1. Introduction
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak was initially reported in December 2019 in Wuhan, China. Since then, it has rapidly spread throughout the world. The coronavirus disease 2019 (COVID-19) has infected ~770 million confirmed cases to date, with about 1% of patients dying from it [1]. The SARS-CoV-2 pandemic has put a significant strain on the current healthcare system and has had a negative social and economic impact. Several recently produced vaccinations have shown remarkable defensive effects against SARS-CoV-2 [2]. However, due to the challenges in commercial accessibility and economic constraints in some locations and areas, the current COVID-19 vaccination programs have not been successfully implemented [3], leading to ongoing viral transmission and outbreaks of variant viruses. Therefore, the creation of a novel antiviral therapy is essential. A safe and effective medicine often takes much time and effort to produce. “Drug repurposing” is regarded as a viable tactic for dealing with the crisis of the SARS-CoV-2 pandemic. Remdesivir, a medication that has a clinically established anti-Ebola virus disease (EVD) action, has been tested on COVID-19, and the results have been promising [4]. In addition, researchers have investigated the effectiveness of chloroquine and its derivatives, which are used to treat malaria, to treat various illnesses like COVID-19, tissue sicknesses, and several cancers like colon, breast, prostate, and bladder [5]. Numerous investigations have assessed how employing immunomodulators can improve the health of severe COVID-19 patients. Consequently, applying naturally occurring bioactive substances and nutraceuticals that include not only immunomodulatory but also antimicrobial and non-toxic effects seems to be a promising cure.
Because of their adaptability, seaweed (marine macroalgae) can be utilized in food and medicine due to their biochemical compositions [6]. Numerous studies have shown that the significant biological and chemical diversity of macroalgae has attracted interest in recent years due to a diversity of active compounds derived from them, including polysaccharides, polyunsaturated fatty acids, pigments, peptides, carbohydrates, vitamins, and polyphenols [7]. These compounds have a variety of properties, including antimicrobial [8], antioxidant [9,10], anti-inflammatory, and immunomodulatory capabilities [11]. They have antiviral properties against a variation of enveloped viral infections, including hepatitis virus [12], human immunodeficiency virus (HIV) [13], herpes simplex virus (HSV) [14], and most recently, SARS-CoV-2 virus [15,16,17,18]. They can also be used as immune boosters [7]. They also help the human gut microbiota by controlling the immune system and metabolism [19,20]. For these reasons, they are called nutritional foods or prebiotics [21]. Worldwide, much research has been performed to identify molecules that can stop the spread, replication, and infection of SARS-CoV-2 using marine bioactive components, such as polyphenolics, carotenoids, and polysaccharides [22,23]. Polysaccharides have received much attention in marine compounds for the development of COVID-19 prevention and therapies.
According to reports, lambda-carrageenan has antiviral properties against SARS-CoV-2 in vitro [24]. Furthermore, healthcare professionals caring for patients with COVID-19 disease showed a 79.8% relative risk decrease in preventing SARS-CoV-2 with an iota-carrageenan nasal spray [25]. Moreover, by obstructing the spike protein’s ability to connect with the host cell’s ACE2 receptor in vitro, several marine sulfated polysaccharides and sulfated galactofucan from Saccharina japonica (formerly Laminaria japonica) (Phaeophyceae) efficiently prevented SARS-CoV-2 entry [26]. According to a recent study by Song et al. [27], the sulfated polysaccharides fucoidan and carrageenan demonstrated noteworthy antiviral properties against SARS-CoV-2 at concentrations ranging from 3.90 to 500 μg/mL.
Fucoidan powders extracted from marine brown seaweed Sargassum spp. may have antiviral properties against SARS-CoV-2 infection in vitro. When DMSO was used as a solvent, fucoidan powder’s antiviral activity increased to 100% protection against SARS-CoV-2 infection [28]. It was determined whether crude polysaccharides made from two seaweed species, Sargassum horneri and Sargassum fusiforme (formerly Hizikia fusiformis), could stop the spread of SARS-CoV-2. These unrefined polysaccharides effectively stopped the spread of SARS-CoV-2, as demonstrated by plaque titration, which showed IC50 values ranging from 0.35 to 4.37 g/mL. With an IC50 of 0.35 g/mL, the crude polysaccharide of S. fusiforme exhibited the most potent antiviral effect. Treatment of Vero-E6 cells with these crude polysaccharides either prior to or following viral infection significantly reduced the number of SARS-CoV-2 spikes, nucleocapsid proteins, and RNA copies of RNA-dependent RNA-polymerase and nucleocapsid that were expressed. According to Kang et al. [16], these findings demonstrate how these crude marine polysaccharides successfully prevent SARS-CoV-2 from spreading by obstructing the virus’s entrance.
According to Binsuwaidan et al. [18], through virucidal activity, inhibition of viral replication, and interference with viral adsorption (% inhibitions of 64%, 33.3%, and 31.1%, respectively), Ulva lactuca, a green macroalgae, showed potent anti-SARS-CoV-2 activity in its aqueous extract. Therefore, ulvan may be a promising compound for preclinical research in the drug development process to combat SARS-CoV-2.
Brown seaweed polysaccharides were reported to have antiviral properties in earlier studies. However, research on the antiviral effect of marine brown seaweeds collected from the coast of Egypt against SARS-CoV-2 is scant or lacking. To the best of our knowledge, none of the seaweed species examined here were shown to have any protective effects against SARS-CoV-2. In this regard, the study’s objectives are to assess the safety of extracted polysaccharides in the treatment of COVID-19 illness and investigate the antiviral activity of polysaccharides (alginate) from two brown seaweed species, Padina boergesenii, and Sargassum euryphyllum, against the SARS-CoV-2 virus. Previous work reported that P. boergesenii was already used for foodstuff, and extracts exhibited hepatoprotective, antidiabetic, and antioxidant activity [15].
2. Materials and Methods
2.1. Collection of Seaweed Samples
Two brown seaweed species were harvested in the summer of 2021 from the seashore in Hurghada, Egypt. The collected seaweed was given to the phycology lab in plastic bags with seawater (Botany Department, Faculty of Science, Tanta University). Seaweeds were cleansed with tap water and then refined water to remove any debris and attached epiphytes. For taxonomical identification, some samples were stored in saltwater containing formalin (0.4%), while the remaining samples were air-dried at 25–30 °C. After being dried and processed into a powder using an electronic mill, the samples were kept at −20 °C until needed. In accordance with Aleem [29], Jha et al. [30], and Guiry and Guiry [31], the seaweed samples were identified as Padina boergesenii Alender and Kraft (Phaeophyceae, Dictyotales, Dictyotaceae) and Sargassum euryphyllum Grunow Tseng and Lu Baoren (Phaeophyceae, Fucales, Sargassaceae).
2.2. Extraction of the Crude Seaweed Polysaccharides
The seaweed powder was boiled in distilled water (1:5 w/v) at 100 °C for 2 h using a reflux condenser under reduced pressure. The hot extract was filtered with a nylon mesh bag (pore size 24 μm), sequentially filtered with 0.45 μM Millipore filters, and then subjected to drying [32].
2.3. Characterization and Identification of Seaweed Polysaccharides
Identification of polysaccharides was carried out using ultraviolet (UV/VIS) spectra and vibrational spectroscopy Fourier transform infrared attenuated total reflectance (FTIR-ATR) at the Marine Algae Lab, Department of Life Sciences, University of Coimbra, Portugal.
2.3.1. Alginate Extraction
Cotas et al. [33] outlined the process as the foundation for sodium alginate extraction. A commercial grinder (TitanMill 300 DuoClean, Cecotec, Valencia, Spain) was used to grind the dried seaweed (3 g) to a particle size of less than 1 cm. The resulting solution of hydrochloric acid (José Manuel Gomes dos Santos, Portugal) was mixed at a ratio of 1.23% (1:30 v:v) (3 mL of hydrochloric acid: 87 mL of distilled water per 3 g of dried seaweed). The mixture was then kept at room temperature (23 °C) for 24 h. The solution was filtered using a Gooch funnel (porosity: G2) under vacuum (Laborport N820, Lisbon, Portugal), and it was then repeated two or three times with distilled water washing. After 24 h at room temperature (23 °C), the residue was alkali extracted in 90 mL of 2% sodium carbonate (Fisher Chemicals, Portugal) (1:30 m:v for the initial weight of the dried biomass). To remove the leftovers from the alginate solution, the extract was once again vacuum-filtered through a cloth filter using a Gooch funnel (porosity: G2). The alginic acid was then precipitated by adding 37% hydrochloric acid (José Manuel Gomes dos Santos, Portugal) to the filtrate (2 mL of 37% hydrochloric acid per 30 mL of the final solution). After being cleaned with ethanol 96% (José Manuel Gomes dos Santos, Portugal) at a 1:3 v:v ratio, the alginate was left in the cold. After discarding the liquid solution, the precipitate was dried for 48 h at 60 °C in an air-force oven (Raypa DAF-135, R. Espinar S.L., Barcelona, Spain).
2.3.2. Physiochemical Properties of Alginate (Viscosity, pH, EC, and TDS)
Alginate solutions (1% m/v) were prepared by dissolving the polysaccharide in distilled water using temperature and magnetic stirring. Then, the solutions were cooled to a temperature of 25 °C. Later, viscosity measurement was carried out using spindles SP2 and SP3 in an IKA Rotavisc Viscometer at a speed of 100 rpm at room temperature for 1 min. A pH/conductivity/TDS meter (Combo HI98129, HANNA instruments, Woonsocket, RI, USA) was used to measure the pH and TDS values of the alginate solutions [34].
2.3.3. UV/VIS Spectrophotometric Profiles of Alginate Solutions
Alginate solutions (1% m/v) prepared for the viscosity analyses were diluted in distilled water (1:2), and UV/VIS absorption spectra were measured in the range of 200–800 nm using a UV-3100PC, UV/VIS Scanning Spectrophotometer (VWR® Radnor, PA, USA) with 1 cm quartz cuvettes [34].
2.3.4. FTIR-ATR Analysis
Fourier transform infrared (FT-IR) attenuated total reflectance (ATR) spectroscopy was employed to characterize the structure of dried extracted alginate [34]. The IR spectra (24 scans) were obtained at room temperature (referenced against air) in the wavenumber range of 400–4000 cm−1 (resolution of 4 cm−1) using a Bruker Alpha II (Bruker, Ettlingen, Germany). Spectra were analyzed with OPUS 7.2 software (Bruker, Ettlingen, Germany).
2.4. SARS-CoV-2 Virus Isolation and Propagation
Utilizing a specimen from an oropharyngeal swab obtained on 18 March 2020 from an Egyptian woman aged 34, Vero-E6 cells (ATCC No. CRL-1586) were used in this study to isolate the highly pathogenic SARS-CoV-2 strain hCoV-19/Egypt/NRC-03/2020. It was isolated from infected humans in Egypt in 2020, characterized as previously described by Kandeil et al. [35] (GISAID accession number: EPI_ISL_430819). All virus experiments were carried out at the Center of Scientific Excellence for Influenza Viruses (CSEIV), Vaccine Development and Virological Tests Unit, National Research Center (NRC), Giza, Egypt.
2.4.1. Cell Line
The African green monkey kidney epithelial (Vero-E6) cells were kindly provided by Dr. Richard Webby, St. Jude Children’s Research Hospital, Department of Virology and Molecular Biology, USA. The cells were cultivated as a confluent sheet in 75 cm2 tissue culture flasks in the DMEM medium, supplemented with 10% fetal bovine serum (FBS) and 2% penicillin/streptomycin.
2.4.2. Detection of Antiviral Activity and Cytotoxicity of Seaweed Polysaccharide Virus Titration by TCID50
Vero-E6 cells were used to prepare a 96-well plate in growth media with 10% FBS and 2% antibiotic/antimycotic and incubated overnight at 37 °C with 5% CO2. Following confluence, 1 log serial dilution of the virus stock was prepared and used in infecting cell monolayers. The virus inoculum was then removed within 1 h of infection, replaced with 160 µL of infection media in each well, and incubated for 72 h. The TCID50 value was calculated using the Reed and Mensch method by observing and counting the cytopathic effect in each well [36].
2.4.3. Determination of Inhibitory Concentration 50% (IC50)
Vero-E6 cells (2.4 × 104) were seeded into each well of 96-well tissue culture plates, and the plates were incubated for the entire night at 37 °C in a humidified environment with 5% CO2. After giving the cell monolayers one wash with 1× PBS, they were exposed to viral adsorption for 1 h at room temperature (RT) using hCoV-19/Egypt/NRC-03/2020 (accession number on GSAID: EPI_ISL_430820). The test compounds were added to the cell monolayers at different concentrations using 100 μL of DMEM. After incubation for 72 h at 37 °C in an incubator with 5% CO2, the cells were fixed for 4 h with 50 μL of 10% formaldehyde and stained for 15 min at room temperature with 0.1% crystal violet in distilled water. After dissolving the crystal violet dye with 100 μL of absolute methanol in each well, the Anthos Zenyth 200 rt plate reader (Anthos Labtec Instruments, Heerhugowaard, The Netherlands) was used to measure the optical density of the color at 570 nm. According to Mostafa et al. [37], the compound’s half-life (IC50) is the amount needed to decrease the virus-induced cytopathic effect (CPE) by 50% in comparison to the virus control.
2.4.4. Viral Plaque Titration Assay
As previously mentioned, the plaque titration method was used to count the number of plaque-forming units in each viral passage [38]. In short, Vero-E6 cells (105 cells/well) were seeded into tissue culture plates with 6 wells. The cells underwent two PBS washes 2–3 days after seeding, or at 90–100% confluence. The viruses were diluted ten times in DMEM, with 100 μL of the undiluted virus mixed with 400 μL of DMEM before being inoculated into Vero-E6 cells. The plates were incubated for 1 h at 37 °C.
The remaining inoculum was then aspirated out of the wells. Then, 3 mL of DMEM overlay medium containing 2% agarose type 1 (Lonza, Basel, Switzerland), 2% antibiotic–antimycotic mixture, and 1 µg/mL TPCK-treated trypsin was immediately added to each well. Afterward, the plates were incubated for three days at 37 °C with 5% CO2. Every day, the plaques were examined under a microscope. One milliliter of 10% formaldehyde was added to each well and left for six hours after clear plaques were visible. For virus inactivation and cell fixation, cells were incubated at room temperature. After discarding the formaldehyde, the plates were cleaned with water and allowed to dry. In 1 mL of the staining solution, a mixture of 1% crystal violet and 20% methanol in distilled water was added to each well to visualize the plaques. The wells were then allowed to incubate for five minutes at room temperature, after which the dye was disposed of, and the wells were cleaned with water and dried. Afterward, the number of viral plaques was determined, and the virus titer was calculated using the formula below:
Plaque-forming unit (PFU)/mL = Number of plaques × Reciprocal virus dilution × Multiplicand number to bring the inoculum volume to 1 mL.
2.4.5. Mode of Action of Virus Inhibition
At three distinct phases of the virus propagation cycle, the potential mechanisms of action of the polysaccharides of the chosen seaweeds’ ability to inhibit viruses were investigated. These mechanisms were based on three primary theories: (i) the prevention of viral replication and budding; (ii) each extract’s capacity to prevent the virus from adhering to infected cells, a process known as membrane fusion that blocks viral entry and is referred to as “viral adsorption”; and (iii) each polysaccharide extract’s direct ability to render the virus inactive (virucidal activity). Furthermore, the antiviral activities that were observed may be explained by the aforementioned mechanism of action alone or in combination to establish how the chosen seaweed polysaccharide interacts with the virus.
Viral Adsorption
For the viral adsorption test, Vero-E6 cells were grown in a 6-well plate with 105 cells/mL for 24 h at 37 °C using the Zhang et al. method [39]. Different concentrations of seaweed polysaccharide were added to a 200 µL medium without any supplements, and the cells were co-incubated for 2 h at 4 °C. After washing the cells three times with free-medium supplements to remove the unabsorbed polysaccharide extract, the virus was diluted and co-incubated with the pretreated cells for one hour. Finally, three milliliters of DMEM supplemented with 2% agarose was added. After allowing the plates to solidify, they were incubated at 37 °C to facilitate the formation of viral plaques. These were then fixed and stained, as previously mentioned, to determine the percentage reduction in the formation of viral plaques compared to control wells where the virus was directly infected into untreated Vero-E6 cells.
Viral Replication
The viral replication assay was conducted in accordance with Kuo et al. [40]. In a 6-well plate, Vero-E6 cells were grown at 37 °C for 24 h (105 cells/mL). After being directly applied to the cells and diluted to 103 PFU/well, the virus was incubated for 1 h at 37 °C. Viral particles that were not absorbed were eliminated by repeatedly washing cells with free-medium supplements three times. After applying the extract at varying concentrations for 1 h, the cell monolayer was supplemented with 3 mL of 2× DMEM medium containing 2% agarose. After allowing the plates to solidify, they were incubated at 37 °C until viral plaques started to appear. After six hours of fixing in a 10% formalin solution, cell monolayers were stained with crystal violet. The control wells contained Vero-E6 cells infected with the virus but not exposed to the extract. Following the counting of plaques, the percentage drop in plaque formation compared to control wells was recorded.
Virucidal Mechanism
Before performing the virucidal assay, Vero-E6 cells (105 cells/mL) were grown for 24 h at 37 °C in a 6-well plate [41]. Serum-free DMEM (200 µL) containing the virus was added to the extracted concentration under test. After one hour of incubation, the mixture was diluted three times with serum-free medium, resulting in a ten-fold dilution increase. This resulted in nearly no extract being left behind, allowing the viral particles to continue growing on Vero-E6 cells. The Vero-E6 cell monolayer was then filled with 100 µL of each dilution. The cell monolayer received a DMEM overlayer after a 1 h contact period. To determine the percentage reduction in the formation of viral plaques in comparison to control wells containing infected cells that were not pretreated with the tested polysaccharide extract, the plates were allowed to solidify before being incubated at 37 °C to allow the formation of viral plaques. The plates were then fixed and stained, as previously mentioned.
2.5. Statistical Analyses
Every experiment was run through three biological replications. For statistical analysis and graphical data visualization, GraphPad Prism 8.01 was utilized. The average of the means of three replicates plus the standard deviation were used to present the data. The obtained data’s non-linear fit was represented by the CC50 and IC50 curves, whose values were determined as the “best-fit value” using GraphPad Prism.
3. Results
3.1. Physicochemical Characterization of Seaweed Polysaccharides (Alginate)
The physicochemical parameters measured are summarized in Table 1. The 1% alginate solutions demonstrated low pH, electrical conductivity, and viscosity.
3.2. UV/VIS Spectra
The spectra do not show any evidence of absorbance peaks in the visible zone (400–700 nm). This can be observed in the spectra below; therefore, it is verified that the samples do not contain pigments like contaminants (Figure 1 and Figure 2). These spectra present even more evidence that the samples are not chemically cleaned. This is because they present absorption in the UV zone, in this case at about 225 and 250 nm. An absorption peak at this wavelength is characteristic of carbonyl and sulfated groups’ absorbance. Absorption in the 210 nm region suggests a strong presence of polysaccharides, while the alginate from S. euryphyllum, owing to its brownish color, exhibits greater absorbance of these compounds compared to P. boergesenii.
3.3. FTIR-ATR
FTIR-ATR was used to analyze the polysaccharides that were extracted (Figure 3 and Figure 4). This spectroscopic method required little material and enabled quick, non-destructive polysaccharide characterization. The acquired spectra were examined and supported by references. FTIR-ATR spectra showed the characteristic absorption bands of alginate (Table 2). Only the extracted alginate spectra showed bands between 1730 and 1710 cm−1, which were proposed to represent the carboxylic acid ester form (C=O). The spectra’s peaks at 1428 cm−1 and 1400 cm−1 were attributed to the symmetric stretching vibrations of alginate carboxyl groups (COOH). The 1280–1230 cm−1 range is associated with fucoidan and other sulfated polysaccharides. The range from 1280 cm−1 to 1230 cm−1 is associated with fucoidan and other sulfated polysaccharides, mainly corresponding to fucoidan, representing peaks with some mild intensity, as shown in Table 2 symbols (+, ++, +++, +++++) to indicate the peaks relative intensity).
The highest peak in both spectra was found in the bands 1030 cm−1 and 1025 cm−1, which correspond to the characteristic alginic acid. The derived alginate spectra’s strong band at 1027 cm−1 may indicate the sample’s high guluronic acid content. Uronic acid residues’ C-O stretching vibration is typically associated with bands that are centered between 950 and 930 cm−1. The carbohydrate anomeric regions ranged from 806 cm−1 to 788 cm−1, with mannuronic acid and guluronic acid residues responsible for each. The main difference between the two samples is the 1082 cm−1 peak in the Padina boergesenii alginate, corresponding to –CO groups. Thus, the UV/VIS and FTIR-ATR spectra support the idea that the extracts are composed mainly of alginate but contain fucoidan and other sulfated polysaccharides.
3.4. Antiviral Activity of Seaweed Polysaccharides Against SARS-CoV-2
Table 3 presents the cytotoxicity concentration 50% (CC50), inhibitory concentration 50% (IC50), and selectivity index (SI) for seaweed polysaccharides on Vero-E6 cells and SARS-CoV-2 virus using the crystal violet assay.
Seaweed polysaccharides’ cytotoxicity concentration 50% (CC50) varied between 587.7 µg/mL for P. boergesenii and 396.4 µg/mL for S. euryphyllum in Vero-E6 cells. The inhibitory concentration 50% (IC50) ranged from 24.2 µg/mL to 29.3 µg/mL for P. boergesenii and S. euryphyllum, respectively, against the SARS-CoV-2 virus. This was also indicated by the selectivity index (CC50)/(IC50), which was 24.3 and 13.5 for P. boergesenii and S. euryphyllum, respectively (Table 3).
P. boergesenii demonstrated potent antiviral activity against SARS-CoV-2 and was therefore more promising than S. euryphyllum, as evidenced by the results, which showed that its polysaccharide had a higher selectivity index than that of S. euryphyllum’s polysaccharide (Figure 5). To confirm P. boergesenii polysaccharide’s efficacy as an anti-SARS-CoV-2 virus agent, further research was conducted to determine its mechanism of action.
Three modes of action of the P. boergesenii polysaccharide were examined (Table 4 and Figure 6 and Figure 7). The results showed that at a concentration of 575 µg/mL of the polysaccharide, the virucidal mechanism was the most effective, followed by viral adsorption, and finally, viral replication, with viral inhibition percentages 68.6% ± 0.8, 57.1% ± 1.4, and 37.2 ± 3, respectively, compared with remdesivir as an antiviral drug (Table 5).
4. Discussion
Human respiratory diseases caused by coronaviruses are responsible for a considerable portion of morbidity and mortality rates worldwide. The ability of the novel coronavirus SARS-CoV-2 to spread from person to person has raised concerns in the wake of its discovery. The public are aware of this severe issue [28]. Antiviral drugs to fight the SARS-CoV-2 virus could be another defense against the new pandemic. This would help contain the infection until sufficient vaccines are produced in sufficient numbers. It is now crucial to find new alternative anti-SARS medications in order to combat the latest strains.
It is believed that algae are rich in bioactive compounds. The use of these bioactive substances in treating various human illnesses is well supported by the available evidence. Furthermore, studies have demonstrated the immunomodulatory and antiviral qualities of sulfated polysaccharides, including ulvan, carrageenan, galactan, alginate, fucan and fucoidan, laminaran, calcium spirulan, and nostaflan. Alginate and carrageenan may open up new possibilities for developing cutting-edge treatment approaches for SARS-CoV-2 and other viral diseases, as numerous studies employing bioinformatic techniques have shown [42,43,44].
Hepatitis C, HIV, herpes simplex, influenza, and most recently, the coronavirus, were all shown to be susceptible to the virucidal effects of sulfated polysaccharides. Worldwide, scientists are investigating and creating vaccines to stop the spread of COVID-19 and SARS-CoV-2 infection. Natural polymers with antiviral qualities, non-toxicity, and biocompatibility, such as chitosan, alginate, gums, and so on, have a good chance of being developed into safe and novel vaccines to stop the spread of infectious diseases, including the COVID-19 disease [45,46].
The present study’s FTIR-ATR and UV/VIS spectra revealed the distinctive absorption bands of alginate containing mannuronic and guluronic acid residues and also the presence of fucoidan and other sulfated polysaccharides (also lowering the viscosity rate of alginate). The C-O stretching vibration of uronic acid residues is generally linked to bands and sulfated esters in the tested brown seaweeds that may have anti-SARS-CoV-2 properties. Sulfated polysaccharides (such as fucoidan) were reported to have a structure similar to heparan sulfates or human glycosaminoglycans due to the presence of uronic acid and sulphate moieties. By attaching itself to viral glycoproteins, this molecular mimicry blocks the entry of the virus [47].
The results of the current study regarding anti-SARS-CoV-2 activity indicated that P. boergesenii and S. euryphyllum exhibited selectivity indices of 24.3 and 13.5, respectively, based on the ratio of CC50 to IC50. This suggests that the polysaccharides from P. boergesenii show a higher level of inhibition against SARS-CoV-2 and present a more promising option than S. euryphyllum (Table 3 and Figure 5).
The polysaccharides from P. boergesenii displayed a distinct mode of action against the hCoV-19/Egypt/NRC-03/2020 strain. Among the three mechanisms tested, the virucidal mechanism demonstrated the highest level of viral inhibition, reducing the viral count from 3.5 × 105 (control) to 1.1 × 105 (after treatment), with an inhibition percentage of 68.6% ± 0.8. This was followed by the viral adsorption mechanism, which achieved an inhibition percentage of 57.1% ± 1.4. Lastly, the viral replication mechanism recorded an inhibition percentage of 37.2% ± 3.0 at the highest tested concentration of 575 µg/mL (Table 4 and Figure 6).
The results supported previous studies that demonstrated the ability of seaweed and abalone viscera’s crude polysaccharides to halt SARS-CoV-2 transmission both before and after infection. Furthermore, by preventing viral entry in vitro, it has been shown that these polysaccharides can stop replication [16]. Additionally, the study’s findings agree with those of Binsuwaidan et al. [18], who discovered that the polysaccharide from Ulva lactuca (ulvan), a green seaweed, exhibited intense anti-SARS-CoV-2 activity through interference with viral adsorption, inhibition of viral replication, and virucidal activity (26, 33, and 31 percent inhibition, respectively). Therefore, ulvan might be a helpful material for preclinical studies searching for a drug to treat SARS-CoV-2.
According to earlier research—an in silico analysis—the main protease complex with laminarin showed the most stable RMSD over a 150 ns MD simulation time. According to Mohammed et al. [48], they might have an inhibitory effect on SARS-CoV-2.
Algae-derived polysaccharides inhibit dengue virus (DENV) replication through the inhibition of cell internalization [49]. Elizondo-Gonzalez [50] discovered that fucoidan, a polysaccharide, inhibited the early stages of Newcastle disease virus infection, in line with this study. Fucoidan may also prevent the internalization of the virus, which would stop SARS-CoV-2 infection. Moreover, PBMCs from COVID-19 recovered patients have demonstrated that fucoidan can support mitochondrial membrane potential recovery. This implies that fucoidan might be a valuable medication that decreases the long-term consequences of the illness [28]. Additionally, our results supported the findings of Yang et al. [51], who found that alginate derivatives, such as Polymannuronate Monophosphate (PMPD), were similar to previously reported heparin analogs and an effective way to block the interaction of spike protein with ACE2. The aforementioned findings show that PMPD has enormous potential for use in preventing SARS-CoV-2 infection.
According to a different study, the seaweed Ecklonia cava subsp. kurome (formerly Ecklonia kurome) crude polysaccharide derivatives 37501, 37502 (alginate), and 37503 have intense anti-SARS-CoV-2 infection activity in cells and can inhibit 3CL protease’s enzymatic activity. The study also demonstrated that alginate disrupted spike protein binding to the ACE2 receptor and could obstruct SARS-CoV-2 replication. Researchers are investigating whether these polysaccharides could prevent SARS-CoV-2 replication in response to the above results [52].
A prior investigation revealed anti-SARS-CoV-2 activity in crude polysaccharides derived from Sargassum horneri and Sargassum fusiforme (Phaeophyceae). Counting the number of plaques following crystal violet staining revealed that crude polysaccharides (CPs; 500 µg/mL) decreased plaque formation by over 98% compared to the virus alone (without CPs). Before SARS-CoV-2 infection, Vero-E6 cells treated with 0.8 µg/mL CP concentrations showed more than 60% reduction in plaque formation [16].
The present work points to possible impacts on viral replication, adsorption, and significant virucidal activity. Numerous studies indicate that these negatively charged substances interfere with the enveloped virus’s early stages of the proliferation cycle, thereby preventing viral infection [53]. According to other studies, sulfated polysaccharides may prevent the production of viral proteins or stop the life cycle at specific points [54]. According to Sepúlveda-Crespo et al. [55], the sulphate residues obstruct the positively charged viral glycoprotein domain and stop the virus from interacting with cells at first. According to Lopes et al. [56], specific sulfated polysaccharides from green macroalgae also stop viral replication, consistent with our findings. Thus, bioactive substances from certain brown algae with antiviral properties may offer a cheap, easily obtainable natural supplement, particularly for managing illnesses brought on by recent or re-emerging viruses. Our research indicates that polysaccharides from certain brown algae may be a unique option for creating easily accessible, reasonably priced antiviral medications and prophylactics.
5. Conclusions
Researchers are motivated to find a more effective solution to this pandemic in light of the limited availability, financial concerns, and poor control and prevention of COVID-19 with these therapies for SARS-CoV-2. Marine seaweeds have potential medical applications because of their diverse biochemical compositions, including polysaccharides, polyunsaturated fatty acids, pigments, peptides, carbohydrates, vitamins, and polyphenols. These substances exhibit antiviral characteristics against various enveloped viral infections, such as the hepatitis virus, the human immunodeficiency virus (HIV), herpes simplex virus (HSV), and more recently, the SARS-CoV-2 virus. The characteristic absorption bands of alginate that exhibited the anti-SARS-CoV-2 effect were visible in UV/VIS and FTIR-ATR spectra. Our findings showed that alginate and fucoidan from P. boergesenii had a more significant antiviral activity against SARS-CoV-2, and these sulfated polysaccharides were a more promising option than the polysaccharides from S. euryphyllum. The distinct modes of action of the P. boergesenii polysaccharide against the SARS-CoV-2 virus demonstrated that, at a concentration of 575 µg/mL, the virucidal mechanism was the most effective of the three mechanisms employed for virus inhibition, followed by the adsorption mechanism, and finally, viral replication. The above findings showed that certain sulfated polysaccharides, such as alginates and fucoidan from brown algae, can be used in the prevention/treatment of SARS-CoV-2 infection. More future research is needed to screen and investigate different metabolites from different seaweed species against the SARS-CoV-2 virus.
Conceptualization, S.G. and S.E.S.; methodology, S.E.S., M.G. and A.M.K.; software, S.G.; validation, S.G.; formal analysis, S.G., A.M.K. and L.P.; investigation, S.G., M.G., J.C. and S.G.; resources, S.G.; writing—original draft preparation, S.G., A.M.K., S.E.S. and M.G.; writing—review and editing, S.G., J.C. and L.P.; visualization, L.P.; supervision, S.G. All authors have read and agreed to the published version of the manuscript.
Data are available from the authors.
The authors declare no conflicts of interest.
Footnotes
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Figure 1 UV spectra of the Padina boergesenii polysaccharide.
Figure 2 UV spectra of the Sargassum euryphyllum polysaccharide.
Figure 3 FTIR-ATR spectra of the Padina boergesenii polysaccharides.
Figure 4 FTIR-ATR spectra of the Sargassum euryphyllum polysaccharides.
Figure 5 Cytotoxicity concentration 50% (CC50), inhibition concentration 50% (IC50), and selectivity index (SI) for the two seaweed polysaccharides and remdesivir as a control drug.
Figure 6 The mode of action of Padina boergesenii polysaccharide (A) and remdesivir control drug (B) against SARS-CoV-2 virus according to virucidal, adsorption, and replication effects.
Figure 7 Mode of action of Padina boergesenii polysaccharide against SARS-CoV-2 virus. (1) Viral adsorption, (2) Viral replication, and (3) Virucidal mechanisms.
Physicochemical characterization of seaweed polysaccharides.
| Seaweed | pH | EC | TDS | Viscosity (cP) |
|---|---|---|---|---|
| Padina boergesenii | 4.21 | 118 | 46 | 3.9 |
| Sargassum euryphyllum | 3.81 | 200 | 108 | 3.6 |
FTIR-ATR band identification and characterization of the brown seaweed polysaccharide.
| Reference Wave Number (cm−1) | Bound | Wave Number Observed (cm−1) | |
|---|---|---|---|
| Padina Boergesenii | Sargassum Euryphyllum | ||
| 1730–1710 | Carboxylic acid ester (C=O) | +++ | +++ |
| 1610–1600 | Asymmetric stretching vibration of carboxyl groups (COOH) | + | + |
| 1428–1400 | Symmetric stretching vibration of carboxyl groups (COOH) | + | + |
| 1280–1230 | Sulfated esters (S=O) | +++ | +++ |
| 1030–1025 | Alginic acid (C-O group) | +++++ | +++++ |
| 950–930 | C-O stretching vibration of uronic acids | ++ | ++ |
| 806 | Guluronic acid residues | + | + |
| 788 | Mannuronic acid residues | ++ | ++ |
+—intensity.
Cytotoxicity concentration 50% (CC50) and inhibition concentration 50% (IC50) for the two seaweed polysaccharides and standard drugs using crystal violet assay.
| Antiviral Compound | CC50 | IC50 | Selectivity Index (SI) |
|---|---|---|---|
| Padina boergesenii polysaccharide | 587.7 | 24.2 | 24.3 |
| Sargassum euryphyllum polysaccharide | 396.4 | 29.3 | 13.5 |
| Standard antiviral drug | 351.8 | 2.34 5 | 150 |
The different mechanisms of action of Padina boergesenii polysaccharide against SARS-CoV-2 virus.
| Mechanism | Conc. µg/mL | Virus Control (PFU/mL) | Viral Count Following | Viral Inhibition |
|---|---|---|---|---|
| Viral adsorption | 575 | 3.5 × 105 | 1.50 × 105 | 57.1% ± 1.4 |
| 550 | 1.60 × 105 | 54.3% ± 1.3 | ||
| 500 | 1.75 × 105 | 50.0% ± 0.6 | ||
| Viral replication | 575 | 3.5 × 105 | 2.20 × 105 | 37.2% ± 3.0 |
| 550 | 2.30 × 105 | 34.3% ± 1.7 | ||
| 500 | 2.40 × 105 | 31.5% ± 6.7 | ||
| Virucidal | 575 | 3.5 × 105 | 1.10 × 105 | 68.6% ± 0.8 |
| 550 | 1.25 × 105 | 64.3% ± 0.7 | ||
| 500 | 1.45 × 105 | 58.6% ± 0.8 |
The different mechanisms of action of remdesivir against SARS-CoV-2 virus.
| Mechanism | Conc. µg/mL | Virus Control (PFU/mL) | Viral Count Following Treatment (PFU/mL) | Viral Inhibition % |
|---|---|---|---|---|
| Viral adsorption | 350 | 3.5 × 105 | 3.15 × 105 | 27.1% ± 1.40 |
| 300 | 2.80 × 105 | 20.0% ± 2.10 | ||
| 250 | 2.55 × 105 | 10.0% ± 1.10 | ||
| Viral replication | 350 | 3.5 × 105 | 0.45 × 105 | 87.1% ± 0.87 |
| 300 | 0.65 × 105 | 81.4% ± 0.73 | ||
| 250 | 0.87 × 105 | 75.1% ± 0.95 | ||
| Virucidal | 350 | 3.5 × 105 | 2.85 × 105 | 42.9% ± 1.90 |
| 300 | 2.50 × 105 | 33.3% ± 1.20 | ||
| 250 | 2.00 × 105 | 18.6% ± 2.30 |
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; Karkour, Ali M 1 ; Shafay, Shimaa El 1 ; GabAllah, Mohamed 2 ; Cotas João 3
; Pereira, Leonel 4
1 Botany and Microbiology Department, Faculty of Science, Tanta University, Tanta 31527, Egypt; [email protected] (A.M.K.); [email protected] (S.E.S.)
2 Center of Scientific Excellence for Influenza Viruses, National Research Centre, Dokki, Cairo 12622, Egypt; [email protected]
3 CFE—Centre for Functional Ecology: Science for People & Planet, Marine Resources, Conservation and Technology—Marine Algae Lab, Department of Life Sciences, University of Coimbra, 3000-456 Coimbra, Portugal; [email protected]
4 CFE—Centre for Functional Ecology: Science for People & Planet, Marine Resources, Conservation and Technology—Marine Algae Lab, Department of Life Sciences, University of Coimbra, 3000-456 Coimbra, Portugal; [email protected], IATV—Instituto do Ambiente, Tecnologia e Vida, 3030-790 Coimbra, Portugal