Use of fucoidan in preparation of medicine for resisting coronavirus in broad spectrum

Abstract

This invention discloses the application of fucoidan in the preparation of broad-spectrum anti-coronavirus drugs, belonging to the field of biomedical technology. Through model experiments with novel coronavirus viroids and porcine epidemic diarrhea virus, this invention demonstrates that fucoidan at concentrations above 69.52 μg / mL and above 372.59 μg / mL can inhibit viral infection, indicating that fucoidan has a broad-spectrum anti-coronavirus effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of fucoidan in the preparation of broad-spectrum anti-coronavirus drugs. Background Technology

[0002] In animals, coronavirus (CoV) infections can cause a variety of serious diseases, posing a significant threat to humans, livestock, and pets. Porcine epidemic diarrhea virus (PEDV) belongs to the class A coronaviruses and primarily infects the intestines of pigs, causing gastroenteritis and diarrhea; its mortality rate in piglets can be as high as 100%. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is characterized by fever as the main symptom, with common clinical manifestations including weakness in the limbs, nasal congestion, rapid breathing, sneezing, muscle and joint pain, nausea, and vomiting; severe cases can lead to death.

[0003] Fucoidan is a polysaccharide found in brown macroalgae and echinoderms (such as sea urchins and sea cucumbers). It contains sulfated L-fucose as the main monosaccharide, with other common monosaccharides including mannose, galactose, glucose, xylose, and glucuronic acid. It possesses a wide range of biological activities, such as antioxidant, anti-inflammatory, anticoagulant, immunomodulatory, and antiviral effects, and is therefore considered a candidate drug for health support and medical applications. The biological activity of fucoidan is closely related to its structure, which includes both the chemical structure of the polysaccharide and the spatial conformation of the polysaccharide chains in solution. As a sulfated esterified heteropolysaccharide, the chemical structure of fucoidan is mainly reflected in its monosaccharide composition, linkage mode, degree of sulfation, and esterification sites. The type and source of the seaweed, as well as the extraction method, have a significant impact on the chemical structure of fucoidan.

[0004] Studies have shown that fucoidan possesses significant anti-SARS-CoV-2 activity. Due to its structural similarity to heparin sulfate in mammalian mucous membranes, it can effectively inhibit viral infection by binding to the S protein of SARS-CoV-2. For example, patent CN111471088B discloses a composition for inhibiting SARS-CoV-2 infection, comprising the polypeptide VDP-4 and fucoidan. The composition achieves its inhibitory effect on viral invasion through the synergistic amplification of the inhibitory effects of VDP-4 and fucoidan on the binding of the SARS-CoV-2 to host cells, and is safe to use with no toxic side effects. Fucoidan is a unique water-soluble polysaccharide with sulfated groups, also known as fucoidan or fucoidan sulfate ester. Patent application CN111588732A discloses the application of fucoidan in the fight against the novel coronavirus. This invention confirms that fucoidan derived from brown algae can prevent the cell membrane of the body from adsorbing and internalizing the SARS-CoV-2 virus by binding to the S protein on the surface of the SARS-CoV-2 virus, thereby preventing the SARS-CoV-2 virus from infecting the body's cells.

[0005] However, to date, there are no studies on the broad-spectrum antiviral effects of fucoidan in coronaviruses. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides two fucoidan-derived broad-spectrum antiviral activities against coronaviruses. These coronaviruses include porcine epidemic diarrhea virus (PEDV) and the novel coronavirus SARS-CoV-2. The broad-spectrum antiviral activity refers to the prevention of coronaviruses, including: the fucoidan can directly interact with the positively charged regions on the viral envelope surface, thereby inhibiting the virus's ability to enter cells; it can also block the interaction between the virus and the receptor through the multi-anionic properties of the receptor, preventing receptor adhesion. This activity of fucoidan can be used to prepare drugs for the prevention of coronaviruses, prebiotics, etc.

[0007] The technical solution of the present invention is as follows:

[0008] The application of fucoidan in the preparation of a broad-spectrum anti-coronavirus drug, wherein the fucoidan is derived from Ascophyllum nodosum (named FUCA) or Undaria pinnatifida (named FUCU).

[0009] Preferably, the fucoidan is a sulfated polysaccharide in which L-fucose residues are linked by repeating a(1,3)- and a(1,4)- bonds.

[0010] Two fucoidan species were derived from *Fuca* and *Fucu*, belonging to the genera *Fuca* (Phaeophyta) and *Fucu* (Lycophyta), respectively. Both fucoidan species are sulfated polysaccharides composed of L-fucose residues linked by repeating α(1,3)- and α(1,4)- bonds, with fucose and sulfate groups as their main components. Furthermore, *Fucu* fucoidan contains galactose, while *Fuca* fucoidan contains galactose, mannose, and a small amount of glucuronic acid.

[0011] The coronaviruses mentioned are the novel coronavirus and porcine epidemic diarrhea virus.

[0012] The novel coronavirus mentioned is a SARS-CoV-2GFP / ΔN virus (GenBank Accession No. MN908947), which is a virus-like particle with transcription and replication capabilities.

[0013] The porcine epidemic diarrhea virus mentioned is rPEDV-GFP (GenBank Accession No. KU558701), which is a recombinant PEDV virus rescued using a reverse genetics operating system.

[0014] Specifically, when the fucoidan is derived from *Tetracentron sinense*, the preparation method of the fucoidan is as follows: *Tetracentron sinense* and water at a mass-to-volume ratio of 1:10 are boiled at 100°C for 2 hours to extract the fucoidan.

[0015] When the fucoidan is derived from wakame seaweed, the fucoidan is prepared by boiling wakame seaweed and water at a mass-to-volume ratio of 1:10 at 100°C for 2 hours to extract the fucoidan.

[0016] This study analyzed the chemical structures of FUCA and FUCU using a series of chemical and spectroscopic methods. These included detecting the composition of the two fucoidan polysaccharides using full-wavelength ultraviolet spectroscopy; analyzing the monosaccharide composition of fucoidan (c) and fucoidan (d) using pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP); and detecting the vibrational peaks of FUCA and FUCU using Fourier transform infrared spectroscopy (FTIR).

[0017] FUCA and FUCU were serially diluted and mixed with the virus. Cells were then infected with the polysaccharide-virus mixture. After 48 hours, the in vitro inhibition efficiency against two coronaviruses was measured, revealing that FUCA and FUCU exhibited good inhibitory ability against viral infection. The IC50 values ​​of FUCA and FUCU against the novel coronavirus were also observed. 50 The concentrations were 48.66 μg / mL and 69.52 μg / mL, respectively. The IC50 against porcine epidemic diarrhea virus (PEDV) was... 50 The values ​​were 287.32 μg / mL and 372.59 μg / mL, respectively.

[0018] The present invention also provides a broad-spectrum anti-coronavirus drug, the active ingredient of which is fucoidan, which is derived from Undaria pinnatifida or Laminaria japonica.

[0019] Specifically, the fucoidan is a sulfated polysaccharide composed of L-fucose residues linked by repeating a(1,3)- and a(1,4)- bonds.

[0020] The coronaviruses mentioned are the novel coronavirus and porcine epidemic diarrhea virus.

[0021] When the fucoidan is derived from *Tetracentron sinense*, the preparation method of the fucoidan is as follows: *Tetracentron sinense* and water at a mass-to-volume ratio of 1:10 are boiled at 100°C for 2 hours to extract the fucoidan.

[0022] When the fucoidan is derived from wakame seaweed, the fucoidan is prepared by boiling wakame seaweed and water at a mass-to-volume ratio of 1:10 at 100°C for 2 hours to extract the fucoidan.

[0023] The beneficial effects of this invention are:

[0024] This invention demonstrates through model experiments with novel coronavirus and porcine epidemic diarrhea virus that fucoidan at concentrations above 69.52 μg / mL and above 372.59 μg / mL, respectively, can inhibit viral infection, indicating that fucoidan has a broad-spectrum anti-coronavirus effect and can be used to prepare broad-spectrum anti-coronavirus drugs. Attached Figure Description

[0025] Figure 1 The ultraviolet spectra show the composition of fucoidan in FUCA(A) and FUCU(B).

[0026] Figure 2 This is the HPLC chromatogram of a monosaccharide standard.

[0027] Figure 3 The monosaccharide composition of FUCA(A) and FUCU(B) was analyzed using the PMP pre-column derivatization method.

[0028] Figure 4 FTIR spectroscopic analysis was performed on FUCA(A) and FUCU(B) samples.

[0029] Figure 5 To demonstrate the inhibitory effect of FUCA and FUCU on SARS-CoV-2 GFP / ΔN-like viruses. 100 TCID... 50 SARS-CoV-2 GFP / ΔN virus was mixed with serially diluted FUCA or FUCU (7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 μg / mL) and incubated with CaCo-2-N. intCells. Viral infection detection was performed using GFP-positive cell counting.

[0030] Figure 6 The inhibitory effects of FUCA and FUCU on PEDV were investigated. 100 TCID... 50 PEDV was mixed with serially diluted FUCA or FUCU (7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 μg / mL) and incubated in Vero cells. Viral infection was detected by counting GFP-positive cells. Detailed Implementation

[0031] Example 1: Preparation of two types of fucoidan

[0032] Powdered *Anoectochilus buergerianum* or *Undaria pinnatifida* was boiled with water at 100°C at a solid-liquid ratio of 1:10 for 2 hours. This process was repeated twice. The extracts from both boilings were then concentrated, and 30% (v / v) calcium chloride was added to the concentrated filtrate to remove alginate. The resulting precipitate was filtered through silica fume, and the resulting solution was dialyzed through a 1 μm membrane and further separated using a 10 kDa ultrafiltration membrane. Finally, the concentrated extract was spray-dried to obtain fucoidan (FUCA) from *Anoectochilus buergerianum* and fucoidan (FUCU) from *Undaria pinnatifida*, respectively.

[0033] Example 2: Structural characteristics of two fucoidan species

[0034] 1. The content of fucoidan prepared in Example 1 was analyzed based on ultraviolet spectroscopy and standard curve.

[0035] Results: The UV full-wavelength scan spectra of FUCU and FUCA are as follows Figure 1 A and Figure 1 As shown in Figure B, both samples exhibited good polysaccharide terminal absorption peaks near 200 nm; in addition, neither sample showed an absorption peak at 260 nm, indicating that neither polysaccharide contained nucleic acids.

[0036] HPLC chromatogram of monosaccharide standard as follows Figure 2 As shown, the HPLC chromatogram of FUCA is as follows: Figure 3 As shown in Figure A, the HPLC chromatogram of FUCU is as follows: Figure 3 As shown in B. The proportions of each monosaccharide residue were calculated, and the results are shown in Table 1. The main monosaccharides contained in FUCA are fucose, galactose, mannose, and a small amount of glucuronate (GlcA), with a relative molar mass ratio of 56.9:7.4:1.5:1.0, while the molar ratio of Fuc:Gal:Man:GlcA in FUCU is 92.7:33.1:1.5:1.0.

[0037] Table 1

[0038]

[0039] The content of uronic acid, total sugar, and sulfate was analyzed using a standard curve. It was found that the total sugar and sulfate content of FUCA were lower than those of FUCU (Table 1). The content of FUCA was 5.3 ± 0.1%, while the content of FUCU was less than 1.0%. The average molecular weights of FUCA and FUCU were 124.3 kDa and 141.7 kDa, respectively.

[0040] 2. Structural characteristics of fucoidan based on infrared spectroscopy analysis

[0041] Results: Infrared spectra of FUCA and FUCU are as follows Figure 4 As shown in A and 4B, at 3447.48cm -1 and 3502.91cm -1 The broad and strong vibrational peaks observed at this point correspond to the OH tensile vibrations of FUCA and FUCU, respectively. 2941.48 cm -1 and 2942.88cm -1 The weak peak at 1733.08 cm⁻¹ is due to the stretching vibration of the CH single bond, which is the methyl absorption peak of fucose. FUCU is at 1733.08 cm⁻¹. -1 The peak at 1636.95 cm⁻¹ indicates the presence of acetyl groups. Furthermore, the peak at 1636.95 cm⁻¹... -1 and 1652.26cm -1 The absorption at this point is due to the asymmetric stretching vibration of the COO- group of the aldehyde acid. 1262.94 cm⁻¹ -1 and 1261.18cm -1 The strong absorption peak at this point is related to the stretching vibration of sulfate S=O, which is consistent with the sulfate content of FUCA and FUCU determined above (Table 1). 1028.77 cm⁻¹ -1 and 1056.05cm -1 The absorption peaks at these locations are related to the COC bonds in the sugar rings of FUCA and FUCU, respectively. Furthermore, in FUCA, the peak at 845.99 cm⁻¹ is also relevant. -1 The absorption peak at 834.24 cm⁻¹ is related to C4-OS, indicating that the sulfate group may exist at the C4 position in this fucoidan. Conversely, FUCU shows an absorption peak at 834.24 cm⁻¹. -1 The peaks at these positions indicate the presence of sulfate at positions C2 and C4.

[0042] Example 3: Effects of two fucoidan species on SARS-CoV-2 virus-like viruses

[0043] Different dilutions (7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 μg / mL) of FUCA or FUCU in DMEM containing 2% FBS were mixed with 100 TCID. 50 Equal volumes (1:1) of SARS-CoV-2 GFP / ΔN virus-like particles (SARS-CoV-2 virus-like particles with replication and transcription functions, established by Professor Ding Qiang's laboratory at Tsinghua University, GenBank Accession No. MN908947) were mixed. At 37°C and 5% CO2, 100 μL of the virus / sample suspension was added to 5 × 10⁻⁶ ppm of the solution. 3 Caco-2-N int The virus was incubated in human-derived Caco-2 cells (stably expressing the SARS-CoV-2N protein, prepared using lentiviral transduction, Ju X, Zhu Y, Wang Y, et al. A novel cell culture system modeling the SARS-CoV-2 lifecycle. Cold Spring Harbor Laboratory, 2020. DOI: 10.1101 / 2020.12.13.422469.) with 100 μL of virus diluted in DMEM containing 2% FBS as a control. After 48 h of incubation, images were observed using a fluorescence microscope to determine the inhibition efficiency.

[0044] Results: FUCA inhibited 50% of SARS-CoV-2 GFP / ΔN virus infection at 48.66 μg / mL, and FUCU inhibited 50% of SARS-CoV-2 GFP / ΔN virus infection at 69.52 μg / mL. Figure 5 This indicates that FUCA and FUCU possess good anti-COVID-19 properties.

[0045] Example 4: Effects of two fucoidan species on PEDV

[0046] FUCA or FUCU were diluted with DMEM serum-free medium (7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 μg / mL), and 100 TCID45 containing 5 μg / mL trypsin was diluted with MMT. 50Porcine epidemic diarrhea virus ZJU / G2 / 2013 (PEDV-GFP, a strain that produces GFP protein by transfecting Vero cells with infectious cDNA of PEDV, GenBank Accession No. KU558701, preserved in our laboratory) was mixed with equal volumes (1:1) of FUCA or FUCU at various dilution gradients. Under conditions of 37°C and 5% CO2, 100 μL of the virus / sample suspension was added to 5 × 10⁻⁶ ppm of the solution. 3 In Vero cells (ATCC, CCL-81), the virus was diluted with 100 μL MMT as a control. After incubation for 48 h, images were observed using a fluorescence microscope to determine the inhibition efficiency.

[0047] The results showed that FUCA inhibited 50% of PEDV-GFP infection at 287.32 μg / mL, and FUCU inhibited 50% of PEDV-GFP infection at 372.59 μg / mL. Figure 6 This indicates that FUCA and FUCU possess good antiviral properties against porcine epidemic diarrhea virus.

Claims

1. Use of fucoidan derived from Ascophyllum nodosum ( Ascophyllum nodosum ) or Undaria pinnatifida ( Undaria pinnatifida ) in the preparation of a medicine for resisting porcine epidemic diarrhea virus. The fucoidan is a sulfated polysaccharide composed of L-fucose residues linked by repeating α(1,3)- and α(1,4)- bonds; The preparation method of the fucoidan is as follows: powdered *Anoectochilus buergerianum* or *Undaria pinnatifida* is boiled with water at 100°C at a solid-liquid ratio of 1:10 for 2 hours. This process is repeated twice. The extracts from both boilings are then concentrated. Calcium chloride (30% by volume) is added to the concentrated filtrate to remove alginate. The resulting precipitate is filtered through silica. The resulting solution is dialyzed using a 1µm membrane and further separated using a 10kDa ultrafiltration membrane. Finally, the concentrated product is spray-dried to obtain fucoidan derived from *Anoectochilus buergerianum* and fucoidan derived from *Undaria pinnatifida*, respectively.

2. A drug for treating porcine epidemic diarrhea virus, characterized in that, The active ingredient is fucoidan, which is derived from Undaria pinnatifida or Laminaria japonica. The fucoidan is a sulfated polysaccharide composed of L-fucose residues linked by repeating α(1,3)- and α(1,4)- bonds; The preparation method of the fucoidan is as follows: powdered *Anoectochilus buergerianum* or *Undaria pinnatifida* is boiled with water at 100°C at a solid-liquid ratio of 1:10 for 2 hours. This process is repeated twice. The extracts from both boilings are then concentrated. Calcium chloride (30% by volume) is added to the concentrated filtrate to remove alginate. The resulting precipitate is filtered through silica. The resulting solution is dialyzed using a 1µm membrane and further separated using a 10kDa ultrafiltration membrane. Finally, the concentrated product is spray-dried to obtain fucoidan derived from *Anoectochilus buergerianum* and fucoidan derived from *Undaria pinnatifida*, respectively.

Images