Formulation and characterization of a novel anti-human endometrial cancer supplement by gold nanoparticles green-synthesized using Spinacia oleracea L. Leaf aqueous extract

2.1 Material

Antimycotic antibiotic solution, dimethyl sulfoxide (DMSO), hydrolyzate, Ehrlich solution, decamplmaneh fetal bovine serum, borax-sulfuric acid mixture, Dulbazolic mixture Modified Eagle Medium (DMED), 4- (Dimethylamino) benzaldehyde, 2,2-diphenyl-1- pikrilhydrazil (DPPH) and phosphate buffer solution (PBS) were supplied from the US Sigma-Aldrich company.

2.2 Green synthesis and chemical characterization of AuNPs

The extract of the S. oleracea leaf plant obtained for the green synthesis method was extracted with distilled water in the microwave.

In a usual synthesis method, 1.5 g of NaOH pellets and 100 mL of HAuCl₄·H₂O (1 mM) were dissolved in distilled water (50 mL) each and properly mixed followed by addition of S. oleracea leaf extract solution (20 mL) under vigorous stirring at 25 °C for 1 h. During this process, the solution color changes to dark yellow color, showing the Au nanoparticles (AuNPs) formation.

The resultant solution was refluxed and let to precipitate; the precipitate subsequently was filtered and washed with acetone, distilled water, and ethanol. To obtain a fine powder of Au nanoparticles, the resultant precipitate was finally dried at 90 °C for 14 h (Jalalvand et al., 2019; Zhaleh, 2019; Shahriari, 2019; Zangeneh, 2019; Soni and Krishnamurthy, 2013).

Different analytical techniques were used for the characterization of Au nanoparticles:

TEM analysis of the Au nanoparticles was performed with JEOL 200 kV by placing them on a carboncoated copper grid.

In the UV–Vis spectroscopy analysis, characteristic absorption bands of Au metal were examined before and after the nanoparticles synthesis process.

The biomolecules related to the Au nanoparticles reduction including secondary metabolites were detected by the FT-IR (Shimadzu IR affinity.1).

2.3 Determination of the antioxidant activities of S. Oleracea green-mediated Au nanoparticles

To study the radical scavenging antioxidant property of our Au salt, S. oleracea leaf extract, and Au nanoparticles, DPPH (2,2-diphenyl-1-picrylhydrazyl) is being used (Hosseinimehr, 2011).

A 39.4% DPPH solution (w/V) was prepared in 1:1 aqueous MeOH. At the same time, different samples of Au salt, S. oleracea leaf extract, and Au nanoparticles of variable concentrations (0–1000 µg/mL) were prepared. The DPPH solution was then added to Au salt, S. oleracea leaf extract, and Au nanoparticles samples and incubated at 37 °C. After 30 min of incubation, the absorbances of the mixtures were measured at 570 nm. MeOH (50 %) and butylated hydroxytoluene (BHT) were considered as negative and positive controls respectively in the study. The antioxidant property of Au salt, S. oleracea leaf extract, and Au nanoparticles was determined in terms of % inhibition and expressed as $$\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\left(\%\right)=\frac{\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{A}.}{\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{A}.}x100$$

2.4 Determination of anti-endometrial cancer effects of S. Oleracea green-mediated Au nanoparticles

In this assay, following human endometrial cell lines and the normal cell line (HUVEC) were used to study the cytotoxicity and anticancer potential of human endometrial over the Au salt, S. oleracea leaf extract, and Au nanoparticles using the common cytotoxicity test i.e., MTT assay in vitro condition:

1)Normal cells: HUVEC.

2)Endometrial cancer cells: Ishikawa, KLE, HEC-1-A, and HEC-1-B.

For culturing the above cells, several materials including penicillin, streptomycin, and Dulbecco’s modified Eagle’s medium (DMEM) were used (Arulmozhi, 2013; Ramyadevi, 2012). The distribution of cells was 10,000 cells/well in 96-well plates. All samples were transferred to a humidified incubator containing 5% CO₂ at 37 °C. After 24 h incubation, all cells were treated with the different Au salt, S. oleracea leaf extract, and Au nanoparticles samples (0–1000 µg/mL) and incubated again for 24 h. Subsequently, they were sterilized by UV-radiation for 2 h. Then 5 mg/mL of MTT was added to all wells and incubated again for 4 h at 37 °C. The percentage of cell viability of samples were determined following the given formula after the measurement of absorbance at 570 nm. $$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}(\%)=\frac{\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{A}.}{\mathit{ControlA}.}x100$$

2.5 Qualitative measurement

The obtained results were loaded into the “SPSS-22” program and evaluated by “one-way ANOVA”, accompanied by a “Duncan post-hoc” check (p ≤ 0.01).

3.1 FE-SEM analysis of S. Oleracea green-mediated Au nanoparticles

The structural morphology of the prepared material was analyzed by the FE-SEM study. Fig. 1 represents the particle shape and size.

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Fig. 1

FE-SEM image of Au nanoparticles mediated using S. oleracea.

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Apparently, assumes wheat-like texture. In a closer look, the small globular particles of Au are seen. Due to high concentration in sampling the figure shows somewhat lump like agglomeration. However, the surface immobilization of biomolecules could not be observed from the images. Also, the FE-SEM picture revealed the 16.7 nm average size and the spherical shape for Au nanomaterials. Several similar observations are noted by Jalalvand et al. (Jalalvand et al., 2019) (Jalalvand et al., 2019); Zangneh et al. (2019) (Zangeneh and Zangeneh, 2019); Zhaleh et al. (Zhaleh, 2019) (Zhaleh, 2019), and Shahriari et al. (Shahriari, 2019) (Shahriari, 2019).

3.2 TEM analysis of S. Oleracea green-mediated Au nanoparticles

TEM was used to present extra information on the size and structure and the morphology, shape and dimension of these NPs (Fig. 2).

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Fig. 2

TEM image of Au nanoparticles mediated using S. oleracea.

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According to the results of TEM images, Au nanoparticles were in the range sizes of ∼11–23 nm. The Au nanoparticles with a high density and spherical shape had been well distributed. Also, some particles are interconnected to each other and formed bigger particles.

3.3 FT-IR analysis of S. Oleracea green-mediated Au nanoparticles

The analysis of the IR spectra of the gold nanoparticles revealed the peaks at 525, 1079, 1326, 1681, and 3351 cm⁻¹ related to the Au-O, C-OH, C = O, C-O, and OH, respectively (Fig. 3). The IR spectra 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 the carboxylic acid group); (III) 1383 cm⁻¹ (C = O stretching of the carboxylic acid group); (IV) 1038 cm⁻¹ (C-OH vibrations of the protein/polysaccharide) (Zangeneh and Zangeneh, 2019; Jalalvand et al., 2019; Zhaleh, 2019; Shahriari, 2019; Zangeneh, 2019).

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Fig. 3

FT-IR spectra of Au nanoparticles mediated using S. oleracea.

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3.4 UV–Vis spectroscopy analysis of S. Oleracea green-mediated Au nanoparticles

Fig. 4 indicates the UV–Vis spectra of gold nanoparticles obtained by green synthesis using S. oleracea aqueous extract.

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Fig. 4

The UV–Vis spectrum of Au nanoparticles mediated using S. oleracea.

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Analysis of the absorption spectrum of the final solution allowed us to more clearly understand this Au⁺–Au⁰ conversion. The band at a wavelength of about 549 nm, which is believed to be due to d-d transitions of the Au⁺ ion, completely disappeared after the green synthesis process. This disappearance was evidence that the Au⁺ cation became completely reduced.

It has been indicated in the work that the SPR band of Au nanoparticles obtained by green synthesis using medicinal plants provides absorption from 510 to 550 nm (Zangeneh and Zangeneh, 2019; Jalalvand et al., 2019; Zhaleh, 2019; Shahriari, 2019; Zangeneh, 2019).

3.5 Cytotoxicity and anti-endometrial cancer potentials of S. Oleracea green-mediated Au nanoparticles

In the recent research, the treated cells with several concentrations of the present HAuCl₄, S. oleracea leaf aqueous extract, and Au nanoparticles were examined by MTT test for 48 h regarding the cytotoxicity properties on normal (HUVEC) and common endometrial cancer cell lines i.e., Ishikawa, KLE, HEC-1-A, and HEC-1-B.

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, S. oleracea leaf aqueous extract, and Au nanoparticles (Figs. 5–9; Table 1).

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Fig. 5

The cytotoxicity potentials of Au salt, S. oleracea, and Au nanoparticles against the HUVEC cell line.

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Fig. 6

The anti-human endometrial cancer potentials of Au salt, S. oleracea, and Au nanoparticles against the HEC-1-B cell line.

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Fig. 7

The anti-human endometrial cancer potentials of Au salt, S. oleracea, and Au nanoparticles against the HEC-1-A cell line.

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Fig. 8

The anti-human endometrial cancer potentials of Au salt, S. oleracea, and Au nanoparticles against the KLE cell line.

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Fig. 9

The anti-human endometrial cancer potentials of Au salt, S. oleracea, and Au nanoparticles against the Ishikawa cell line.

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In the case of endometrial cancer cell lines, the viability of them reduced dose-dependently in the presence of HAuCl₄, S. oleracea leaf aqueous extract, and Au nanoparticles. The IC50 of S. oleracea, and AuNPs against HEC-1-B cell line were 484 and 324 µg/mL, respectively; against Human HEC-1-A cell line were 458 and 316 µg/mL, respectively; against KLE cell line were 460 and 335 µg/mL, respectively; and against Ishikawa cell line were 468 and 341 µg/mL, respectively. The best finding of anti-endometrial cancer potentials was determined in the HEC-1-A cell line.

Among the different parameters of metallic nanoparticles such as, size, texture and nature of surface functions, the size effect is most essential in the anticancer assay using standard cancer cell lines. Previous reports revealed that the anticancer activity increases with a decrease in particle size based on their better penetration ability over the cell lines. It has been surveyed that particle size lower than 50 nm displays better activity in the corresponding cancer cell lines (Namvar, 2014).

As shown in Figs. 3 and 4 of our study, the average size of gold nanoparticles synthesized by S. oleracea leaf aqueous extract is 16.7 nm.

In the anticancer effects of Au nanoparticles, they have been used to treat several cancers including human lung cancer, mammary carcinoma, uterus cancer, lung epithelial cancer, Lewis lung carcinoma, colon cancer, and human glioma (Singh, 2018).

3.6 Antioxidant capacities of S. Oleracea green-mediated Au nanoparticles

Now, turning our attention to investigate the bioactivity of S. oleracea leaf aqueous extract green-synthesized Au nanoparticles, a concentration-dependent DPPH radical scavenging effect of nanomaterial was observed against BHT as a reference. DPPH process is widely applied to determine the free radical scavenging effect of different antioxidant materials. The DPPH scavenging abilities are known to be because of the hydrogen donating activities of antioxidant materials. When DPPH results are examined, it is observed that it has increased in a dose-dependent manner (Zangeneh and Zangeneh, 2019; Jalalvand et al., 2019; Zhaleh, 2019; Shahriari, 2019; Zangeneh, 2019; Soni and Krishnamurthy, 2013).

The interaction between S. oleracea leaf aqueous extract green-synthesized Au nanoparticles and DPPH might have occurred by transferring electrons and hydrogen ions (Zangeneh and Zangeneh, 2019; Jalalvand et al., 2019; Zhaleh, 2019; Shahriari, 2019; Zangeneh, 2019). The scavenging capacity of the S. oleracea leaf aqueous extract green-synthesized Au nanoparticles and BHT at different concentrations, expressed in percentage inhibition, has been shown in Fig. 10.

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Fig. 10

The antioxidant potentials of Au salt, S. oleracea, Au nanoparticles, and BHT against DPPH.

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In the antioxidant test, the IC50 of S. oleracea, butylated hydroxytoluene and Au nanoparticles were 365, 210, and 194 µg/mL, respectively (Table 2).

Several studies have indicated that the antioxidant properties of Au nanoparticles green-mediated by medicinal herbs is the most among all metallic nanoparticles. So far notable antioxidant properties of Au nanoparticles green-mediated by many medicinal herbs such as Camellia sinensis, Allium noeanum Reut. ex Regel, Thymus vulgaris, Gundelia tournefortii L., and Falcaria vulgaris have been proved (Zangeneh and Zangeneh, 2019; Jalalvand et al., 2019).

Au nanoparticles green-mediated by medicinal herbs show higher antioxidant effects against free radicals formation into the living system (Jalalvand et al., 2019; Zhaleh, 2019). The Au nanoparticles green-formulated have excellent redox properties and have a significant role in free radicals deactivating (Zhaleh, 2019; Shahriari, 2019; Zangeneh, 2019).

Previous researches have indicated that flavonoids and phenolic compounds attached to the metallic nanoparticles have significant antioxidant properties (Reuter, 2010; Gultekin, 2016; Rehana, 2017; Del Mar 2016; Jeong, 2012; Sankar, 2014). Previously it has been revealed that S. oleracea is full of antioxidant compounds including sinapic acid, luteolin, patuletin, resins, saponins, catechin, flavones, flavonols, cardiac glycosides, daidzein, quercetin, flavonoids, apigenin, steroids, kaempferol, kaempferol, and terpenes (Raut, 2010). Many studies were performed in the nanobiotechnology branch using several ethnomedicinal plants, but no study is present on S. oleracea leaf aqueous extract green-synthesized Au nanoparticles.

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