated using the following formula [22,23]:

DPPH scavenging effect ( % ) [ ( A 0 A 1 ) / A 0 ] × 100 .

2.3 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) (MTT) test protocol

To carry out this research study, Caco-2, COLO 320, DLD-1‎, HCT-15‎, HCT-116‎, and HT-29 colorectal cancer cell lines were used. To assess the cytotoxic effect, different concentrations of NPs were prepared and used in the experiment. The survival percentage of cells was studied after 24, 48, and 72 h. Cells in 1640-RPMI culture medium enriched with 10% fetal bovine serum and streptomycin (50 µg/mL) and (50 IU/mL) penicillin were placed in an incubator at 37°C with 5% CO2. The degree and humidity of 95% were cultivated. After five to six passages, the cells were in the logarithmic phase of growth. After passaging the cells and counting them with a Marienfeld hemocytometer, 3 × 104 cells was poured into each well of the 96-well plate. For 24 h, the cells were placed in the CO2 incubator of Mamrat Company with the culture medium containing serum, so that the cells stick to the bottom of the well. Then, the wells were washed with PBS buffer and treated with 100 µL of fresh medium and 20 µL of the desired concentrations, and each treatment was repeated three times. Then, the plates were placed in a CO2 incubator at a temperature of 37 degrees for 24, 48, and 72 h. After the aforementioned times, it was removed from the previous environment and washed with buffer. Then, 100 µL of fresh medium without serum and 10 µL of MTT solution (Sigma) were removed and added to wells and placed in a CO incubator for 2 h. After this period of time, the supernatant of the wells was discarded and 200–150 mL of dimethyl sulfoxide (Shikma Company) was added to each well for cell lysis. Then, the light absorption belonging to each well at a wavelength between 570 and 590 nm was read by the Dlagnostic device and used to calculate the viability of the cells through the following formula:

Cell viability ( % ) = Sample A Control A × 100

In this research, different concentrations of NPs were used as treatment groups and a group without NPs as a negative control. At the end, the treated cells were photographed using a Zeiss light microscope (Axiostar Plus) equipped with a Canon (Japan) camera [22,23].

Statistical analysis of the data was performed by SPSS24 software; according to the results of the Shapiro–Wilk test, all the data had a normal distribution. Statistical analysis of the data was performed with one-way analysis of variance tests (p < 0.05).

3 Results and discussion

3.1 Optimization of green synthesis of vanadium NPs

To obtain the optimization condition for the synthesis of vanadium NPs, the synthesis was run at different conditions, including different temperatures (25, 35, 45, 55°C), times (1, 2, 3, 4 h), volumes of extract (30, 40, 50, 60 mL), and NaVO3 concentration (0.02, 0.03, 0.04, 0.05 M). The highest yield was obtained for temperature of 35°C, time of 2 h, volume extract of 40 mL, and concentration of 0.04 for NaVO3. The solid-state stability of NPs was checked by the color change, the yellow color of NP was stable, and no change was observed in the color of NPs.

3.2 X-ray diffraction (XRD) results of vanadium NPs

Vanadium NP XRD pattern is indicated in Figure 1. According to the results, the NPs are crystalized in the V2O5 formula. The obtained data are matched to joint committee on powder diffraction standards no: 85–2422 as the standard data. The values of 22.82, 25.18, 27.82, 31.02, 31.87, 40.88, 45.00, and 47.78 for two thetas are indexed for the planes of the signals at (001), (101), (110), (301), (011), (002), (411), and (600) respectively. Similar signals have been reported for V2O5 [24].

Figure 1 
                  XRD pattern of V NPs.
Figure 1

XRD pattern of V NPs.

3.3 SEM results of vanadium

In recent years, SEM imaging is developed as a sufficient technique to study the morphology of the material. In this method, the surface is scanned using an electron beam with a specific energy [25,26,27]. The image is produced by the collected data with the detector. The resolution of the images depends on the synthesis condition of NPs [28,29,30,31,32]. Figure 2 (a–d) reveals the field emission scanning electron microscopy (FE-SEM) pictures of V NPs. The images show a spherical morphology. Vanadium NPs are aggregated, which is known as a familiar property of metallic NPs [24,25,26,27]. The aggregation of vanadium NPs was observed for all selected optimization conditions of the synthesis. The NPs are produced in an average size of 26.26 nm. Vanadium NP, which was synthesized using green chemistry approaches, ranged in size from 15 to 90 nm [28,29,30,31,32].

Figure 2 
                  FE-SEM pictures of V NPs.
Figure 2

FE-SEM pictures of V NPs.

3.4 Fourier transform infrared (FT-IR) results of vanadium NPs

FT-IR spectrum of V NPs is shown in Figure 3. The peaks at 430 and 709 cm−1 are attributed to V–O and V–O–V bonds. The bands at 821 and 1,018 cm−1 are attributed to V2O5 crystal [33]. The other peaks in the regions of 1,400–1,700 and 2,850–3,300 cm−1 belong to the organic functional groups of C═O, C═C, C–H, and C–O, which are bending to the surface of V NPs and play an important role to cap and reduce the synthesis of V NPs.

Figure 3 
                  FT-IR spectrum of V NPs.
Figure 3

FT-IR spectrum of V NPs.

3.5 Energy-dispersive X-ray spectroscopy (EDS) results of vanadium NPs

Figure 4 reveals the EDS diagram of V NP. The elemental analysis of NPs revealed the presence of vanadium by the signals at 0.6, 4.8, and 5.4 for VLα, VKα, and VKβ, respectively. The signals below 0.3 KeV confirm the presence of oxygen and carbon; similar signals for vanadium NPs have been reported previously [30].

Figure 4 
                  EDS analysis of V NPs.
Figure 4

EDS analysis of V NPs.

3.6 Antioxidant and anticancer results of vanadium NPs

In the recent research, V NP radical scavenging capacity is expressed as a percentage inhibition, which is shown in Figure 5.

Figure 5 
                  Antioxidant potentials of butylated hydroxytoluene (BHT) and V NPs.
Figure 5

Antioxidant potentials of butylated hydroxytoluene (BHT) and V NPs.

There are many studies about biological and therapeutic effects of the NPs [34,35,36,37,38,39,40,41]. Nam et al. designed smart gold NPs to accumulate in the relatively acidic environment of the cell. Considering that the size of these particles was very small (10 nm), their entry into the internal environment of cancer cells was easily done. These NPs had both negative and positive charges on their surface. The electrostatic state between the charges of the cell caused the accumulation of NPs in the space inside the cell. The entry of NPs into the cell was monitored using a dark field microscope, and it was observed that this accumulation and their photothermic effects cause the destruction of cancer cells [34]. In the study of Qiu et al., compounds of cathalocyanine NPs were prepared and synthesized to deliver hydrophobic photosynthesizers to the desired tissue in the photodynamic therapy method. It is one of the effective drug delivery ways to the target tissue, which was used to treat cancer. Gold NPs stabilized with phthalocyanine had a size of about 2–4 nm, and phthalocyanine was present in nano-dimensions on the surface of the NPs. In a method that can be referred to as cancer treatment using a Trojan horse, the compounds of this NP were incubated with a cancer cell line, and it was observed that these NPs are effectively absorbed by the cells. This caused significant cell death. The effectiveness of photodynamic therapy with NPs was twice as high as when the uterus (HeLa) was not treated with NPs. In the end, this research showed that gold compounds can be a very suitable tool for delivering hydrophobic photosynthesizers used in photodynamic therapy of cancer cell culture [35]. Of course, the research of Samberg et al. (2009) showed that uncoated silver NPs have the ability to induce toxic effects on various human cells, but on the other hand, if these NPs are covered with a layer of carbon, these toxic effects will not be observed [36]. In the study of Zeng et al., a drug delivery system, including acid, cholic Poly(lactic-co-glycolic acid) (PLGA), and vitamin E TPGS, was designed to deliver and control the stability of docetaxel to the target tissue in order to treat uterine cancer. Cholic acid consists of steroid units and three hydroxyl groups. This substance can cause better biocompatibility in the substances that come with it. The triple complex of these materials (cholic acid-PLGA–vitamin E) has a high ability to enter the cell and also has anti-tumor effects. Therefore, this substance can be used as a modern biological molecular substance in pharmacology and cancer treatment with high efficiency [37]. In the research of Ma et al. who used poly(ε-caprolactone)-poly(lactic acid)-triglycerides NPs for the chemotherapy of uterine cancer, docetaxel was loaded on these NPs and its cellular uptake and toxicity in HeLa cells were compared with the commercial formulation of Taxotere. These NPs entered HeLa cells well and were much more effective in reducing the number of tumor cells [38]. Also, in Qiu et al.‘s study, a new biodegradable NP called TPGS-b-PCL-ran-GA was used for simultaneous drug delivery to investigate its synergistic effect on uterine cancer treatment. Experiments conducted with this method on HeLa cells of naked mice showed that the combination of these drugs with various NPs can effectively and simultaneously deliver them to HeLa cells. As a result of this experiment, it was seen that HeLa cell life significantly decreased (p < 0.01). Also, using this mechanism can reduce the growth of tumors and even eradicate them [35].

The cytotoxicity of metallic NPs is affected by the variation in NP size [42]. NPs indicated a necessary activity on reactive oxygen species (ROS) generation, lactate dehydrogenase activity, and cell viability in a size-dependent manner in several cell lines [42]. It is evident that surface reactivity, volume ratio, and surface area can be changed with particle size [43]. In addition, deposition velocity, attachment efficiency, mass diffusivity, and sedimentation velocity of NPs over the solid or biological surfaces are considerably affected by particle size [43,44,45,46,47]. Particle size can also affect the interaction of mammalian cells [45,46]. Different studies have been carried out to assess the NP size effect on several cell lines. In support of this statement, Carlson et al. worked with  55 and 15 nm hydrocarbon-coated NPs and found that the 15 nm NPs can generate more ROS compared with 55 nm NPs in a macrophage cell line [48]. Using different cell lines (SGC-7901, MCF-7, HepG2, and A549), Liu et al. found that 5 nm NPs were more toxic than 50 and 20 nm NPs [47].

In this research, the colorectal and HVEC normal cell lines were treated with V NPs using MTT assay. The results of HUVEC did not reveal any cytotoxicity effect of V NPs (Figures 69).

Figure 6 
                  The anti-colorectal cancer potentials of NPs against Caco-2 and COLO 320.
Figure 6

The anti-colorectal cancer potentials of NPs against Caco-2 and COLO 320.

Figure 7 
                  The anti-colorectal cancer potentials of NPs against DLD-1‎ and HCT-15.
Figure 7

The anti-colorectal cancer potentials of NPs against DLD-1‎ and HCT-15.

Figure 8 
                  The anti-colorectal cancer potentials of NPs against HCT-116‎ and HT-29.
Figure 8

The anti-colorectal cancer potentials of NPs against HCT-116‎ and HT-29.

Figure 9 
                  The cytotoxicity potentials of NPs on healthy cells (HUVEC).
Figure 9

The cytotoxicity potentials of NPs on healthy cells (HUVEC).

Cancer cell line viability decreased dose dependently in the presence of V NPs. The IC50 values of V NPs were 149, 125, 173, 83, 131, and 105 µg/mL against Caco-2, COLO 320, DLD-1‎, HCT-15‎, HCT-116‎, and HT-29 cell lines, respectively (Figures 69).

4 Conclusion

In summary, the green synthesis of vanadium NPs was performed successfully using a green chemistry approach using a plant extract. The NPs were recognized by chemical techniques such as FE-SEM, XRD, FT-IR, and EDS. V NPs were formed in a spherical morphology with an average size of 26.26 nm.

The findings of the research revealed the benefits of V NPs to cure colorectal cancer. A dose-dependent vanadium NPs were observed for the malignant cancer cell line viability. Furthermore, the NPs exhibited a better antioxidant activity against DPPH compared to BHT as the positive.


# Yang Nie and Huifang Chen contributed equally to this work.


  1. Funding information: This study was supported by Medical Science and Technology Research Foundation of Guangdong Province (No. B2018184), Foundation of Guangdong Provincial Bureau of Traditional Chinese Medicine (No. 20192063, 20201247), Natural Science Foundation of Guangdong Food and Drug Vocational College (No. 2017YZ001, 2018ZR024), Science and Technology Plan Foundation of Huizhou City (No. 2018Y277), and Guangdong Health Committee Fund (Project No.: a2021203.2021).

  2. Author contributions: All authors had the same role in conceptualization, data curation, formal analysis, acquisition, investigation, methodology, project administration, resources, software, supervision, validation, visualization, writing – original draft, and writing – review and editing.

  3. Conflict of interest: There is no conflict of interest.

  4. Ethical approval: The conducted research is not related to either human or animal use.

  5. Data availability statement: The datasets generated during and/or analyzed during this study are available from the corresponding author upon a reasonable request.

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Received: 2023-05-25
Revised: 2023-06-30
Accepted: 2023-07-17
Published Online: 2023-10-27

© 2023 the author(s), published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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