This research was conducted to develop effective and safe marine-derived antiviral compounds against norovirus. The ethyl acetate (EtOAc)-extract from
Eisenia bicyclis
exhibited strong antiviral activity against murine norovirus (MNV) as a norovirus surrogate. Among the phlorotannins from
E. bicyclis
, dieckol (DE) and phlorofucofuroeckol-A (PFF) were known to possess the strongest antibacterial activity. In this study, DE and PFF were evaluated for antiviral activity against MNV. DE and PFF exhibited strong anti-MNV activity with 50% effective concentration (EC
50
) of 0.9 μM. However, PFF exhibited more effective antiviral activity against MNV with higher selective index (668.87) than that of DE (550.60), due to its lower cell toxicity against RAW 264.7. This is the first report on the anti-MNV activity of phlorotannins from seaweed. The results obtained in this study suggest that the phlorotannins could be used as a potential source of natural antiviral agents.
INTRODUCTION
Norovirus, a genus in the family Caliciviridae, is a group of non-enveloped viruses that have a single-stranded, positive sense RNA genome (
Atmar 2010
). Norovirus infection can usually be caused by contaminated water or food, and it spreads via human contact with infected materials through the fecal-oral route (
Choi et al. 2014
). This infection has been recognized as a leading cause of epidemics with the symptoms of vomiting, diarrhea, mild fever, abdominal cramping, and nausea in the community (
La Rosa et al. 2013
). According to the
Centers for Disease Control and Prevention (2011)
, over 70% of water-related gastroenteritis patients and over 50% of patients due to food-consumption resulted from norovirus infection in USA. Likewise, norovirus causative issues have been a great concern over the world during the recent years.
Norovirus has the characteristics such as low infectious dose, prolonged shedding period, strong stability, great diversity, and frequent genome mutations (
Lee et al. 2014
). Norovirus could be effectively reduced by using disinfectants such as alcohols and povidone idodines (
Belliot et al. 2008
). However, chemical sanitizers can cause various side effects in humans, such as fever and itching (
Choi et al. 2014
). Recently, attempts have been made to find sustainable solutions from medicinal plants and marine organisms (
Balunas and Kinghorn 2005
,
Jain et al. 2008
,
Choi et al. 2014
). Among marine organisms, chitosan and chitosan oligosaccharides were reported to be able to effectively reduce the infectivity of human enteric viral surrogates (feline calicivirus [FCV] F-9) (
Su et al. 2009
,
Davis et al. 2012
). However, there are limitations in the progress of this study, due to inefficient cell culture amplification process for norovirus (
Guix et al. 2007
,
Lay et al. 2010
). Recent studies revealed that the structure and genetic relatedness of murine norovirus (MNV) to human norovirus makes this virus a promising and relevant surrogate for studying the environmental survivability of human norovirus (
Cannon et al. 2006
,
Zhang et al. 2012
).
In this study, we investigated the possibility of using the marine alga
Eisenia bicyclis
extract and its ingredients as an alternative antiviral agent against MNV. The brown algae have also been reported to exhibit several antimicrobial activities against pathogenic bacteria and FCV (
Eom et al. 2013
,
Choi et al. 2014
,
Lee et al. 2014
). Since inadequate scientific research findings are available on the antiviral activity from marine organisms against MNV, the present study with MNV as a norovirus surrogate may have great contribution to the development of effective antiviral agents to control human norovirus.
MATERIALS AND METHODS
In the late September of 2013,
E. bicyclis
was purchased from Ulleung Trading Co. (Ulleung, Korea). A voucher specimen was refrigerated at -80°C. Dried
E. bicyclis
was triturated into powder with electronic grinder (HMF-1000A; Hanil Electronics, Seoul, Korea). The dried
E. bicyclis
powder (1.0 kg) was extracted with methanol (MeOH; 10 L × 3) at 68°C for 3 h (3 times), and the solvent was evaporated using rotary evaporator (Eyela Co., Tokyo, Japan) under vacuum at 45°C. The combined crude MeOH extract (164.3 g) was suspended in 10% MeOH (1.0 L), and then it was fractionated in turn with
n
-hexane (Hexane; 1.0 L × 3), dichloromethane (DCM; 1.0 L × 3), ethyl acetate (EtOAc; 1.0 L × 3), and
n
-butanol (BuOH; 1.0 L × 3) solution, to yield Hexane-soluble extract (42.3 g), DCM-soluble extract (2.5 g), EtOAc-soluble extract (23.8 g), BuOH-soluble extract (26.5 g), and H
2
O-soluble extract (69.1 g).
Phlorofucofuroeckol-A (PFF)
Fig. 1A
and dieckol (DE)
Fig. 1B
were isolated from the EtOAc-soluble extract of
E. bicyclis
by using Sephadex LH-20 and RP-18 open column chromatography, as a part of previous research, along with several other phloroglucinol derivatives (
Eom et al. 2013
) (
Fig. 1
).
Structures of phlorofucofuroeckol-A (A) and dieckol (B) isolated from Eisenia bicyclis.
RAW 264.7 cells, obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL, Grand Island, NY, USA) with 1% penicillin (100 U mL
-1
; Gibco BRL)-streptomycin (100 μg mL
-1
; Gibco BRL) and heat inactivated 10% fetal bovine serum (Gibco BRL) at 37°C in 5% CO
2
atmosphere. MNV-1 was obtained from Dr. Gwang Pyo Ko at Seoul National University (Seoul, Korea) with the permission of Dr. Herbert W. Virgin at the Washington University School of Medicine (St. Louis, MO, USA); and it was propagated in RAW 264.7 cells. RAW 264.7 cells were harvested three times by using freezethawing method. The virus stock was stored at -80°C for further use.
The cytotoxicity of
E. bicycilis
extracts was determined by quantifying RAW 264.7 cells viability using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. The medium containing RAW 264.7 cells were cultured into a 96-well plate at a density of 3 × 10
5
cells mL
-1
. The 50% cytotoxicity concentration (CC
50
) value was examined following 24 h of exposure of RAW 264.7 cell monolayers to different concentrations of extracts. For control, equal volume of medium was added and incubated at 37°C in 5% CO
2
atmosphere. Formazan solubilizing solution was added after incubation. Absorbance of each well was measured at 540 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). The optical density of formazan formed by untreated cells was taken as 100% of viability.
The antiviral activity of
E. bicyclis
extracts against MNV was evaluated by tissue culture infectivity dose 50% (TCID
50
), according to the method used by
Kim et al. (2010)
. The antiviral effective concentration was expressed as 50% effective concentration (EC
50
) value, which is defined as the concentration of the sample required to exhibit only 50% of cytopathogenic effects caused by the virus (
Goldman and Prabhakar 1996
,
Goodfellow et al. 2005
,
Choi et al. 2014
). Concentration of 5 log TCID
50
mL
-1
MNV suspensions was treated with identical volumes of serially diluted extracts at room temperature for 24 h. The mixtures were added to a monolayer of RAW 264.7 cells in a 96 well plate. After incubating at 37°C for 24 h, the cells were fixed with formaldehyde and stained with crystal violet solution to be monitored for cytopathic effects. Untreated control cells were suspended with maintenance medium (DMEM; Gibco BRL), instead of extracts or phlorotannins. The relative effectiveness for inhibiting viral infection inducing cell death is defined as the selectivity index (SI; CC
50
EC
50
-1
).
Data were represented as the means for three replicate samples and expressed as the mean ± standard deviation. A student’s t-test and one-way ANOVA were used by SPSS ver. 12.0 (SPSS Inc., Chicago, IL, USA). p < 0.05 was considered statistically significant. The CC
50
and EC
50
were calculated by regression analysis for the dose-response curves.
RESULTS AND DISCUSSION
In recent years, norovirus has gained a lot of attention concerning the management of food safety, since 50% of all outbreaks related to food-poising were caused by norovirus across the nations (
Centers for Disease Control and Prevention 2011
). Although various biological activities of marine organism have been reported, there are still many unknown potentials that could be utilized. We previously reported that
E. bicyclis
methanolic extract exhibit antiviral activity against FCV (
Choi et al. 2014
). However, as described above, FCV may not be the most suitable surrogate for evaluating the efficacy of disinfectants against human norovirus (
Cannon et al. 2006
,
Zhang et al. 2012
). Therefore, the antiviral activity of
E. bicyclis
extracts against MNV as a norovirus surrogate was evaluated. Cytotoxicity of
E. bicyclis
extracts was evaluated by determining CC
50
value using RAW 264.7 cells. It has been proposed the following cutoff points to determine a cytotoxicity of plant extracts toward normal cells: 1) significant or strong cytotoxicity: <100 μg mL
-1
; 2) moderate cytotoxicity: 100 μg mL
-1
to <300 μg mL
-1
; 3) low cytotoxicity: 300 μg mL
-1
to <1,000 μg mL
-1
; 4) no cytotoxicity: <1,000 μg mL
-1
(
Kuete and Efferth 2015
). Thus, the MeOH extract and its solvent-soluble extracts showed no severe cytotoxicity towards RAW 264.7 cells with the CC
50
values of 322.48 to 2,146.42 μg mL
-1
(
Table 1
). The cytotoxicity of
E. bicyclis
methanolic extract against RAW 264.7 cells was higher than that of our previous report using CrFK cells (CC
50
= 410) (
Choi et al. 2014
). This result could be due to the difference in toxic susceptibility of host cells to the methanolic extract. Antiviral activity of
E. bicyclis
extracts, inhibiting viral infection of host cells, was evaluated by measuring EC
50
value as described in MATERIALS AND METHODS.
E. bicyclis
methanolic extract showed an antiviral activity against MNV in RAW 264.7 cells with EC
50
value of 42.34 μg mL
-1
(
Table 1
). It has been reported that
E. bicyclis
methanolic extract exhibit EC
50
value of 80 μg mL
-1
against FCV (
Choi et al. 2014
). Then, the antiviral activity of the methanolic extract was evaluated by SI analysis, as described above. It is desirable to have a high SI giving maximum antiviral activity, with minimal cell toxicity as well as higher possibility of application (
Oh et al. 2013
). The SI value of
E. bicyclis
methanolic extract against MNV is 22.27, while SI value of the same extract against FCV is 5.13 (
Choi et al. 2014
). Thus,
E. bicyclis
methanolic extract exhibits higher inhibiting effectiveness against MNV infection in host cells, compared to that of FCV. In addition, the SI value is similar to that of green tea (
Camelia sinensis
) methanolic extract with SI value of 18.57 against FCV (
Oh et al. 2013
).
In vitroantiviral activities of the methanolic extract and its solvent-soluble extracts fromEisenia bicyclisagainst murine norovirus in RAW 264.7 cells using the mixed treatment assay
aCC50: mean (50%) value of cytotoxic concentration. bEC50: mean (50%) value of effective concentration. cSI: selective index, CC50 EC50-1. dMeOH, methanolic extract; Hexane, n-hexane-soluble extract; DCM, dichloromethane-soluble extract; EtOAc, ethyl acetate-soluble extract; BuOH, n-butanol-soluble extract; H2O, water-soluble extract. Means (±standard error of mean) within the same row having different letters are significantly different (p < 0.05).
To elucidate an active substance(s) responsible for the antiviral activity against MNV, the methanolic extract was further partitioned as Hexane, DCM, EtOAc, BuOH, and H
2
O soluble extracts. The antiviral activity of solvent-soluble extracts was also evaluated by the SI analysis. Accordingly, EtOAc extract showed the highest SI value, followed by H
2
O, BuOH, DCM, and Hexane soluble extract (
Table 1
). These results clearly showed that EtOAc extract has strong antiviral activity with low cytotoxicity. Thus, the extract will be a good choice for the search of novel antiviral compounds against MNV.
Several phlorotannins, phloroglucinol derivatives, from the EtOAc-soluble extract of
E. bicyclis
were previously isolated. Of these, DE and PFF exhibited the strongest antibacterial activity against pathogenic microbes such as
Staphylococcus aureus
and
Propionibacterium acnes
(
Eom et al. 2013
,
Lee et al. 2014
). The structural characteristics of DE and PFF have been reported earlier (
Eom et al. 2013
) (
Fig. 1
). Therefore, in the present study, the antiviral activity of these compounds against MNV in RAW 264.7 cells was demonstrated, as described above. The CC
50
values of DE and PFF were calculated to be 495.71 μM and 579.02 μM, respectively (
Table 2
). DE and PFF inhibited the infection of MNV in RAW 264.7 cells with an EC
50
value of 0.90 μM. Consequently, the calculated SI values of DE and PFF were 550.60 and 668.87, respectively, which is suggesting that PFF can more effectively inhibit MNV infection in host cells compared to that of DE (
Table 2
). Compared to epigallocatechin gallate (a catechin from green tea) with SI value of 26.67 against FCV (
Oh et al. 2013
), the present study showed SI values of DE and PFF against MNV were about 20 and 25 times higher, respectively, than that of epigallocatechin gallate. This finding clearly highlights the existence of potential antiviral compounds against MNV in edible brown alga
E. bicyclis
.
In vitroantiviral activities of isolated compounds from the EtOAc-soluble extracts ofEisenia bicyclisagainst murine norovirus in RAW 264.7 cells using the mixed treatment assay
aCC50: mean (50%) value of cytotoxic concentration. bEC50: mean (50%) value of effective concentration. cSI: selectivity index, CC50 EC50-1.
Recent studies have revealed the antiviral effect of plant-derived compounds against MNV (
Li et al. 2013
). However, to our knowledge, there has been no previous report on marine-derived polyphenols exhibiting antiviral activity against MNV.
Choi et al. (2014)
reported that
E. bicyclis
methanolic extract possess an anti-norovirus activity which inhibits FCV infection in CrFK cells with EC
50
value at the concentration of 80 μg, but MNV belongs to the human norovirus unlike FCV. In addition, MNV is demonstrated as enteric pathogens of mice and humans, respectively, whereas FCV infects via nasal, oral, and conjunctival routes (
Radford et al. 2007
,
Howell and D’Souza 2013
). MNV has been increasingly used as a surrogate for human norovirus in virucidal efficacy evaluations (
Howell and D’Souza 2013
). Our results provide evidence that the phlorotannins (DE and PFF) could provide a way to develop effective marine-derived antiviral agents against human norovirus. However, further antiviral experiments should be performed to delineate how phlorotannins deactivate MNV and the mechanism of action. Generally, although it is not fully understood, the antiviral action mechanism of natural compounds against human norovirus or its surrogates is usually supposed to be the prevention of the viral attachment to host cells (
Howell and D’Souza 2013
).
CONCLUSION
The EtOAc-soluble extract from
E. bicyclis
exhibited the strongest antiviral activity against MNV among five solvent-soluble extracts. The antiviral activity of EtOAc-soluble extract against MNV may also be correlated with their marine-derived polyphenolic components. Therefore, previously isolated phlorotannins, such as DE and PFF, were evaluated for antiviral activity against MNV. DE and PFF demonstrated strong anti-MNV activity with low cytotoxicity against RAW 264.7 cells. The results of the present investigation are expected to contribute to the development of an alternative phytotherapeutic agent against MNV, as natural disinfectant for the improvement in food safety.
Acknowledgements
This research was a part of the project titled ‘Development of manufactured goods and oyster,Crassostrea gigasfree from norovirus’, funded by the Ministry of Oceans and Fisheries, Korea. Graduate students were supported by the special fund of Pukyong National University donated by the SKS Trading Co. in Lynnwood, WA, USA in memory of the late Mr. Young Hwan Kang, who had inspiration and a deep concern for fishery science.
Belliot G.
,
Lavaux A.
,
Souihel D.
,
Agnello D.
,
Pothier P.
2008
Use of murine norovirus as a surrogate to evaluate resistance of human norovirus to disinfectants
Appl. Environ. Microbiol.
74
3315 -
3318
DOI : 10.1128/AEM.02148-07
Cannon J. L.
,
Papafragkou E.
,
Park G. W.
,
Osborne J.
,
Jaykus L. A.
,
Vinjé J.
2006
Surrogates for the study of norovirus stability and inactivation in the environment: a comparison of murine norovirus and feline calicivirus
J. Food Prot.
69
2761 -
2765
2011
Updated norovirus outbreak management and disease prevention guidelines
MMWR Recomm. Rep.
Centers for Disease Control and Prevention
60
1 -
18
Choi Y.
,
Kim E.
,
Moon S.
,
Choi J. -D.
,
Lee M. -S.
,
Kim Y. -M.
2014
Phaeophyta extracts exhibit antiviral activity against feline calicivirus
Fish. Aquat. Sci.
17
155 -
158
Davis R.
,
Zivanovic S.
,
D’Souza D. H.
,
Davidson P. M.
2012
Effectiveness of chitosan on the inactivation of enteric viral surrogates
Food Microbiol.
32
57 -
62
DOI : 10.1016/j.fm.2012.04.008
Eom S. -H.
,
Lee D. -S.
,
Kang Y. M.
,
Son K. -T.
,
Jeon Y. -J.
,
Kim Y. -M.
2013
Application of yeast Candida utilis to ferment Eisenia bicyclis for enhanced antibacterial effect
Appl. Biochem. Biotechnol.
171
569 -
582
DOI : 10.1007/s12010-013-0288-x
Eom S. -H.
,
Lee M. -S.
,
Lee E. -W.
,
Kim Y. -M.
,
Kim T. H.
2013
Pancreatic lipase inhibitory activity of phlorotannins isolated from Eisenia bicyclis
Phytother. Res.
27
148 -
151
DOI : 10.1002/ptr.4694
Goldman A. S.
,
Prabhakar B. S.
1996
Immunology overview in medical microbiology
University of Texas Medical Branch at Galveston
Galveston, TX
1 -
29
Goodfellow I. G.
,
Evans D. J.
,
Blom A. M.
,
Kerrigan D.
,
Miners J. S.
,
Morgan B. P.
,
Spiller O. B.
2005
Inhibition of coxsackie B virus infection by soluble forms of its receptors: binding affinities, altered particle formation, and competition with cellular receptors
J. Virol.
79
12016 -
12024
DOI : 10.1128/JVI.79.18.12016-12024.2005
Guix S.
,
Asanaka M.
,
Katayama K.
,
Crawford S. E.
,
Neill F. H.
,
Atmar R. L.
,
Estes M. K.
2007
Norwalk virus RNA is infectious in mammalian cells
J. Virol.
81
12238 -
12248
DOI : 10.1128/JVI.01489-07
Howell A. B.
,
D’Souza D. H.
2013
The pomegranate: effects on bacteria and viruses that influence human health
Evid. Based. Complement. Alternat. Med.
2013
606212 -
Jain R.
,
Sonawane S.
,
Mandrekar N.
2008
Marine organisms: Potential source for drug discovery
Curr. Sci.
94
292 -
Kim K. -L.
,
Lee D. -S.
,
Park M. -S.
,
Eom S. -H.
,
Lim K. -S.
,
Kim J. -S.
,
Lee D. -H.
,
Kang C. -K.
,
Kim Y. -M.
,
Lee M. -S.
2010
Antiviral activity of seaweed extracts against feline calicivirus
Fish. Aquat. Sci.
13
96 -
101
Kuete V.
,
Efferth T.
2015
African flora has the potential to fight multidrug resistance of cancer
Biomed. Res. Int.
2015
914813 -
La Rosa G.
,
Fratini M.
,
Libera S. D.
,
Iaconelli M.
,
Muscillo M.
2013
Viral infections acquired indoors through airborne, droplet or contact transmission
Ann. Ist. Super. Sanità.
49
124 -
132
Lay M. K.
,
Atmar R. L.
,
Guix S.
,
Bharadwaj U.
,
He H.
,
Neill F. H.
,
Sastry K. J.
,
Yao Q.
,
Estes M. K.
2010
Norwalk virus does not replicate in human macrophages or dendritic cells derived from the peripheral blood of susceptible humans
Virology
406
1 -
11
DOI : 10.1016/j.virol.2010.07.001
Lee J. -H.
,
Eom S. -H.
,
Lee E. -H.
,
Jung Y. -J.
,
Kim H. -J.
,
Jo M. -R.
,
Son K. -T.
,
Lee H. -J.
,
Kim J. H.
,
Lee M. -S.
,
Kim Y. -M.
2014
In vitro antibacterial and synergistic effect of phlorotannins isolated from edible brown seaweed Eisenia bicyclis against acne-related bacteria
Algae
29
47 -
55
DOI : 10.4490/algae.2014.29.1.047
Lee S. -G.
,
Cho H. -G.
,
Paik S. -Y.
2014
Molecular epidemiology of norovirus in South Korea
BMB Rep.
48
61 -
67
Li D.
,
Baert L.
,
Uyttendaele M.
2013
Inactivation of food-borne viruses using natural biochemical substances
Food Microbiol.
35
1 -
9
DOI : 10.1016/j.fm.2013.02.009
Oh E. -G.
,
Kim K. -L.
,
Shin S. B.
,
Son K. -T.
,
Lee H. -J.
,
Kim T. H.
,
Kim Y -M.
,
Cho E. -J.
,
Kim D. -K.
,
Lee E. -W.
,
Lee M. -S.
,
Shin I. -S.
,
Kim J. H.
2013
Antiviral activity of green tea catechins against feline calicivirus as a surrogate for norovirus
Food Sci. Biotechnol.
22
593 -
598
DOI : 10.1007/s10068-013-0119-4
Radford A. D.
,
Coyne K. P.
,
Dawson S.
,
Porter C. J.
,
Gaskell R. M.
2007
Feline calicivirus
Vet. Res.
38
319 -
335
DOI : 10.1051/vetres:2006056
Su X.
,
Zivanovic S.
,
D’Souza D. H.
2009
Effect of chitosan on the infectivity of murine norovirus, feline calicivirus, and bacteriophage MS2
J. Food Prot.
72
2623 -
2628
Zhang X. -F.
,
Dai Y. -C.
,
Zhong W.
,
Tan M.
,
Lv Z. -P.
,
Zhou Y. -C
,
Jiang X.
2012
Tannic acid inhibited norovirus binding to HBGA receptors, a study of 50 Chinese medicinal herbs
Bioorg. Med. Chem.
20
1616 -
1623
DOI : 10.1016/j.bmc.2011.11.040