RETRACTED ARTICLE: In vitro antioxidant, anticancer, anti-inflammatory, anti-diabetic and anti-Alzheimer potentials of innovative macroalgae bio-capped silver nanoparticles

Abstract

The antagonistic side effects of chemical medications led to the search for safe strategies such as biogenic agents. Correspondingly, this study aims to create biogenic, appropriate, auspicious and innovative therapeutic agents like Galaxaura elongata {GE}, Turbinaria ornata {TO} and Enteromorpha flexuosa {EF} macroalgae-based silver nanoparticles (Ag-NPs). The Ag⁺ reduction and the creation of Ag[GE]-NPs, Ag[TO]-NPs and Ag[EF]-NPs have been validated using UV–visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM) and zeta potential analysis, and the chemical composition of macroalgae crude extracts was estimated through gas chromatography–mass spectrometry (GC–MS). Further, macroalgae-based Ag-NPs were tested for their free radical scavenging activity DPPH, ABTS, anticancer activity in human liver carcinoma (HepG2) cell line, distinctive inflammation forms and elevated α-amylase. Results showed that the biosynthesized Ag-NPs have unique mechanical and physicochemical characters attributed to their high relative surface area, nanosized dimensions and spherical shape. At dose of 200 µg/mL, the DPPH radical scavenging capacity was maximized with Ag[TO]-NPs (67.26%); however, Ag[EF]-NPs was the most potent as ABTs scavenger (97.74%). Additionally, Ag[GE]-NPs had the maximum proteinase inhibitory action with 59.78%. The 1000 µg/mL of Ag[GE]-NPs, Ag[TO]-NPs and Ag[EF]-NPs revealed significant inhibitions of cell growth of HepG2 resulting in cell viabilities 5.92%, 4.44% and 11.33%, respectively. These findings suggest that macroalgae bio-capped Ag-NPs have magnificent biological potentials for safe biomedical applications.

Introduction

The main drawback in most diseases is oxidative stress, which is imbalance between free radical production and reactive metabolites. This imbalance initiates the damage of imperative biomolecules and cells with a potential impression on the overall organisms. The removal of such free radicals by protective mechanisms is known collectively as an antioxidant defense mechanism (Perry et al. 2011; Ahmed et al. 2017a, b). The antioxidant defense mechanism plays an essential role in depleting free radicals by synchronizing their damaging action of free radicals. They mainly attack macromolecules causing cell impairment (Matough et al. 2012; Rosenzweig et al. 2019; Madkour 2020). Furthermore, free radicals are disproportionately created in diabetes by glucose oxidation in addition to nonenzymatic protein glycation (Povova et al. 2012; Mehta et al. 2012).

Pathophysiological conditions such as oxidative stress, cytotoxicity and inflammation play a main role in several chronic diseases. The damaged degrees of free radicals and the synchronized demolition of antioxidant defense mechanisms can cause precarious impairment of cellular organelles and enzymes, preeminent lipid peroxidation and insulin resistance development. These consequences of oxidative stress can evolve diabetes mellitus (DM) complications (Suganthy and Pandima Devi 2016). Similarly, oxidative stress and diminished acetylcholine (ACh) levels have a substantial role in the Alzheimer’s disease (AD) pathogenesis (Ames and Gold 1992; Guyton and Kensler 1993). Accordingly, the treatment of elevated oxidative stress blocks the route of different diseases (Rajendran et al. 2021).

In order to overcome the recent global problems, health problems and wastes which have increased substantially in many parts of the world, innovative ways must be developed. Hence, nanotechnology occupies the interest of most researcher and so as to create trustworthy nanomaterial to solve those growing problems (Badawi and Zaher 2021; Badawi, et al. 2021a; Badawi, et al. 2021b). Previous studies focused on the synthesis of nanomaterials by chemical and physical approaches. Nevertheless, these techniques are mostly expensive, need extensive complicated laboratory procedures, and consume toxic chemicals (Badawi, et al. 2021a; Dawadi et al. 2021).

Consequently, the plant- and alga-originated particles such as flavonoids, tannins, polyphenols, alkaloids, and polysaccharides are highly accepted for their wide-ranging biological roles and have an immense therapeutic possibility in nano-medicines for various types of diseases (Ahmed et al. 2014; Watkins et al. 2015; Saratale et al. 2018b; Barani et al. 2021; Javad Farhangi et al. 2021). So, the biosynthesis of NPs from plant and algal extracts is currently the greatest manipulated technique because it is eco-friendly, obtainable, cost-saving, biocompatible and safe (Pillai et al. 2020; Mahmoud et al. 2020, 2021; Barani et al. 2021; Hassanisaadi et al. 2021; Es-Haghi et al. 2021; Mohammadzadeh et al. 2022; Hassanisaadi et al. 2022).

The green synthesis processes of Ag-NPs utilize numerous crude extracts of different plant parts, such as fruit peels (Saratale et al. 2018a), leaves (Raman et al. 2012; Kumar et al. 2016), flowers and gums (Velmurugan et al. 2016) and roots (Rao et al. 2016) as well as of macroalga (Mahmoud et al. 2020, 2021), which are the most recently used methods in nanomaterial biosynthesis. The seaweeds used for green biosynthesis of inorganic nanoparticles (NPs) have a variety of bioactive constituents, which are broadly implicated in different biotechnological fields and biological applications (Fahim et al. 2008; Ahmed et al. 2008, 2014; Lomartire and Gonçalves 2022). Recently, researcher has validated that those distinctive compounds express valuable properties such as antioxidant, anti-diabetic, anticancer, anti-arthritic, antimicrobial, antiviral, water treatment and photo-bioreactor (Ahmed 2010a, b; Ahmed and Ahmed 2014; Abd-Ellatef et al. 2017; Ahmed et al. 2017a, b; Badawi et al. 2022).

Moreover, the created biosynthesized Ag-NPs are widely recommended for using in medical fields as anti-inflammatory (Prabakaran and Mani 2019), antioxidant and anticancer potentials (Gomaa 2017; Saratale et al. 2018a; Hashemzadeh et al. 2021), anti-diabetic (Rajaram et al. 2015; Popli et al. 2018), hepatoprotective (Mahmoud et al. 2021), renocardio protective (Mahmoud et al. 2020) and anti-Alzheimer potentials (Popli et al. 2018).

Regardless of the previous consideration, the biosynthesis of Ag-NPs still defies ongoing challenges as little yields, period spending and high cost. Hence, the procurement of biological, safe, available and inexpensive materials with high reducing/stabilizing potentials and production of Ag-NPs with good biological, chemical and physical properties is now a high urgency for medical field. Marine macroalgae such as Galaxaura elongata (G. elongata), Turbinaria ornata (T. ornata) and Enteromorpha flexuosa (E. flexuosa) crude extracts are innovative and promising green reducing and capping agents, and they contain various vitamins, minerals, organic and inorganic ingredients that could help human health (Pádua et al. 2004; Rao and Boominathan 2015; Ramkumar et al. 2017; Ahmed et al. 2020; Fathy et al. 2020; Abdelrheem et al. 2021; Abdel Azeem et al. 2021; Mohamed et al. 2022).

The applications of G. elongata, T. ornata and E. flexuosa macroalgae-based Ag-NPs for different in vitro effects were not investigated by previous publications. So, we need to assess whether these macroalgae-biosynthesized Ag-NPs have in vitro antioxidant, anticancer, anti-inflammatory, anti-diabetic and anti-Alzheimer or not. Accordingly, the present investigation was designed to create biogenic Ag-NPs from different types of crude extracts of G. elongate, T. ornata and E. flexuosa and also to originate biomedical agents with safe influences. As these bioactive Ag-NPs have distinctive biological, chemical and physical features due to their heightened relative surface area, size, distribution shape and quantum impacts as proved by ultraviolet–visible (UV–Vis) spectroscopy, Fourier transform infrared spectroscopy (FTIR) and gas chromatography (GC)-mass spectrum (MS), scanning electron microscope (SEM) and zeta potential analysis. These biosynthesized Ag-NPs are tested to assess their antioxidant activity by detecting the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2’-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) scavenging activities and anticancer effects on human liver carcinoma cell line (HepG2) as well as anti-inflammatory (anti-proteinase and protein denaturation), anti-diabetic (alpha amylase enzyme inhibition) and anti-Alzheimer activities.

Materials and methods

Materials

Natural materials

The macroalgae samples (G. elongata, T. ornata and E. flexuosa) were collected from the Egyptian Red Seashores, identified in Phycology lab, Botany and Microbiology Department, Faculty of Science, Beni-Suef University, Beni-Suef, Egypt, according to the description of Chapman and Chapman 1980 & Robert 1989.

Chemicals

Silver nitrate was purchased from Sigma-Aldrich Chemicals, USA, DPPH, ABTS, α-amylase, acetyl cholinesterase enzyme and dimethyl sulfoxide (DMSO).

Methods

The methods of the preparation, characterization and applications of Ag[GE]-NPs, Ag[TO]-NPs and Ag[EF]-NPs are summarized in Fig. 1.

Fig. 1.

The outline of the experiments operated in this study

Biosynthesis of Ag-NPs by different forms of Egyptian macroalgae

Collection, extraction of G. elongata and biosynthesis of Ag [GE]-NPs

Initially, fresh G. elongata (J. Agardh) a red macroalga sample was collected and consecutively washed with tap and distilled water. Subsequently, the samples were dried at room temperature (25 °C) for one-week air drying with continuous stirring. Then, the dried macroalgae samples were milled through electric mixer until it turned into powder. Moreover, this powder was stored in dark place and finally became ready for different extraction methods.

Typically, 2 g of G. elongata powder was added into 100 mL of 95% ethanol. The combination was left on stirrer for 24 h to be extracted and filtered by Whatman no.1 filter paper, and a 50 mL volume of extract was finally added gradually into 450 mL 10^–3 M of AgNO₃ solution. The reduction progression of Ag⁺ to Ag⁰ NPs was associated with the change in solution color from pale yellow to reddish brown (Abdel Azeem et al. 2021).

Collection and extraction of T. ornata and biosynthesis of Ag[TO]-NPs

The brown macroalga T. ornata (Turn.) J. Agardh var ornate toylor was collected and washed with running tap water and distilled water; they were dried at room temperature ( 25 °C) and milled by electrical mixer until turned into powder, and then the powdered were stored in dark place. Last of all Ag[TO]-NPs were created according to the methods of (Deepak et al. 2017; Abdel Azeem et al. 2021).

Collection and extraction of E. flexuosa and biosynthesis of Ag [EF]-NPs

Primarily, E. flexuosa (L.) Nees. (green macroalga) was collected, washed several times with tap and distilled water and dried at room temperature (25 °C ). It was converted into powder by a mixer. Consequently, it was stored in a dark place and stand for various extraction methods.

The extraction of macroalgae samples were done according to the methods of (Yousefzadi et al. 2014; Abdel Azeem et al. 2021). Moreover, 50 mL of E. flexuosa extract was gently added into 450 mL of 10⁻³ M of AgNO₃ solution for Ag [EF]-NPs biosynthesis. Lastly, the color of solution was converted to brown as a sign of Ag [EF]-NPs formation and the formed NPs were preserved in a dark place at room temperature (Yousefzadi et al. 2014).

Purification and drying Ag-NPs solution

The Ag[GE]-NPs, Ag[TO]-NPs and Ag[EF]-NPs solution was centrifuged at 12,000 rpm for 15 minutes, washed repeatedly with deionized water, dried at 50 ⁰C and saved for various applications (Arunachalam et al. 2012).

Characterization of biosynthesized Ag-NPs

UV–Vis spectra analysis

The bioreduction of Ag⁺ to Ag⁰ was detected by visual judgment through appearance of brown color and by appraisal the UV–Vis spectrum of the reaction, at a wavelength of 250–800 nm in PerkinElmer UV WinLab UV–Vis spectrophotometer at the Nano-photonic Lab in Beni-Suef University, Beni-Suef, Egypt.

Fourier transform infrared spectroscopy (FTIR)

Fourier transform infrared spectroscopy spectral capacities were done to individualize functional groups in the biosynthesized Ag-NPs responsible for reducing and capping the bulk AgNO_3. The formed Ag-NPs solution was analyzed using the FTIR instrument (VERTEX70 FT-IR spectrometer coupled to a RAM 11 FT-Raman module).

Gas chromatography–mass spectrometry (GC–MS) analysis

The chemical nature of crude extract of G. elongata, T. ornata and E. flexuosa was scrutinized by GC–MS chemical analysis assay. The investigation used a coupled Varian gas chromatography/mass spectrometry. The ionized voltage was 70 eV, with a mass range of m/z 39–400 amu. The ingredients were categorized by paralleling their mass spectra with the spectra of products in the library search report. The isolated peaks were categorized by matching with data from the mass spectra library (National Institute of Standard and Technology) and matched with authenticated compounds and available data (Adams 2007). The quantitative determination was carried out based on peak area integration.

Scanning electron microscope (SEM)

The morphological imaging SEM was performed using EDX-equipped JSM-6510.

Zeta potentials

Zeta potentials were scrutinized using Dynamic Light Scattering (DLS) at fixed angle of 173° at 25 °C.

In vitro biological analysis

DPPH free radical scavenging activity

The free radical scavenging capacity of macroalgae-based Ag-NPs on DPPH was evaluated at different concentrations {12.5, 25, 50, 100 and 200 µg/mL} of Ag[GE]-NPs, Ag[TO]-NPs and Ag[EF]-NPs and gallic acid as a standard. The Ag[GE]-NPs, Ag[TO]-NPs, Ag[EF]-NPs and gallic acid were mixed with 1.0 mL of methanol containing DPPH (4.0 mL of methanol per 4.0 mg of DPPH) radicals (0.2 mM). The mixture was forcefully shaken and left for 30 min in the dark, and the sample was diluted to be measured at the absorbance of 517 nm (Sousa et al. 2008). All assays were run in triplicate and averaged.

The scavenging ability was calculated using Eq. (1):

$$DPPHscavenging\mathbf{\%}=\frac{\mathit{A}\mathit{o}-\mathit{A}\mathit{s}}{\mathit{A}\mathit{o}}\times \mathit{100}$$ 1

where A_s is the sample absorbance and A₀ is the blank absorbance which contains all reagents except for the test samples.

The calculation of IC₅₀ was done after charting of DPPH scavenging. Equation of straight lines was verified, and the IC₅₀ was considered “x” in equation y = mx + b where y = 50 and values of m and b were given in the Trendline equation. The graphical slope of DPPH inhibition % was considered (Rahdar, et al. 2021).

ABTS free radical scavenging activity

In vitro antioxidant activity of macroalgae-based Ag-NPs was assessed by ABTS assay methods (Phull et al. 2016), the stock solution consisting of 7.4 mM ABTS and 2.6 mM potassium persulfate (Brand-Williams et al. 1995). The working mixtures were practiced by mixing the two stock solutions in equivalent volume and were permitted to react in the dark at room temperature for 12 h. The dilution process was done for the solution by mixing ABTS solution with methanol to obtain an absorbance of 0.736 ± 0.01 at 734 nm. The fresh solution was prepared for each ABTS assay. Then, 1.0 mL of different concentrations [12.5, 25, 50, 100 and 200 µg/mL] of Ag[GE]-NPs, Ag[TO]-NPs and Ag[EF]-NPs solutions and gallic acid as a standard reference was mixed with 1.0 mL of ABTS solution. After 10 min of incubation at room temperature, they were measured at the absorbance 734 nm. Finally, the inhibition percentage was calculated by Eq. (2):

$$\mathrm{ABTS}-\mathrm{scavenging}\mathrm{activity}\%=[1-\left(\frac{\mathrm{As}}{\mathrm{Ao}}\right)]\times 100$$ 2

Anticancer assessment of Ag-NPs against the HepG2 cell line

The cell viability potentials of macroalgae-based Ag-NPs were verified by 3-[4,5-Dimethylthiazol-2-yl] 2,5-diphenyltetrazolium bromide (MTT) which is a yellow water-soluble tetrazolium salt manipulating HepG2 cells similar to the approach of Sriranjani et al. 2016.

The cell viability percentage was estimated to get anticancer potential of the macroalgae-based Ag-NPs using calculated by Eq. (3):

$$Cellviability\mathbf{\%}=\frac{\mathrm{OD}\mathrm{of}\mathrm{thetreated}\mathrm{sample}}{\mathrm{OD}\mathrm{of}\mathrm{untreated}\mathrm{control}}\times 100$$ 3

where OD is the optical density ratio obtained from the spectrophotometer.

Evaluation of in vitro anti-inflammatory activity

Anti-proteinase action

The anti-proteinase activity of macroalgae-based Ag-NPs was measured in accordance with the methods of (Oyedapo and Famurewa 1995; Sakat et al. 2010), along with trivial modification. The reaction mixture (1.0 mL) included 0.06 mg trypsin, 0.5 mL 20 mM Tris–HCl buffer pH 7.4 and 0.5 mL of different Ag[GE]-NPs, Ag[TO]-NPs and Ag[EF]-NPs with various concentrations (12.5, 25, 50, 100 and 200 μg/mL). This combination was incubated at 37⁰C for 5 min, and 0.5 mL of 0.8% (w/v) casein was supplemented. The mixture was then incubated for an additional 20 min, and 1.0 mL of 70% perchloric acid was added to block the reaction. The confused suspension was centrifuged, and the supernatant was read at an absorbance of 210 nm compared to blank buffer. These procedures were done in triplicate. The proteinase inhibitory activity ratio was calculated by Eq. (4):

$$\mathrm{Proteinase}\mathrm{inhibition}\%=\frac{\mathrm{Ao}-\mathrm{As}}{\mathrm{Ao}}\times 100$$ 4

Effect on protein denaturation

Protein denaturation analysis was completed as explained by Elias and Rao 1988, with slight modifications. Moreover, 1.0 mL of different concentrations of Ag [GE]-NPs, Ag[TO]-NPs, Ag[EF]-NPs and standard diclofenac sodium {12.5, 25, 50, 100 and 200 µg/mL} was admixed with 1.0 mL of fresh egg albumin solution and incubated at 27 ± 1⁰C for 15 min. Denaturation was generated by keeping the reaction mixture for 10 min in water bath at 70⁰C. The turbidity was assessed spectrophotometrically at 660 nm.

The inhibition ratio of denaturation was estimated from the blank where no samples were added. Every test was done in triplicate, and the mean was appropriated. The protein denaturation activity was calculated by Eq. (5):

$$\mathrm{Protein}\mathrm{denaturation}\%=\frac{\mathrm{Ao}-\mathrm{As}}{\mathrm{Ao}}\times 100$$ 5

In vitro anti-diabetic α-amylase inhibitory assay

The inhibition of α-amylase activity of biogenic Ag-NPs was finalized by assessing the maltose amount released during the test. This process is similar to Bhutkar and Bhise 2012, with some modifications. Different concentrations of Ag[GE]-NPs, Ag[TO]-NPs and Ag[EF]-NPs {12.5, 25, 50, 100 and 200 µg/mL} solution were pre-incubated with 100 μL of α-amylase solution (1.0 U/mL) at room temperature for 30 min; 100 μL of starch solution (1% w/v) was more supplemented to the mixture and incubated at room temperature for 10 min, and 100 μL of iodine solution was added to the mixture to stop the reaction; then, the solution was incubated for 5 min. Blank was provided where the identical enzyme quantity and the Ag-NPs were substituted by sodium phosphate buffer retained at a pH value of 6.9 and acarbose behaved as a positive control, and this procedure was done in triplicate. Finally, the absorbance of the tested samples was measured at 540 nm and the α-amylase inhibition percentage was estimated using Eq. (6):

$$\alpha -\mathrm{amylase}\mathrm{inhibition}\%=\frac{\mathrm{Ao}-\mathrm{As}}{\mathrm{Ao}}\times 100$$ 6

Anti-acetyl cholinesterase activity

Acetyl-cholinesterase inhibition activity by innovative biogenic Ag-NPs was operated as stated by the procedures of Ellman et al. 1961, with little adjustments by adding 1.5 mL of 0.1 M phosphate buffer [Na2HPO4/NaH2PO4, pH 8.0], 50 µl of AChE source (600 U/mL) diluted 1:120, 50 µL of Ag[GE]-NPs, Ag[TO]-NPs and Ag[EF]-NPs and standard donepezil with different concentrations {12.5, 25, 50, 100 and 200 µg/mL}. Moreover, 10 µL of [0.075 M] acetylcholine iodide was then added and finally read at different periods at 412 nm. The experiment was done in triplicate. The AChE inhibition percentage was calculated using Eq. (7).

$$\mathrm{Inhibition}\%\mathrm{of}\mathrm{AChE}=[1-\frac{\mathrm{\Delta }\mathrm{A}\mathrm{sample}}{\mathrm{\Delta }\mathrm{A}\mathrm{blank}}]\times 100$$ 7

Statistical analysis

Data were given as mean ± standard error of 3 replicates using one-way analysis of variance (ANOVA) followed by Duncan^’s test α = 0.05.

Results and discussion

An itemized study on biosynthesis, characterization and in vitro biological potentials of macroalgae-based Ag-NPs from the crude extract of G. elongata, T. ornata or E. flexuosa was elucidated in this paper.

Characterization

The UV–Vis, GC–MS and FTIR analysis were applied to analyze and characterize the marine macroalgae-based Ag-NPs properties.

Formation of macroalgae-based silver nanoparticles

After the addition of G. elongata, T. ornata and E. flexuosa crude extracts to AgNO₃ initiate Ag-NPs formation, the change in color from colorless to brown or dark brown is the main sign of the reduction of Ag⁺ to Ag⁰ and formation of Ag-NPs as shown in Fig. 2.

Fig. 2.

Observation of macroalgae-based Ag-NPs synthesis: (A) formation of Ag[GE]-NPs; (B) formation of Ag[TO]-NPs; (C) formation of Ag[EF]-NPs

Optical properties (UV–visible spectroscopy)

The spectral optical absorption of Ag-NPs is ruled by SPR that shows a shift in the direction of the red or blue end hinges on the NPs size, structure, the surrounding dielectric medium, and aggregation (Mahmudin et al. 2015). The biosynthesis of Ag-NPs was confirmed by color alteration from light yellow to brown and UV–Vis spectroscopy using SPR in the range of 250–800 nm. The SPR peak represents different morphological features of the NPs, for example, the shape, size, and stability. The bands depicted in Fig. 3 are paralleled to the absorption by colloidal Ag-NPs in the [400–450 nm] region due to the SPR excitation which is good standard for choice of metal NPs ranging from 1 to 100 nm (Njagi et al. 2011; Arunachalam et al. 2012). The SPR spectrum of [GE]-NPs, Ag [TO]-NPs and Ag[E]-NPs is 440 nm, 436 nm and 434 nm, respectively, which indicate that the biosynthesized Ag-NPs have strong SPR, round and spherical in shape (Palithya et al. 2021).

Fig. 3.

UV–Vis spectra of different forms of Ag[GE]-NPs, Ag[TO]-NPs and Ag[EF]-NPs

Fourier transform infrared spectroscopy (FTIR)

The spectra of FTIR of biogenic Ag-NPs by G. elongata, T. ornata and E. flexuosa demonstrate absorption peaks located in the 500–4000 cm⁻¹ region. Table 1 presents the assignment of the observed bands in the three spectra. These spectra show the intense band at 3280, 3303 and 3285 cm⁻¹ for Ag[GE]-NPs, Ag[TO]-NPs, and Ag[EF]-NPs, respectively, as depicted in Fig. 4, which is ascribed to the strong stretching hydroxyl O–H and H-bond of alcohols and phenols. The bands at 2119, 2132 and 2136 cm⁻¹ for Ag[GE]-NPs, Ag[TO]-NPs and Ag[EF]-NPs, respectively, correspond to C≡C stretch of alkynes. The bands at 1637, 1636.75 and 1636.7 cm⁻¹ for Ag[GE]-NPs, Ag[TO]-NPs, and Ag[EF]-NPs, respectively, to indicate to the N–H bend of primary amines. The bands at 1095.7 and 1043.7 cm⁻¹ of Ag[GE]-NPs are due to C–N stretch of aliphatic amines. Finally, the bands at 604.2, 602.9 and 599.7 cm⁻¹ for Ag[GE]-NPs, Ag[TO]-NPs and Ag[EF]-NPs, respectively, are related to the C–Cl stretch of alkyl halides. Also, the variation in the transmittance level suggests that biomolecules of the metal nanoparticles were functionalized with macroalgae extract. Thus, the carbonyl group can bind metal from amino acid residues and proteins, indicating that the proteins will possibly build a layer covering the metal nanoparticles (i.e., capping Ag-NPs) to prevent clusters and thereby stabilize the medium (Shankar et al. 2004; Ragupathi et al. 2012; Jeeva et al. 2014).

Table 1.

FTIR of different forms of macroalgae biosynthesized Ag-NPs

Ag[GE]-NPs	Ag[TO]-NPs	Ag[EF]-NPs	Bond	Assignment	Reference
604.275	602.907	599.766	C–Cl stretch	Alkyl halides	Anandalakshmi et al. (2016)
1043.476	––––––-	–––––––	C–OH	Primary strong alcohol	Jeeva et al. (2014)
1095.721	––––––-	–––––––	C–O stretch	Aliphatic amines	Shankar et al. (2004)
1637.730	1636.755	1636.764	C = O	Aromatic group	Ragupathi et al. (2012)
2119.551	2132.089	2136.005	C≡C Stretch	Alkynes	Devaraj et al. (2013)
3280.078	3303.658	3285.372	O–H stretch, H-bonded	Alcohols, phenols	Abideen and Sankar (2015)
3780.047	3779.339	3779.970	O–H Stretch	Flavonoids and proteins	Anandalakshmi et al. (2016)

Fig. 4.

FTIR of (A) Ag [GE]-NPs, (B) Ag[TO]-NPs and (C) Ag[EF]-NPs

Gas Chromatography–Mass Spectrometry (GC–MS) analysis of macroalgae crude extract

The chemical composition of G. elongata, T. ornata and E. flexuosa extracts was detected by GC–MS analysis. Data show the occurrence of multifarious phytochemicals components. The principal constituent concentrations of more than 1% of the total composition are displayed in Tables (2, 3, and 4) and Fig. 5(A, B, and C). The mass spectra of the compounds were compared with the standard library data bases and were characterized and identified.

Fig. 5.

GC–MS of the crude ethanolic extract of (A) G. elongata, (B) T. ornata and (C) E. flexuosa

Table 2 represents the fourteen bioactive compounds with ratio more than 1% from methanolic extract of G. elongata with their chemical constituents, while Fig. 5A. shows the abundance them with the retention time.

Table 2.

GC–MS analysis of the crude methanolic extract of G. elongata

No	Retention time	Chemical constituents	Area % (higher than 1%)
1	3.025	• Toluene	8.91
2	3.088	• Toluene	7.39
3	4.227	• Ethylbenzene	4.87
4	4.359	• o-Xylene, • Ethylbenzene • P-xylene	4.48
5	4.765	• Benzene propanoic acid, octyl ester, • Benzyl glycolate, • Carbamic acid, (4-nitrophenyl)-, phenylmethyl ester	4.765
6	5.972	• Diethylmalonic acid, monochloride, phenethyl ester, • Succinic acid, di(2-phenylethyl) ester, • Dimethylmalonic acid, ethyl 2-phenethyl ester	7.33
7	6.55	• p-Toluic acid, 2-phenylethyl ester • Styrene • Thiourea, N,N'-dimethyl	3.52
8	18.24	• Decane, 2,3,7-trimethyl, Heptacosane • Tridecane, 1-iodo	1.04
9	21.216	• Nonanoic acid, methyl ester • Decanoic acid, methyl ester • Hexadecanoic acid, methyl ester	7.44
10	21.502	• 1H-Pyrrolo[3,2,1-i,j]quinolin-2-one,2,4,5,6-tetrahydro-4,4,6-trimethyl-6-phenyl- • 4,5-Diamino-6-[3,4,5-trimethoxyanilino]pyrimidine • 2-(4-Acetylphenylamino)-1,4-naphthoquinone	1.5
11	21.673	• 5–Acetamido-5-propyldecane • 4-Acetamido-4-ethyloctane • Undecanoic acid	6.73
12	21.748	• 5,8-Methano-3H-2-benzopyran-3-one, • 4,4a,5,6,7,8-hexahydro-exo-4-(4-methoxyphenyl)-1-phenyl- • Dipropetryn 2-(1-propenyl)-6-methylphenol	3.43
13	23.464	• Cyclododecanol • 1H-Indene, octahydro-, cis- cis-11-Tetradecen-1-ol	1.16
14	30.514	• 2,5-Dibromobenzoic acid • 7,10-Dimethylbenzo(a)pyrene • Perhydro-htx, 1-acetyl-, acetate(ester)	1.26

Data shown in Table 3 represent the details of the bioactive compounds of T. ornata which has fifteen compounds more than 1%; Fig. 5B reveals the chromatogram of the identified compounds by GC–MS. Additionally, Table 4 shows the only thirteen bioactive compounds more than 1% in GC–MS analysis of E. flexuosa which is depicted in Fig. 5C.

Table 3.

GC–MS analysis of the crude methanolic extract of T. ornata

No	Retention time	Chemical constituents	Area % (higher than 1%)
1	2.487	• Furan, tetrahydro-2,4-dimethyl-, cis N- Aminopyrrolidine • Silane, ethenyltrimethyl-	1.48
2	2.544	• 2(3H)-Furanone, dihydro-5-methyl- Piperazine • Piperazine • Furan, tetrahydro-2,5-dimethyl-, cis	1.69
3	2.562	• Tetrahydropyran • Tetrahydropyran • Piperazine	3.1
4	2.796	• 1,2-Propanedione, 1-phenyl-Pyridine, 2-ethenyl-2-Methyl-3-thiosemicarbazide	3.45
5	3.019	• 1,5-Hexadien-3-yne, 2-methyl- • 1,5-Hexadien-3-yne, 2-methyl- • Toluene	1.14
6	4.341	• Ethylbenzene • o-Xylene • p-Xylene	34.92
7	4.438	• Ethylbenzene • Benzene, 1,3-dimethyl- • p-Xylene	10.65
8	4.719	• Ethylbenzene • Ethylbenzene • Ethylbenzene	6.02
9	4.747	• Ethylbenzene • p-Xylene • 1,6-Heptadiyne	3.11
10	4.77	• 1-Propanol, 2-chloro-Ethylbenzene • Ethylbenzene	10.17
11	5.972	• 2,4-Nonadiyne • Benzene, 1-ethyl-4-methyl- • Benzene, 1-ethyl-4-methyl-	9.11
12	6.55	• 3-Heptadien-5-yne, 2,4-dimethyl- • Benzene, 1,2,3-trimethyl- • Benzene, 1,2,3-trimethyl-	6.12
13	7.105	• 1,5-Dimethyl-2-pyrrolecarbonitrile • 3-Pyridinecarbonitrile, 1,4-dihydro-1-methyl • 1,5-Dimethyl-2-pyrrolecarbonitrile	2.37
14	7.196	• trans-.beta.-Ocimene • (1S)-2,6,6-Trimethylbicyclo[3.1.1] • hept-2-ene • trans-.beta.-Ocimene	2.36
15	26.84	• yrido[3,2-d]pyrimidin-4(3H)-one, 3-hydroxy-2-methyl- 1H-Indole-2-carboxylic acid, 5-chloro-, ethyl ester • (2-Oxo-3,4-dihydro-2H-benzo[1,4]thiazin-3-yl)acetic acid, methyl ester	1.72

Table 4.

GC–MS analysis of the crude methanolic extract of E. flexuosa

No	Retention time	Chemical constituents	Area % (higher than 1%)
1	2.453	• 1-Benzyl-3-phenyl-1H-1,2,4-triazol-4-oxide • 1-(2-Hydroxyimino-2-phenyl–ethyl)-pyrrolidine-2,5-dione • 1H-Pyrimidine-2,4-dione, 1-benzyloxymethyl-5-bromo-6-methyl-	3.35
2	3.054	• 1,5-Heptadien-3-yne • 10-Phenyldecanoic acid • Benzene, undecyl	3.39
3	4.227	• 3-Benzylthio-5-hydroxy-6,6-dimethy • l-1,6-dihydro-1,2,4-triazine • 1H-1,2,3-Triazole-4,5-dimethanol, 1-(phenylmethyl) • Benzyl isopentyl ether	1.02
4	4.341	• 1,2-Benzisoxazole • 1,5-Heptadien-3-yne • Pyridine, 4-[2-(phenylmethyl)-2H-1,2,3,4-tetrazol-5-yl]	14.68
5	16.037	• 1-Methoxybicyclo[2,2,2]oct-5-en-2-yl methyl ketone • 2,4-Dichloro-N-(4-ethoxy-phenyl)-3-methyl-benzenesulfonamide • 3,6-Dimethyl-6-hepten-4-yn-3-ol	1.09
6	17.965	• 1-Octadecanol • n-Nonadecanol-1 • 1-Hexadecanol	7.56
7	20.214	• 2-Pentadecanone, 6,10,14-trimethyl	1.40
8	21.221	• Nonanoic acid, methyl ester • Hexadecanoic acid, 15-methyl-, methyl ester • Decanoic acid, methyl ester	1.48
9	21.702	• Pentadecanoic acid • alpha.-D-Glucopyranose, 4-O-.beta-D-galactopyranosyl- • Tetradecanoic acid	43.55
10	23.470	• 6-Nonen-1-ol, acetate, (Z)- 1,1'-Bicyclopropyl, 2,2,2',2'-tetramethyl-1H-Imidazole-4-propanamine	3.30
11	27.819	• Nicotinamide, 5-chloro-2-(4-fluorophenoxy)-4,6-dimethyl-N-phenethyl- • Imidazo[1,2-a]pyridine, 6-bromo-2-thiophen-2-yl- 4-Bromophenoxathiin]	8.79
12	27.882	• 4-Bromophenoxathiin • 3,5-Dibromobenzoic acid • 2-(p-Methoxyphenyl)-4-quinolinecarboxamide	4.70
13	28.019	• Imidazo[1,2-a]pyridine, 6-bromo-2- thiophen-2-yl- 1,3-Dithiolo[4,5-c]pyridine-6-carbonitrile, 4,7-dichloro-2-thioxo- 2,4,6,8-Tetrathiatricyclo[3.3.1.1(3,7)]decane-1-carboxylic acid, 3,5 –dimethyl	5.08

Zeta potential analysis for macroalgae biosynthesized Ag-NPs

Zeta potentials of macroalgae-based Ag-NPs were assessed by DLS as illustrated in Fig. 6(A, B and C) . The obtained data recommend that zeta potential of macroalgae-based Ag-Nps is stable colloid. Ag[GE]-NPs, Ag[TO]-NPs and Ag[EF]-NPs have a zeta potential of [-24.4, -20.8 and -18.3] mV, respectively. These high negative potential values support long-term stability and appropriate colloidal nature of Ag-NPs as a result of negative repulsion (Mukherjee et al. 2014).

Fig. 6.

Zeta potentials and SEM images of biosynthesized (A,D) Ag[GE]-NPs, (B,E) Ag[TO]-NPs, and (C,F) Ag[EF]-NPs

Scanning electron microscope.

The image analysis was performed using analytical low vacuum SEM (JEOL, JSM-6490LA, Japan) at 20 kV. Figure 6(D-F) reveals the SEM images of Ag[GE]-NPs, Ag[TO]-NPs and Ag[EF]-NPs, which show almost spherical NPs with non-uniform size.

The particle size of Ag[GE]-NPs ranges from 30 to 90 nm (Fig. 6D), while Ag[TO]-NPs particle size range from 20 to 60 nm. Comparably, the Ag[EF]-NPs particle size is diverse from 30 to 90 nm with a mean value of 55 nm.

In vitro potentials of marine macroalgae-based Ag-NPs

Antioxidant Activity of macroalgae-based Ag-NPs

Antioxidants are essential in alleviating many syndromes like inflammation, diabetes mellitus, and cancer due to oxidative stress suppression. FTIR and GC–MS analysis confirmed that G. elongata, T. ornata and E. flexuosa are reducing and stabilizing agents for Ag-NPs biosynthesis consisting of phenol, flavonoids, terpenoids which significantly scavenged DPPH and ABTS.

DPPH scavenging activity

The DPPH radicals react with appropriate reducing agents, wherein the electrons are paired off and the solution color disappears stoichiometrically hanging on the number of electrons taken up as explained by Subhasree et al. 2009. Herein, the color solution is increasingly converted from purple to yellow, diphenyl picryl hydrazine, the free radical scavenging was found to be ameliorated with increased macroalgae-based Ag-NPs concentrations.

The DPPH scavenging activity for the tested biogenic NPs exhibited an enhancement in a concentration-dependent manner. The antioxidant potential of macroalgae-based Ag-NPs could be assigned to the presence of flavonoids and phenols originating from macroalgae crude extract as explained in the FTIR analysis. Hence, the decrease in DPPH radical absorbance was because of the radical scavenging by electron supply. As observed in the results of the current study, all treatments including Ag[GE]-NPs, Ag[TO]-NPs and Ag[EF]-NPs have a significant inhibitory action against the DPPH radical recording of 63.69%, 67.26% and 58.92%, respectively, compared with standard gallic acid 72.96% at 200 µg/mL (Fig. 7A).

Fig. 7.

Impacts of Ag[GE]-NPs, Ag[TO]-NPs, Ag[EF]-NPs and gallic acid on [A] DPPH and [B] ABTS radical scavenging. Values are mean ± SE, n = 3, using Duncan’s test p < 0.05

The greater antioxidant activity is signified by the lower IC₅₀ value, shown by the IC₅₀ calculations for gallic acid 82.58 µg/mL which is followed by Ag[GE]-NPs 98.37 µg/mL, Ag[TO]-NPs 98.68 µg/mL, and finally, Ag[EF]-NPs 140.38 µg/mL, These findings are in concurrence with the previous investigations of Popli et al. (2018) and Saratale et al. (2018a).

ABTS scavenging activity

Free radicals are extremely reactive species that are created by cells in immune function and respiration. This reaction is fetal because generates cardiovascular disorders, cancer, atherosclerosis and inflammatory complaints (Chang et al. 2012). The ABTS scavenging activity of macroalgae biosynthesized NPs exhibited an increase in a dose dependent manner. Except for the highest concentration the effect of Ag[GE]-NPs was the most potent and was analogous to the effect of gallic acid. The antioxidant proficiency of 200 μg/mL of Ag[GE]-NPs, Ag[TO]-NPs and Ag[EF]-NPs shows the highest ability to scavenge ABTS (Fig. 7B).

The IC₅₀ values for Ag[GE]-NPs 48.12 µg/mL, Ag[TO]-NPs 50.63 µg/mL and Ag[EF]-NPs 50.9 µg/mL are near the gallic acid 55.37 µg/mL. This finding is more significant than those conveyed in preceding investigations (Gomaa 2017; Sudha et al. 2017; Saratale et al. 2018a; Salem et al. 2022) which confirm the scavenging potentials of biogenic Ag-NPs.

Anticancer activity against the HepG2 cell line

The anticancer activity of Ag[GE]-NPs, Ag[TO]-NPs and Ag[EF]-NPs against HepG2 cell line was scrutinized by MTT assay utilizing different doses of macroalgae-based Ag[GE]-NPs, Ag[TO]-NPs and Ag[EF]-NPs (0.97, 1.9, 3.9, 7.8, 15.6, 31.25, 62.5, 125, 250, 500 and 1000 μg/mL). The results indicate that macroalgae biosynthesized Ag-NPs possess significant anticancer activity affected by time and concentrations as illustrated in Fig. 8. Interestingly, these data displayed the highest concentrations of Ag[GE]-NPs, Ag[TO]-NPs and Ag[EF]-NPs (1000 µg/mL) exhibited a significant inhibitions of cell growth of HepG2 resulting in cell viabilities of 5.92%, 4.44% and 11.33%, respectively. The results propose that macroalgae biosynthesized NPs are more efficient in a dose reliant manner as it contain functional biological groups which hinder the growth of HepG2. Accordingly, the anticancer effects of Ag-NPs are associated with interactions of NPs and cellular functional proteins which ultimately cause cellular changes (A´valos et al. 2018). Moreover, the IC₅₀ values for all treatment agents seem to be equal as indicated Ag[GE]-NPs 104.15 µg/mL, Ag[TO]-NPs 104.81 µg/mL and Ag[GE]-NPs 104.91 µg/mL.

Fig. 8.

Anticancer activity of different concentrations of Ag[GE]-NPs, Ag[TO]-NPs and Ag[EF]-NPs against HepG2 cell line. Values are mean ± SE, n = 3, p < 0.001, using Duncan’s test

In vitro α-amylase inhibitory activity

Alpha-amylase is the key enzyme associated with starch breakdown and carbohydrate also liberates sugar into the bloodstream, which is an auxiliary cause of the increase in blood glucose level and eventually diabetes. The current finding reveals that macroalgae-based Ag [GE]-NPs, Ag[TO]-NPs and Ag[EF]-NPs powerfully inhibit the α-amylase enzyme. The standard acarbose is complex oligosaccharide that obstructs the carbohydrates digestion. It inhibits the action of pancreatic amylase in the starch breakdown (Narkhede et al. 2011). The reaction mechanisms involved in inhibiting α-amylase by protein inhibitors are not understood. However, some recommendations exist that protein (flavanols) may cause conformational structural changes (Kim et al. 2000). The FTIR and GC–MS analyses of crude extracts of G. elongata, T. ornata and E. flexuosa have shown the presence of flavonoids, phenols, proteins, aliphatic amines and tannins.

Figure 9 illustrates the impacts of macroalgae-based Ag-NPs on enzyme inhibition. These results verify that macroalgae-based Ag-NPs exhibit an inhibitory impact on α-amylase in a dose-dependent way and when Ag[GE]-NPs, Ag[TO]-NPs and Ag[EF]-NPs dose was 200 μg/mL, the inhibitory action was 64.20%, 67.46% and 67.46%, respectively, while inhibition caused by standard acarbose was 73.25%.

Fig. 9.

The α-amylase inhibitory activity of Ag[GE]-NPs, Ag[TO]-NPs, Ag[EF]-NPs and acarbose. Values are mean ± SE, n = 3, using Duncan’s test p < 0.05

The α-amylase IC₅₀ value for acarbose was 74.66% µg/mL which is near to Ag[EF]-NPs 87.33 µg/mL, Ag[TO]-NPs 90.0 µg/mL and lastly Ag[GE]-NPs with 102.55 µg/mL; this finding is reliable with those reported in previous studies (Rajaram et al. 2015). Subsequently, it could be elucidated from these results that macroalgae-based Ag-NPs possibly will be utilized as a substitution to acarbose because they have a prospective inhibitory influence on α-amylase and do not have toxic side effects when they are used in an appropriate doses.

Evaluation of in vitro anti-inflammatory activity

Proteinase inhibitory action

Neutrophils are a common brilliant supply of serine proteinase and are concentrated in lysosomes. Leukocytes proteinase formerly shared a vital part in the tissue damage progression for the duration of inflammatory reactions and proteinase inhibitors offered a noteworthy defense. Proteinase inhibitory process is evaluated via the decreased IC₅₀ as given by standard diclofenac sodium 86.82 µg/mL which is close to Ag[GE]-NPs 90.8 µg/mL, Ag[EF]-NPs 125.24 µg/mL and finally 127.01 µg/mL, biosynthesized Ag[GE]-NPs, Ag[TO]-NPs and Ag[EF]-NPs generate significant anti-inflammatory actions using membrane stabilization and inhibiting protein denaturation in a dose dependent behavior as revealed in Fig. 10. Maximum inhibitions are shown at 59.78%, 44.40%, and 47.38% at 200 μg/mL. Diclofenac sodium displayed the maximum inhibition 64.28% at 200 μg/mL. However, the effects of Ag [GE]-NPs were the most potent and were comparable to the effect of diclofenac sodium. These findings are in harmony with Prabakaran and Mani 2019.

Fig. 10.

Anti-proteinase action of Ag[GE]-NPs, Ag[TO]-NPs, Ag[EF]-NPs and diclofenac sodium. Values are mean ± SE, n = 3, using Duncan’s test p < 0.05

The inhibitory efficiency of biogenic Ag-NPs on protein denaturation, cyclooxygenase and 5-lipoxygenase indicates that G. elongate, T. ornata or E. flexuosa macroalgae-based Ag-NPs are capable of hindering the creation of prostaglandins and leukotrienes, which assigns the anti-inflammatory properties of macroalgae-based Ag-NPs.

Inhibition of albumin denaturation

Protein denaturation is a mislaying progression of tertiary and secondary protein structures in response to external pressure or composites, (e.g., heating, strong base or acid, a concerted inorganic salt or organic solvents). Furthermore, biotic proteins lose their biological function once denatured.

The albumin denaturation inhibition process is evaluated via the decreased IC₅₀ as given by standard diclofenac sodium 105.41 µg/mL which is higher than Ag[EF]-NPs 126.87 µg/mL, Ag[TO]-NPs 135.27 µg/mL and Ag[GE]-NPs 163.53 µg/mL; the inhibition activity of 200 μg/mL of Ag[GE]-NPs, Ag[TO]-NPs and Ag[EF]-NPs was 57.46%, 54.8% and 59.46%, respectively, which approaches 200 μg/mL of standard diclofenac sodium which offered 64.28% as depicted in Fig. 11. The current study^’s findings illustrate that macroalgae-based Ag-NPs have marked in vitro anti-inflammatory effects on protein denaturation. These findings are more ameliorated than the findings of Mani et al. 2015; Prabakaran and Mani 2019.

Fig. 11.

Protein denaturation (fresh egg albumin) activity of Ag[GE]-NPs, Ag[TO]-NPs, Ag[EF]-NPs and diclofenac sodium. Values are mean ± SE, n = 3, using Duncan’s test p < 0.05

Acetyl cholinesterase (AChE) inhibition percentage

Alzheimer^’s disease is a developing syndrome that accompanies with AChE deficiency which damages nerve cells in different parts of the brain including memory, learning, language, and reasoning. Thus, the treatment of this disease must have the potency to inhibit AChE. When biogenic Ag-NPs cooperate with AChE proteins, they inhibit AChE proteins and show that NPs have binding affinity to ChE. This interaction may occur because of the lithiophilicity of the NPs and hydrophobicity nature of the enzyme ChE molecule (Wang et al. 2009; Rajakumar et al. 2017; Moawad et al. 2020).

The IC₅₀ values for AChE inhibition indicated that Ag[EF]-NPs is the best with 19.78 µg/mL, while the standard donepezil value is 71.4 µg/mL which is near the values of Ag[TO]-NPs 76.14 µg/mL and Ag[GE]-NPs 71.92 µg/mL. Figure 12 illustrates the inhibitory effects of Ag[GE]-NPs, Ag[TO]-NPs and Ag[EF]-NPs on AChE with the results of 73.75%, 70.53% and 63.46%, respectively, and the reference standard donepezil was of 83.6%. The increase in AChE inhibition was concentration dependent. The effect of Ag [GE]-NPs was the most potent and was comparable to that of donepezil.

Fig. 12.

AChE inhibition ratio Ag[GE]-NPs, Ag[TO]-NPs, Ag[EF]-NPs and diclofenac sodium. Values are mean ± SE, n = 3, using Duncan’s test p < 0.05

Conclusion

Based on the obtained results, the marine macroalgae crude extracts of G. elongate, T. ornata or E. flexuosa have been efficiently used as a natural, non-toxic, low-cost, bio-reducing and capping agents for Ag-NPs biosynthesis. The reduction of Ag⁺ and formation of Ag⁰ have been verified by UV–Vis spectroscopy and FTIR analyses. The formed Ag-NPs were shaped in sphere having a crystalline nature with size ranging from 20 to 25 nm. Moreover, our findings powerfully advocate that the G. elongate, T. ornata or E. flexuosa biogenic Ag-NPs have many potent in vitro biological activities including antioxidant, anticancer, anti-inflammatory, anti-diabetic and anti-Alzheimer. These biogenic Ag-NPs may be used as medication in near future. However, further in vivo studies in animals and clinical studies are required to ensure their safeties and efficacies before their approval for treatment of diseases in human beings. One limitation for the clinical use of these innovative macroalgae bio-capped Ag-NPs as antioxidant, anticancer, anti-inflammatory, anti-diabetic and anti-Alzheimer is that most studies supporting their efficiency are performed in vitro or in vivo on animal models. Analogously, there are concerns regarding the possible side effects associated with their use in medical fields on human beings. Addressing these productions may help apply these medications in the future in clinical.

Author Contributions

M.N.A., O.M.A., K.N.M.E. and M.S. contributed to the conceptualization; M.N.A. and O.M.A. contributed to the approaches; M.N.A. and O.M.A helped in the assessment; M.N.A., O.M.A., K.N.M.E. and M.S. contributed to the resources; M.N.A. was involved in writing—original draft preparation; O.M.A., K.N.M.E. and M.S. contributed to writing—review and editing.

Funding

Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

Data Availability Statement

Supporting data presented in this paper are available on request from the corresponding author.

Declarations

Ethical Approval

All animal approaches are in harmony with the standard guidelines of the Experimental Animal Ethics Committee of Faculty of Science, Beni-Suef University, Egypt (Ethical Approval number: BSU/FS/020–99).

Consent to Participate

We understand that all information we provide for this study will be treated confidentially.

Consent to Publish

All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.