Inhibitory Activities of Marine Sulfated Polysaccharides Against

Food &
View Article Online

View Journal
Function
Linking the chemistry and physics of food with health and nutrition
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: S. Song, H. Peng,
Q. Wang, Z. Liu, X. Dong, C. Wen, C. Ai, Y. Zhang, Z. Wang and B. Zhu, Food Funct., 2020, DOI:
10.1039/D0FO02017F.
Volume 9
This is an Accepted Manuscript, which has been through the
Food &
Number 1
January 2018
Pages 1-658
Royal Society of Chemistry peer review process and has been
accepted for publication.
Function
Linking the chemistry and physics of food with health and nutrition Accepted Manuscripts are published online shortly after acceptance,
rsc.li/food-function
before technical editing, formatting and proof reading. Using this free
service, authors can make their results available to the community, in
citable form, before we publish the edited article. We will replace this
Accepted Manuscript with the edited and formatted Advance Article as
soon as it is available.
You can find more information about Accepted Manuscripts in the

Information for Authors.
Please note that technical editing may introduce minor changes to the
text and/or graphics, which may alter content. The journal’s standard
ISSN 2042-650X Terms & Conditions and the Ethical guidelines still apply. In no event
PAPER
Xian Wu, Hang Xiao et al.
A metabolite of nobiletin, 4' -demethylnobiletin and
shall the Royal Society of Chemistry be held responsible for any errors
atorvastatin synergistically inhibits human colon cancer cell
growth by inducing G0/G1 cell cycle arrest and apoptosis
or omissions in this Accepted Manuscript or any consequences arising
from the use of any information it contains.
rsc.li/food-function
Page 1 of 17 Food & Function
View Article Online
Inhibitory activities of marine sulfated polysaccharides against DOI: 10.1039/D0FO02017F
SARS-CoV-2
Shuang Song1, Haoran Peng2, Qingling Wang3, Zhengqi Liu1,4, Xiuping Dong1,
Chengrong Wen1, Chunqing Ai1, Yujiao Zhang1, Zhongfu Wang5, Beiwei Zhu1,*
Food & Function Accepted Manuscript

1National Engineering Research Center of Seafood, School of Food Science and Technology,
Published on 24 August 2020. Downloaded on 8/26/2020 3:11:39 AM.
Collaborative Innovation Center of Seafood Deep Processing, Dalian Polytechnic University,
Dalian 116034, China
2 Department of Biomedical Defense, Faculty of Naval Medicine, Naval Medical University
(Second Military Medical University), Shanghai 200433, China
3 CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology,
Chinese Academy of Sciences, Beijing 100101, China
4 Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food
Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, China
5 Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education
and Provincial Key Laboratory of Biotechnology, College of Life Sciences, Northwest University,
Xi’an 710069, China

Food & Function Page 2 of 17
View Article Online

Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory
DOI: 10.1039/D0FO02017F
syndrome coronavirus 2 (SARS-CoV-2) has spread around the world at an

unprecedented rate. In the present study, 4 marine sulfated polysaccharides were
screened for their inhibitory activity against SARS-CoV-2, including sea cucumber
sulfated polysaccharide (SCSP), fucoidan from brown algae, iota-carrageenan from
red algae, and chondroitin sulfate C from shark (CS). Of them, SCSP, fucoidan, and

carrageenan showed significant antiviral activities at concentrations of 3.90~500
μg/mL. SCSP exhibited the strongest inhibitory activity with IC50 of 9.10 μg/mL.
Furthermore, a test using pseudotype virus with S glycoprotein confirmed that SCSP
could bind to the S glycoprotein to prevent SARS-CoV-2 host cell entry. The three
antiviral polysaccharides could be employed to treat and prevent COVID-19.
View Article Online
1. Introduction DOI: 10.1039/D0FO02017F
COVID-19 is caused by severe acute respiratory syndrome associated coronavirus-2

(SARS-CoV-2). This infectious disease has spread to most countries in the world
since the first report in December 2019. By now, it has infected over 15 million
people and killed more than 638,000. The ongoing COVID-19 pandemic has become

a major threat to public health and social stability. Effective therapies to treat or
prevent COVID-19 are urgently needed.
Some natural sulfated polysaccharides have already been reported for their
potent inhibitory effects on virus infection. Depolymerized fucosylated chondroitin
sulfate extracted from sea cucumber showed in vitro activity against human
immunodeficiency virus type-11. Chondroitin sulfate E from squid cartilage has been
considered as a potent antiviral agent against T-cell leukaemia virus type I2 and the
flavivirus dengue3. Iota-carrageenan has been reported to inhibit the infection of some
human viral pathogens, e.g. papilloma virus4 and influenza A virus5. It has been
suggested that the sulfated polysaccharide could bind to virus envelope protein so to
inhibit virus entry into the host cell3.
Like other pathogenic enveloped viruses, SARS-CoV-2 uses unique envelope
glycoprotein, the spike (S) glycoprotein, for host cell receptor recognition and
binding, and subsequent viral and host cell membrane fusion6. Thus, interacting with
the S glycoprotein could stop virus entry into the host cell. It is exciting to know that
heparin, a highly sulfated polysaccharide, could bind tightly to the SARS-CoV-2
spike glycoprotein7, and the administration of heparin is associated with lower
mortality in patients with COVID-198. However, the medication safety of heparin is a
concern because of its strong anticoagulant activity9.
In order to find safe and effective antiviral agent to fight COVID-19, 4 marine
sulfated polysaccharides were screened for their inhibitory activities against SARS-
CoV-2 in the present study, including sea cucumber sulfated polysaccharide (SCSP),
fucoidan from brown algae, iota-carrageenan from red algae, and chondroitin sulfate
C from shark (CS). SCSP was extracted from Stichopus japonicus which is a kind of
View Article Online

sea cucumber consumed in Asian countries for thousands of years, and it has been
DOI: 10.1039/D0FO02017F
proved to be beneficial for health in vivo10, 11. Fucoidan from brown algae and CS
have been used in medicines and nutritional supplements for their healthy functions12,
13. Carrageenan is in worldwide commercial use as an additive for foods. Thus, these
polysaccharides are generally considered as safe for oral administration. In the present
study, we investigated their inhibitory activities against SARS-CoV-2, and found

SCSP, fucoidan and carrageenan as promising inhibitors of SARS-CoV-2 infection.
2. Materials and methods

2.1. Materials
Sea cucumber sulfated polysaccharide (SCSP) was extracted from Stichopus japonicus
as we previously reported11. Fucoidan, iota-carrageenan and chondroitin sulfate C
sodium (CS) were bought from Qingdao Brightmoon Seaweed Group Co., Ltd
(Qingdao, China), Aladdinn Industrial Corporation (Shanghai, China), and ChromaDex,
Inc (Irvine, California, USA). Standard monosaccharides including L-fucose (Fuc), D-
glucuronic acid (GlcA), D-galacturonic acid (GalA), D-galactose (Gal), D-glucose
(Glc), L-arabinose (Ara), D-ribose (Rib), D-rhamnose (Rha), D-mannose (Man), and D
-xylose (Xyl) were purchased from Sigma-Aldrich (MO, USA).
2.2 Characterization of polysaccharides
The uronic acid content was determined through the carbazole-sulfuric acid method
with GlcA as a standard. The sulfate content was measured by the barium chloride-
gelatine method using anhydrous potassium sulfate as a standard.
Monosaccharide compositions of these polysaccharides were analyzed according
to our previously reported method14. Briefly, polysaccharides were hydrolyzed at
120 °C with 2 M trifluoroacetic acid for 3 h, and the free monosaccharides were labeled
with 1-phenyl-3-methyl-5-pyrazolone (PMP). Then the PMP derivatives were detected
by a LXQ linear ion trap mass spectrometer equipped with an electrospray ion source
(ESI) and a photodiode array detector (PAD). A Silgreen ODS C18 (250 × 4.6 mm, 5
View Article Online

μm) was applied and kept at 30 °C, and the mobile phase was 20 mM ammonium
DOI: 10.1039/D0FO02017F
acetate-acetonitrile (83:17, v/v) with a flow rate of 1.0 mL/min.
2.3 Virus neutralization assay
The SARS-CoV-2 strain was isolated from COVID-19 patients in Shanghai, China.
Vero E6 cells were cultured in DMEM media supplemented with 10% FBS and 1%

antibiotic in saturated humidity at 37 °C with 5% CO2. SARS-CoV-2 neutralization
assay was carried out in biological safety protection third-level laboratory of Naval
Medical University. Briefly, Vero E6 cells (8 000～10 000 cells /well) were seeded in
96-well plates and incubated for 12 h. Then 300 FFU (focus forming unit) virus in 100
μL DMEM medium containing 2% FBS was incubated with polysaccharides for 30 min
at 37 °C. After that, the mixture replaced the medium in the well to infect Vero E6 cells.
After incubation for 24 h, the virus protein expression was measured by indirect
immunofluorescent assay (IFA). The serum from COVID-19 patient in recovery stage
was used as the primary antibody, and Alexa Fluor® 488-conjugated anti-human IgG
(ThermoFisher, USA) was the secondary antibody. After fluorescent antibody reactions,
DAPI was used to stain the nuclear DNA of live cells. Images of the cells were captured
using a Cytation 5 Imaging Reader (BioTek, USA).
2.4 Pseudovirus preparation and neutralization assay
SARS-CoV-2 pseudovirus with S glycoprotein was prepared and neutralized as

previously described with minor modification15. Briefly, the full-length coding
sequence of HCoV-19 spike was cloned into the pCAGGS vector and named as
pCAGGS-HCoV-19-S. The construct was verified by direct DNA sequencing. Then
the plasmids of pCAGGS-HCoV-19-S and pNL4-3.luc.RE were co-transfected into
HEK 293T cells. After 48 h culture, the supernatant was harvested and centrifuged to
obtain pseudovirus. The 50% tissue culture infectious dose (TCID50) was determined
by infection of Huh7 cells.
For pseudovirus neutralization assay, 100 TCID50 /well pseudovirus in medium
without FBS was incubated with polysaccharides for 30 min at room temperature. Then
View Article Online

the mixtures (100 μL) were used to infect Huh7 cells rinsed with PBS. After 5DOI:h,10.1039/D0FO02017F
100
μL medium with 5% FBS was added. EK1 peptide was used as the positive control.
After incubation for another 48 h, luciferase activity was measured using a GloMax 96
Microplate luminometer (Promega, USA).
3. Results and discussion

3.1 Characterization of polysaccharides
Because the bioactivities are greatly dependent on structural features, the

compositions of polysaccharides were determined and provided in Table 1. Compared

to the monosaccharides in their well-known main structures11, 16-18 (Fig. 1), some
other monosaccharides were also detected. SCSP are mainly comprised of fucosylated
chondroitin sulfate (Fig. 1A) and fucoidan (Fig. 1B)11. Fucoidans from sea cucumber
and from brown algae have the same backbone (Fig. 1B), but some fucoidans from
brown algae contain a large proportion of Gal16. Among these polysaccharides, SCSP
has the highest sulfate content (25.8%), followed by fucoidan (22.8%) and CS
(20.4%). Compared with those reported11, 16, the chemical compositions of these
polysaccharides varied a little possibly due to changes of isolation procedures or
materials.
3.2 Inhibitory effects of polysaccharides against SARS-CoV-2
The activity of the 4 polysaccharides against SARS-CoV-2 was screened. As shown in

Fig. 2, SCSP, fucoidan and carrageenan inhibited SARS-CoV-2 infection on Vero E6
cells at various concentrations. The representative images of immunofluorescent assay
are shown in Fig. 3. Among these polysaccharides, SCSP showed the strongest antiviral
activity with IC50 of 9.10 μg/mL. Fucoidan could also inhibit SARS-CoV-2 infection
at a concentration as low as 15.6 μg/mL. Iota-carrageenan could prevent the infection
at concentrations ≥ 125 μg/mL, and virus aggregation could be observed in the samples
treated with carrageenan (Fig. 3) possibly due to its gelling property. But no obvious
inhibitory effect was observed for CS, and this is consistent with the recent report that
View Article Online

CS shows no competitive binding to S protein19. In addition, the effects ofDOI:
these 4
10.1039/D0FO02017F
polysaccharides on the cell viability were also evaluated, and none of them
demonstrated cytotoxicity (Fig. 4).
SCSP with the highest sulfation degree showed the best inhibitory effect, followed
by fucoidan from seaweed. Kwon et al recently reported that fucoidans from the
seaweed Saccharina japonica had great antiviral activity against SARS-CoV-219. Then

SCSP could be a better candidate drug against SARS-CoV-2 for its more potent activity.
Considering the poor oral bioavailability of polysaccharides, it is better to deliver these
bioactive polysaccharides by a nasal spray or inhaler. Because fucoidan and iota-
carrageenan are economical, it is also promising to apply them in foods and daily
chemical products against SARS-CoV-2. Of note, the antiviral activities of these
polysaccharides were not positively associated with the sulfate content because highly
sulfated CS showed no obvious activity. According to the previous research on the
interaction of virus envelope protein with heparin, besides a high charge density, a high
level of structural flexibility was also required for the polysaccharides to bind to S
glycoprotein20. Thus, the inhibition activity against SARS-CoV-2 of the
polysaccharides may be also influenced by their structural flexibility.
3.3 Inhibitory effect of SCSP against pseudotype virus with S glycoprotein
It has been revealed that before SARS-CoV-2 host cell entry, S glycoprotein will bind
to heparan sulfate (HS) chains of host cell surface proteoglycans21. Therefore, S
glycoprotein is the most possible target for the sulfated polysaccharides with structures
like HS. Thus, pseudotype virus with S glycoprotein of SARS-CoV-2 was used to
evaluate the antiviral activity of SCSP, and the result was shown in Fig. 5. SCSP
demonstrated significant inhibitory effect against pseudotype virus at 100 and 1000
μg/mL. This result confirmed that SCSP interacted with the S glycoprotein of SARS-
CoV-2 to inhibit SARS-CoV-2 infection. A well-known sulfated polysaccharide,
heparin has also been reported to prevent SARS-CoV-2 infection by binding to the S
glycoprotein of SARS-CoV-27.
View Article Online
4. Conclusion DOI: 10.1039/D0FO02017F
In summary, 4 sulfated polysaccharides from marine organisms were screened for

the activities against SARS-CoV-2, and 3 of them showed significant antiviral activities,
including SCSP from sea cucumber, fucoidan from brown algae, and iota-carrageenan.
SCSP demonstrated the strongest inhibitory activities. Further experiment using

pseudotype virus with S glycoprotein indicates that SCSP binds to the S glycoprotein
to prevent SARS-CoV-2 host cell entry. Fucoidan and iota-carrageenan are also
promising in application because they are economical and safe. The three bioactive
polysaccharides deserve further efforts for application in the fight against COVID-19.
Conflicts of interest
The authors have no potential financial conflicts of interest.
Acknowledgements
This work was funded by the National Key Research and Development Program
of China (No. 2019YFD0902005) and National Natural Science Foundation of China
(No. 31972084).
References
1. N. Huang, M. Y. Wu, C. B. Zheng, L. Zhu, J. H. Zhao and Y. T. Zheng, The depolymerized
fucosylated chondroitin sulfate from sea cucumber potently inhibits HIV replication via
interfering with virus entry, Carbohydrate Research, 2013, 380, 64-69.
2. A. Jinno-Oue, A. Tanaka, N. Shimizu, T. Mori, N. Sugiura, K. Kimata, H. Isomura and H.
Hoshino, Inhibitory effect of chondroitin sulfate type E on the binding step of human T-cell
leukemia virus type 1, AIDS Research and Human Retroviruses, 2013, 29, 621-629.
3. D. Kato, S. Era, I. Watanabe, M. Arihara, N. Sugiura, K. Kimata, Y. Suzuki, K. Morita, K. I.
Hidari and T. Suzuki, Antiviral activity of chondroitin sulphate E targeting dengue virus
envelope protein, Antiviral Research, 2010, 88, 236-243.
4. C. B. Buck, C. D. Thompson, J. N. Roberts, M. Müller, D. R. Lowy and J. T. Schiller,
Carrageenan is a potent inhibitor of papillomavirus infection, PLoS Pathog, 2006, 2, e69.
5. A. Leibbrandt, C. Meier, M. König-Schuster, R. Weinmüllner, D. Kalthoff, B. Pflugfelder, P.
View Article Online

Graf, B. Frank-Gehrke, M. Beer and T. Fazekas, Iota-carrageenan is a potent inhibitorDOI:
of 10.1039/D0FO02017F
influenza A virus infection, PloS One, 2010, 5, e14320.
6. A. Sternberg and C. Naujokat, Structural features of coronavirus SARS-CoV-2 spike protein:
Targets for vaccination, Life Sciences, 2020, 118056.
7. S. Y. Kim, W. Jin, A. Sood, D. W. Montgomery, O. C. Grant, M. M. Fuster, L. Fu, J. S.
Dordick, R. J. Woods and F. Zhang, Characterization of heparin and severe acute respiratory
syndrome-related coronavirus 2 (SARS-CoV-2) spike glycoprotein binding interactions,
Antiviral Research, 2020, 104873.
8. L. Ayerbe, C. Risco and S. Ayis, The association between treatment with heparin and survival

in patients with Covid-19, Journal of Thrombosis and Thrombolysis, 2020, 1-4.
9. E. Dunne, M. O'Halloran, E. Porter, J. Bonello, L. Farrugia, C. V. Sammut and P. Schembri-
Wismayer, Heparin as an anticoagulant for the dielectric measurement of blood, IEEE
Transactions on Dielectrics and Electrical Insulation, 2019, 26, 229-234.
10. Z. Z. Zhu, B. W. Zhu, Y. J. Sun, C. Q. Ai, L. L. Wang, C. R. Wen, J. F. Yang, S. Song and X.
L. Liu, Sulfated polysaccharide from sea cucumber and its depolymerized derivative prevent
obesity in association with modification of gut microbiota in high‐fat diet‐fed mice, Molecular
Nutrition & Food Research, 2018, 62, 1800446.
11. Z. Z. Zhu, B. W. Zhu, Y. J. Sun, C. Q. Ai, S. F. Wu, L. L. Wang, S. Song and X. L. Liu,
Sulfated polysaccharide from sea cucumber modulates the gut microbiota and its metabolites
in normal mice, International Journal of Biological Macromolecules, 2018, 120, 502-512.
12. R. V. Usoltseva, S. D. Anastyuk, I. A. Ishina, V. V. Isakov, T. N. Zvyagintseva, P. D. Thinh,
P. A. Zadorozhny, P. S. Dmitrenok and S. P. Ermakova, Structural characteristics and
anticancer activity in vitro of fucoidan from brown alga Padina boryana, Carbohydrate
Polymers, 2018, 184, 260-268.
13. G. Jiao, G. Yu, J. Zhang and H. S. Ewart, Chemical structures and bioactivities of sulfated
polysaccharides from marine algae, Marine Drugs, 2011, 9, 196-223.
14. H. X. Wang, J. Zhao, D. M. Li, C. R. Wen, H. M. Liu, S. Song and B. Zhu, Comparison of
polysaccharides of Haliotis discus hannai and Volutharpa ampullacea perryi by PMP-HPLC-
MSn analysis upon acid hydrolysis, Carbohydrate Research, 2015, 415, 48-53.
15. H. Sun, Y. Li, P. Liu, C. Qiao, X. Wang, L. Wu, K. Liu, Y. Hu, C. Su and S. Tan, Structural
basis of HCoV-19 fusion core and an effective inhibition peptide against virus entry, Emerging
Microbes & Infections, 2020, 1-19.
16. J. Gao, L. Z. Lin, Z. J. Chen, Y. J. Cai, C. Q. Xiao, F. B. Zhou, B. G. Sun and M. Zhao, In
vitro digestion and fermentation of three polysaccharide fractions from Laminaria japonica
and their impact on lipid metabolism-associated human gut microbiota, Journal of Agricultural
and Food Chemistry, 2019, 67, 7496-7505.
17. T. Mikami, H. Kitagawa. Biosynthesis and function of chondroitin sulfate, Biochimica et
Biophysica Acta, 2013, 1830, 4719-4733.
18. J. Necas, L. Bartosikova. Carrageenan: a review. Veterinrn Medicna, 2013, 58, 187-205.
19. P. S. Kwon, H. Oh , S. Kwon, W. H. Jin, F. M. Zhang, K. Fraser, J. J. Hong. Sulfated
polysaccharides effectively inhibit SARS-CoV-2 in vitro. Cell Discovery, 2020, 6, 50.
20. R. M. Marks, H. Lu, R. Sundaresan, T. Toida, A. Suzuki, T. Imanari, M. J. Hernáiz , R. J.
Linhardt. Probing the interaction of dengue virus envelope protein with heparin: assessment of
glycosaminoglycan-derived inhibitors. Journal of Medicinal Chemistry, 2001, 44, 2178-2187.
View Article Online

21. J. F. W. Chan, K. H. Kok, Z. Zhu, H. Chu, K. K. W. To, S. Yuan and K. Y. Yuen, Genomic
DOI: 10.1039/D0FO02017F
characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with
atypical pneumonia after visiting Wuhan, Emerging microbes & Infections, 2020, 9, 221-236.

View Article Online

Table 1. The chemical compositions of sea cucumber sulfated polysaccharide DOI:
(SCSP),
10.1039/D0FO02017F
fucoidan, iota-carrageenan, and chondroitin sulfate C (CS).

Polysaccharides Monosaccharide molar ratio Sulfate content Uronic acid content
% %
SCSP GlcN:GalN:Glc:Gal:Fuc= 25.8±2.4 16.5±0.5
1.0:1.7:1.1:1.8:16.0
Fucoidan Rha:Gal:Fuc= 1.0:12.6:6.3 22.8±0.7 4.7±0.4

Carrageenan Glc:Gal=1.0:5.2 10.4±0.5 0.9±0.1
CS GlcN:GalN:Glc:Gal= 20.4±0.3 42.7±4.2

3.3:6.7:1.0:5.2
View Article Online

DOI: 10.1039/D0FO02017F
Figure Captions
Fig. 1 Chemical structures of fucosylated chondroitin sulfate (A), fucoidan (B), iota-
carrageenan (C), and chondroitin sulfate C (D).
Fig. 2 Inhibitory activities of sea cucumber sulfated polysaccharide (SCSP), fucoidan,

iota-carrageenan, and chondroitin sulfate C (CS) against SARS-CoV-2.
Fig. 3 Representative images of immunofluorescent assay of sea cucumber sulfated

polysaccharide (SCSP), fucoidan, carrageenan and chondroitin sulfate C (CS) against
SARS-CoV-2. Indirect immunofluorescent assay with the antibody against SARS-

CoV-2 nucleocapsid protein (in green) and DAPI staining for the DNA of the live
Vero E6 cells (in blue) were performed.
Fig. 4 The effects of sea cucumber sulfated polysaccharide (SCSP), fucoidan,

carrageenan and chondroitin sulfate C (CS) on the viability of Vero E6 cells.
Fig. 5 Inhibitory activities of sea cucumber sulfated polysaccharide (SCSP) against

pseudotype virus with the spike glycoprotein.
Page 13 of 17
Fig. 1
Food & Function
DOI: 10.1039/D0FO02017F
View Article Online

Fig. 2
Food & Function
DOI: 10.1039/D0FO02017F
View Article Online

Page 14 of 17
Page 15 of 17
Fig. 3
Food & Function
DOI: 10.1039/D0FO02017F
View Article Online

Fig. 4
Food & Function
DOI: 10.1039/D0FO02017F
View Article Online

Page 16 of 17
Page 17 of 17
Fig. 5
Food & Function
DOI: 10.1039/D0FO02017F
View Article Online

Inhibitory Activities of Marine Sulfated Polysaccharides Against

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Inhibitory Activities of Marine Sulfated Polysaccharides Against

Uploaded by

Copyright:

Available Formats

Food &

View Article Online

You can find more information about Accepted Manuscripts in the

View Article Online