Inhibition of SARS-CoV-2 Virus Entry by the Crude Polysaccharides of Seaweeds and Abalone Viscera In Vitro

Much attention is being devoted to the potential of marine sulfated polysaccharides as antiviral agents in preventing COVID-19. In this study, sulfated fucoidan and crude polysaccharides, extracted from six seaweed species (Undaria pinnatifida sporophyll, Laminaria japonica, Hizikia fusiforme, Sargassum horneri, Codium fragile, Porphyra tenera) and Haliotis discus hannai (abalone viscera), were screened for their inhibitory activity against SARS-CoV-2 virus entry. Most of them showed significant antiviral activities at an IC50 of 12~289 μg/mL against SARS-CoV-2 pseudovirus in HEK293/ACE2, except for P. tenera (IC50 > 1000 μg/mL). The crude polysaccharide of S. horneri showed the strongest antiviral activity, with an IC50 of 12 μg/mL, to prevent COVID-19 entry, and abalone viscera and H. fusiforme could also inhibit SARS-CoV-2 infection with an IC50 of 33 μg/mL and 47 μg/mL, respectively. The common properties of these crude polysaccharides, which have strong antiviral activity, are high molecular weight (>800 kDa), high total carbohydrate (62.7~99.1%), high fucose content (37.3~66.2%), and highly branched polysaccharides. These results indicated that the crude polysaccharides from seaweeds and abalone viscera can effectively inhibit SARS-CoV-2 entry.


Introduction
Since the first identified case in December 2019, COVID-19, caused by the SARS-CoV-2 virus, has spread worldwide, causing tremendous fear and a long recession. Approximately 119 million cases of COVID-19 and more than 2.65 million deaths have been reported [1]. On 11 and 18 December 2020, and 27 February 2021, respectively, the U.S. Food and Drug Administration issued an emergency use authorization (EUA) for the Pfizer-BioNTech (indicated for individuals aged 16 years or older), Moderna, and Janssen (both vaccines are indicated for individuals aged 18 years or older) COVID-19 vaccines for the prevention of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [2]. However, a total of eight (Pfizer-BioNTech, Moderna, Janssen, AstraZeneca-Oxford, Sinovac Biotech, Gamaleya, CanSino Biologics, and Sinopharm) vaccines are now available for public use, in limited quantities, in at least 122 countries [3]. To date, at least 359 million doses of coronavirus vaccines have been administered around the world [3,4]. According to Bloomberg, the latest vaccination rate is an average of 9,245,747 doses per day worldwide, and they estimate that it will take another 3.3 years to cover 75% of the population with a two-dose vaccine at the current rate [3,4]. Although vaccination

Trace Elements in the Crude Polysaccharides
Results for the analysis of minerals and trace element concentrations in the CPs are outlined in Table 3. In all samples, Ca, K, Mg, and Na were the most abundant elements and Cd, Ga, Li, Pb, and Sr were not detected. Some mineral and trace elements (K > B > Ti > In > Fe > Al > Cr > Zn > Ba > Ni) were highest in CPPT, whereas, As, Bi, and Cu were not detected. Titanium (Ti, 19,386.2 µg/g of sample) and nickel (Ni, 39.4 µg/g) were found only in CPPT. Calcium (Ca), sodium (Na), and manganese (Mn) were the highest in CPCF (282,637.1 µg/g, 94,248.6 µg/g, 343.2 µg/g, respectively) compared to other CPs, but Ca, Na, and Mn were lowest in CPPT (62,308.7 µg/g), CPSH (424.7 µg/g), and CPUP (1.1 µg/g), respectively. Arsenic (As) was detected in small concentrations (ranging from 1.9 to 33.1 µg/g) in all CPs except for CPCF, fucoidan, and CPPT. Magnesium (Mg), silver (Ag), and bismuth (Bi) were the most abundant in CPLJ (17,807.9 µg/g, 926.3 µg/g, and 740.1 µg/g, respectively) but potassium (K) was the lowest (876.0 µg/g) compared to other CPs.

Molecular Weight Distribution of Crude Polysaccharides
Molecular weight (MW) was estimated using gel permeation chromatography (GPC) and the MW distribution is presented in Figure 1. The major and minor peaks of all CPs were detected around 11 and 20 min, respectively. Among the CPs, CPSH displayed the highest peak with a retention time around 11 min, followed by fucoidan, CPHF, CPAV, CPLJ, CPUP, CPPT, and CPCF, respectively. However, CPCF displayed the highest peak with a retention time of around 20 min, and CPPT and CPUP were detected around 19 and 21 min, respectively. Moreover, in CPAV, a different peak that was not present in the seaweed polysaccharide results was detected at around 12 min. The average polysaccharide molecular weight in the CPs was over 800 kDa, and some CPs were distributed in ranges of 1~10 kDa when compared to pullulan standard peaks. The molecular weight of fucoidan was also over 800 kDa. , and 800 kDa were used as standards; (B) fucoidan and the crude polysaccharides from seaweed and abalone viscera were analyzed by gel permeation chromatography (GPC) using an Ultrahydrogel 500 column. The mobile phase was water. An evaporative light scattering detector (ELSD) was used, the injection volume was 20 μL, and the flow rate was 0.5 mL/min at 45 °C.

Cytotoxicity Assay
The viability of HEK293/ACE2 cells was assessed using a Cell Titer-Glo Luminescent cell viability assay kit (Promega, Madison, WI, USA). When the HEK293/ACE2 cells were treated with each CP at the final concentration range from 1 ng/mL to 1 mg/mL (serial diluted 1/10) for 96 h, most of the CPs did not show cytotoxicity ( Figure 2). However, CPAV, CPCF, and CPPT were slightly cytotoxic at a concentration of 1 mg/mL. CPAV showed notable cytotoxicity when applied at a concentration of 1 mg/mL, following a degree of cytotoxicity by CPPT, CPCF, CPHF, CPLJ, CPUP, CPSH, and fucoidan. Nevertheless, the CC50 of all CPs was over 500 μg/mL. When the cells were treated with CP, the cell growth tended to increase temporarily at low concentrations of all CPs.

Inhibition of Viral Infection
The testing of the inhibition of viral infection by CPs was performed with a SARS-CoV-2 pseudovirus (COV-PS02; Creative Diagnostics, Shirley, NY, USA). As shown in Figure 2, all CPs, except CPPT, inhibited SARS-CoV-2 pseudovirus infection of HEK293/ACE2 cells at various concentrations. After viral exposure, the infection rate was determined by GFP (green fluorescent protein) fluorescence imaging, which is repre- Figure 1. Gel permeation chromatogram for 10 mg/mL of the crude polysaccharides from seaweeds and abalone viscera: (A) pullulan samples of molecular weight 1.3, 6, 12, 22, 50, 110, 200, 400, and 800 kDa were used as standards; (B) fucoidan and the crude polysaccharides from seaweed and abalone viscera were analyzed by gel permeation chromatography (GPC) using an Ultrahydrogel 500 column. The mobile phase was water. An evaporative light scattering detector (ELSD) was used, the injection volume was 20 µL, and the flow rate was 0.5 mL/min at 45 • C.

Cytotoxicity Assay
The viability of HEK293/ACE2 cells was assessed using a Cell Titer-Glo Luminescent cell viability assay kit (Promega, Madison, WI, USA). When the HEK293/ACE2 cells were treated with each CP at the final concentration range from 1 ng/mL to 1 mg/mL (serial diluted 1/10) for 96 h, most of the CPs did not show cytotoxicity ( Figure 2). However, CPAV, CPCF, and CPPT were slightly cytotoxic at a concentration of 1 mg/mL. CPAV showed notable cytotoxicity when applied at a concentration of 1 mg/mL, following a degree of cytotoxicity by CPPT, CPCF, CPHF, CPLJ, CPUP, CPSH, and fucoidan. Nevertheless, the CC50 of all CPs was over 500 µg/mL. When the cells were treated with CP, the cell growth tended to increase temporarily at low concentrations of all CPs.

Discussion
The SARS-CoV-2 virus can enter a host cell by the interaction of the COVID-19 spike glycoprotein and the ACE2 receptor or another receptor, neuropilin-1 (NRP1), of the host cell [23,24]. In previous studies, sulfated polysaccharides were reported to effectively inhibit SARS-CoV-2 entry by interfering with the interaction of the spike protein with the ACE2 receptor of the host cell in vitro [8,25,26]. Based on these studies, we chose various edible seaweeds (U. pinnatifida, L. japonica, H. fusiforme, S. horneri, C. fragile, and P. tenera) known to be high in sulfated polysaccharides and the viscera of the abalone, which eats such seaweeds, and extracted the crude polysaccharides after removing alginic acid with CaCl 2 . Seaweeds are known to produce different types of polysaccharides (laminaran, alginate, carrageenan, agar, porphyran, xylan, mannitol, fucoidan, etc.) and contain numerous components that can exert antioxidant, anti-inflammatory, and antiviral effects [7,10].
As SARS-CoV-2 is highly infectious and pathogenic, we used a SARS-CoV-2 pseudovirus (COV-PS02, Creative Diagnostics) to investigate whether fucoidan or the CPs of seaweeds and abalone viscera could inhibit host cell infiltration, through interaction with the COVID-19 spike glycoprotein and the ACE2 receptor of the host cell in vitro. Most of the CPs of seaweeds and CPAV effectively inhibited SARS-CoV-2 entry, like the previously reported sulfated polysaccharides [8,25,26]. None of the CPs showed toxicity below 1 mg/mL of CPs. Among the tested polysaccharides, CPSH showed the strongest antiviral activity with an IC50 of 12 µg/mL to prevent COVID-19 entry. CPAV and CPHF could also inhibit SARS-CoV-2 infection with an IC50 of 33 µg/mL and 47 µg/mL, respectively. These CPs (from S. horneri, abalone viscera, and H. fusiforme) have common properties such as high molecular weight (>800 kDa), high total carbohydrate (99, 63, and 94%, respectively), and higher fucose content (66, 37, and 42%, respectively) than other CPs. However, the sulfate ion content (9.8, 0.5, and 20.4%, respectively) of these CPs was somewhat lower than that of others (fucoidan and CPUP had over 30% sulfate ion content). These results indicate that the molecular weight, total polysaccharide content, and fucose content were important factors in the inhibitory effect of the CPs against SARS-CoV-2 host cell entry. According to previous studies [8,25], RPI-27, a high molecular weight (~100 kDa), branched polysaccharide from Saccharina japonica, was shown to have higher antiviral activity (EC50 = 8.3 µg/mL) than RPI-28 (EC50 = 16 µg/mL), which is a lower molecular weight (~12 kDa). Sulfated galactofucan (195.0 kDa) and glucuronomannan (7.0 kDa) strongly inhibited interaction between the SARS-CoV-2 S-glycoprotein and heparin (IC50 values are 27 and 231 nM, respectively).
Interestingly, according to our present results, CPAV also displayed high antiviral activity compared to the CPs from seaweeds. The CPAV was lower in total carbohydrate, sulfate ion, and fucose content than CPSH and CPHF, while the galactose and rhamnose contents were higher than the same two CPs. In gel permeation chromatography, a different peak in CPAV was detected at around 12 min compared to other CPs. Abalones are primarily fed various seaweeds including E. maxima, L. japonica, U. rigida, C. flaccida, G. gracilis, and U. lactuca for the ocean-rearing period [15][16][17][18] and these seaweeds are broken down by commensal marine microorganisms and digestive enzymes in viscera. In this present study, the abalone viscera came from 5-year-old abalone raised with L. japonica produced by Dashimachonbok Fisheries (Wando, Jeollanam-do, South Korea). The monosaccharide composition of CPAV was similar to CPLJ, but the CPAV displayed higher antiviral activity (IC50 = 33 µg/mL) than CPLJ (IC50 = 105 µg/mL). The reason is thought to be that the CPAV had higher total carbohydrate, fucose, and galactose content, and the amount of high molecular weight polysaccharides was higher than the CPLJ. Abalone viscera is composed of polysaccharides, peptides, and other bioactive molecules with various biological activities, including free radical scavenging, antioxidant, antibacterial, anti-tumor, and immunomodulatory activities [19,[27][28][29]. It is presumed that the synergistic effect of these bioactive compounds and high molecular weight polysaccharides resulted in the strong antiviral activity.

Seaweed Collection
The algae Laminaria japonica, Hizikia fusiforme, sporophyll of Undaria pinnatifida, Sargassum horneri, Porphyra tenera, and Codium fragile were cultured in Wando, Jeollanam-do, South Korea. Laminaria japonica, Hizikia fusiforme, and sporophyll of Undaria pinnatifida were collected in May 2020, and Porphyra tenera was collected in January 2020, and Sargassum horneri and Codium fragile were collected in August 2020. The fresh seaweed (20 kg) was immediately washed with tap water in order to remove salt, epiphytes, and sand attached to the surface of the samples and then dried. The fresh abalone (Haliotis discus hannai) viscera were purchased from Dashimachonbok (Wando, jeollanam-do, South Korea), washed, and dried by lyophilization. The dried seaweed and abalone viscera were crushed, ground into a powdered form, passed through a 100-mesh sieve, and then stored at −20 • C. Fucoidan was obtained from Haerim Fucoidan (Wando, Jeollanam-do, South Korea).

Preparation of the Crude Polysaccharides
Each dried powder (2 kg) of seaweed and abalone viscera was treated twice at 100 • C with 40 L of water for 6 h, and the hot solution (80 L) was separated from the algae residues by successive filtration through cotton wool. The extracts were concentrated to about 4 L under reduced pressure and then 2% CaCl 2 and 1.2 L of 85% (v/v) ethanol were added to eliminate the alginate. The extracts were centrifuged at 10,000 g and the supernatants were filtered through Whatman's filter papers (150 mm, 541). The filtered solution was dialyzed against distilled water and concentrated to about 1L using an ultrafiltration hollow fiber cartridge (10,000 NMWC, 31.8 L × 3.2 cm O.D., Cytiva, Marlborough, MA, USA). Finally, the CP was lyophilized and stored at −20 • C.

Total Carbohydrate Content
The total sugar content was analyzed using the phenol−sulfuric acid method [33]. D-glucose was used as the standard. Serially diluted standards were prepared at 20, 40, 80, 160, 320, and 640 µg/mL. Two hundred microliters of eluted CPs or eluted standards were transferred into 10 mL test tubes containing 0.5 mL of 5% phenol, followed by the addition of 2.5 mL of 18 M H 2 SO 4 . Each tube was mixed thoroughly and incubated for 30 min. The absorbance was measured at 480 nm with a Varioskan LUX (Thermo Fisher Scientific, Waltham, MA, USA). Serially diluted standards were calculated to obtain the standard curve.

Determination of Sulfate Contents
The sulfate content of CPs was determined by the BaCl 2 -gelatin turbidity method [21]. Gelatin solution (0.5%) was prepared in hot water (60~70 • C) and stored at 4 • C overnight. Barium chloride (2 g) was dissolved in the gelatin solution and allowed to stand for 2~3 h at 25 • C. To the CP solution (0.2 mL), 3.8 mL of 4% trichloroacetic acid was added, followed by 1 mL of the BaCl 2 -gelatin solution, and the absorbance was measured at 360 nm by UV spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) after incubation for 20 min at room temperature. A standard curve was prepared with a potassium sulfate solution between approximately 20 and 200 µg/mL.

Total Phenolic Content Analysis
The determination of total phenolics was carried out spectrophotometrically using Folin-Denis' reagent [22]. Briefly, 0.5 mL of CP or standard (gallic acid) were diluted in 0.5 mL of water. Thereafter, 0.5 mL of Folin-Denis' reagent was added and let stand for 5 min. Finally, 0.5 mL of saturated Na 2 CO 3 solution was added, and after 30 min the absorbance was measured at 700 nm with a Varioskan LUX (Thermo Fisher Scientific, Waltham, MA, USA). Gallic acid was used as the standard prepared in different concentrations ranging from 0 to 100 µg/mL.

BCA Assay
The CPs were prepared at 2 mg/mL Each sample (10 µL) was added to a 96-well plate in triplicate, and deionized water was added to bring the total volume to 20 µL. Blanks were prepared with 20 µL of deionized water in triplicate. Fresh BCA solution and 4% copper solution were prepared according to the manufacturer's instructions, and 180 µL was added to each well, which were mixed by shaking and then incubated at 37 • C for 30 min. A standard curve was made with bovine serum albumin (BSA; 0, 0.02, 0.04, 0.06, 0.08, and 0.1 mg/mL), and the absorbance was read at 562 nm (A 562nm ) with a Varioskan LUX (Thermo Fisher Scientific, Waltham, MA, USA).

Monosaccharide Analysis of Crude Polysaccharides
Each CP (0.1 g) was treated with 50 mL of 2 M trifluoroacetic acid at 110 • C for 8 h. After the hydrolysis of the polysaccharide, 1 mL of reaction medium was dried with a vacuum concentrator, and 450 µL of 0.3 M NaOH was added to resuspend the sample. The PMP derivatization of monosaccharides was carried out according to a previous method [34] with minor modifications. The hydrolyzed samples were mixed with 450 µL of 0.5 M PMP solution (in methanol). The mixtures were incubated at 70 • C for 30 min in a water bath, then cooled to room temperature and neutralized with 450 µL of 0.3 M HCl. Chloroform (1.0 mL each) was added and mixed, then the organic phase was carefully removed. The extraction process was repeated three times and the aqueous layer was passed through a 0.45 µm syringe filter before HPLC analysis. Standard solutions of the eight monosaccharides (arabinose, fucose, galactose, glucose, mannose, rhamnose, and xylose; 20 mM) were also treated as described above. The chromatographic analysis was performed on an LC-20AD HPLC system (Shimadzu, Kyoto, Japan) consisting of a binary pump (LC20AD XR; Shimadzu, Japan), an automatic injection pump (SIL-20AC XR Prominence Autosampler; Shimadzu, Japan), a degasser, a column oven controller, and a photodiode array detector (PDA; Shimadzu, Japan). The monosaccharides were separated on a reverse-phase Sunfire C 18 column (5 µm particle size, 250 × 4.6 mm I.D., Waters, Milford, MA, USA) coupled to a C 18 guard column (5 µm particle size, 15 × 4.6 mm I.D.). The injection volume was 20 µL with a flow rate of 1.0 mL/min at 25 • C. Mobile phase A was 40% acetonitrile with 0.05 M KH 2 PO 4 (pH 6.9) and mobile phase B was 15% acetonitrile with 0.05 M KH 2 PO 4 (pH 6.9); gradient elution was performed at 92-83-92% B at 0-10-40 min, respectively. Each sample was analyzed in duplicate.

Analysis of Minerals and Trace Elements
The crude polysaccharides were acid-digested using Sineo Microwave Digestion (MDS-8, Shanghai Sineo Microwave Chemistry Technology, Shanghai, China). Briefly, 0.500 g of each crude polysaccharide was digested with nitric acid (Supra Pure Metal 65%). Approximately 10 mL of nitric acid was added to a Teflon vessel and 0.500 g of each crude polysaccharide was allowed to predigest for 20 min before 4 mL of nitric acid and 1 mL of hydrogen peroxide (30%) were added. The pre-digested seaweed samples were capped. The Teflon vessels and their contents were subjected to microwave digestion, which was operated at four steps of temperature: 130 • C for 10 min, 150 • C for 5 min, 180 • C for 5 min, and a holding temperature of 200 • C for 10 min. At the end of the digestion, all seaweed samples were allowed to cool at room temperature. Then, the digests inside the vessels were quantitatively transferred into 50 mL polyethylene centrifuge vials.

Estimation of Average Molecular Weights
The molecular size distribution of the various CPs from seaweeds and abalone viscera was determined by gel permeation chromatography (GPC) using a LC-20AD HPLC system (Shimadzu, Kyoto, Japan) consisting of a binary pump (LC20AD XR; Shimadzu, Japan), an automatic injection pump (SIL-20AC XR Prominence Autosampler; Shimadzu, Japan), a degasser, a column oven controller, and an evaporative light scattering detector (ELSD; Shimadzu, Japan). An Ultrahydrogel 500 (300 × 7.8 mm) column (Waters Co., Miliford, MA, USA) in combination with a guard column (Ultrahydrogel, 6 × 40 mm, Waters, Milford, MA, USA) was used to maximize the resolution. Standard pullulans including 1300, 6000, 12,000, 22,000, 50,000, 110,000, 200,000, 400,000, and 800,000 Da were used as molecular mass markers. The injection volume was 20 µL and eluted with distilled water at 45 • C with a flow rate of 0.5 mL/min. Each sample was analyzed in duplicate.

Cytotoxicity Assay
Assays of the cytotoxicity of the crude polysaccharides were performed using a Cell Titer-Glo Luminescent cell viability assay kit. After the HEK293/ACE2 cells were treated with each 20 µL sample of crude polysaccharides at various concentrations (10-fold serial dilution in range of 1 ng/mL to 1 mg/mL, final D.W 5%) for 96 h, 4 µL/well of Cell titer Glo reagent was added and the vessel was shaken for 2 min at 700 rpm. After 10 min, the mixture was read with a luminometer and cell viability was calculated as follows: Cell Viability (%) = RLU sample RLU conc. × 100 (1) where the RLU sample is the luminescence of the experimental sample and RLU conc. is the luminescence of the control. Cytotoxicity was calculated as follows: The 50% cytotoxic concentration for crude polysaccharides was determined using nonlinear regression analysis with Graph-Pad Prism software (Graph-Pad, San Diego, CA, USA). Each sample was analyzed in duplicate wells and repeated three times.

Inhibition of Viral Infection
To investigate the inhibitory effect of the crude polysaccharides on viral infection, HEK293/ACE2 was seeded in 384 well plates at a concentration of 2 × 10 3 cells/well using DMEM and incubated at 37 • C, 5% CO 2 for 24 h. When HEK293/ACE2 cells had grown to a density of 30~40% in the 384-well plate, the cells were treated with a mixture of 20 µL of the particular crude polysaccharides (serial diluted 1/10 in a concentration range of 1 ng/mL to 1 mg/mL, final D.W 5%) and 1 µL of SARS-CoV-2 pseudovirus (titer: 1.0E + 07 TU/mL) to induce viral infection. After 96 h incubation, the viral infection rate was analyzed by scanning GFP fluorescence with an MBD ASFA scanner (MBD Biotech., Suwon, South Korea). To calculate the cell penetration inhibition of the SARS-CoV-2 pseudovirus, the GFP fluorescence area of infected cells was analyzed with an MBD cell analyzer and was calculated as follows: % inhibition = (GFP AREA sample) (GFP AREA conc.) × 100 (3) where the GFP AREA sample is the GFP area (µm 2 ) of the experimental sample and GFP AREA conc. is the GFP area (µm 2 ) of the control. Antiviral activity was calculated as follows: Antiviral activity (%) = 100 − % inhibition Each sample was analyzed in triplicate and the plots were made with Graph-Pad Prism software (Graph-Pad, San Diego, CA, USA).

Statistical Analysis
All experiments were performed in duplicate wells and repeated three times. The area of fluorescent cells infected with the virus was calculated using the MBD Cell Analyzer and the half maximal inhibitory concentrations (IC 50 ) were calculated using nonlinear regression analysis of GraphPad Prism 9 software by plotting log (inhibitor) versus normalized response (variable slope). The equation corresponds to: where Y is the response, X is the logarithm of doses or concentrations, and HillSlope describes the steepness of the family of curves.

Conclusions
Crude polysaccharides from seaweeds and abalone viscera were screened for antiviral activity against SARS-CoV-2, and most of them showed significant antiviral activity except for CPPT. They can be divided into three groups (strong, medium, and weak inhibition) according to their inhibitory activity. CPSH is alone in the strong inhibition group, CPAV, CPHF, and CPCF are in the medium inhibition group, and CPLJ, CPUP, and fucoidan (Haerim) are in the weak inhibition group, respectively. These bioactive polysaccharides from edible seaweeds and abalone viscera can be examined as therapeutic agents for inhibiting COVID-19 entry through further studies such as the purification and identification of bioactive compounds.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.