Anti-human colon cancer properties of a novel chemotherapeutic supplement formulated by gold nanoparticles containing Allium sativum L. leaf aqueous extract and investigation of its cytotoxicity and antioxidant activities

2.1 Material

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

2.2 Synthesis of AuNPs

To obtain the aqueous extract of the plant, 250 gr of the dried branches of the A. sativum leaves were poured in a container containing 2000 mL boiled water, and the container lid was tightly closed for 4 h. Then, the content of the container 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 extract of the A. sativum leaves was prepared for the green synthesis method was extracted with distilled water in the microwave. Green synthesis of gold nanoparticles was started with such a process combination of 100 mL HAuCl₄·H₂O at concentrations of 10⁻³ M and 400 mL of A. sativum leaf aqueous isolate (20 μg / mL) in a cylindrical flask.

The reaction mixture was kept under magnetic stirring for 12 h at room temperature. At the end of the reaction time, the dark yellow colored colloidal solution of Au was formed. The solution was centrifuged at 10,000 rpm for 15 min. The precipitate was sprayed with water and then resuspended (Hemmati et al., 2019).

Various analytical techniques were used to characterize the Ag nanoparticles:

The biomolecules related to the Au nanoparticles reduction including secondary metabolites were detected by the FT-IR (Shimadzu IR affinity.1). In the UV–Vis spectroscopy analysis, characteristic absorption bands of Au metal were examined before and after the nanoparticles synthesis process. TEM and FE-SEM analysis of the Au nanoparticles was performed with JEOL 200 kV by placing them on a carbon-coated copper grid.

2.3 Investigation of the antioxidant capacity of AuNPs

The DPPH method is a common method for the assessment of the antioxidant activity of plant species and metallic nanoparticles. It is based on trapping the free radicals of the material, called DPPH, using antioxidant agents which reduce the absorption rate at 517 nm wavelength. When the DPPH solution is mixed with a material that can donate hydrogen atom, radical resuscitation is formed, which is followed by color reduction. This reaction eliminates the purple color; whose index is the formation of an absorption band at 517 nm (Hosseinimehr et al., 2011).

To determine the radical scavenging activity of the HAuCl₄, A. sativum, and AuNPs, 1 mL of 50 µm DPPH was combined with 1 mL of variable concentrations (0–1000 µg/mL) of HAuCl₄, A. sativum, and AuNPs. Then, they were transferred to the 37 °C for 1 h. The samples absorption rate was determined at 517 nm by a spectrophotometer, and the antioxidant activity was calculated by the below formula (Hosseinimehr et al., 2011): $$\%\text{Inhibiton}=[(A_{\mathit{blank}}-A_{\mathit{sample}})/A_{\mathit{blank}}]\times 100$$

The blank sample contained 1 mL methanol and 1 mL HAuCl₄, A. sativum, and AuNPs, and a sample of 1 mL DPPH (Hosseinimehr et al., 2011).

Calculation of half-maximal inhibitory concentration (IC50) is a suitable method for comparison of the activity of pharmaceutical materials. In this method, the measurement and comparison criterion is the concentration in which 50% of the final activity of the drug occurs. In this experiment, the IC50 of various repeats is estimated and compared with the IC50 of BHT, which is introduced as the antioxidant activity index. The closer is the obtained value to the IC50 of BHT, the stronger is the antioxidant activity of the material. The graph of the IC50 of the extract was produced by drawing the percent inhibition curve versus the extract concentration. First, three stock samples with variable concentrations (0–1000 µg/mL) of HAuCl₄, A. sativum, and AuNPs 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. BHT, with different concentrations, was considered positive control. All experiments were performed in triplicate (Hosseinimehr et al., 2011).

2.4 Measurement of cell toxicity of AuNPs

To determine the anti-colon cancer effects of the HAuCl₄, A. sativum, and AuNPs, we used following cell lines according to the MTT assay:

• (1)

  HUVEC: Normal cell line.

• (2)

  HT-29: Colorectal adenocarcinoma.

• (3)

  HCT 116: Colorectal carcinoma.

• (4)

  HCT-8 [HRT-18]: Ileocecal colorectal adenocarcinoma.

• (5)

  Ramos.2G6.4C10: Burkitt's lymphoma.

They were then cultured as a monolayer culture in 90% RPMI-1640 medium and 10% fetal serum and supplemented with 200 mg/mL streptomycin, 125 mg/mL penicillin, and 8 mg/mL amphotericin B. The culture was then exposed to 0.5 atmospheric carbon dioxide at 37 °C, on which the tests were performed after at least ten successful passages. MTT assay a method used to investigate the toxic effects of various materials on various cell lines, including non-cancer and cancer cells. To evaluate the cell toxicity effects of the compounds used in this research, the cells were transferred from the T25 flask to the 96-well flasks. In each cell of the 96-cell flasks, 7000 cells of cancer and fibroblast cell lines were cultured, and the volume of each cell was eventually increased to 100 µL. Before the treatment of the cells in the 96-well flak, the density of cells was increased to 70%, so the 96-well flasks were incubated for 24 h to obtain the cell density of 7 × 10³. Next, the initial culture medium was discarded, and variable concentrations (0–1000 µg/mL) of HAuCl₄, A. sativum, and AuNPs were incubated at 37 °C and 0.5 CO₂ for 24, 48, and 72 h. Then, 20 µL MTT was added to each well after a certain amount of time. Next, 100 µL DMSO solvent was added to each well. They were then kept at room temperature for 25 min and read at 490 and 630 nm by a microtitre plate reader (Arulmozhi et al., 2013).

The cell lines were treated with the hydroalcoholic extract (1.25 mg/mL), which inhibited about 20% of the cell growth. Annexin/PI method was used to determine the apoptosis level in the treated and control cell lines using a flow cytometry machine. To perform experiment, the cell lines were treated with a variable concentration (0–1000 µg/mL) of HAuCl₄, A. sativum, and AuNPs for 24 h. Cells were irrigated with phosphate-buffered saline (PBS). After centrifugation, buffer binding was added to the obtained precipitate. Then, 5 µL Annexin V dye was added and incubated for 15 min at 25 °C. Cells were washed with the binding solution, following which 10 µL PI dye was added. Finally, cell analysis was done by a flow cytometry machine according to the below formula (Arulmozhi et al., 2013): $$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}(\%)=\frac{\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{A}.}{\mathit{Control}A.}\times 100$$

2.5 Statistical analysis

The obtained results were analyzed by SPSS (version 20) software using one-way ANOVA, followed by Duncan post-hoc test (P ≤ 0.01).

3.1 UV–visible spectroscopy

At the start of the experiment, immediately the solution light color turned dark yellow, showing the formation of AuNPs, characterized by the UV–Vis spectrum with λmax of around 523 nm. (Fig. 1). Produced AuNPs were collected by centrifugation for analysis in subsequent studies. In the previous studies, the criteria of gold nanoparticles synthesizing were the dark yellow color similar to our studies (Zhaleh et al., 2019; Tahvilian et al., 2019).

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Fig. 1

The UV–Vis spectrum of biosynthesized gold nanoparticles.

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Previous studies have indicated that for the biosynthesis of gold oxide, there should also be a peak with at a wavelength range of 510–550 nm (Tahvilian et al., 2019).

Zhaleh et al. (2019) indicated the Gundelia tournefortii L. green-formulated Au nanoparticles have a clear peak at a wavelength of 528 nm. Zangneh et al. assessed Falcaria genus green-synthesized Au nanoparticles with a high peak in the wavelength of 535 nm (Zhaleh et al., 2019).

3.2 FT-IR analysis

In the recent study, FT-IR characterization was carried out on a Thermo Scientific Nicolet iS5 Infrared Spectrometer using the KBr pellet method over the range 400–4000 cm⁻¹.

The analysis of the IR spectrum of the gold nanoparticles revealed the peaks at 514, 1048, 1384–1612, and 3326 cm⁻¹ related to the Au-O, C—O, C⚌C and C⚌O, and OH, respectively (Fig. 2). The IR spectrum investigated for the gold nanoparticles revealed the absorption peaks at (I) 3287 cm⁻¹ (OH group of alcohols and phenols); (II) 1623 cm⁻¹ (C⚌O group of carboxylic acid group); (III) 1383 cm⁻¹ (C⚌C stretching of carboxylic acid group); (IV) 1038 cm⁻¹ (C—O vibrations of the protein/polysaccharide) (Hemmati et al., 2019; Varma, 2012).

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Fig. 2

FT-IR spectrum of biosynthesized gold nanoparticles.

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3.3 FE-SEM and TEM analysis

The image of FE-SEM of gold nanoparticles is revealed in Fig. 3. The gold nanoparticles looked as an agglomerated structure. The OH groups present in A. sativum leaf could be responsible for agglomeration.²² Also, the image of FE-SEM revealed the average size of 19 nm and the spherical shape for gold nanoparticles. Many similar observations are observed by Shahriari et al., Zhaleh et al. (2019), and Hemmati et al. (2019) (Zhaleh et al., 2019; Hemmati et al., 2019).

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Fig. 3

FE-SEM image of gold nanoparticles.

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Also, the AuNPs average size (19 nm) was determined through images of TEM (Fig. 4). The TEM image histogram plot revealed the bioformulated gold nanoparticles particle size distribution ranges from 7 to 27 nm. The previous researches have reported the ranges of 10–50 nm and spherical shape for biosynthesized of gold nanoparticles (Zhaleh et al., 2019).

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Fig. 4

TEM image of gold nanoparticles.

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3.4 Antioxidant properties of gold nanoparticles against DPPH

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. Some of these changes cause disease. Problems such as heart problems, muscle failure, diabetes and cancer are all caused by these free radicals.²³ Antioxidants act like a broom against free radicals, destroying free radicals and regenerating damaged cells. Laboratory evidence has shown that antioxidants can prevent cancer (Singh et al., 2018).

In the current experiment, the DPPH free radical scavenging potential of A. sativum leaf aqueous extract and AuNPs in many concentrations revealed impressive prevention similar to BHT. The IC₅₀ of A. sativum leaf aqueous extract, BHT, and AuNPs were 421, 259, and 231 µg/mL, respectively (Fig. 5; Table 1). Metallic gold nanoparticles also have excellent potential to inhibit DPPH. Following our ingredients, in many studies, the mutual effect has been monitored to increase the antioxidant capacities between herbs and HAuCl₄ against DPPH (Singh et al., 2018; Tahvilian et al., 2019).

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Fig. 5

The antioxidant properties of HAuCl₄, A. sativum, AuNPs, and BHT against DPPH.

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In general, metal nanoparticles have very extraordinary antioxidant effects, among which the antioxidant effects of gold nanoparticles have been more special. In the recent study, the potent free radical scavenging property of the nanoparticles is noticed at IC₅₀ value 231 µg/mL. This may be due to their ability to donate electrons or hydrogen ions to DPPH free radicals to neutralize them. When the concentration is increased, radical scavenging is also increased proportionally. Moreover, the plant contains most of the bioactive compounds which have been attributed to their antioxidant ability (Singh et al., 2018; Tahvilian et al., 2019).

Thomson and Ali (2003) indicated that A. sativum is rich of antioxidant compounds such as 3-vinyl-6H-1,3-dithiin, methyl methanesulfinate, dimethyl sulfide, methyl propyl disulfide, S-propyl cysteine sulfoxide, 3-vinyl-6H-1,2-dithiin, dipropyldi sulfide, dimethyl disulfide, 3-vinyl-4H-1,2-dithiin, allyl methyl thiosulfinate, diallyl disulfide, S-methyl cysteine sulfoxide, S-allyl cysteine sulfoxide, allyl methane sulfinate, dipropyl sulfide, dially trisulfide, diallyl tetrasulfide, methyl allyl trisulfide, and diallyl thiosulfinate, and the excellent antioxidant properties of A. sativum leaf aqueous extract are related to these compounds (Hosseinimehr et al., 2011). Several studies were carried out in the nanotechnology field using various medicinal plants, but still, no report is available on gold nanoparticles synthesized using A. sativum leaf aqueous extract.

3.5 Cytotoxicity potential of gold nanoparticles

In our study, the treated cells with several concentrations of the present HAuCl₄, A. sativum, and AuNPs were examined by MTT test for 48 h regarding the cytotoxicity properties on normal (HUVEC), colorectal adenocarcinoma (HT-29), colorectal carcinoma (HCT 116), ileocecal colorectal adenocarcinoma (HCT-8 [HRT-18]), and Burkitt's lymphoma (Ramos.2G6.4C10) cell lines (Figs. 6-10; Table 2). The absorbance rate was determined at 570 nm, which indicated extraordinary viability on normal cell line (HUVEC) even up to 1000 μg/mL for HAuCl₄, A. sativum, and AuNPs.

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Fig. 6

The cytotoxicity properties of HAuCl₄, A. sativum, and AuNPs against HUVEC cell line.

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Fig. 7

The anti-colorectal adenocarcinoma properties of HAuCl₄, A. sativum, and AuNPs against HT-29 cell line.

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Fig. 8

The anti-colorectal carcinoma properties of HAuCl₄, A. sativum, and AuNPs against HCT 116 cell line.

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Fig. 9

The anti-ileocecal colorectal adenocarcinoma properties of HAuCl₄, A. sativum, and AuNPs against HCT-8 [HRT-18] cell line.

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Fig. 10

The anti-Burkitt's lymphoma properties of HAuCl₄, A. sativum, and AuNPs against Ramos.2G6.4C10 cell line.

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In the case of colon cancer cell lines, the viability of them decreased dose-dependently in the presence of gold salt, A. sativum, and AuNPs. The IC50 of A. sativum and AuNPs against HT-29 cell line were 417 and 269 µg/mL, respectively; against Human HCT 116 cell line were 361 and 225 µg/mL, respectively; against HCT-8 [HRT-18] cell line was 402 and 250 µg/mL, respectively; and against Ramos.2G6.4C10 cell line were 380 and 236 µg/mL, respectively. The best result of cytotoxicity property of gold nanoparticles against the above cell lines was seen in the case of the HCT 116 cell line.

In the present study, the cytotoxicity of the gold nanoparticles on the normal cell line, HUVEC, was weaker than those on the colon cancer cell lines. The reason of the resent phenomenon is related to the cancer cell structure (Arulmozhi et al., 2013; Ramyadevi et al., 2012; Reuter et al., 2010). The cancer cell is characterized by: acceleration of the cell cycle; genomic alterations; invasive growth; increased cell mobility; chemotaxis; changes in the cellular surface; secretion of lytic factors, etc (Gultekin et al., 2016; Rehana et al., 2017). Morphological and functional characteristics of the malignant cell. Morphologically, the cancerous cell is characterized by a large nucleus, having an irregular size and shape, the nucleoli are prominent, the cytoplasm is scarce and intensely colored or, on the contrary, is pale (Del Mar Delgado-Povedano et al., 2016). The nucleus of neoplastic cells plays through its changes a main role in the assessment of tumor malignancy. Changes concern its surface, volume, the nucleus/cytoplasm ratio, shape and density, as well as structure and homogeneity (Jeong et al., 2012; Sankar et al., 2014). Ultrastructural characteristics are related to nucleus segmentation, invaginations, changes in chromatin, such as heterochromatin reduction, increase of interchromatin and perichromatin granules, increase of nuclear membrane pores, formation of inclusions, etc. The nucleolus is characterized by hypertrophy, macro- and microsegregation, its movement towards the membrane, numerical increase and formation of intranuclear canalicular systems between the nuclear membrane and the nucleolus (Namvar et al., 2014). Mitoses are characteristic of malignant cells. The number of mitoses increases, atypical mitosis forms with defects in the mitotic spindle appear, which results in triple or quadruple asters and dissymmetrical structures and atypical forms of chromosomes. Nuclear changes explain the presence of different cell clones and genetic anomalies associated with these changes. In intensely anaplastic tumors, the presence of gigantic nuclei and multinucleate cells expresses abnormal divisions. These morphological characteristics reflect the changes occurring at metabolic level, with the augmentation of structures in relation to cell division and the attenuation of structures associated to other metabolisms (Katata-Seru et al., 2018; Sangami and Manu, 2017). The above reasons are caused that the cytotoxicity of the gold nanoparticles on HUVEC was weaker than those on the colon cancer cell lines.

Among the various agents of metallic nanoparticles such as, surface functions nature, texture, size, and morphology, the size efficacy is most necessary in the anticancer test. Previous researches indicated that the anticancer property enhances with reducing in size of particle based on their better penetration property over the cell lines. It has been determined that the size of particle lower than 50 nm demonstrates better effect in the corresponding cancer cell lines (Namvar et al., 2014). As can be observed in Figs. 4 and 5 of the recent study, the sizes of Au nanoparticles synthesized by A. sativum leaf aqueous extract are at the ranges of 7–27 nm.

About the anticancer capacities of Au nanoparticles, they have applied to cure various cancers including human glioma, Lewis lung carcinoma, uterus cancer, human lung cancer, colon cancer, lung epithelial cancer, and mammary carcinoma (Katata-Seru et al., 2018).

Likely the significant anti-colon cancer potentials of gold nanoparticles synthesized by A. sativum aqueous extract against colon cancer cell lines are linked to their antioxidant activities. Similar researches have revealed the antioxidant materials such as metallic nanoparticles especially gold nanoparticles and ethnomedicinal plants reduce the volume of tumors by removing free radicals (Sangami and Manu, 2017). In detail, the high presence of free radicals in the normal cells makes many mutations in their DNA and RNA, destroys their gene expression, and then accelerates the proliferation and growth of abnormal cells or cancerous cells. The free radicals high presences in all cancers such as skin, throat, ovarian, testicular, bladder, colon, small intestine, gastrointestinal stromal, stomach, breast, lung, vaginal, prostate, pancreatic, liver, gallbladder, hypopharyngeal, fallopian tube, thyroid, esophageal, parathyroid, bile duct, and rectal cancers indicate the significant role of these molecules in making angiogenesis and tumorigenesis (Beheshtkhoo et al., 2018; Radini et al., 2018). Many researchers reported that gold nanoparticles synthesized by ethnomedicinal plants with high antioxidant compounds have a remarkable role in removing free radicals and growth inhibition of all cancerous cells (Oganesvan et al., 1991).