Green synthesis of silver nanoparticles using Pimpinella anisum seeds: antimicrobial activity and cytotoxicity on human neonatal skin stromal cells and colon cancer cells

Abstract

Background: The present study focused on a simple and eco-friendly method for the synthesis of silver nanoparticles (AgNPs) with multipurpose anticancer and antimicrobial activities.

Materials and methods: We studied a green synthesis route to produce AgNPs by using an aqueous extract of Pimpinella anisum seeds (3 mM). Their antimicrobial activity and cytotoxicity on human neonatal skin stromal cells (hSSCs) and colon cancer cells (HT115) were assessed.

Results: A biophysical characterization of the synthesized AgNPs was realized: the morphology of AgNPs was determined by transmission electron microscopy, energy dispersive spectroscopy, X-ray powder diffraction, and ultraviolet-vis absorption spectroscopy. Transmission electron microscopy showed spherical shapes of AgNPs of P. anisum seed extracts with a 3.2 nm minimum diameter and average diameter ranging from 3.2 to 16 nm. X-ray powder diffraction highlighted the crystalline nature of the nanoparticles, ultraviolet-vis absorption spectroscopy was used to monitor their synthesis, and Fourier transform infrared spectroscopy showed the main reducing groups from the seed extract. Energy dispersive spectroscopy was used to confirm the presence of elemental silver. We evaluated the antimicrobial potential of green-synthesized AgNPs against five infectious bacteria: Staphylococcus pyogenes (29213), Acinetobacter baumannii (4436), Klebsiella pneumoniae (G455), Salmonella typhi, and Pseudomonas aeruginosa. In addition, we focused on the toxicological effects of AgNPs against hSSC cells and HT115 cells by using in vitro proliferation tests and cell viability assays. Among the different tested concentrations of nanoparticles, doses < 10 µg showed few adverse effects on cell proliferation without variations in viability, whereas doses >10 µg led to increased cytotoxicity.

Conclusion: Overall, our results highlighted the capacity of P. anisum-synthesized AgNPs as novel and cheap bioreducing agents for eco-friendly nanosynthetical routes. The data confirm the multipurpose potential of plant-borne reducing and stabilizing agents in nanotechnology.

Keywords: Pimpinella anisum seeds; antibacterial; biosafety; cancer; green nanotechnology; metal nanoparticles.

Figure 1

Green synthesis of silver nanoparticles using the aqueous extract of Pimpinella anisum seeds: (A) seed extract postreaction with AgNO₃; (B) P. anisum seed extract alone.

Figure 2

Pimpinella anisum-synthesized silver nanoparticles: UV-vis spectra recorded over different time intervals, until 196 h. Abbreviations: UV-vis, ultraviolet-visible; h, hours.

Figure 3

FTIR spectrum of dried powder of Pimpinella anisum-synthesized silver nanoparticles. Abbreviation: FTIR, Fourier transform infrared spectroscopy.

Figure 4

XRD confirmed the crystalline nature Pimpinella anisum-synthesized silver nanoparticles. Abbreviation: XRD, X-ray powder diffraction.

Figure 5

TEM of Pimpinella anisum-synthesized silver nanoparticles. Abbreviation: TEM, transmission electron microscopy.

Figure 6

EDS showing the presence of ag in Pimpinella anisum-synthesized silver nanoparticles. Abbreviation: EDS, energy dispersive spectroscopy.

Figure 7

Antibacterial effect of Pimpinella anisum seed extract (A) (1a, 2a, 3a, 4a, and 5a corresponding to 10 µL, 20 µL, 30 µL, 40 µL, and 50 µL, respectively) and anise-fabricated silver nanoparticles (1b, 2b, 3b, 4b, and 5b). (B) Bar diagram representation of zone of inhibition, two-tailed t-test (data are presented as mean ± SE, n=5). *P<0.05.

Figure 8

Cell viability (A) and proliferation (B) in hSSCs exposed to different doses of Pimpinella anisum-synthesized silver nanoparticles (AgNPs) (2–10 µg/mL). Note: (A) scale bars= 10 µm. (B) Alamar blue assay showing the proliferation (%) decline in a dose dependent manner. Mean ± SE, n=10. ***P<0+.0005. Abbreviation: hSSCs, human neonatal skin stromal cells.

Figure 8

Cell viability (A) and proliferation (B) in hSSCs exposed to different doses of Pimpinella anisum-synthesized silver nanoparticles (AgNPs) (2–10 µg/mL). Note: (A) scale bars= 10 µm. (B) Alamar blue assay showing the proliferation (%) decline in a dose dependent manner. Mean ± SE, n=10. ***P<0+.0005. Abbreviation: hSSCs, human neonatal skin stromal cells.

Figure 9

Cell viability (A) and proliferation (B) in HT115 exposed to different doses of Pimpinella anisum-synthesized silver nanoparticles (AgNPs) (2–10 µg/mL). Notes: AgNPs effects on hT115 cell viability and proliferation, (A) Fluorescence images of hSSCs cells (± AgNPs, exposure of different concentration [2–10 µg/mL]) stained with acridine orange/ethidium bromide to analyse the apoptosis and viability. Scale bars= 10 µm. (B) Alamar blue assay showing the proliferation (%) decline in a dose dependent manner. Data are presented as mean ± SE, n=10. *P<0.05; ***P<0.0005.. Abbreviations: AgNPs, silver nanoparticles; hT115, colon cancer cell line.