CHARACTERIZING SILVER NANOPARTICLES BIOSYNTHESIZED FROM SALVIA ROSMARINUS AND ASSESSING THEIR IN VITRO ANTIFUNGAL AND CYTOTOXIC ACTIVITIES AGAINST PHYTOPATHOGENS AND CERVICAL CELLS

H. Rizwana, N. A. Bokahri, S. A. Rashed, S. A. Shehri, M. A. Awad, N. Merghani and H. Tabasuum

¹Department of Botany and Microbiology, College of Science, King Saud University, P. O Box 22452, Riyadh‒11495, Kingdom of Saudi Arabia

²King Abdullah Institute for Nanotechnology, King Saud University, Riyadh 11451, Saudi Arabia.

³ Central Laboratory, Female Center for Medical Studies and Scientific Section, King Saud University, Riyadh, KSA, Saudi Arabia

⁴Department of Clinical Laboratory Sciences, College of Applied Medical Science.

 King Saud University, P. O. Box 22452, Riyadh-11495, Riyadh- Saudi Arabia

*For correspondence: hrizwana@ksu.edu.sa

ABSTRACT

Nanotechnology is significantly revolutionizing world agriculture through engineered nanomaterials, which contribute to enhancing agricultural production by controlling fungal and bacterial phytopathogens and, consequently, minimizing crop losses. In this study green silver nanoparticles (AgNPs) were biosynthesized using aqueous extracts of organically grown Salvia rosmarinus leaves.The AgNPs were examined and characterized with ultraviolet–visible spectroscopy, dynamic light scattering, energy-dispersive X-ray spectroscopy, transmission electron microscopy, and Fourier transform infrared spectroscopy. Their antifungal activity was evaluated against an array of fungal phytopathogens. In addition, the AgNPs were tested for their cytotoxic properties using microculture tetrazolium assays against the cervical cancer HeLa cell line. The nanoparticle size ranged between 7 and 58 nm, and the AgNPs significantly inhibited Fusarium oxysporum (61%), followed by Alternaria alternata (50%). The in vitro cytotoxicity assay against HeLa cells showed potent inhibition with a median inhibitory concentration of 11.28 ± 0.33 µg/mL. S. rosmarinus biosynthesized AgNPs demonstrate significant antifungal and antiproliferative activities against plant pathogens and HeLa cells.

Key words: Salvia rosmarinus, silver nanoparticles (AgNPs), phytopathogens, cytotoxicity

http://doi.org/10.36899/JAPS.2022.3.0478
Published first online October 19. 2021

INTRODUCTION

Plant pathogens in general and fungi in particular cause immense loss to plants by drastically lowering their yield, which affects the global economy. In addition, the quality of edible parts (fruits and vegetables) of plants is affected, raising serious concerns regarding human health. In the past few years, most agricultural research has aimed to increase the crop yield to meet the growing demands of the rising global population (Nellemann et al., 2009; Savary et al., 2012). Fungal pathogens, such as Fusarium spp., Colletotrichum spp., Penicillium spp., Botrytis cinerea, Alternariaalternata, and Aspergillus sp., cause many devastating postharvest diseases in fruits and vegetables, leading to a 25%–60% loss of total production (Gonzalez -Estrada et al., 2018). Postharvest spoilage due to fungi is predominantly controlled by applying chemical fungicides (Dukare et al., 2018). Although most fungicides are effective against fungal pathogens, their use is still questionable due to harmful effects on human health and the environment. Therefore, new eco-friendly biological fungicides that are less harmful to human health and the environment are required.

Nanotechnology is a novel technology that has opened new avenues and concepts that are applicable in various fields, such as medicine, pharmacology, chemistry, physics, and, recently, food sciences (Balaure et al., 2017; Sinha et al., 2017). Nanotechnology involves the synthesis and application of nanosized particles (1 -100 nm), at this size, particles exhibit unique properties that are not present in their original form (Sandoval, 2009; Bajpai et al., 2018).). Many materials are used to prepare nanoparticles, such as metals and their oxides, lipids, emulsions, and ceramics. Recently, due to less toxicity and low cost, plants are also being explored extensively to prepare nanoparticles.

Silver nanoparticles (AgNPs) have drawn tremendous attention and increasing interest due to their high conductivity, catalytic activity, chemical stability, localized surface plasma resonance, and antimicrobial and anti-inflammatory activities (Ahmad et al., 2003; Ahmed et al., 2016). AgNPs are used for drug delivery, diagnosis, and tissue regeneration (Naidu et al., 2015); as postharvest/biological coatings (Balamurugan et al., 2017) and antimicrobials (Qasim et al., 2018); and in the textile (Gokarneshan and Velumani, 2017), cosmetic (Naidu et al., 2015), and food (Carbone et al., 2016) industries.Green synthesis refers to the process of using plant extracts and microorganisms for nanoparticle synthesis. The various bioactive compounds in plants serve as reducing agents that render them safe and eco-friendly.

Salvia rosmarinus Spenn., commonly known as rosemary (Rosmarinus officinalis L), belongs to the Lamiaceae family and is a native of the Mediterranean region (de Macedo et al., 2020). It is an herb with fragrant leaves that are used in culinary settings as flavoring agents all over the world (Panda, 2009; Ibarra, 2010). S. rosmarinus has antiangiogenic (Kayashima and Matsubara, 2012), antibacterial (Georgantelis et al., 2007), and hepatoprotective properties (Raskovic et al., 2014). S. rosmarinus extracts have also shown anticancer properties against prostrate, colon, and skin cancer cell lines (Mirghaed and Yadollahi, 2013). In addition, the European Union has approved rosemary extracts (E3920 in EU additive regulation no. 1129/2011), and the European Food Safety Authority has proposed rosemary extracts as feed additives in the antioxidant class (Aguilar et al., 2008).

In this study, AgNPs were synthesized using aqueous leaf extracts of S. rosmarinus leaves, grown in an organic manner in Saudi Arabia. The biosynthesized AgNPs were characterized and evaluated for their cytotoxic and antifungal activities.

MATERIALS AND METHODS

Plant material and chemicals used: Fresh, disease free leaves of S. rosmarinus were provided by Dr. Sara Al Rashid and identified by plant taxonomist Professor Dr. Najat Al Bukhari. All the reagents used in the experimental work were of analytical grade and were obtained from Sigma-Aldrich.

Pathogens: Alternaria. alternata, Fusarium solani, F. oxysporum, F. graminearum, Macrophomina phaseolina, and Trichoderma harzianum were provided by the Department of Plant Protection at the College of Food and Agricultural Sciences, King Saud University, Riyadh, Saudi Arabia.

Preparation of aqueous leaf extracts: S. rosmarinus leaves were washed under running tap water to remove any adhering visible impurities and soil particles. The leaves were chopped and dried (25°C), and 10 g of roughly chopped leaves were added to a beaker containing 100 mL of distilled water. This mixture was heated at 60°C for 20 min. After cooling, the supernatant was filtered through Whatman filter paper (No. 1) and centrifuged at 5000 rpm for 5 min. The supernatant was collected and used to synthesize AgNPs.

Synthesis of green Ag nanoparticles: AgNP synthesis was performed as described by Jain and Mehata (2017) with slight modifications. Briefly, a fixed volume of AgNO₃ powder (0.0085 gm) was dissolved in 25 mL of distilled water to prepare 1 mM AgNO₃ solution. Next, 1 mL of aqueous leaf extract of S. rosmarinus was added to 5 mL of 1 mM AgNO₃ solution, mixed thoroughly on a magnetic stirrer, and observed for any color changes. A change in color from light yellow to colloidal brown indicated the formation of AgNPs. The biosynthesized AgNPs were assessed by ultraviolet–visible (UV–Vis) spectroscopy, field emission scanning electron microscopy (FE-SEM), dynamic light scattering (DLS), transmission electron microscopy (TEM), and Fourier transform infrared spectroscopy (FTIR).

Characterization of biosynthesized AgNPs

UV–Vis spectroscopy: V–Vis spectroscopy (Thermo Scientific 1500, USA) was performed on the biosynthesized AgNPs to assess the reduction of Ag⁺ ions and the formation of AgNPs (colloidal-brown solution). The absorbance of the reaction mixture was measured at a wavelength of 200–700 nm.

Zetasizer: The biosynthesized AgNP samples were diluted with pure water, and the average size of the biosynthesized AgNPs was measured using a Nano–ZS-90 zetasizer (Malvern).

TEM: AgNP samples were prepared by carefully adding the biosynthesized AgNPs on a copper-coated grid. The average size of the AgNPs was determined by observing the samples under a JEM-1400 Plus transmission electron microscope (JEOL).

EDX analysis by scanning electron microscopy: The elemental composition (energy-dispersive X-ray spectroscopy [EDX]) of biosynthesized AgNPs was determined. A thin film of AgNPs was prepared on a glass slide by adding 8 µL of the suspension dropwise. The suspension was air-dried, coated with platinum, and observed by FE-SEM (JSM-7610F; JSM, Japan).

Fourier transform infrared spectroscopy (FTIR): The biomolecules present in the S. rosmarinus aqueous leaf extract and the biosynthesized AgNPs were analyzed by a Nicolet-6700 FTIR spectrometer (Thermo Scientific). The absorption spectrum was obtained at a scan range of 400–4000 cm^‒1 with a KBr pellet.

In vitroantifungal activity of the biosynthesized AgNPs: The inhibitory activity of the biosynthesized AgNPs was tested against six phytopathogenic fungal strains. In vitro mycelial inhibition was tested using potato dextrose agar medium. Roughly, 15 mL of sterilized PDA was poured into a sterilized petri dish and allowed to solidify. Test fungi were separately grown in PDA plates for 5 days and later used to remove a 6 mm mycelial plug for an in vitro assay. To test the effects of AgNPs on fungal growth, 1 mL of the biosynthesized AgNP suspension was added to sterile petri dishes, followed by 15 mL of PDA. The mixture was gently swirled and allowed to solidify. After solidification, the 6 mm mycelial plug removed from the periphery of test plates was placed in the center of the petri dishes in an upside-down manner. The dishes were incubated at 25°C ± 2°C for 7 days, and the diameter (mm) of the fungal colony was measured. Control dishes received only PDA (without AgNPs). The experiment was run in triplicate.

The effect of AgNPs on mycelial growth inhibition was calculated using the following formula:

Inhibition (%) = (Dfc − Dft)/Dfc × 100,

where Dfc is the average increase in mycelial growth in controls, Dft is the average increase in mycelial growth at each treatment.

Cytotoxicity (MTT assay): The cytotoxicity of the biosynthesized AgNPs was examined using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, as described by Siddiqui et al. (2010). The human cervical cancer cell line (HeLa cells) was obtained from the American Type Culture Collection (USA). Briefly, HeLA cells were plated in 96-well plates at a density of 1 × 10⁴ cells/well and allowed to settle for 24 h prior to treatment. The cells were treated with various concentrations of the biosynthesized AgNPs and prepared by twofold serial dilution. After incubating the plates for 24 h, MTT was added to the wells and the plates were further incubated for 4 h. Next, 200 µL of DMSO was added to the reaction mixture in each well and thoroughly mixed. All experiments were run in triplicate. Absorbance of the plates was measured at a wavelength of 550 nm, and the results were expressed as percentage cell viability. The median inhibitory concentration (IC50) was assessed using GraphPad Prism version 7, and a graph showing the dose-dependent response was generated using regression analysis.

RESULTS

Formation of S. rosmarinus AgNPs: Visual examination and UV–Vis spectral analysis: The formation of S. rosmarinus AgNPs was visually observed when the colorless AgNO₃ solution gradually transformed to a brown solution with an orange tint upon addition of the S. rosmarinus aqueous leaf extract. The color change indicated the formation of AgNPs due to surface plasmons. Fig. 1 shows the brown-colored solution, which indicates the formation of AgNPs. The process was gradual and started within the first few minutes of adding the S. rosmarinus aqueous leaf extract and took approximately 1 h to complete.

The AgNP formation was further confirmed and measured with a UV–Vis spectrophotometer to obtain a surface plasmon resonance (SPR) band. The SPR band gradually increased in intensity for up to 50 min and then stopped after 1 h. The absorption band at 450 nm indicates the formation of AgNPs (Fig. 2).

Dynamic light scattering- particle size determination: The average size of the biosynthesized S. rosmarinus AgNPs was 56 nm, and the polydispersity index (PDI) was 0.285 (Fig. 3).

Transmission electron microscopy (TEM): Microphotographs showed uniform distribution of biosynthesized S. rosmarinus AgNPs. The particles were spherical in shape and separated in a uniform manner. The mean particle size was 7–58 nm (Fig. 4).

Energy-dispersive spectrum (EDX) of AgNPs: EDX analysis of the biosynthesized S. rosmarinus AgNPs showed the presence of Ag, which is evident in the spectrum (Fig. 5). An absorption peak with an intense signal was observed at 3 KeV, corresponding to the presence of AgNPs due to SPR. The presence of other elements was also observed in the spectrum.

Fouriertransform infrared spectroscopy (FTIR) of the extract and biosynthesizedS. rosmarinus nanoparticles: The IR bands of both the S. rosmarinus aqueous leaf extract and synthesized S.rosmarinus AgNPs are shown in Fig. 6 and 7, respectively. Some common bands were observed in both spectra. The S. rosmarinus aqueous leaf extract revealed bands at 3402 cm⁻¹ (corresponding to the O–H stretching of phenols) and 1605 cm⁻¹ (corresponding to the C=C stretching of conjugated alkene). In addition, the 2 peaks at 1267 and 1039 cm⁻¹ were due to C–O stretching. However, the IR spectrum of the S. rosmarinus AgNPs exhibited the disappearance of certain bands at 1524 (N–O stretching), 1405 (N–H bending vibration), and 813 cm⁻¹ (C–H bending vibration), which were quite evident in the IR spectrum of the S. rosmarinus aqueous leaf extract. New peaks were also observed at 2928 and 1386 cm⁻¹ in the spectrum of biosynthesized AgNPs. In addition, peaks of the S. rosmarinus aqueous leaf extract shifted from 3402, 1605, and 1386 cm⁻¹ to 3426, 1620, and 1068 cm⁻¹, respectively, in the biosynthesized AgNPs and were tremendously narrower with weak transmittance (less intense).

Antifungal activity: The biosynthesized AgNPs exhibited variable antifungal activity against phytopathogenic fungi. The highest antifungal activity was seen against F. oxysporum, followed by A. alternata and F. solani (61%, 50%, and 43%, respectively). However, poor mycelial inhibition was observed in other fungal strains (Fig. 8 and 9).

Cytotoxicity of AgNPs by MTT assay: To determine the in vitro cytotoxic activity of the biosynthesized AgNPs, MTT assay was performed. HeLA cells were treated with different concentrations (3.125–100 µg/mL) of biosynthesized AgNPs, as previously described. Fig. 10 shows the percentage growth inhibition of HeLa cells and their viability when treated with biosynthesized AgNPs compared to control cells (untreated). The cancer cell inhibition was dose dependent: cell viability decreased with an increase in the AgNP concentration. Significant inhibition began at a concentration of 25 µg/mL, as only 19% of HeLa cells were viable, which dropped to a maximum of 5% cell viability at 100 µg/mL. The IC50 value was 11.28 ± 0.33 µg/mL.

DISCUSSION

This study demonstrated AgNP synthesis from S. rosmarinus aqueous leaf extracts. The appearance of a brown color marks the bioreduction of silver ions to AgNPs, while the UV–Vis spectrophotometer monitors the bioreduction process through the formation of a characteristic SPR peak. AgNPs show potent SPR activity in aqueous solutions (Shanker et al., 2004). The biosynthesized AgNPs showed an SPR peak at 450 nm, which was broad and pronounced, indicating that the AgNPs were polydispersed in nature. Characteristic AgNP peaks (λmax) are observed between 400 and 500 nm (Sastry et al., 1997). The absorption spectrum of AgNPs is highly sensitive to several factors (Jain and Mehata, 2017). The SPR position, shape, and size are affected by the dielectric medium, the size and shape of AgNPs, and the surroundings medium (Kelly et al., 2003; Zhao et al., 2008; Wani et al., 2010; Dada et al., 2017). S. rosmarinus aqueous leaf extracts contain phenolic compounds, flavonoids, and amides, which are responsible for the bioreduction of silver ions to AgNPs. Similar findings related to the SPR peak from S. rosmarinus AgNPs have also been previously reported (Ghaedi et al., 2015).

DLS analysis of the biosynthesized S. rosmarinus AgNPs showed that the PDI and average size of AgNPs were 56 and 0.28 nm, respectively, indicating that the S.rosmarinus AgNPs are stable. PDI values greater than 0.7 indicate that the sample has a broad size (Roy et al., 2017). Since the PDI was lower than 0.7 in this study, the biosynthesized AgNPs are of considerably good quality. Our findings agree with those of previous reports in which PDI values of 0.398 and 0.7, respectively, were observed, since the plant extracts formed a wide range of coatings around the nanoparticles (De Aragao et al., 2016; Roy et al., 2017).

TEM microphotographs showed that the biosynthesized S. rosmarinus AgNPs were roughly spherical. The average size was 7–58 nm. In addition, the AgNPs were polydispersed, well separated, and distributed in a uniform manner. Their small size observed in the TEM microphotographs, in comparison to the DLS spectrum, is due to the physical state in which the sample was measured. As TEM requires dry samples (nanoparticles) and DLS requires hydrated particles, the hydrodynamic volume is larger in the hydrated state, which contributes to a large size of the nanoparticles (Gao et al., 2008). Biomolecules that are present in the S. rosmarinus leaf extract (aqueous) also caused capping of the AgNPs, which is evident in the microphotographs. Similar findings were previously reported in which various organic bioactive compounds present in plant extracts formed a thin coating around the nanoparticles, facilitated bioreduction, stabilized the biosynthesized nanoparticles, and sometimes also resulted in the agglomeration of a few nanoparticles (Mallikarjuna et al., 2014; Aritonang et al., 2019).

The EDX spectrum of the S. rosmarinus AgNPs showed a prominent peak at 3 KeV which indicates the presence of silver. The peak in this region is attributed to SPR, which ascertains AgNP formation (Das et al., 2013; Mallikarjuna et al., 2014). Besides, the silver peak, peaks related to other elements were also observed in the spectrum. These peaks arise due to elements that are present in the leaf extracts. The platinum peak is due to the coating used on the sample.

FTIR analysis was used to identify the potential biomolecules responsible for bioreduction and capping of silver ions during AgNP synthesis. The broad peaks in the S. rosmarinus aqueous leaf extract spectrum at 3402 cm⁻¹ indicated that the extract was rich in phenols. However, after AgNP synthesis, the disappearance and narrowing of the certain peaks in the IR spectrum indicated that the biomolecules (functional groups) served a purpose in the bioreduction and synthesis of S. rosmarinus AgNPs. In addition, comparison of the IR spectra of biosynthesized S. rosmarinus AgNPs and leaf extract showed that several peaks observed in the leaf extract were shifted in the IR spectrum of synthesized AgNPs. Shifts in peaks from 1461 to 1386 cm⁻¹ and from 3402 to 3426 cm⁻¹, suggests the involvement of bending vibration of N–H (amines) or alcoholic groups in Ag reduction. The peak at 1405 cm⁻¹, due to N–H bending in the amine group in the S. rosmarinus aqueous leaf extract, serves as a capping and stabilizing agent, as previously reported by Jyoti et al. (2016). Thus, FTIR analysis of biosynthesized AgNPs in this study evidently indicates the presence of phenols, aliphatic amines, terpenoids, and flavonoids. These molecules seem to surround the AgNPs and serve as strong binding sites for the AgNPs during synthesis. S. rosmarinus leaf extracts are rich in important secondary metabolites, such as flavonoids, phenols and carnosol derivatives, which play a key role in capping, reduction, and in providing stability (Shah et al., 2014). Previous studies suggest that the presence of functional groups on the surfaces of biosynthesized AgNPs are from S. rosmarinus aqueous leaf extracts and other plants (Fierascu et al., 2014; Prasannaraj and Venkatachalam, 2017; Femi-Adepoju et al., 2019).

AgNPs biosynthesized from S. rosmarinus aqueous leaf extracts show significant antifungal activity against phytopathogenic fungi but in a variable manner. The potent antifungal activity of certain biosynthesized nanoparticles has been previously reported (Gupta et al., 2014; Bahrami-Teimoori et al., 2017; Al-Zubaidi et al., 2019). The exact mode of action of AgNPs as antifungals is unclear. However, studies have shown that AgNPs adhere to fungal hyphae and conidia and potentially penetrate the cell membrane, which disrupts cell integrity (Srikar et al., 2016). Another perspective regarding significant antifungal activity involves interference with ergosterol synthesis, which directly affects the integrity of cell structures and leads to cell death (Radhakrishnan et al., 2018; Roy et al., 2019). In addition, the large surface area of AgNPs induces increased reactive oxygen species (ROS) and free-radical production and the leakage of DNA and proteins, resulting in cellular damage (Ogar et al., 2015; Dakal et al., 2016; Ibrahim et al., 2020). Previous studies have shown strong in vitro inhibition of fungi, such as Bipolaris sorokiniana, Magnaporthe grisea, and Rhizoctonia solani, when treated with nanoparticles biosynthesized from plants (Elgorban et al., 2016; Lopez et al., 2018). Recently, the in vitro inhibition of Aspergillus oryzae and C. albicans by Rosmarinus officinalis AgNPs was reported (Ghaedi et al., 2015).

Our findings showed potent cytotoxic activity of S. rosmarinus AgNP against HeLa cells, which could be due to the diverse bioactive compounds attached to the AgNPs and the nanosize that enables effective cell penetration. AgNPs induce cytotoxicity by disturbing the cell cycle of cancer cells and inhibiting cell proliferation (Dziedzic et al., 2016). A micronucleus (MN) assay recently revealed the genotoxic potential of AgNPs as they induced chromosomal damage and abnormalities during mitosis (Sahu et al., 2016). Biosynthesized nanoparticles cause HeLa cells to shrink, decrease in density, and lose cell adhesion capability (Al Sheddi et al., 2018). Similar dose-dependent cytotoxic effects and cellular changes have been reported for S. rosmarinus AgNPs and some other green AgNPs against various cancer cell lines (Vivek et al., 2012; Sulaiman et al., 2013; Suman et al., 2013; Al Sheddi et al., 2018. Thus, the antiproliferative activity of S. rosmarinus AgNPs could be due to their capability to induce ROS generation and apoptosis (Stroh et al., 2004; Farah et al., 2016).

rosmarinus is a rich source of carnosol, carnosic acid, and rosmarinic acid. The two major components of rosemarinic acid and carnosic acid reportedly induce high cytotoxicity in breast cancer cell lines at an IC50 of 24.08–31.87 and 12.50 μg/mL, respectively (Yesil-Celiktas et al., 2010). S. rosmarinus aqueous leaf extracts and carnosic acid exert antitumorigenic effects and promote apoptosis (Huang et al., 1994; Petiwala et al., 2013). Our findings are consistent with all previous studies, as the S. rosmarinus aqueous leaf extracts have demonstrated significant antiproliferative activity against several human cancer cell lines.

Conclusion: The biosynthesized AgNPs demonstrated significant antifungal and antiproliferative activities against plant pathogens and HeLa cells. Based on the findings of this study, AgNPs synthesized from S. rosmarinus leaf extracts can be applied in postharvest technology and for crop protection against harmful fungal pathogens. In addition, S. rosmarinus AgNPs can be further explored for their anticancer properties against various cancer cell lines.

Acknowledgments: This study was supported by a grant from the Researchers Support Project. The authors would like to extend their sincere appreciation to the Researchers Supporting Project (number: RSP-2021/229), King Saud University, Riyadh, Saudi Arabia for the funding of this work.

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