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Saudi Journal of Biological Sciences logoLink to Saudi Journal of Biological Sciences
. 2022 Jan 3;29(4):2772–2781. doi: 10.1016/j.sjbs.2021.12.056

Comparative study of antifungal activity of two preparations of green silver nanoparticles from Portulaca oleracea extract

Fatimah Al-Otibi 1,, Shahad A Alfuzan 1, Raedah I Alharbi 1, Abdulaziz A Al-Askar 1, Rana M AL-Otaibi 1, Hajar F Al Subaie 1, Nadine MS Moubayed 1
PMCID: PMC9073049  PMID: 35531187

Abstract

The green silver nanoparticles (green AgNPs) exhibit an exceptional antimicrobial property against different microbes, including bacteria and fungi. The current study aimed to compare the antifungal activities of both the crude aqueous extract of Portulaca oleracea or different preparations of green AgNPs biosynthesized by mixing that aqueous extract with silver nitrate (AgNO3). Two preparations of the green AgNPs were synthesized either by mixing the aqueous extract of P. oleracea with silver nitrate (AgNO3) (normal AgNPs) or either irradiation of the AgNPs, previously prepared, under 60Co γ-ray using chitosan (gamma-irradiated AgNPs). Characterization of different AgNPs were tested by Zeta potential analyzer, Ultraviolet (UV) Visible Spectroscopy, and Fourier-Transform Infrared (FTIR) spectrometry. Three different plant pathogenic fungi were tested, Curvularia spicifera, Macrophomina phaseolina, and Bipolaris sp. The antifungal activities were evaluated by Transmission Electron Microscope (TEM) for either the crude aqueous extract of P. oleracea at three doses (25%, 50%, and 100%) or the newly biosynthesized AgNPs, normal or gamma-irradiated. With a few exceptions, the comparative analysis revealed that the irradiated green AgNPs at all three concentrations showed a relatively stronger antifungal effect than the normal AgNPs against all the three selected fungal strains. UV–visible spectroscopy of both preparations showed surface plasmon resonance at 421 nm. TEM results showed that both AgNPs were aggregated and characterized by a unique spherical shape, however, the gamma-irradiated AgNPs were smaller than the non-irradiated AgNPs (0.007–0.026 µM vs. 0.009–0.086 µM). TEM photographs of the fungal strains treated with the two AgNPs preparations showed flaccid structures, condensed hyphae, and shrunken surface compared with control cells. The data suggested that the biosynthesized P. oleracea AgNPs have antifungal properties against C. spicifera, M. phaseolina, and Bipolaris sp. These AgNPs may be considered a fungicide to protect different plants against phytopathogenic fungi.

Keywords: Antifungal activity, Aqueous extract, Green silver nanoparticles, Gamma irradiation, Portulaca oleracea, Plant pathogenic fungi

1. Introduction

Portulaca oleracea L., which is commonly known as Purslane, the duckweed or the little hogweed, a flowering plant and a member of the Portulacaceae family (order Caryophyllales), comprises 30 genera and about 500 species including some small herbaceous plants and herbs such as Portulaca pilosa and Avonia ustulate (Zhou et al., 2015). It's a widespread annual succulent edible herb that was a traditional folk medicine that acted as a vermifuge, febrifuge, and antiseptic in the North African, Asian, and European countries, besides, the tropical and subtropical regions (Zhou et al., 2015, Irsathy et al., 2012).

The chemical analysis of the P. oleracea revealed a high content of phytochemicals. Previous studies reported that its extract contains different proteins, soluble carbohydrates, inorganic acids, flavonoids, alkaloids, cardiac glycosides, coumarins, tannins, saponin, and anthraquinone glycosides (Achilonu et al., 2018, Nagarani et al., 2014). The leaves of P. oleracea contain a high level of magnesium, omega-3 fatty acids, and α-linolenic acid (Nagarani et al., 2014, Rahimi et al., 2019). Previous studies haven't reported the full medicinal properties of the P. oleracea. Despite that, some recent studies showed that its crude extract exhibited antibacterial (Mousavi et al., 2015), antifungal (Du et al., 2017), antiviral (Li et al., 2019), anti-inflammatory (Allahmoradi et al., 2018), antiulcerogenic, antioxidant, and wound-healing properties (Achilonu et al., 2018). Another study showed that the ethanolic extract of P. oleracea inhibited the growth of the pathogenic yeast Candida albicans (Soliman, et al., 2017).

Macrophomina phaseolina (M. phaseolina) is a soil-borne fungal pathogen with heterogeneous host septicity. It causes seedling blight, damping off, and rot (collar, stem, basal stem, and root) in many plant species (Mayék-Pérez et al., 2001). Bipolaris sp. is a common phytopathogenic fungi that cause leaf blights and spots, melting outs, root and foot rots, and other diseases in high-value field crops, such as rice, maize, wheat, sorghum, and other host plants (Manamgoda et al., 2014). Curvularia spicifera (C. spicifera) is another phytopathogen that causes the leaf blights, and rotting of the fruit and stem in date and ornamental palms. It occasionally affects wheat, rice, barley, and other cereals (Qostal et al., 2019).

The green silver nanoparticle is a recent technique that synthesis silver nanoparticles (AgNPs) in combination with plant-derived phytochemicals (AlSalhi et al., 2019). Previous studies reported that the green AgNPs exhibited different antimicrobial activities (Mallmann et al., 2015, Qing et al., 2018). The relative ease of preparation of green AgNPs, the low cost, and eco-friendly applications are the most reported advantages (Ahmed and Mustafa, 2020, Abbasi et al., 2019, Singh et al., 2019).

In the current study, we compared the antifungal activity of either the normal or gamma-irradiated silver nanoparticles (AgNPs) synthesized in combination with the aqueous extract of P. oleracea. Different fungal species were used, included C. spicifera, M. phaseolina, and Bipolaris sp. Moreover, the Fourier-transform infrared (FTIR) spectroscopy was applied to identify the phytochemical composition of the synthesized AgNPs. Besides, the transmission electron microscopy (TEM) characterized the ultrastructural damage induced by the two green AgNPs preparations.

2. Materials and methods

2.1. Sample’s collection and preparation of the P. oleracea aqueous extract

P. oleracea was collected from a local garden in Buraydah governorate, Al-Qassim Region, Saudi Arabia. The fresh leaves were washed carefully with deionized water, air-dried at room temperature, then sliced into small pieces with sterile scissors. An amount of 2.5 g of the fresh sliced leaves were soaked in deionized water at 10% (w/v). The leaves were boiled by using a hot plate supplied with a magnetic stirrer (Fig. 1). The mixture was then cooled and filtered by Whatman® qualitative filter paper (Sigma-Aldrich, St. Louis, Missouri, USA). The filtrate was clear and colorless with an acidic pH of 4.5. The extract was preserved aseptically in glass bottles at 4 °C for further use.

Fig. 1.

Fig. 1

Schematic representation of the aqueous extract preparation from the P. oleracea leaves. A) Leaves slicing, B) Boiling in deionized water, C) extract filtration, D) Ready-to-use aqueous extract of P. oleracea leaves.

2.2. Synthesis of the green AgNPs using P. oleracea aqueous extract

The microwave-assisted extraction technique was employed to biosynthesize silver nanoparticles (Joseph and Mathew, 2014, Seku et al., 2018). AgNPs were prepared either normally or by gamma-irradiation at three different concentrations (100%, 50%, and 25%). For example, for the synthesis of the normal AgNPs, we mixed 1 mL of the P. oleracea aqueous extract with 1 mM of silver nitrate (AgNO3) solution (100%), as described previously (Mohammed et al., 2018). The mixture was then boiled with continuous stirring for a few minutes until the colorless aqueous extract turned yellowish-brown because of the extracellular synthesis of nanoparticles (Guilger-Casagrande and de Lima, 2019). The synthesis of the gamma-irradiated AgNPs was by irradiating the normal AgNPs under 60Co γ-ray using chitosan (Nhien et al., 2018). The radiating of the normal AgNPs solution was optimized at a fixed dose rate of 40.9 Gy/min and at different time points. Then, stabilization of the got AgNPs took place in a dilute irradiation-degrade chitosan solution (Phu et al., 2014).

2.3. Fungal specimens

The fungal strains were identified and obtained from the Department of Botany and Microbiology, College of Sciences, King Saud University, Riyadh, Saudi Arabia. The fungi included C. spicifera (NCBI Taxonomy ID:145392, accession number: MT497471.1), M. phaseolina (NCBI Taxonomy ID: 35725, accession number: MN128590), and Bipolaris sp. (NCBI Taxonomy ID: 339742, accession number: MN978926). The fungal isolates were cultured and maintained on potato dextrose agar. The strains were stored at 4 °C or sub-cultured once a month for further uses.

2.4. Antifungal screening

The antifungal activity of the synthesized green AgNPs of the crude aqueous extract of P. oleracea was investigated using the poisoned food technique (Gakuubi et al., 2017). Briefly, one mL of different AgNPs at the three different doses (100%, 50%, and 25%) was, aseptically, poured into sterile Petri dishes. Then, the volume of 19 mL of molten potato dextrose agar was poured and solidified. At nine days old, either C. spicifera, M. phaseolina, or Bipolaris sp. cultures were seeded, separately, into the central area of the Petri dishes for more seven days at 25 ± 2 °C. An untreated control petri dish was used for comparison. The percentage of the inhibition in mycelial growth (IMG) was calculated as follows:

%IMG=dC-dTdCX100

where dC and dT are the mean diameters of the mycelial growth in the control and treated plates, respectively (Costa et al., 2015).

2.5. Characterization of biosynthesized green AgNPs

The characteristics of the synthesized green AgNPs were determined by the UV–visible spectroscopy, Libra S22 (Biochrom PVT, Cambridge, UK) at a resolution of 200–800 nm. The size and morphological characteristics of AgNPs were determined by TEM, JEM-1011 (JEOL, Peabody, MA, USA). FTIR spectroscopy by Nicolet 6700 FT-IR (Thermo Fisher Scientific, MS, USA) which is supplied with a beam splitter and a detector, where the configuration of the functional constituents in AgNPs preparations were analyzed by OMNIC software (Alotibi and Rizwana, 2019). Finally, the Zeta potential analyzer, Zeta sizer Nanoseries HT (Malvern Panalytical, Malvern, UK) was used to detect the surface charge of AgNPs in the solution.

2.6. Statistical analysis

As all experiments were carried out in triplicates, the means and standard deviations were calculated using GraphPad Prism 6. Significant differences between results were evaluated by IBM SPSS Statistics 22.0 by the analysis of variance tool, One-way ANOVA. The significance levels were set at P < 0.05.

3. Results

3.1. The biogenic properties of AgNPs of P. oleracea aqueous extract:

The morphological, chemical and physical properties of the synthesized AgNPs of P. oleracea had been tested. Characterization of normal and gamma-irradiated AgNPs using UV–visible spectroscopy showed a strong peak at approximately 421 nm, which could be because of the surface plasmon resonance (SPR) band of AgNPs. There were characteristic differences observed in the absorption spectra of both AgNPs (Fig. 2).

Fig. 2.

Fig. 2

UV–visible spectra of AgNPs assessed by Libra S22 A) spectra of normal AgNPs, B) spectra of gamma-irradiated AgNPs. UV, ultraviolet.

Results from TEM revealed the morphological characteristics of the synthesized AgNPs. Both types of AgNPs were aggregated and characterized by a unique spherical shape. The image analysis estimated the size of the gamma-irradiated AgNPs to range from 0.007 to 0.026 µM (Fig. 3A) where the size of the normal AgNPs ranges from 0.009 to 0.086 µM (Fig. 3B).

Fig. 3.

Fig. 3

TEM microphotographs of the synthesized green silver nanoparticles of P. oleracea extract by JEM-1400, the diameter of the particles was calculated in µM with a magnification of 200,000×. (A) Normal AgNPs (without irradiation) and (B) Gamma-irradiated AgNPs. TEM; Transmission Electron Microscopy.

FTIR spectroscopic analysis of the synthesized AgNPs revealed almost similar estimated composition (Fig. 4). In the normal AgNPs, FTIR showed medium absorption peaks at 3267, 2181, and 1635 cm−1 which are corresponding, respectively, to alcohol, alkyne, and alkylamines, besides four weak sharp peaks at 2160, 2150, 1987, and 1959 cm−1 which correspond to azide, thiocyanate, ketones, and aromatic compounds, respectively (Fig. 4A; Table 1). Similarly, FTIR analysis of the gamma-irradiated AgNPs showed medium absorption peaks at 3260, 2207, 2173, 2042, 2026, and 1634 cm−1 which are corresponding to alcohol, alkyne, thiocyanate, and alkylamines, besides four weak sharp peaks at 2159, 2104, 1988, and 1960 cm−1 which correspond to azide, alkyne, and aromatic compound, respectively (Fig. 4B; Table 1).

Fig. 4.

Fig. 4

Fourier-transform infrared (FTIR) spectrum of synthesized AgNPs of P. oleracea extract. A) Normal AgNPs, B) Gamma-irradiated AgNPs.

Table 1.

The functional group analysis by FTIR.

Tested material Absorption (cm−1) Appearance Group Compound Class
Normal AgNPs 3267 Medium, broad O─H stretching Alcohol, intramolecular bonded
2181 Medium C≡C stretching Alkyne, disubstituted
2160 Weak N═N═N stretching,
S─C≡N stretching
Azide or Thiocyanate
2150 Weak C═C═O stretching ketene
1987, 1960 Weak C─H bending Aromatic compound, overtone
1635 Medium, sharp C═C stretching,
N─H bending
Alkene or Amine
2011 Medium None



Gamma-irradiated AgNPs 3261 Medium, broad O─H stretching Alcohol, intramolecular bonded
2207 Medium C≡C stretching Alkyne, disubstituted
2173 Medium S─C≡N stretching Thiocyanate
2159 Weak N═N═N stretching Azide
2104 Weak C≡C stretching Alkyne, monosubstituted
2042, 2026 Medium N=C=S stretching Isothiocyanate
1988, 1960 Weak C─H bending Aromatic compound, overtone
1634 Medium, sharp C═C stretching,
N─H bending
Alkene or Amine

The Zeta potential analysis detected the mean diameter size of the normal P. oleracea AgNPs to be 117.4 nm with a polydispersity index (PdI) value of 0.167 and an intercept of 0.901 which was represented by one specific peak at 100% intensity and size of 142.9 ± 58.3 nm (Fig. 5A). Unlikely, the average particle size of the gamma-irradiated P. oleracea extracts AgNPs was smaller at Z-average of 69.09 nm, with a PdI value of 0.243 and an intercept of 0.869 which was represented by two peaks at the sizes of 94 ± 41.3 nm (95%) and 15 ± 3.1 nm (5%) (Fig. 5B).

Fig. 5.

Fig. 5

The intensity percentage describing the particle size distribution of biosynthesized AgNPs of P. oleracea extract. A) Normal AgNPs, B) Gamma-irradiated AgNPs. UV, ultraviolet; PdI, polydispersity index.

3.2. Antifungal activity of silver nanoparticles of aqueous extract of P. oleracea

Both normal and irradiated silver nanoparticle preparations of P. oleracea extract inhibited the mycelial growth of C. spicifera, M. phaseolina, and Bipolaris sp. Different concentrations didn’t show any markable differences in the percentage of inhibition of the mycelial growth of the three fungal strains. Comparative analysis revealed that, with a few exceptions, the irradiated green AgNPs at all three concentrations showed a relatively stronger antifungal effect than the normal AgNPs against all the three selected fungal strains (Table 2). The control plates (untreated) showed confluent normal mycelial growth for all fungi (Fig. 6).

Table 2.

Antifungal activity of Normal and Irradiated AgNPs of P. oleracea extract.

Fungal species Curvularia spicifera
Macrophomina phaseolina
Bipolaris sp.
Growth* (mm) % IMG Growth* (mm) % IMG Growth* (mm) % IMG
Control 88.0 ± 0.0 0.00 88.0 ± 0.0 0.00 88.0 ± 0.0 0.00
Normal AgNPs 16.5 ± 5.0 81.25 15.5 ± 4.5 82.39 25.8 ± 9.8 70.68
12.8 ± 0.3 85.45 16.5 ± 1.0 81.25 26.3 ± 1.8 70.11
16.0 ± 0.5 81.82 14.0 ± 0.0 84.09 20.3 ± 1.3 76.93
Gamma-irradiated AgNPs 17.8 ± 1.3 79.77 14.3 ± 2.3 83.75 19.0 ± 1.5 78.41
9.5 ± 2.0 89.20 9.8 ± 0.8 88.86 17.5 ± 6.0 80.11
17.0 ± 3.5 80.68 13.0 ± 0.5 85.23 15.0 ± 4.0 82.95

*Means ± SD of triplicates.

Fig. 6.

Fig. 6

In vitro antifungal activity of three different doses of either normal or gamma-irradiated AgNPs by the poisoned food technique, against. (A)Carvabaria spicifera, (B)Macrophomina phaseolina and (C)Bipolaris sp.

TEM microphotographs revealed the morphological changes induced by different AgNPs treatments against the strains of C. spicifera, M. phaseolina, and Bipolaris sp. (Fig. 7). The untreated strains showed the mycelia with intact to a normal tubular structure (left panel). The fungi treated with the normal AgNPs showed distended and flaccid structures with condensed hyphal branches and rough/shrunken surfaces (middle panel). Similarly, distorted structures were observed for all the three fungal strains treated with the gamma-irradiated green AgNPs (right panel).

Fig. 7.

Fig. 7

TEM microphotographs showing the antifungal activity of the synthesized AgNPs of P. oleracea extract by JEM-1400 with a magnification of 200,000×. The left panel shows the untreated strains with normal mycelia and tubular structure, the middle panel shows the effect of normal AgNPs treatment and the effect on the hyphal and surface structures, while the right panel shows the gamma-irradiated green AgNPs. A)Carvabaria spicifera, B)Macrophomina phaseolina, and C)Bipolaris sp. TEM; Transmission Electron Microscopy.

4. Discussion

Deserts of the Arabian Peninsula countries are rich sources of many wild plants and herbs of medical importance (Chaurasia and Gharia, 2017). The antifungal activity of P. oleracea different extracts was well-studied against different bacterial and fungal species (Mousavi et al., 2015, Du et al., 2017). Multiple studies suggested that different preparations of plant extracts using ethanol, chloroform, or other solvents could affect the pharmacological and morphological properties of the growth of some species such as Trichophyton sp. (Oh et al., 2000) and Candida ablicans (Soliman et al., 2017) more than the aqueous extract (Chaurasia and Gharia, 2017). However, recent studies showed that aqueous extract of P. oleracea leaves which had a greater content of phenolic and flavonoids compounds 210.4 ± 1.15 and 36.7 ± 0.79 mg/mL) could inhibit the growth of different fungal and bacterial strains belong to Aspergillus sp. (El-Desouky, 2021), Helicobacter pylori, Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus mutan, and Escherichia coli (Cho et al., 2008, Sun et al., 2015). In the study conducted by Sun et al., (2015), the aqueous extract of P. oleracea leaves (0.25 g/ml) was more effective against Escherichia coli as it showed larger inhibition zone (19.2 ± 0.19 mm) than the ethanolic extract (19.2 ± 0.19 mm).

In the present study, the antifungal activity of silver nanoparticles of the aqueous extract of P. oleracea was investigated. The synthesis of AgNPs followed the eco-friendly green route method to create two different preparations of nanoparticles, including the normal and gamma-irradiated AgNPs, to screen their inhibitory effect on the mycelial growth of the selected fungal strains (C. spicifera, M. phaseolina, and Bipolaris sp.). The results of the UV-spectrum analysis showed that the biosynthesized AgNPs presented an excitonic absorption edge at 400–500 nm which confirmed their formation. Regarding that, a previous study showed that synthesized AgNPs had a unique band of SPR at 400–500 nm compared to AgNO3 such as the AgNPs of Cornus officinalis extract (Wang et al., 2020) and Coptidis rhizome (Sharma et al., 2018). Besides, the analysis of TEM images confirmed the morphological differences in the sizes of both AgNPs where the gamma-irradiated AgNPs were smaller than the normal AgNPs. In agreement with these findings, a previous study used TEM and dynamic light scattering (DLS) experiments to screen the characteristics of Bombyx mori silk AgNPs irradiated with gamma and showed that it was smaller in size despite it induced more reduction in the mycelial growth (Madhukumar et al., 2017). The phytochemical screening, by FTIR spectroscopy, revealed a remarkable differentiation in the bioactive compound’s composition (aliphatic and aromatic) in both AgNPs preparations which might be considered key factors to explain the mechanism of the antifungal activities. Similar findings were reported in the study conducted by Afify et al. (2017), where the FTIR analysis of gamma-irradiated AgNPs showed absorption peaks at 3460, 2850–2900, 1650, 1430, and 1370 cm−1 which are assigned to ─OH, ─CH, C═O, C≡C and C═O stretching vibrations, respectively, which suggested their integrations in the AgNPs synthesis which increase the interaction with other compounds through bonding with the O and N atoms (Afify et al., 2017).

Multiple studies had reported the effectiveness of green AgNPs biosynthesized by plants that suggested them as promising alternatives or natural fungicides to shield plants against different phytopathogenic fungi and overcome the toxic effect imposed by chemical pesticides (Kim et al., 2012). Studies showed that exposure to green AgNPs caused serious cellular deformation by interacting with unsaturated fatty acids which increases the permeability of the cell membranes resulted in the loss of water, salts, proteins, and some intracellular components which affect the vitality and survival of the fungal cells (Durán et al., 2016, Zhang et al., 2018). In the present study, treatments with both preparations of green AgNPs, synthesized by different concentrations of aqueous extract of P. oleracea, showed a strong reduction in the mycelial growth of the selected pathogenic fungi. The comparative analysis showed that gamma-irradiated AgNPs exhibited relatively better antifungal activity than the normal green AgNPs. As mentioned before, these variations are thought to be due to the differences in concentrations of the bioactive compounds (aliphatic and aromatic compounds) in both AgNPs preparations (Afify et al., 2017). Other factors such as the method of extraction, pH, solubility, volatility, growth medium composition, and the natural characteristics of the tested species might contribute to the variation in the antifungal activities (Martínez-martínez et al., 2012, Mohammed et al., 2018). In agreement with our findings, similar studies showed the antifungal activity of colloidal gamma-irradiated AgNPs against Corticium salmonicolor (Phu et al., 2010), Colletotrichum sp. (Lamsal et al., 2011), Macrophomina phaseolina, A. alternata, F. oxysporum, Trichoderma harzianum. and Geotrichum candidum induced by treatment with AgNPs synthesized from the Amaranthus retroflexus leaf extract (Bahrami-Teimoori et al., 2017).

TEM microphotographs of the fungal species treated with both AgNPs showed different morphological changes in the fungal mycelium compared to untreated controls that endorse their antifungal activity, particularly the gamma-irradiated AgNPs. Gamma-irradiated AgNPs were relatively stronger than normal AgNPs as it caused a greater mycelial reduction in the growth M. phaseolina compared with C. spicifera and Bipolaris sp. A previous study showed that AgNPs prepared at the 1:10 to the aqueous extract of P. oleracea showed 12 mm and 19 mm zone of inhibition against the fungal species of Candida albicans and Saccharomyces cerevisiae, respectively (Jannathul Firdhouse and Lalitha, 2015). The data suggested that AgNPs of P. oleracea, particularly the gamma-irradiated, possess a strong antifungal property and has the potential to be used as a promising antifungal agent against plants against phytopathogenic fungi.

5. Conclusion

The results confirm to the antifungal activity of the aqueous extract of P. oleracea leaves and the AgNPs prepared from it against different strains including, C. spicifera, M. phaseolina, and Bipolaris sp. The results revealed that gamma-irradiated AgNPs of P. oleracea extract might be more active and possess a relatively greater antifungal activity than the normal AgNPs against the selected phytopathogenic fungi. Further studies are required to understand the molecular mechanism by which green AgNPs of P. oleracea extract inhibits the mycelial growth of different fungi, besides, the medicinal properties of the P. oleracea which have not been fully explored.

Author’s contribution

The conception and study design were performed by F.A. and S.A.A. R.I.A. and N.M.S.M. were responsible for the literature search. Both of R.M.A. and H.F.A. collected the samples, performed the methodology. The experimental design, data acquisition and analysis, statistical analysis, editing and reviewing of the intellectual contents were carried out by F.A. R.I.A. and A.A.A. prepared the manuscript. F. A. reviewed and edited the manuscript, besides acting as a guarantor and corresponding author. All authors have read and agreed to the published version of the manuscript.

Funding

This research project was supported by a grant from the Researchers Supporting Project number (RSP-2021/114), King Saud University, Riyadh, Saudi Arabia.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors would like to extend their sincere appreciation to the Research Supporting Project number: RSP-2021/114, King Saud University, Riyadh, Saudi Arabia.

Footnotes

Peer review under responsibility of King Saud University.

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