Silver Nanoparticles Green Synthesis from Catharanthus roseus Flowers and Effect on A549 Lung Cancer Cells

Abstract

Silver nanoparticles (AgNPs) have extensive applications in nanomedicine due to their physicochemical properties and interactions with biomolecules. One way of obtaining them is through green synthesis with plant extracts containing reducing and capping agents, such as Catharanthus roseus (L.) G Don. This plant has pharmacological applications due to its phytocompounds with biological activity. The C. roseus flowers are rich in alkaloids and phenolic compounds, and have the potential to synthesize AgNPs but have never been used with this purpose. In this study, AgNPs were synthesize with the aqueous extract from the C. roseus flowers, and their cytotoxicity evaluated on A549 lung tumor cells through MTT assay. The AgNP synthesis could be seen visually from the solution's color change and through UV-visible spectroscopy to detect a peak between 400 - 530 nm. FEG microscopy showed AgNPs with variable shapes and sizes. In vitro cytotoxicity assay against A549 cells showed that the C. roseus extract have low toxicity to the lineage, and AgNPs reduced half the viable cells at the 1.5% concentration, demonstrating high cytotoxicity. Therefore, AgNPs from C. roseus were successfully obtained and showed significant cytotoxic activity on A549 lung cancer cells.

Keywords:
Green synthesis; nanotechnology; silver nanoparticles; sustainability; cytotoxicity

HIGHLIGHTS

• The aqueous extract of Catharanthus roseus (L.) G Don flowers can successfully produce silver nanoparticles (AgNPs) in a simple and fast way.

• The syntethised AgNPs presented higher cytotoxic activity against A549 lung cancer cells compared to the C. roseus flower extract.

INTRODUCTION

Nanomedicine is one of the fields where nanotechnology has extensive applications with nanomaterials and devices designed for diagnosis, treatment, pain relief, and overall preservation and improvement of health. The application of silver nanoparticles (AgNPs) in nanomedicine is possible due to their physicochemical properties, interactions with biomolecules on both the surface and inside of the cells, size, and natural transport uptake mechanisms of the drug by the diseased cells [¹-⁷].

The green synthesis of metallic nanoparticles is an eco-friendly alternative to the use of precursor chemicals and solvents like sodium borohydrate, ascorbate, hydrazine, and sodium citrate [², ⁹]; and possible generation of toxic byproducts. This technique uses biological agents such as plants and microorganisms or their parts to synthesize nanoparticles both extracellular and intracellular. In this approach, plants extracts are a great resource for nanoparticles synthesis by providing natural reducing and capping agents such as monosaccharides, sapogenins, flavonoids, and other phytochemicals [⁷-¹¹].

Catharanthus roseus (L.) G Don (Family: Apocynaceae) is an herbaceous plant known by her alkaloids vincristine and vimblastine with antineoplastic properties, besides other several active phytocompounds, such as organic acids, flavonoids, tannins, saponins, glycosides and terpenoids. Originated from Madagascar, this plant has now worldwide distribution, and is widely cultivated as an ornamental plant and herbal medicine for antibacterial, antifungal, wound healing, antiplasmodial, antioxidant, antiviral, and anticancer activities [⁵, ⁷, ¹⁰-¹² ].

Reports have confirmed AgNPs synthesis using Catharanthus roseus (L.) G Don leaf [⁵] and root extracts [¹⁴], but the use of C. roseus flowers have not yet been reported. The C. roseus flowers contain the anthocyanidin pigment rosinidin and are especially rich in alkaloids and phenolic compounds (Table 1) [¹⁰, ¹⁶-²¹]. The phytochemical composition of C. roseus provides flowers with great potential to synthesize AgNPs.

Therefore, the present work aimed the green synthesis of silver nanoparticles (AgNPs) using Catharanthus roseus (L.) G Don aqueous flowers extract and cytotoxicity evaluation on A549 tumor cells.

Table 1
Alkaloids and phenolic compounds on C. roseus flowers

Class	Compound Name	References
Alkaloids	14′,15′-Didehydrocyclovinblastine 17-Deacetoxycyclovinblastine 17-Deacetoxyvinamidine Cathachunine Catharanthine Catharoseumine Clovinblastine Cycloleurosine Leurosidine Leurosine Methylvingramine Ocyclovinblastine Perivine, Tricin (Flavones) Vinamidine Vindoline Vingramine,	[16-21]
Phenolic Compounds	4-O-caffeoylquinic acid Hirsutidin-3-O-(6-O-p-coumaroyl) Hirsutidin-3-O-glucosides Isorhamnetin-3-O-(6-O-rhamnosyl-galactoside) Isorhamnetin-3-O-(6-O-rhamnosyl-glucoside) Kaempferol-3-O-(2,6-di-O-rhamnosyl-galactoside) Kaempferol-3-O-(2,6-di-O-rhamnosyl-glucoside) Kaempferol-3-O-(6-O-rhamnosyl-galactoside) Kaempferol-3-O-(6-O-rhamnosyl-glucoside) Malvidin-3-O-(6-O-p-coumaroyl) Malvidin-3-O-glucosides Petunidin-3-O-(6-Op-coumaroyl) Quercetin-3-O-(2,6-di-O-rhamnosyl-galactoside)	[14-17, 21]

MATERIAL AND METHODS

Preparation of C. roseus extract

Catharanthus roseus (L.) G Don, Apocynaceae, flowers were collected from Palmeira, Parana, Brazil (25° 26′ 3″ S, 49° 59′ 60″ W; 867 m above sea level). Representative samples can be found at the Federal University of Technology - Paraná of Dois Vizinhos Herbarium under the register DVPR000082. The fresh flowers were de-stemmed, displayed on kraft paper, then dried in a forced air circulation oven at 60º for 24 hours. The extract was prepared using 2.5 grams of the dried flowers in 100 mL distilled water; the mixture was heated at 80 ± 5°C for 20 minutes, and then filtered with a paper filter. The extract concentration was adjusted to 1%, 1.5%, 2%, and 2.5% with volumetric flasks.

Green synthesis of silver nanoparticles

1 mL C. roseus flower extract was mixed with 9 mL AgNO3 1mM. The solution was exposed to heat (70 ± 5°C) and UV-light (365 - 405 nm) for 10 minutes. The conversion of silver nitrate (AgNO₃) into silver nanoparticles (AgNPs) was accompanied by the solution color change and record absorbance at 200-800 nm using a UV-visible spectrophotometer (Genesys 10S UV-Vis) [²²].

Characterization of green synthesized silver nanoparticles

AgNPs were characterized by Field Emission Gun-Scanning Electron Microscopy (FEG-SEM) (Mira 3 / Tescan), UV-visible spectrophotometry (Genesys 10S UV-Vis), Dynamic light scattering (DLS) with Polydispersion Index (PDI) and Zeta Pontential (Zetasizer Nano ZS90).

Cell Viability Assay

A549 cells were maintained at 37 °C under 5% CO2 and 100% humidity in DMEM and supplemented with 10% fetal calf serum and antibiotics (200 μl/mL penicillin G). After sufficient growth, the cells detached using trypsin, plated in 96-cluster well culture plates, and incubated for 24 h at 37 °C under 5% CO2. Later, the medium was removed and the cells exposed to AgNPs (from 0,1 to 50% v/v) and C. roseus flower extract (from 0,004 to 3% m/v) for 24 h (n=8 for each concentration). As negative control, only culture medium was used. After the incubation period the liquid was removed from each well and added MTT reagent, incubated for 1 h and then discarded. 100 μL of isopropanol was added and the absorbance was measured using a microplate reader at a wavelength of 570 nm [²³].

RESULTS AND DISCUSSION

Green synthesis of silver nanoparticles

The silver nanoparticles (AgNPs) synthesis from the Catharanthus roseus (L.) G Don flower extract was seen visually from the graduating change of the solution’s color from a yellowish transparent to reddish-brown presented in Figure 1. The formation of the AgNPs was monitored through UV-Visible spectral analysis recorded between 200-800 nm, and the appearance of a peak around 420 nm confirmed the synthesis (Figure 2). Different concentrations of extract tested (1%, 1.5%, 2%, 2.5%) produced similar results, therefore the smallest was chosen.

Figure 1
Process of AgNPs synthesis with C. roseus flower extract and AgNO using UV-light and heat evidencing graduate solution’s color change yellowish transparent to reddish-brown that indicated the successful synthesis.

Figure 2
UV-Vis absorption spectra of AgNPs synthesized from C. roseus flowers.

As reported in the literature [¹, ⁸, ²⁴, ²⁵], the color change from transparent to a brownish color and UV-visible spectroscopy techniques used to track the formation of AgNPs through an increase in the absorbance between 400 and 530 nm is possible due to the surface plasmon resonance of the nanometal [²⁶]. These techniques are often used as an indicator of the formation of nanoparticles. The AgNP synthesis was made using heat, UV-light, and UV-light and heat simultaneously; during the process was observed a faster change of color with UV-light plus heat indicating an optimization of the AgNP formation. Through UV-Vis spectroscopy was possible to track the complete synthesis in approximately 10 min (Figure 2).

The biosynthesis mechanism of AgNPs by green synthesis is not fully understood but likely related to the chemical composition of the extracts, in which metabolites such as monosaccharides, sapogenins, polyphenols, and others might convert silver ions to elemental silver, and they might act as stabilizing agents for the nanoparticles as well [⁵-¹⁰]. Studies have shown that proteins might also be involved in the synthesis process [²⁵].

The diagram presented in Figure 3 illustrates a possible synthesis mechanism of AgNPs accomplished in four main steps. It starts with the reduction of silver ion (Ag⁺) to molecular silver (Ag⁰), followed by a nucleation process. The third step consists of growth with coalescing metallic silver atoms into AgNPs, and finally, the fourth step is the stabilization that prevents further growth and agglomeration and might involve the extract metabolites.

Figure 3
The schematic diagram represents a possible mechanism of AgNPs synthesis from C. roseus flower extract. (1) Reduction of Ag+ to Ag0 by extract components; (2) Nucleation of molecular silver, starting the AgNP formation; (3) Growth with coalescing metallic silver atoms into AgNPs; (4) Stabilization of the AgNP by extract components and physical forces.

Characterization of green synthesized silver nanoparticles

FEG-SEM confirm the synthesis of the AgNP through photomicroscopy and inform about their morphology, size, and distribution. The results obtained showed nanoparticles with diameter ranging from 70 to 100 nm and different shapes such as spherical, rod, triangle, rhombic, and prism-like structures (Figure 4). The different AgNPs sizes and shapes can be the result of a number of factors such as precursor concentration, extract composition, time of incubation, pH, temperature, as well as method of preparation [⁹, ²⁷-²⁹].

Figure 4
FEG microscopy of the synthesized AgNPs. (A) x20.0 k/scale. (B) x62,3 k/scale.

To confirm the hydrodynamic diameter of nanoparticles in solution and size distribution profile, the Dynamic Light Scattering (DLS) and Polydispersion Index (PDI) was used. The Figure 5 shows one of the DLS and PDI results, where it is observed only one population of AgNPs with average size of 158,4 nm and PDI of 0,210. After three repetitions the average size was 161 nm (± 31,05) and PDI of 0,230 (± 0,016). The ideal value for the DLS is under 1 micrometer and for the PDI under 0,300 [³⁰, ³¹]. Therefore, the results confirm the synthesis of homogeneous nanoparticles.

The zeta potential is related to the stability of the synthesized AgNPs. The zeta potential - 16,6 mV (± 2,15) was verified by using water as dispersant (Figure 6). The negative potential value shown by the AgNPs could be due to the extract components that contain negative charge. The ideal values to affirm the AgNPs electro stability should be around - 30 mV or + 30 mV [⁹, ³⁰, ³² ]. Nonetheless the zeta potential alone is not enough to determine the overall stability, and other factors such as the presence of stabilizing agents in the solution have to be taken under consideration.

Figure 5
DLS and PDI of the synthesized AgNPs.

Figure 6
Zeta potential of the synthesized AgNPs.

Cell Viability Assay

In vitro cytotoxicity studies against A549 cells were performed with different concentrations of C. roseus extract and AgNPs with MTT assay to measure cell viability and proliferation. The obtained results are presented in Figure 7. It is observed that none of the extract flower concentrations tested in this study exhibited a reduction of viable cells equal or superior to 50%, indicating low cytotoxicity from aqueous extract of C. roseus flowers in the given conditions on A549 cellular lineage.

The results for the AgNPs showed a concentration-dependent decrease in the cell viability until 2,5%, with a decreasing percentage of viable cells observed as the concentration of AgNPs increased. A reduction of half the viable cells was observed around 1.5% of AgNPs, indicating high cytotoxicity. Others studies about the effects of green synthesized AgNPs on lunger cancer cells also demonstrated high cytotoxic activities [⁷, ³⁴].

AgNPs are mainly known for their potent antibacterial activity [⁴, ⁶], but also possess cytotoxic activity and potential as agents for cancer therapy, as shown by Yesilot and Aydin [³⁵] in a review article. These activities are possible due to the AgNPs’ small size that enables them to enter the cells and capacity to reduce the activity of antioxidant enzymes from mitochondrial respiratory chain, production of free radicals, and disruption of the oxidative state of the cells leading to their death by oxidative stress [⁹, ³⁴, ³⁵].

The small size of AgNPs also implies a large surface area that allows the coordination of a vast number of ligants with diagnosis and therapeutic finalities that can be explored for medical applications, giving AgNPs great potential as caring agents for targeted and controlled release of drugs, markers and others [⁶]. But further studies assessing potential efficacy, biosafety, and biodistribution of AgNPs are necessary.

Therefore this study demonstrated that the synthesis of silver nanoparticles from C. roseus is possible, simple, fast, and ecofriendly. The AgNPs presented higher cytotoxic activity against A549 lung cancer cells than the extract in the tested concentrations, meaning that this activity is mainly attributed to the AgNPs and not the extract components.

Figure 7
Cell viability of A549 lunger cancer human cells after treatment with C. roseus extract and AgNPs at 24 h determined via MTT reduction assay. Results presented as mean ± standard error of mean (n=8) and analysed using one-way ANOVA followed by Tukey’s post hoc test of significance p < 0.001 (***) compared to negative control.

CONCLUSION

The present study showed that the aqueous extract of Catharanthus roseus (L.) G Don flowers successfully produce silver nanoparticles (AgNPs) in a simple, fast, and ecofriendly process; with high cytotoxic activity against A549 lung cancer cells. Therefore, the AgNPs obtained from the aqueous extract of Catharanthus roseus (L.) G Don flowers demonstrate to be a potential candidate for further studies evolving cancer therapy.

Acknowledgments:

State University of Ponta Grossa (UEPG); C-Labmu (UEPG); Araucaria Foundation; CNPq for providing instruments, facilities and other items used in this study, and for the incentives for research and scientific production in Brazil.

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• Funding:

  This research received no external funding

Edited by

Editor-in-Chief:

Paulo Vitor Farago

Associate Editor:

Jane Manfron Budel

Publication Dates

• Publication in this collection
  20 Oct 2023
• Date of issue
  2023

History

• Received
  19 Dec 2022
• Accepted
  07 Aug 2023