Introduction

For over 2000 years, silver has been used in the medical field to treat a wide array of pathogens, parasites and physiological disorders. In recent years, nanoparticles are being increasingly used for several purposes. They represent an important tool in several fields, including physical, biological, and pharmaceutical applications [1, 2]. In particular, silver nanoparticles (AgNPs) have been used as antimicrobials on several microorganisms and as cytotoxic against different cancer cells. There are chemical and physical methods to synthesize AgNPs. Chemical methods are toxic and harmful to the health of living organisms, if compared to physical methods, which, however, require high-energy inputs [1, 2]. Notably, the synthesis nanoparticles using metal and metal oxide using different plant part extract as reducing and stabilizing agents is rapid, cheap and not require the employ of toxic reagents nor high-energy inputs [3, 4].

Medicinal plant-based synthesis of AgNPs is highly appreciated nowadays. Plants are capable of easily reducing metals, both on their surface and in various organs and tissues remote from the ion penetration site, in a size <100 nm [5]. Using “green synthesis” routes, extensive studies have been performed on AgNPs fabricated exploiting the reducing potential and capping agents from medicinal plant parts such roots, seeds, leaves, flowers, stems, and fruits [6–8]. Similarly, also different genera of fungi can be used for AgNPs synthesis [9, 10].

In recent times, plant-mediated, synthesized AgNPs have been used as newer and safer tools for prevention and enhancement of mosquito vector control [11, 12], while other studies reported the potential importance of green synthesis of AgNPs with antimicrobial activity [13, 14].

Colorectal cancer (CRC) is the third most common predominant cancer type, with high mortality rates worldwide [15]. Current chemotherapeutic agents used for CRC treatment are expensive, with low efficiency, and induce severe side effects in non-cancerous tissues, due to their high toxicity.

Nowadays, nanoparticles have been seen to be beneficial due to the selective delivery of oligonucleotides to tumor cells. Besides, certain nanoparticles exhibited some interesting capability to reverse multidrug resistance, which is one of the major difficulties in chemotherapy. However, they must be properly designed in order to maximize the efficacy [16, 17]. Previous studies have showed that Ag-based nanoparticles induce apoptosis in human cancer [18].

Nevertheless, the present study focused on a green synthesis route to harvest AgNPs at room temperature using Pimpinella anisum seed extract. Moreover, in the mainstream of previous studies, a single clone of a tumor model was studied, which confined the generalizability of their findings. Here, we conducted a comparative assessment in two clones of CRC. Our data revealed that AgNPs greatly affected CRC proliferation in a minimum dose. Moreover a cell cycle analysis revealed that the sensitivity of the AgNPs could vary depending on the tumor clones.

Materials and Methods

Plant Materials and Chemicals

Plant material was collected from the Al-Madina region, Saudi Arabia, while silver nitrate was purchased from Sigma Aldrich, USA.

Preparation of P. anisum Seed Extract

P. anisum seeds were rinsed thoroughly under tap water and then with double distilled water to remove dust and other physical contaminants. Seeds were then dried at room temperature and ground to powder. 20 g of powder was soaked in 100 ml of double distilled water. Filtration was done after 24 h using Whatman No. 1 filter paper.

Synthesis of Ag Nanoparticles and Characterization

For the biosynthesis of AgNPs, 4 ml of seed extract was mixed with 100 ml of 3 mM AgNo3 solution at room temperature. After 172 h a dark brown colored solution was obtained from a yellow colored solution. This reaction exhibited and confirmed the formation of AgNPs. Furthermore, synthesis was confirmed by Ultraviolet–Visible (UV–vis) spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and energy dispersive spectrophotometry (EDS).

Cell Proliferation Analysis

The effect of AgNPs on cellular proliferation was evaluated using Alamar blue assay (BUF012B; AbD Serotec, UK). 0.01 × 106 cells/well were seeded in 96-well plates with routine culture medium and incubated for 12–24 h at 37 °C. The medium contained Dulbecco’s Modified Eagle Medium (DMEM) supplemented with d-glucose 4500 mg/L, 4 mM l-glutamine, and 110 mg/L sodium pyruvate; 10% fetal bovine serum (FBS); 1 × penicillin–streptomycin (Pen-strep); and non-essential amino acids (all purchased from Gibco-Invitrogen, USA). The medium was removed, and various concentrations of AgNPs, diluted in a complete culture medium, were added. Control wells were contained in the medium alone. Cell proliferation was determined at 48 and 96 h following addition of AgNPs. Alamar Blue (1:10) was added to each well, and the plates were incubated at 37 °C for 4 h. After incubation, plates were read using a spectrophotometric microplate reader (Biotek Synergy 2; Biotek Instruments, USA) and the relative fluorescence unit (RFU) was recorded.

Cell Viability Analysis

To determine the cell viability, cells were stained with a dual fluorescent staining solution (1 µl), containing 100 µg/ml AO and 100 µg/ml EB (AO/EB, Sigma, St. Louis, MO). Cells were washed with PBS and gently mixed with AO/EB (1:100) dye solution for one minute: Later, cells were observed under a Nikon Eclipse Ti fluorescence microscope. Cells cultured without AgNPs were considered as the experiment control. Acridine Orange/Ethidium Bromide (AO/EB) staining uses a combination of two dyes to visualize cells with aberrant chromatin organization. Differential uptake of AO/EB allowed the identification of viable and non-viable cells. Acridine Orange was specifically used to visualize the number of apoptotic cells.

Cell Cycle Analysis by Flow-Cytometery

Cell pellets were collected and washed in phosphate-buffered saline (PBS), centrifuged, re-suspended and fixed in 1 mL of 70% alcohol and incubated in −20 °C overnight. Then, tubes were centrifuged and the pellets were washed twice in PBS. After washing the pellets, they were treated with riboneuclease (50 µl; 100 µg/ml stock solution) and stained with propidium iodide (PI; 200 µl (from 50 µg/ml stock solution). Tubes were incubated at room temperature for 30 min and were analyzed by a flow cytometery, Beckman Coulter, Navios. Staining was detected in the respective fluorescence channel (FL3; PI maximum emission of 605 nm) and the outcome data were analyzed by using the KLuza software. Differences among cell mortality and apoptosis data as a function of the tested treatment and dose were analyzed using unpaired t test (P < 0.05).

Statistical Analysis

Statistical analyses and graphing were performed using Microsoft excel 2010 and GraphPad Prism 6.0 software (GraphPad, San Diego, CA, USA). P-values were calculated using the unpaired t test and ANOVA (RM one-way ANOVA, with the Greenhouse-Geisser corrections).

Results and Discussion

Figure 1a shows the brownish yellow colored seeds of P. anisum in a dried condition, Fig. 1, shows the P. anisum seeds extract alone, and Fig. 1c shows the yellow color solution had changed to brown, which indicated the formation of Ag+ to AgNPs [19]. UV–visible absorption spectrum of the AgNPs synthesized from P. anisum seeds in water suspension after 170 h exhibited a peak at 442 nm (Fig. 2); this is linked to the fact that AgNPs exhibit an unusual optical phenomenon, called surface Plasmon resonance, due to the cumulative oscillation of the conducting metal surface electrons in resonance with non-particulate radiation [20, 21]. Figure 3 shows the FTIR spectrum of AgNPs, where peaks were observed, with interactions at 815, 1034, 1300, 1642, 2925, 3287, and 3779 cm−1. In agreement with earlier study, the peaks obtained were due to the presence of carboxyl (–C=O), hydroxyl (–OH), and amine (–NH)-stretching vibrations of molecules [22, 23].

Fig. 1
figure 1

(a) Pimpinella anisum seeds after drying, (b) aqueous extract of P. anisum seeds, (c) AgNPs biosynthesized using the aqueous extract of P. anisum seeds

Full size image
Fig. 2
figure 2

Absorption spectrum of Ag nanoparticles synthesized from P. anisum seeds after 170 h

Full size image
Fig. 3
figure 3

FTIR spectrum of AgNPs synthesized using P. anisum seed extract

Full size image

Figure 4 shows TEM of the anise-synthesized AgNPs. Most green-fabricated AgNPs were spherical in shape and well dispersed, with an average size of as around 80–85 nm. Similar results were also observed in plant seed extracts of Nyctanthes arbor-tristis, which had a diameter size of 50–80 nm [24]. Figure 5a shows the EDX analysis of P. anisum seed extract, showing a strong signal in the C-K plant region. Figure 5b shows the EDX analysis of AgNPs; it exhibited a very strong signal in the silver region and it also confirmed the formation of AgNPs. Metallic silver ion behavior was noted, with the typical absorption peak at 3 keV due to surface Plasmon resonance [25].

Fig. 4
figure 4

TEM of AgNPs synthesized using P. anisum seed extract a) AgNPs formed after 170 h exposure to AgNO3 20 kV ×270,000, 0.5 μm b 20 kV ×270,000, 0.5 μm higher magnification

Full size image
Fig. 5
figure 5

EDX of P. anisum seed extract (a) and AgNPs (b)

Full size image

In vitro cytotoxicity potential of AgNPs was investigated on CRC cell lines. In order to rule out a cell line-based bias, we selected two models that were derived from colorectal adenocarcinoma, from HCT8 (ATCC: CCL-244™) and SW620 (ATCC: CCL-227™). Alamar Blue assay revealed cell proliferation and viability of metabolically active cells. Both CRC clones were exposed to AgNPs in different doses (0.05–25 μg/mL) for 48 and 96 h. After 48 h, significant reductions in proliferation were observed at >3.2 μg (Figs. 6, 7) in both cell lines, it was consistent at 96 h. Moreover, the viability was affected in SW620 compared to HCT8, particularly in the apoptotic cells, with more dead cells observed in SW620, at 4 μg (Figs. 6, 7). Likewise, it was confirmed by cell cycle analysis that there more apoptotic cells were found at Sub G1 phase in SW620 cells. SW620 cells were sensitive to AgNP at 3 μg (Fig. 7). Interestingly, we observed that the cells were arrested at G2/M phases in both groups at 1.5 μg (Figs. 6, 7). However, further studies are needed to show the effect of AgNP in cell proliferation. A previous study suggested that AgNPs were valid anticancer agents that acted in a p53-dependent manner and decreased the growth and viability of HCT116 colon cancer cells and increased apoptosis [18].

Fig. 6
figure 6

Impact of Ag nanoparticles on SW620 cell viability, proliferation and cell cycle: a fluorescence images of SW620 cells (control and Ag nanoparticles exposure at 2 and 4 µg/ml) stained with acridine orange/ethidium bromide to analyze the apoptosis and viability. Scale bar 10 µm. b Alamar blue assay showing the proliferation (%) decline in a dose dependent manner (25–0.05 µg/ml). Data are presented as mean ± SE, n = 10. c Flow cytometry based cell cycle analysis of SW620 cells (control and Ag nanoparticle exposure at 1.5 and 3 µg/ml) stained with propidium iodide. d Data showing the percentage (%) of population in each cell cycle phase, data represent as mean ± SE, n = 3. *p < 0.05; **p < 0.005; ***p < 0.0005

Full size image
Fig. 7
figure 7

Impact of Ag nanoparticles on HCT8 cell viability, proliferation and cell cycle: a fluorescence images of HCT8 cells (Control and Ag nanoparticles exposure to 2 and 4 µg/ml) stained with acridine orange/ethidium bromide to analyze the apoptosis and viability. Scale bars 10 µm. b Alamar blue assay showing the proliferation (%) decline in a dose dependent manner (25–0.05 µg/ml). Data are presented as mean ± SE, n = 10. c Flow cytometry based cell cycle analysis of HCT8 cells (control and Ag nanoparticles exposure to 1.5 and 3 µg/ml) stained with propidium iodide. d Data showing the percentage (%) of population in each cell cycle phase, data represent as mean ± SE, n = 3. *p < 0.05; **p < 0.005; ***p < 0.0005

Full size image

Likewise, our study also revealed the same in two other CRC clones. Moreover, not all cell clones showed comparable AgNP sensitivity. For example, nickel–zinc (Ni–Zn) ferrite nanoparticles led to cytotoxicity in epithelial cancer cells. Ni-Zn ferrite nanoparticles behaved differently to HepG2, HT29, and MCF7 cells. Viability rates indicated that HepG2 cells were more sensitive than MCF7 or HT29 cells [26]. AgNPs are certainly soluble and that prevents their agglomeration or their being entrapped in a matrix. Therefore, AgNPs could be good candidates for the development of broad-spectrum antiviral and bactericidal composites, due to their efficiency in small doses, as well as their negligible toxicity [12, 27].

Furthermore, our anise-synthesized AgNPs represent an effective eco-friendly process to produce nano-drug compounds for pharmacological and clinical applications. Overall, the attaining of AgNPs could represent an alternative to fight recurrent drawbacks caused by chemotherapy agents. However, conclusive safety and efficacy have not been broadly confirmed in in vivo models, therefore, using AgNPs needs further investigations before starting to use them in clinical applications.

Conclusions

Green synthesized AgNPs have exhibited greater cytotoxicity on colorectal adenocarcinoma CRC cells. AgNPs have selectively killed cancer cells through suppression of proliferation, cell cycle arrest at the G2/M phase, and induction of apoptosis. Interestingly, we have confirmed that SW620 are more sensitive than HCT8. However, we need further investigation to understand the detailed mechanism of AgNPs. Overall, our findings strongly suggest that AgNPs merit further investigation as cancer chemotherapeutic agents or combination nano drugs in future anti-cancer therapy.