Green synthesis and chemical characterization of a novel anti-human pancreatic cancer supplement by silver nanoparticles containing Zingiber officinale leaf aqueous extract

2.1 Material

Dimethyl sulfoxide (DMSO), Antimycotic antibiotic solution, hydrolysate, decamplmaneh fetal bovine serum, Ehrlich solution, 4-(Dimethylamino) benzaldehyde, DPPH, carbazole reagent, borax-sulphuric acid mixture, Dulbecco's Modified Eagle Medium (DMED), and phosphate buffer solution (PBS) were purchased from Sigma-Aldrich (USA).

2.2 Preparation of Zingiber officinale leaf aqueous extract

At the beginning of the aqueous extracting, the fresh and healthy parts of Zingiber officinale leaf were collected. After shade drying in a mixer, 50 g of powdered plant sample was extracted with distilled water with the increase of polarity at a ratio of 1:15 (v/v). In the end, for concentrating, the rotary evaporator was used (Ghashghaii et al., 2017).

2.3 Synthesis of AgNPs

Green synthesis of silver nanoparticles was started with such a process combination of 100 mL of AgNO₃·H₂O at concentrations of 1 mM and 10 mL of Zingiber officinal 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 black colored colloidal solution of Ag was formed. The solution was centrifuged at 10,000 rpm for 15 min. The precipitate was sprayed with water and then resuspended (Ahmeda et al., 2020).

2.4 The characterization of AgNPs

Field Emission Scanning Electron Microscopes (FE-SEM), Transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), and Ultraviolet–visible (UV–Vis) spectrophotometry were employed to characterize the composition, structure, and morphology of the silver nanoparticles. Silver nanoparticles have been verified using UV–Vis spectroscopy nm (Jasco V670 Spectrophotometer) at a scanning scale of 350–650 nm wavelength. The FT-IR spectrophotometer (Shimadzu IR Affinity.1) has been used to track the organic compounds engaged in removing silver nanoparticles. With “FE-SEM (Fe-SEM ZEISS EVO18)” analysis and “TEM (TEM FEI-TECNAI G2-20 TWIN)”, the morphological properties of silver nanoparticles were examined in terms of form and thickness.

2.5 Determination of the antioxidant property of AgNPs

The organic chemical compound DPPH stands for 2,2-diphenyl-1-picrylhydrazyl as an abbreviation. This is a crystalline powder of dark colors, composed of stable free-radical molecules. DPPH has two main laboratory research applications: one of the chemicals monitors the radicals, particularly the common analysis of antioxidants, and the next of the paramagnetic electron resonance signal position and strength (Zangeneh et al., 2020).

The above DPPH was added to the various concentrations of AgNO₃, Zingiber officinale leaf aqueous extract, and silver nanoparticles and all samples were transferred to an incubator at the temperature of 37 °C. After 30 min incubating, the absorbance’s were measured at 517 nm. Acceding to the following formula, the antioxidant properties of AgNO₃, Zingiber officinale leaf aqueous extract, and silver nanoparticles were determined in detail (Zangeneh et al., 2020): $$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{V}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\left(\%\right)=\frac{\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{A}.}{\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{A}.}\times 100$$

2.6 Determination of anti-pancreatic cancer effects of silver nanoparticles

The following cell lines have been utilized in this research to precisely invest the cytotoxicity and anti-pancreatic of the AgNO₃, Zingiber officinale leaf aqueous extract, and silver nanoparticles using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay:

1. Normal cell line

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     HUVEC.

2. Pancreatic cancer cell lines

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     PANC-1

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     AsPC-1

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     MIA PaCa-2

For culturing the above cells streptomycin, penicillin, and Dulbecco’s modified Eagle’s medium (DMEM) were used. Cell density in 96-well plates was 10,000 cells/right. Both samples were then moved at a temperature of 37 °C to a humidified incubator of %5 CO₂. After 24 h incubating, all cells were treated with several concentrations of AgNO₃, Zingiber officinale leaf aqueous extract, and silver nanoparticles, then incubated for 24 h. AgNO₃, Zingiber officinale leaf aqueous extract, and silver nanoparticles were sterilized using the radiation of UV for 2 h. $$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}(\%)=\frac{\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{A}.}{\mathit{Control}A.}\times 100$$

2.7 Qualitative measurement

The findings reported 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 The characterization of silver nanoparticles

3.1.1

3.1.1 UV–Vis analysis

Fig. 1 shows a 414 nm absorption band linked to the AgNPs surface plasmon resonance (SPR). We may also note that the SPR band decreases, by growing the volume of aqueous isolate liquid Zingiber officinale strip. It indicates that utilizing a higher concentration of Zingiber officinale extract aqueous extract reduces the average size of AgNPs, and raises the density of AgNPs.

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Fig. 1

The UV–Vis spectrum of silver nanoparticles green-synthesized using Zingiber officinale leaf. ABS. Absorbance.

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Shasha Han et al. reported Gundelia tournefortii L. aqueous extract synthesized AgNPs peaked at 440 nm in the UV–Vis spectrum (Han et al., 2020). Mohammadi et al. observed the peak of silver nanoparticles containing Phoenix dactylifera seed ethanolic extract at the wavelength of 438 nm (Mohammadi et al., 2020). Han et al. recorded 430 nm of the absorption coefficient for silver nanoparticles utilizing the polyol process (Han et al., 2020). Zangeneh et al. revealed the absorbance at 462 nm for silver nanoparticles synthesized by Spinacia oleracea L. (Zangeneh, 2019a). Ahmeda et al. studied Melissa officinalis leaf aqueous extract mediated synthesis of AgNPs. Absorption of the continuum was detected at 462 nm (Ahmeda et al., 2020). Zangeneh et al. recorded Stachys lavandulifolia leaf extract induced AgNP as well as absorption maximum was detected at 440 nm (Ahmeda et al., 2020).

3.1.2

3.1.2 FE-SEM and TEM analysis

The FE-SEM image of Zingiber officinale leaf aqueous extract mediated silver nanoparticles is shown in Fig. 2. As understood from Fig. 2, AgNPs show an agglomerated structure and indicates particulate sizes from 15 to 31 nm for biosynthesized AgNPs. Silver nanoparticles can usually be agglomerated and the hydroxyl groups present in the Zingiber officinale leaf aqueous extract are thought to be responsible for the observed agglomeration (Zangeneh, 2019b; Zangeneh, 2020).

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Fig. 2

FE-SEM image of silver nanoparticles green-synthesized using Zingiber officinale leaf.

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TEM analysis is preferred to detect detailed grain size, size distribution, and morphology of nanoparticles (Katata-Seru et al., 2018). TEM image shows that biosynthesized silver nanoparticles have a particle size distribution between 12 and 30 nm (Fig. 3). Thus, the size of the nanoparticles we obtained was smaller than the literature (Katata-Seru et al., 2018).

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Fig. 3

TEM image of silver nanoparticles green-synthesized using Zingiber officinale leaf.

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3.1.3

3.1.3 FT-IR analysis

The FT-IR analysis was performed to identify the antioxidant compounds which may be responsible for reducing the Ag+ ions in the Zingiber officinale extract, as well as those that may be concerned with stabilizing the synthesis of AgNPs. The solution was centrifuged at 10,000 rpm for 15 min. The precipitate was sprayed with water and then resuspended (Yang et al., 2017; Judith Vijaya et al., 2017). The composition of the AgNPs suggests that the presence of a maximum at 484 cm⁻¹ is part of the Ag-O tension change in the current case. Thus, the IR spectroscopic approach has been used as a suitable method to identify bioactive components in the field of natural products.

Also, this technique is a valuable tool for detecting the existence of secondary metabolites over the AgNPs in plants. As per the findings, the occurrence of common-IR bars was linked to the nature of different organic compounds in an aqueous extract of Zingiber officinale extract. In addition, a band at 2071 cm⁻¹ referred to aliphatic “C—H” stretching; peaks at 3416 cm⁻¹ linked to “O—H” stretching; peaks at 1079, 1204 and 1287 cm⁻¹ may be compared to “—C—O” stretching and peaks at 1391 and 1612 cm⁻¹ refer to “C⚌C” stretching and “C⚌O” stretching occurring in phenolic and flavonoid compounds (Fig. 4). These peaks may be known for the existence of numerous substances at the Zingiber officinale such as flavonoid, phenolic, and carboxylic substances previously recorded.

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Fig. 4

FT-IR spectra of silver nanoparticles green-synthesized using Zingiber officinale leaf.

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3.2 Antioxidant potential of silver nanoparticles green-synthesized by Zingiber officinale leaf aqueous extract

Plants have impressive antioxidant capabilities. One alternative to increase the plants' antioxidant ability is to combine them with metallic salts. Recent experiments have shown that their antioxidant function improves significantly as the leaves are mixed with zinc, iron, silver, gold, copper and titanium (Taghavi Fardood and Ramazani, 2016). Several studies have indicated that the antioxidant properties of silver nanoparticles green-synthesized by medicinal plants are the most among all metallic nanoparticles.

In our study, a significant concentration-dependent DPPH radical scavenging effect was demonstrated by the Zingiber officinale leaf aqueous isolate and silver nanoparticles close to BHT. The connection with both the Zingiber officinale leaf aqueous isolate and silver nanoparticles and DPPH may have happened from the conversion of electrons and hydrogen ions into 2,2-diphenyl-1-picrylhydrazyl radical, forming a controlled DPPH complex [24–26]. The IC50 values of Zingiber officinale leaf aqueous extract, BHT, and silver nanoparticles were 275, 203, and 172 μg/mL, respectively (Fig. 5, Table 1).

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Fig. 5

The antioxidant properties of AgNO₃, Zingiber officinale leaf, silver nanoparticles, and BHT against DPPH.

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The oxidative efficacy of Zingiber officinale leaf aqueous isolate can be due to the presence of various phytochemicals, which are thought to work dynamically and synergistically to neutralize the “reactive oxygen species (ROS)” and the “reactive nitrogen species (RNS)” (Rajesh et al., 2018; Kaur et al.,2016; Reuter et al., 2010). In an earlier analysis, compounds such as ethylate, bis (2- ethylhexyl) phthalate, cinnamic acid, nepitrine, gallic acid, oleylic alcohol, isorhamnetine-3-O-rutinoside, acteoside and quercetine were found in Zingiber officinale analysis (Demirci Gültekin et al., 2016). These biologically active substances were shown to sustain redox homeostasis through multi-step antioxidant reactions, including initiation, aggregation, splitting, and free radicals (Rehana et al., 2017).

3.3 Cytotoxicity anti-pancreatic cancer potentials of silver nanoparticles synthesized in green with Zingiber officinale leaf aqueous extract

Treated cells with specific concentrations of the present AgNO₃, Zingiber officinale leaf aqueous extract, and silver nanoparticles were tested by 48 h MTT study for cytotoxicity, regular anti-pancreatic (HUVEC) and pancreatic (PANC-1, AsPC-1, and MIA PaCa-2 cell lines of cancer (Figs. 6–9, Table 2). The absorbance limit of AgNO₃, Zingiber officinale leaf aqueous extract, and silver nanoparticles was measured at 570 nm, indicating exceptional viability even up to 1000 μg/mL on a standard cell line (HUVEC).

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Fig. 6

The anti-human pancreatic cancer properties of AgNO₃, Zingiber officinale leaf, and silver nanoparticles against PANC-1 cell line.

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Fig. 7

The anti-human pancreatic cancer properties of AgNO₃, Zingiber officinale leaf, and silver nanoparticles against AsPC-1 cell line.

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Fig. 8

The anti-human pancreatic cancer properties of AgNO₃, Zingiber officinale leaf, and silver nanoparticles against MIA PaCa-2 cell line.

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Fig. 9

The cytotoxicity properties of AgNO₃, Zingiber officinale leaf, and silver nanoparticles against HUVEC cell line.

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In typical human pancreatic, their cell viability decreased dose-dependently with various concentrations (1–1000 μg/mL) of AgNO₃, Zingiber officinale leaf aqueous extract, and silver nanoparticles current.

There were 443 and 295 μg/mL for specific human pancreatic cancer cell lines, 479 and 312 μg/mL for Zingiber officinale leaf aqueous extract and 312 μg/mL for silver nanoparticles against PANC-1 cell lines, and 359 and 220 μg/mL for MIA PaCa-2 cell lines, respectively. The strongest findings of the cytotoxicity and anti-human pancreatic cancer ability of silver nanoparticles against the above cell lines were shown in the PANC-1 cell line, and BHT against DPPH.

The chemotherapeutic results of metallic nanoparticles, in particular iron, zinc and silver nanoparticles in vitro and in vivo settings, have been confirmed by multiple studies to date. Various cell lines including Cos-7 monkey fibroblasts, BRL3A rat liver cells, human epidermal keratinocytes, and rodent lung epithelial cell lines have been studied for iron nanoparticles' cytotoxicity properties, which have shown promise [30]. More work has also shown that iron oxide nanoparticles produced utilizing medicinal plant extracts have great potential for cytotoxicity toward human cell counts for leukemia (Jurkat cells), cervical cancer (HeLa cells), liver cancer (HepG2 cells), and breast cancer (MCF-7 cells) (Delgado-Povedano et al., 2016). The anticancer effects of silver nanoparticles green-synthesized by medicinal plants have been confirmed in the previous studies (Jeong et al., 2012; Sankar et al., 2014). It has been suggested in the previous research that silver nanoparticles green-synthesized by Annona quamosal leaf have excellent potential for anti-breast cancer against the MCF-7 cell line (Mahmoudi et al., 2012; Namvar et al., 2014). In another study, the anti-liver cancer properties of silver nanoparticles containing Piper longum leaf against Hep-2 cell lines were proved (Vivek et al., 2012; Justin Packia et al., 2012). In the study of Suman et al. was clarified the anti-cervix cancer effects of silver nanoparticles containing natural compounds (Morinda citrifolia) against the HeLa cell line. In the previous study, the silver nanoparticles killed all HeLa cells in high doses (Suman et al., 2013).

Similar researches have revealed the antioxidant materials such as metallic nanoparticles especially silver 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 results of many studies have shown that both AgNPs and Ag⁺ generated by AgNPs are involved in the chemotherapeutic cascade in different ways:

AgNPs are needed to have an acceptable surface outside the mitochondria for the univalent transfer of oxygen from either the electron transport chain to the superoxide. Ag+ attaches to DNA and proteins that interact with their functions (Beheshtkhoo et al., 2018). Silver nanoparticle chemotherapeutic effects have been found to rely on several factors relevant to their physical characteristics, such as surface coloring, shape and thickness. To the current scale, it has been reported that tiny silver nanoparticles will migrate from the cell membrane and remove from its tumor cells. The above potential is substantially reduced in bigger sizes (Suman et al., 2013). Likely significant anti-pancreatic cancer potentials of silver nanoparticles synthesized by Zingiber officinale leaf aqueous extract against pancreatic (PANC-1, AsPC-1, and MIA PaCa-2) tumor layers are linked to their antioxidant function. Engineering work has shown that antioxidant substances such as metallic nanoparticles, particularly silver nanoparticles and ethnomedicinal herbs, reduce cancer density by eliminating free radicals (Sangami and Manu, 2017).

The free radical’s high presences in all cancers like gallbladder, breast, rectal, stomach, liver, gastrointestinal stromal, bile duct, esophageal, pancreatic, small intestine, colon, parathyroid, bladder, thyroid, testicular, prostate, vaginal, fallopian tube, ovarian, hypopharyngeal, throat, lung, and skin cancers indicate a significant role of these molecules in making angiogenesis and tumorigenesis (You et al., 2012). Many researchers reported that silver nanoparticles synthesized by ethnomedicinal plants have a remarkable role in removing free radicals and growth inhibition of all cancerous cells (Beheshtkhoo et al., 2018; Radini et al., 2018).