Synergistic Effects of Sulfated Polysaccharides from Mexican Seaweeds against Measles Virus

Hindawi Publishing CorporationBioMed Research InternationalArticle ID 8502123

Research ArticleSynergistic Effects of Sulfated Polysaccharides from MexicanSeaweeds against Measles Virus

Karla Moraacuten-Santibantildeez1 Lucia Elizabeth Cruz-Suaacuterez2 Denis Ricque-Marie2 DanielRobledo3 Yolanda Freile-Pelegriacuten3 Mario A Pentildea-Hernaacutendez1 Cristina Rodriacuteguez-Padilla1 and Laura M Trejo-Avila1

1Laboratorio de Inmunologıa y Virologıa Facultad de Ciencias Biologicas Universidad Autonoma de Nuevo Leon CiudadUniversitaria CP 66455 San Nicolas de los Garza NL Mexico2Programa Maricultura Facultad de Ciencias Biologicas Universidad Autonoma de Nuevo Leon Ciudad Universitaria CP 66455San Nicolas de los Garza NL Mexico3Cinvestav Unidad Merida Km 6 Carretera Antigua a Progreso Cordemex AP 73 97310 Merida YUC Mexico

Correspondence should be addressed to Laura M Trejo-Avila lauratrejohotmailcom

Received 18 March 2016 Revised 11 May 2016 Accepted 16 May 2016

Academic Editor Ibrahim M Banat

Copyright copy Karla Moran-Santibanez et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Sulfated polysaccharides (SPs) extracted from five seaweed samples collected or cultivated in Mexico (Macrocystis pyrifera Eiseniaarborea Pelvetia compressa Ulva intestinalis and Solieria filiformis) were tested in this study in order to evaluate their effect onmeasles virus in vitro All polysaccharides showed antiviral activity (as measured by the reduction of syncytia formation) and lowcytotoxicity (MTT assay) at inhibitory concentrations SPs from Eisenia arborea and Solieria filiformis showed the highest antiviralactivities (confirmed by qPCR) and were selected to determine their combined effect Their synergistic effect was observed at lowconcentrations (00274 120583gmL and 0011120583gmL of E arborea and S filiformis SPs resp) which exhibited by far a higher inhibitoryeffect (96 syncytia reduction) in comparison to the individual SP effects (50 inhibition with 0275120583gmL and 0985 120583gmL of Earborea and S filiformis resp) Time of addition experiments and viral penetration assays suggest that best activities of these SPsoccur at different stages of infection The synergistic effect would allow reducing the treatment dose and toxicity and minimizingor delaying the induction of antiviral resistance sulfated polysaccharides of the tested seaweed species thus appear as promisingcandidates for the development of natural antiviral agents

1 Introduction

LatinAmerica has an important and diverse group of seaweedspecies [1] Recent data on seaweed management in thisregion have described the main harvest and aquaculturetaking place in Argentina Brazil Chile Peru andMexico [2]One of the goals of seaweeds exploitation is to diversify theirapplication by screening their diverse bioactive compoundswhich remain unexplored in nutraceutical and pharmaceu-tical areas [3] Different chemical compounds have beenisolated from algae including polysaccharides which havebeen subjected to a variety of studies due to their extensivebioactivities and applications [4]

An increasing number of biological activities of seaweedpolysaccharides have been reported in the last decadeswhere sulfated polysaccharides (SPs) are among the moststudied compounds [5] SPs include a complex group ofmacromolecules with numerous activities such as antioxidant[6 7] antitumor [8 9] anticoagulant [6] anti-inflammatory[6 10] and antiviral [11 12]

Antiviral activity of SPs was first reported in 1958 [13] andover the years a substantial research has been focused on thisfield SP can be obtained from each of the three main classesof seaweed fucoidans and alginates from brown algal speciesagaroids and carrageenans from red macroalgae and ulvansfrom green seaweeds [14]

2 BioMed Research International

0

20

40

60

80

100

120

Untreated 1 5

sy

ncyt

ia co

unt o

r vira

l RN

A co

pies

Concentration (120583gmL)

Syncytia reduction assayqPCR assay

(a)

0

20

40

60

80

100

120

Untreated 1 5

sy

ncyt

ia co

unt o

r vira

l RN

A co

pies



(b)

Figure 1 Confirmation of antiviral activity of Eisenia arborea (a) and Solieria filiformis (b) SPs at their best inhibitory concentrations bysyncytia reduction and qPCR assays Syncytia count and viral RNA copies number are given in of the untreated control values

2000 1800 1600 1400 1200 1000 800 600

1210ndash1260 804846929

D

C

B

A

1639

(cmminus1)

(a)

2000 1800 1600 1400 1200 1000 800 600

C

B

124414101627

1039ndash1041

A

(cmminus1)

(b)

Figure 2 (a) Infrared spectra of (A) Solieria filiformis aqueous extract (B) 120580-carrageenan (C) 120581- carrageenan and (D) 120582-carrageenan (b)Infrared spectra of (A) Eisenia arborea aqueous extract (B) fucoidan and (C) alginic acid

Fucoidans have shown a potent antiviral activity againstnumerous enveloped viruses including herpes simplex virustype 1 (HSV-1) [15] human immunodeficiency virus [16]influenza A virus [17] and different kind of paramyxovirusessuch as Newcastle disease virus (NDV) and canine distempervirus (CDV) [18 19] In vitro and in vivo antiretroviral effectsof alginates preventing syncytium formation and reducingthe P24 core antigen level have been demonstrated [20]Antiviral activity of carrageenans has been demonstrated invitro against human papillomavirus (HPV) acting mainly onthe inhibition ofHPV virions binding to cells and also in vivoby preventing infection by different HPV genotypes [21 22]Recently antiviral activity against NDV of ulvan from Ulvaclathrata cultivated in Mexico has been reported [23]

Nowadays combining multiple drugs is a primaryapproach for improving antiviral effects within the antiviraldrug therapy field The advantages of multidrugs combina-tion are the reduction of individual drugs doses a decrease inthe side effects of antiviral agents and the prevention of drug-resistant viruses emergence Drug combination theories pro-vide an ideal tool for this purpose to understand the benefitsof multidrugs combinations therapy [24]

Measles virus (MeV) belongs to the Paramyxoviridaefamily of Mononegavirales is a nonsegmented negative-strand RNA virus and causes a highly contagious disease[25] Although preventable by vaccination measles stillremains one of the causes of death among young childrenworldwide [26] Many new antiviral drugs have been licensed

BioMed Research International 3

7

1

4

3

12

6

59

2

11

10 8

O O OO

O

OH

OH 1

234

6

5

78

10

9

12

11

TSP-d4

OSO3minus

HOCH2CH3

250

240

230

220

210

200

190

180

170

160

150

140

130

120

110

100

90 80 70

60

50

40

30 20 10 0

minus10

minus20

105 100 95 90 85 80 75 70 65

(ppm)

O3minusSO

10466

9451

8084

8025

7948

7927

7743

7727

7451

7233

7219

7167

6379

(a)

[MMM][GMM]

Carbonyl signalof uronic acids

250

240

230

220

210

200

190

180

170

160

150

140

130

120

110

100

90 80 70

60

50

40

30 20 10 0

minus10

minus20

(ppm)

TSP-d4

18783

17768

10293

10281

8082

7890

7407

7279

7251

6604

4683

(b)

Figure 3 (a) Spectrum and expansion 13C-NMR of the aqueous extract of S filiformis (b) 13C-NMR spectrum of the aqueous extract of Earborea


Table 1 Cytotoxic effect antiviral activity and selectivity index ofSPs

Algaea CC50(120583gmL)b IC

50(120583gmL)c SId

Macrocystis pyrifera gt1500 100 gt1500Eisenia arborea gt1500 0275 gt545454Pelvetia compressa gt1500 100 gt1500Ulva intestinalis gt1500 36 gt4167Solieria filiformis gt1500 0985 gt152284aAlgal sulfated polysaccharide extract bConcentration of test compound(120583gmL) that reduced Vero cell viability by 50 cConcentration of a testcompound that reduced the number of MeV syncytia in Vero cells by 50dSelectivity index value

in recent years most of which are used for the treatment ofHIV infections [27] The investigation of natural antiviralsisolated frommarine sources is an interesting approach in thedevelopment of new antiviral agents In the present study wetested the antiviral activity of SPs isolated from five Mexicanseaweeds againstMeVThe aimof this researchwas to developnew candidates of antiviral drugs that could help to controlviral infection diseases

2 Materials and Methods

21 Antiviral Agents

211 Collection of Seaweed Five species of macroalgae werecollected from the Mexican coasts and tested for this studythree brown seaweeds from Baja California (Macrocystispyrifera Eisenia arborea and Pelvetia compressa) one greenseaweed from Southern Baja California (Ulva intestinalis)and one red seaweed from Yucatan (Solieria filiformis)

Macrocystis pyrifera (Linnaeus) C Agardhwas collectedin Bahıa de Ensenada (Manto Jantay) in front of the Sal-sipuedes beach (31983ndash116815) in January 2013 Eiseniaarborea J E Areschougand Pelvetia compressa (J Agardh)De Toni were collected in the Escalera Zone North of PuntaChina (31520ndash116650) in December 2014-January 2015 Thegreen algaUlva intestinalis (Linnaeus) was collected from thewater drainage channel of the Gran Mar shrimp farm on theBaja California West coast (24434ndash111584) in August 2014

Solieria filiformis (Kutzing) P W Gabrielson a red sea-weed considered as a potential source of 120580-carrageenan [28]was obtained from an aquaculture facility at the TelchacMarine station-CINVESTAV Yucatan (Mexico) where it isperiodically cultivated in bimonthly cycles in semiopen tanksas part of an IntegratedMultitrophic aquaculture systemThesample used came from a batch cultured from April to May2014

Once harvested the brown and green algae samples werewashed in seawater to eliminate sand shells and epibiontsand dried under shade while the cultivated red algae waswashedwith freshwater and dried in an oven at 60∘C Prior toextraction the samples were cut into small 2-3 cm pieces andground to pass through a 05mm sieve (Turbomolino Pulvex200 mill)

212 Extraction and Purification of Sulfated PolysaccharidesPolysaccharides extraction was performed after extraction ofpolyphenols [29] Briefly 10 g of alga powder was washedwithdistillated water and dried at room temperature overnightThe washed powder was extracted with 200mL 50 vvethanol and sonicated for 30min at room temperaturefollowed with an extraction period in a bath shaker at70∘C during 2 hours The samples were centrifuged for15min (2500 rpm)The pellet was used for the polysaccharideextraction according to the procedure described by Takoet al 2000 and Ale et al 2012 [30 31] Briefly 200mL of01M HCl was added to the algae pellet and heated for 1hour at boiling temperature and centrifuged at 3500 rpmfor 10 minutes The supernatant was recovered and absoluteethanol was added (4 1) for polysaccharides precipitationOnce precipitated the polysaccharides were separated fromthe aqueous medium by centrifugation at 3500 rpm for 10minutes the supernatant was discarded and the pellet waswashed three times with 96 ethanol to remove residualpigments and finally resuspended in a minimum amountof distilled water for a 72-hour dialysis with stirring Thedialyzed product was precipitated with absolute ethanol(4 1) Polysaccharide extracts were lyophilized and weighedto calculate their yield

213 Characterization of Selected Polysaccharide Extracts

(1) FT-IR Spectra Analysis IR spectra of aqueous extractedpolysaccharides from Solieria filiformis and Eisenia arboreawere obtained using diffuse reflectance infrared Fouriertransform spectroscopy (DRIFTS) Scans were performed atroom temperature in the infrared region between 4000 and400 cmminus1 on a Thermo Nicolet Nexus 670 FT-IR spectrome-terThe infrared spectra of commercial available carrageenan(120580-carrageenan C1138 120581-carrageenan C1013) fucoidan fromFucus vesiculosus (F5631) alginic acid (A7003) from Sigma-Aldrich (St LouisMOUSA) and 120582-carrageenan fromCelticColloids Inc (B Blakemore) were included for comparison

(2) NMR Spectra Analysis 13C-NMR spectra were acquiredon a Varian 600 spectrometer The extracts were exchangedtwice with 998 deuterium oxide (D2O) with intermediatelyophilization and dissolved at 10mgmLminus1 in D2O Sodium[3-trimethylsilyl 221015840331015840-2-H4] propionate (TSP-d4) wasused as an internal reference to 000 ppm

(3) Carbohydrate Determination For determination of totalsugars in the samples acid hydrolysis of the extracts wasperformed A solution with 25mg of polysaccharide extractin 100mL of 1MH

2SO4was prepared and boiled for 3 hours

subsequently an aliquot of 1mL of each extract was takenAnthrone reagent (5mL) was added to the aliquot placedin a water bath for 12 minutes and cooled down at roomtemperature Absorbance was read at 630 nm Quantificationwas performed against a calibration curve of a stock solutionof fucose

(4) Sulfate Content Determination The analysis was per-formed using the turbidimetric method of Jackson and


Table 2 Synergistic effects of SPs on MeV infection

Compoundscombination

Compound concentration (120583gmL) relative syncytia formation in presence ofthe different SPs combinations SD CI Description

Eisenia arborea Solieria filiformisIC75-IC75

298 3027 345 44 1059 AntagonismIC75-IC50

298 0985 264 56 308 AntagonismIC75-IC25

298 0011 33 73 147 AntagonismIC50-IC75

0275 3027 284 41 371 AntagonismIC50-IC50

0275 0985 32 59 188 AntagonismIC50-IC25

0275 0011 4 32 0001 SynergismIC25-IC75

001 3027 225 66 185 AntagonismIC25-IC50

001 0985 167 71 031 SynergismIC25-IC25

001 0011 394 25 005 Synergism

2

minus2

minus2

2

SfEa

Comb

log (FaFu)

log (D)

(a)

1

05

00 05 1

A

B

E50-S25E25-S50E25-S25

(b)

Figure 4 Analysis of Eisenia arborea and Solieria filiformis combinations (a) Median-effect plot for combinations of Eisenia arborea andSolieria filiformis was generated with the CompuSyn software (119865

119886 affected fraction 119865

119906 unaffected fraction 119863 concentration of SP used Sf

Solieria filiformis SP Ea Eisenia arborea SP Comb Eisenia arborea and Solieria filiformis combinations) (b) Normalized isobologram plotsfor Sf and Ea at nonconstant combination ratios For each SP different combinations of various concentrations based on IC

25 IC50values

were tested and combination index (CI) values were determined using the CompuSyn software CI values represented by points below thelines indicate synergy

McCandless 1978 [32] Briefly this quantification of sulfateswas determined by measuring turbidity as barium sulfatewhen adding 12mL of TCA 8 and 06mL of 001 reactionreagent (agarosebarium chloride) to the sample reactionwas homogenized by stirring for 35 minutes The turbiditywas determined at 500 nm in a ShimadzuUV-Vis spectropho-tometer 1601 The calibration curve was performed withpotassium sulfate (K

2SO4) with a concentration of 0 to 100 120583g

of SO4

minus2mL SP extracts of Solieria filiformis and Eiseniaarboreawere weighed (7mg) and 1mL of 1N HCl was addedand heated at 105∘C for 12 hours in a thermoblock (Lab line)

A dilution was performed with 10mL of deionized watersamples were then filtered using a microfilter with Whatmanpaper of 12 120583m and an aliquot of 11mL of the samples wastaken for quantificationAnalysiswas performedby triplicate

22 Cells and Virus Vero cells were grown at 37∘C in a5 CO

2atmosphere in Dulbeccorsquos Modified Eagle Medium

Nutrient Mixture F-12 (DMEMF12 Gibco Invitrogen USA)supplemented with 5 fetal bovine serum (FBS GibcoInvitrogen USA) and 1 antibiotic (Gibco Invitrogen USA)


0

20

40

60

80

100

120

UntreatedConcentration (120583gmL)


sy

ncyt

ia co

unt o

r vira

l RN

A co

pies

E50S25 E25S50 E25S25

Figure 5 Antiviral activity confirmation by qPCR of the RNAextracted from Vero cells infected with MeV and cultivated inpresence of synergistic SPs combinations E

25and E

50are the SPs

concentrations corresponding to IC25

and IC50

values of Eiseniaarborea SPs S

25and S

50concentrations correspond to the respective

IC values of Solieria filiformis SP

0

20

40

60

80

100

120

Untreated minus60 0 15 30 60 120

sy

ncyt

ia co

unt

Time (minutes)

Solieria filiformisEisenia arborea

Figure 6 Time of addition experiments Antiviral activity of SPwas tested at different times of infection and analyzed by syncytiainhibition assays SPs were added at 60min before infection and0 15 30 60 and 120min after infection The data are expressedas relative syncytia count () compared to that of untreated virus-infected control cells which was defined as 100 The data shownare the mean plusmn SD of triplicate experiments

Measles virus (Edmonston strain) was purchased fromATCC (ATCC VR-24) Virus was propagated on Verocells and viral titers were determined by cytopathogeniceffect and expressed as 50 tissue culture infectious dose(TCID50)mL Aliquots of viral stock were stored at minus80∘Cuntil use

23 Cytotoxicity Assays The effect of SPs on cell viabilityof Vero cells was determined by MTT assay The cells werecultured in 96-well plates at a density of 15 times 104 cellswellat 37∘C in an atmosphere of CO

2 After 1 day of incubation

increasing concentrations of SPs diluted in DMEM were

0

20

40

60

80

100

120

Untreated 1 5

sy

ncyt

ia co

unt



Figure 7 Effect of SPs on viral penetration Vero cells were infectedwith MeV at 4∘C in the absence of SPs and then shifted to 37∘Cto permit penetration of the adsorbed virus in the presence of SPsAntiviral effect of SPs was evaluated using syncytia inhibition assaysThe data shown are the mean plusmn SD of triplicate experiments

added after 48 h of incubation the media were replaced with22120583L of 25mgmL MTT dissolved in phosphate-bufferedsaline (PBS) After 1 h 30min 150 120583L of DMSO was addedand incubated at room temperature for 15min The opticaldensity (OD450 nm)wasmeasured using amicroplate reader(Multiskan FC Thermo USA) Cell viability was expressedby percentage as the mean value of three independentexperiments considering control cells absorbance as 100viable CC

50was the concentration of the test substances that

inhibited the Vero cells growth by 50 compared with thegrowth of the untreated cells

24 Syncytia Reduction Assays The antiviral activity of theSPs was evaluated by syncytia reduction assays Vero cellsseeded in 12-well plates were treated with different con-centrations of SPs (001ndash5120583gmL) and infected with MeV(1 times 1035 TCID50 of Edmonston strain) at the same timeAfter virus adsorption for 1 h at 37∘C the medium wasremoved and monolayers were washed with PBS after whichthe corresponding concentrations of SPs were added againEach concentration was tested using three culture wellsper PS concentration per experiment the experiments wereperformed by triplicate After incubation of 48 or 72 h at37∘C in a 5 CO

2incubator monolayers were fixed with

methanol acetone (1 1) and stained with 1 crystal violetSyncytia were counted and the result was expressed as a per-centage of the number of syncytia observed in viral controlmonolayers (untreated cultures) IC

50was determined from

dose-response curves The selectivity index (SI) values werecalculated as CC

50IC50 SPs showing the best SI were selected

for the subsequent experiments

25 Quantitative Real-Time PCR Total RNA was isolatedfrom treated Vero cells using RNAzol RT (MRC IncUSA) Reverse transcription was performed using the HighCapacity cDNA Reverse Transcription Kit (Applied Biosys-tems USA) and the viral genome was amplified with spe-cific primers (MeVF 51015840 GAGGGTCAAACAGAGTCGAG 31015840


MeVR 51015840 CGGTTGGAAGATGGGCAG 31015840) that amplifieda 95 nt fragment The real-time PCR was carried out usingSensiFAST SYBR No-ROX Kit (BIOLINE USA) and theChromo4 Real-Time PCR Detector (Bio-Rad USA) withthe following procedures 95∘C for 2min followed by 50cycles of 95∘C for 2 s 60∘C for 10 s and 72∘C for 20 s Thenumber of viral copies was calculated by using a standardcurve Serial 10-fold dilutions of a synthetic oligonucleotideencompassing the target measles gene were used to establishthe standard curves

26 Evaluation of SPs Synergy Potential synergistic effectsof selected SPs on MeV infection were evaluated usingsyncytia reduction assays Each combination was tested onits corresponding IC

75 IC50 and IC

25values The synergistic

effect of SPs was calculated by using a combination index(CI) described previously by Chou [33] and CompuSynsoftware CI was calculated from the data as a measureof the interaction among drugs CI values lower than 09indicate synergy CI values from 09 to 11 indicate an additiveeffect and CI values higher than 11 indicate antagonismCombinations with synergistic antiviral effect were selectedand qPCR assays were performed in order to confirm theinhibitory effect as described above

27 Time of Addition Assay Vero cell monolayers wereinfected with MeV SPs were added at a concentration of5 120583gmL at different times of infection 60min before infec-tion and 0 15 30 60 and 120min after infection Thereafterfor each treatment cells were incubated with SP for 1 h andthen washed three times with PBS Monolayers were fixedwithmethanol acetone after incubation for 48 or 72 h at 37∘Cand 5 CO

2and stained with 1 crystal violet syncytia were

counted subsequently

28 Viral PenetrationAssay Virus penetration intoVero cellswas evaluated according to the method reported by Huangand Wagner [34] with some modifications [18] Vero cellmonolayers precooled at 4∘C for 3 h were infected with MeVat 4∘C for 1 h in the absence of SP After washing three timeswith ice-cold PBS different concentrations of SP were addedto the monolayers and the temperature was shifted to 37∘CAfter 1 h of incubation at 37∘C the cells were treated with40mM citrate buffer (pH 30) to inactivate unpenetratedviruses Buffer was replaced by culture medium and the cellswere incubated for 48 or 72 h at 37∘C and 5CO

2and stained

with 1 crystal violet syncytia were counted subsequently

29 Statistical Analysis The variables (tested by triplicate ineach experiment that were in turn repeated at least threetimes) were submitted to a one-way analysis of variancefollowed by Dunnettrsquos test (SPSS software 120572 = 005)CC50

and IC50

values were determined by probit regressionanalysis

3 Results

31 Cytotoxicity and Antiviral Activity of SPs TheMTT assayindicated no cytotoxicity for any of the SPs at concentrationsfrom 01 to 1500 120583gmL up to 2 days (data not shown)

Antiviral activity of SPs against MeV was evaluated bysyncytia reduction inhibition assays at concentrations of 00101 1 and 5 120583gmL of each compound (data not shown)All tested compounds showed significant antiviral activitybut only compounds with the best SI values were selectedfor the subsequent experiments As shown in Table 1 SPsof Eisenia arborea and Solieria filiformis exhibited antiviralactivity at the lowest concentrations (IC

500275120583gmL and

0985 120583gmL resp) without cytotoxic effect at concentrationsof 01 to 1500120583gmL Therefore SPs of Eisenia arborea andSolieria filiformiswere selected based on their SI and antiviralactivity for the combination experiments

Antiviral effect of selected SPs was confirmed by qPCRassays as shown in Figure 1 Inhibitory effect of Eiseniaarborea and Solieria filiformis SP was tested at the bestinhibitory concentrations (1120583gmL and 5 120583gmL for bothSPs) Results of qPCR assays were consistent with the resultsobserved by syncytia reduction inhibition assays

32 Characterization of SPs Infrared spectroscopy has beenused for the qualitative characterization of carrageenans andhas proven to be a valuable tool for the characterizationof sulfated oligosaccharides [35] FT-IR and NMR spectraanalyses of selected SPs extracts were performed The FT-IRspectrum of Solieria filiformis extract (Figure 2(a)) indicatesthe presence of a typical 120580-carrageenan type Character-istics signal bands are indicated 36 anhydrogalactose-2-sulfate (804 cmminus1) characteristic of 120580-carrageenan galactose-4-sulfate (846 cmminus1) signal present in 120581- and 120580-carrageenanThe signal between 1210 and 1260 cmminus1 is common to all typesof compounds containing sulfate13C-NMR spectroscopy has been highly recommended

for distinguishing the polysaccharides of the agar and car-rageenan group [36] Spectrum and expansion 13C-NMR ofthe S filiformis extract (Figure 3(a)) showed signals at 20and 60 ppm typical of residual ethanol Carbohydrates sig-nals (6379ndash10466 ppm) observed particularly two upfield-shifted signals (9451 and 10466 ppm) suggest that themolecule has two anomeric carbons Overall its spectrumshowed the presence of the 120580-carrageenan The next assign-ment is the mapping of the carbon signals of the moleculeCarbons of 2-sulfate-36-anhydrogalactose are 9451 (C1)7744 (C2) 8025 (C3) 8084 (C4) 7949 (C5) and 7233(C6) ppm [37] Carbons of 4-sulfate-galactose are 10466(C1) 7168 (C2) 7927 (C3) 7451 (C4) 7727 (C5) and6379 (C6) ppm [37] Sulfate content of S filiformis showed2114 (plusmn0056) of total sulfate and total polysaccharidedetermination resulted in 91 of polysaccharide

The FT-IR spectrum of Eisenia arborea extract (Fig-ure 2(b)) indicates the presence of a mixture of fucoidanand alginic acid Characteristics signal bands are indicatedcarboxylate vibrations (1627 and 1410 cmminus1) can be attributedto uronic acids Stretching vibrations at 1039ndash1041 cmminus1 can


be assigned to pyranose ring from guluronic andmannuronicacid residues The broad band at 1244 cmminus1 indicates thepresence of sulfated ester groups which are characteristicin fucoidans 13C-NMR spectrum of E arborea extract(Figure 3(b)) showed typical signals of alginate ranging from6604 to 17768 ppmThe signal at 6604 ppm is characteristicof carbon-2 of guluronic acid (G) [38] The signals at 72517279 7407 7890 8082 10281 10293 and 17768 ppmcorrespond to repeating blocks of mannuronic (M) andguluronic acid [39]The signals at 10281 and 10293 ppmmayindicate the presence of two repeating units one of MMMand another of GMM [39] Sulfate content of E arboreashowed 1285 (plusmn0346) of total sulfate

33 Combined Antiviral Effect of SPs The combined effectof SPs of Eisenia arborea and Solieria filiformis on MeVinfections was examined each SP was tested at differentconcentrations combining its corresponding IC

25 IC50 and

IC75

values E25 E50 and E

75correspond to IC

25 IC50 and

IC75

values of Eisenia arborea SPs and S25 S50 and S

75

correspond to the respective values of Solieria filiformis SP(Table 2) Syncytia reduction assay results were expressedin relative syncytia percentage according to the number ofsyncytia in viral control Best inhibitory effect was observedfor E50-S25combination

The evaluation of drug synergism based on a median-effect equation has been extensively used in the literatureCI values of SPs combinations were calculated as describedpreviously using the CompuSyn software and are givenin Table 2 Median-effect and the normalized isobologramgenerated with the software determined the presence of threesynergistic combinations represented by points below thelines at normalized isobologram (Figure 4)

Results showed strong synergistic effects at low concen-trations combinations (E

50-S25 E25-S50 and E

25-S25) and

antagonism at high concentrations combinations (E25-S75

E50-S50 E50-S75 E75-S25 E70-S50 and E

75-S75) Combinations

with synergistic effect were selected and qPCR assays wereperformed As shown in Figure 5 the inhibitory effect ofthe synergistic combinations was confirmed These datawere consistent with results observed by syncytia reductioninhibition assays

34 Effect of SPs on Viral Infection at Different Times ofAddition In order to determine which step of the MeV cyclewas targeted by SPs ldquotime of additionrdquo experiments wereperformed in Vero cells infected withMeV and exposed to PSat different times of infectionThemost efficient inhibition byS filiformis was observed in early phases of infection 0 and15min after infection (Figure 6) syncytia inhibition beforeinfection and 30min after infection was not significant Earborea showed the most efficient inhibition 1 hour beforeinfection and 0 and 15min after infection At 30 60 and120min after infection a minimal syncytia inhibition by Earborea was still observed

35 Effect of Fucoidan on Viral Penetration into Host CellsViral penetration assays were performed to determine

whether entry events downstream of virus binding wereinhibited by SPs Vero cells were plated and incubated withMeV at 4∘C for 1 h to allow virus binding but preventviral internalization Unbound virus was inactivated and SPs(1 120583gmL or 5120583gmL) were added to the cells and incubatedat 37∘C Figure 7 shows that SP from S filiformis (5 120583gmL)significantly decreased viral infection by 58 while SPs fromE arborea (5 120583gmL) decreased viral infection only by 24when compared with the findings in infected cells in theabsence of treatment

4 Discussion

Since the first studies by Gerber in 1958 showing the inhi-bition of mumps and influenza B virus by marine algaepolysaccharides increased efforts and research have beencarried out in this field [13] Previous studies have alsodemonstrated no cytotoxicity of SPs isolated from certainseaweed species [40] The absence of cytotoxicity to the hostcells is one of the principal challenges in the development ofnew antivirals

Eisenia arborea an edible brown alga used in folkmedicine in Japan is the kelp species with the largest andmost southerly latitudinal distribution on the North PacificEast Coast [41 42] Researches on Eisenia biological activitieshave been focused on the evaluation of their polyphenoliccompounds [43] To our knowledge the antiviral effects ofEisenia arborea extracts have never been tested before Inthis study the extract of Eisenia arborea is rich in fucoidansand alginates and also showed the best SI of the five sea-weed extracts (Table 1) Previous chemical characterizationof Mexican E arborea extracts also reported the presenceof alginates with higher yields than the one reported inthis study [44] Alginates with antiviral effects have beenpreviously tested against HIV IAV and HBV showing apotent antiviral activity [4] Antiviral activity of fucoidan hasbeen also reported in vitro and in vivo against many RNAand DNA viruses such as HIV HSV1-2 dengue virus andinfluenza virus [39 45ndash47]

Macrocystis pyrifera has been harvested since 1956 alongthe Pacific coast of Baja California and exported to theUnitedStates for the production of alginates [48] SPs extracts ofMexican Macrocystis pyrifera showed a significant antiviraleffect but were not selected for subsequent assays becauseof their IC

50value (Table 1) Previous studies with crude

dialyzed extracts ofMacrocystis pyrifera have shown antiviraleffects against VSV with the fucoidan being responsible forthese results [49]

In this study antiviral effects of the extract from Solieriafiliformis display the second lowest IC

50among the analyzed

extracts In vitro studies have reported antiviral properties ofcarrageenans againstDNAandRNAviruses [21 50] Recentlyit has been shown that carrageenan (Rong Yuan FFI CoLtd) can inhibit influenza virusASwineShandong7312009H1N1 (SW731) responsible for the influenza pandemic of2009 Carrageenans can significantly inhibit SW731 replica-tion by interfering with different steps of viral replicationincluding adsorption transcription and expression of the


viral proteins however they act especially by inhibiting theinteractions between the viral receptor (HA) and the targetcell [51] Sulfate content analysis and total polysaccharidedetermination of S filiformis extract resulted in 2114(plusmn0056) total sulfate and 91 polysaccharide these data areconsistent with previous reports [52] Degree of sulfation hasa major impact on the antiviral activity of polysaccharidesincluding carrageenans [53]

In relation to the combination therapy approach usedin this study results showed a strong synergistic effect atlow concentrations combinations of SPs and antagonism athigh concentrations combinations Our results determinedthat low concentrations combinations (00274 120583gmL and0011 120583gmL of E arborea and S filiformis resp) exhibitedthe higher inhibitory effect (96) in comparison to theindividual effect of SP (50 of inhibition with 0275 120583gmLand 0985 120583gmL of E arborea and S filiformis resp) Syn-ergistic effect observed in this study has been also reportedfor the sulfated polysaccharides from Fucus vesiculosus incombination with AZT against HIV [54] Furthermore thiseffect has been also observed with acyclovir in combina-tionwith 3 19-isopropylideneandrographolide against herpessimplex virus (wild type) and drug-resistant strains Lowconcentrations of these compounds were required for acomplete inhibition of DNA replication and late proteinsynthesis of HSV-1 wild type and drug-resistant HSV-1 [55]The combined effect of nitazoxanide with neuraminidaseinhibitors against influenza A viruses tested in vitro suggeststhat regimens that combine neuraminidase inhibitors andnitazoxanide exert synergistic anti-influenza effects [56] Incontrast antagonistic effects at high concentrations wereobserved in our study this antagonism of SPs was previouslyobserved in a combination of ulvan and fucoidan againstNDV infection [23] Particular chemical features of SPs likechain ramifications could explain antagonism effects of SPsMoreover carbohydrate to carbohydrate interactions couldbe responsible to adhesion events these aggregates have beenpreviously observed in marine sponges [57]

To understand if a synergistic effect was related to differ-ent modes of action of the tested SPs viral penetration andtime of addition assays were performed Results suggestedthe possibility that SP from S filiformis inhibits postbindingevents because best inhibition effect was observed at 0 and 15minutes after viral infection (Figure 6) To support this idea aviral penetration assay was performed (Figure 7) and resultsshow the best antiviral effect after viral adsorption Ourresults are in agreement with those observed by Elizondo-Gonzalez et al [18] who demonstrated the ability of fucoidanfromC okamuranus to be responsible for the antiviral activityagainst Newcastle disease virus suggesting that fucoidaninhibits viral penetration into host cells must probably byblocking the F protein

Similar results were also observed by Bouhlal et al [58]who suggested that carrageenans can inhibit DENV repli-cation by interfering viral entrance but they also suggestedthat SPs could avoid viral adsorption into the cell as a secondmode of action This mode of action could be similar to themechanism observed with SPs of E arborea Alginates andfucoidan of E arborea were able to show the best antiviral

effect 1 hour before infection and this effect lasted up to 0ndash15minutes after infection Although both SPs from S filiformisand E arborea exhibited antiviral activity at 0 and 15minafter infection only E arborea showed inhibitory effect at60minThis result suggests the capability of these SPs to avoidviral adsorption to the cell these data were confirmed by viralpenetration assays where we observed less antiviral activityafter viral attachment to the cell More recent studies havedemonstrated that fucoidans exhibit their antiviral activitywhen the compound is present during the virus adsorptionperiod by blocking the interaction of viruses to the cells [59]

SPs tested in this study exhibit the best antiviral effectat different stages of infection viral penetration and viraladsorption (S filiformis and E arborea resp) Multiple-drug antiviral therapy with two or more drugs that targetdifferent proteins or act in different stages of infection maydecrease drug resistance and may enhance clinical outcomesby allowing a reduction of individual drug doses thusdecreasing dose-related drug toxicity [60]

5 Conclusions

In this study sulfated polysaccharides from Mexican sea-weed showed antiviral activity against measles virus Dueto the lack of cytotoxicity at inhibitory concentrations asindicated by the selectivity index potential application canbe found for these SPs Eisenia arborea and Solieria filiformisextracts showed the higher antiviral activity andwere selectedto determine their combined effect Synergistic effect wasobserved at the lowest concentrations tested for each SP ofthese species Results suggest that SPs combined in this studyare acting at different level of first stages in viral infectionSynergistic therapeutic effect allows dose and toxicity reduc-tion and would minimize or delay the induction of antiviralresistance Sulfated polysaccharides of Mexican seaweed arepotential candidates for the development of new antiviraldrugs that can help to control viral infection diseases

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

The authors thank E Hernandez E Caamal and C Chavezfor chemical analytical support M Maldonado for polysac-charide extraction support I Beamonte R Marcos andR Ojeda for seaweed collection support S Salcedo forthe confirmation of seaweeds species identification and KLedezma for cytotoxicity assays support This work wassupported by Consejo Nacional de Ciencia y Tecnologıa(CONACYT) Mexico (Project no 10002-255075)

References

[1] R OrsquoDor P Miloslavich and K Yarincik ldquoMarine biodiversityand biogeographymdashregional comparisons of global issues anintroductionrdquo PLoS ONE vol 5 no 8 Article ID e11871 2010


[2] C Rebours E Marinho-Soriano J A Zertuche-Gonzalez et alldquoSeaweeds an opportunity forwealth and sustainable livelihoodfor coastal communitiesrdquo Journal of Applied Phycology vol 26no 5 pp 1939ndash1951 2014

[3] L Wang X Wang H Wu and R Liu ldquoOverview on biologicalactivities and molecular characteristics of sulfated polysaccha-rides from marine green algae in recent yearsrdquo Marine Drugsvol 12 no 9 pp 4984ndash5020 2014

[4] A Ahmadi S Zorofchian Moghadamtousi S Abubakar andK Zandi ldquoAntiviral potential of algae polysaccharides isolatedfrom marine sources a reviewrdquo BioMed Research Internationalvol 2015 Article ID 825203 10 pages 2015

[5] S Kraan ldquoAlgal polysaccharides novel applications and out-lookrdquo in CarbohydratesmdashComprehensive Studies on Glycobiol-ogy and Glycotechnology InTech 2012

[6] C M P G Dore M G D C Faustino Alves L S E PofırioWill et al ldquoA sulfated polysaccharide fucans isolated frombrown algae Sargassum vulgare with anticoagulant antithrom-botic antioxidant and anti-inflammatory effectsrdquoCarbohydratePolymers vol 91 no 1 pp 467ndash475 2013

[7] B W S Souza M A Cerqueira A I Bourbon et alldquoChemical characterization and antioxidant activity of sulfatedpolysaccharide from the red seaweed Gracilaria birdiaerdquo FoodHydrocolloids vol 27 no 2 pp 287ndash292 2012

[8] V Suresh N Senthilkumar R Thangam et al ldquoSepara-tion purification and preliminary characterization of sulfatedpolysaccharides from Sargassum plagiophyllum and its in vitroanticancer and antioxidant activityrdquo Process Biochemistry vol48 no 2 pp 364ndash373 2013

[9] P Shao X Chen and P Sun ldquoChemical characterizationantioxidant and antitumor activity of sulfated polysaccharidefrom Sargassum hornerirdquo Carbohydrate Polymers vol 105 no1 pp 260ndash269 2014

[10] C O Coura I W F de Araujo E S O Vanderlei et alldquoAntinociceptive and anti-inflammatory activities of sulphatedpolysaccharides from the red seaweed Gracilaria corneardquo Basicand Clinical Pharmacology and Toxicology vol 110 no 4 pp335ndash341 2012

[11] C A Pujol S Ray B Ray and E B Damonte ldquoAntiviral activityagainst dengue virus of diverse classes of algal sulfated polysac-charidesrdquo International Journal of Biological Macromoleculesvol 51 no 4 pp 412ndash416 2012

[12] H H A Gomaa and G A Elshoubaky ldquoAntiviral activity ofsulfated polysaccharides carrageenan from some marine sea-weedsrdquo International Journal of Current Pharmaceutical Reviewand Research vol 7 no 1 pp 34ndash42 2016

[13] P Gerber J D Dutcher E V Adams and J H Sherman ldquoPro-tective effect of seaweed extracts for chicken embryos infectedwith influenza B or mumps virusrdquo Experimental Biology andMedicine vol 99 no 3 pp 590ndash593 1958

[14] M F De Jesus Raposo A M B De Morais and R M S CDe Morais ldquoMarine polysaccharides from algae with potentialbiomedical applicationsrdquoMarine Drugs vol 13 no 5 pp 2967ndash3028 2015

[15] S F Mohamed and F A Agili ldquoAntiviral sulphated polysac-charide from brown algae Padina pavonia characterizationand structure elucidationrdquo International Journal of ChemTechResearch vol 5 no 4 pp 1469ndash1476 2013

[16] T T T Thuy B M Ly T T T Van et al ldquoAnti-HIV activityof fucoidans from three brown seaweed speciesrdquo CarbohydratePolymers vol 115 pp 122ndash128 2015

[17] M Kim J H Yim S-Y Kim et al ldquoIn vitro inhibitionof influenza A virus infection by marine microalga-derivedsulfated polysaccharide p-KG03rdquoAntiviral Research vol 93 no2 pp 253ndash259 2012

[18] R Elizondo-Gonzalez L E Cruz-Suarez D Ricque-MarieE Mendoza-Gamboa C Rodriguez-Padilla and L M Trejo-Avila ldquoIn vitro characterization of the antiviral activity offucoidan from Cladosiphon okamuranus against NewcastleDisease Virusrdquo Virology Journal vol 9 no 1 article 307 2012

[19] L M Trejo-Avila M E Morales-Martınez D Ricque-Marie etal ldquoIn vitro anti-canine distemper virus activity of fucoidanextracted from the brown algaCladosiphon okamuranusrdquoVirus-Disease vol 25 no 4 pp 474ndash480 2014

[20] G Meiyu L Fuchuan X Xianliang L Jing Y Zuowei andG Huashi ldquoThe potential molecular targets of marine sulfatedpolymannuroguluronate interfering with HIV-1 entry interac-tion between SPMG and HIV-1 rgp120 and CD4 moleculerdquoAntiviral Research vol 59 no 2 pp 127ndash135 2003

[21] C B Buck C D Thompson J N Roberts M Muller D RLowy and J T Schiller ldquoCarrageenan is a potent inhibitorof papillomavirus infectionrdquo PLoS Pathogens vol 2 no 7 pp0671ndash0680 2006

[22] A Rodrıguez K Kleinbeck O Mizenina et al ldquoIn vitro andin vivo evaluation of two carrageenan-based formulations toprevent HPV acquisitionrdquo Antiviral Research vol 108 no 1 pp88ndash93 2014

[23] J A Aguilar-Briseno L E Cruz-Suarez J-F Sassi et al ldquoSul-phated polysaccharides from Ulva clathrata and Cladosiphonokamuranus seaweeds both inhibit viral attachmententry andcell-cell fusion in NDV infectionrdquoMarine Drugs vol 13 no 2pp 697ndash712 2015

[24] Y Koizumi and S Iwami ldquoMathematical modeling of multi-drugs therapy a challenge for determining the optimal com-binations of antiviral drugsrdquo Theoretical Biology amp MedicalModelling vol 11 p 41 2014

[25] W J Moss and D E Griffin ldquoMeaslesrdquoThe Lancet vol 379 no9811 pp 153ndash164 2012

[26] W J Moss and D E Griffin ldquoGlobal measles eliminationrdquoNature Reviews Microbiology vol 4 no 12 pp 900ndash908 2006

[27] G Antonelli and O Turriziani ldquoAntiviral therapy old andcurrent issuesrdquo International Journal of Antimicrobial Agentsvol 40 no 2 pp 95ndash102 2012

[28] D Robledo and Y Freile-Pelegrın ldquoProspects for the cultiva-tion of economically important carrageenophytes in SoutheastMexicordquo Journal of Applied Phycology vol 23 no 3 pp 415ndash4192011

[29] J Xi D Shen S Zhao B Lu Y Li and R Zhang ldquoChar-acterization of polyphenols from green tea leaves using ahigh hydrostatic pressure extractionrdquo International Journal ofPharmaceutics vol 382 no 1-2 pp 139ndash143 2009

[30] M Tako E Yoza and S Tohma ldquoChemical characterizationof acetyl fucoidan and alginate from commercially culturedCladosiphon okamuranusrdquo Botanica Marina vol 43 no 4 pp393ndash398 2000

[31] M T Ale J D Mikkelsen and A S Meyer ldquoDesignedoptimization of a single-step extraction of fucose-containingsulfated polysaccharides from Sargassum sprdquo Journal of AppliedPhycology vol 24 no 4 pp 715ndash723 2012

[32] S G Jackson and E L McCandless ldquoSimple rapid tur-bidometric determination of inorganic sulfate andor proteinrdquoAnalytical Biochemistry vol 90 no 2 pp 802ndash808 1978


[33] T-C Chou ldquoTheoretical basis experimental design and com-puterized simulation of synergism and antagonism in drugcombination studiesrdquo Pharmacological Reviews vol 58 no 3pp 621ndash681 2006

[34] A S Huang and R R Wagner ldquoPenetration of herpes simplexvirus into human epidermoid cellsrdquo Proceedings of the Societyfor Experimental Biology and Medicine vol 116 no 4 pp 863ndash869 1964

[35] P Volery R Besson and C Schaffer-Lequart ldquoCharacterizationof commercial carrageenans by Fourier transform infraredspectroscopy using single-reflection attenuated total reflectionrdquoJournal of Agricultural and Food Chemistry vol 52 no 25 pp7457ndash7463 2004

[36] V L Campo D F Kawano D B da Silva Jr and I CarvalholdquoCarrageenans biological properties chemical modificationsand structural analysismdasha reviewrdquo Carbohydrate Polymers vol77 no 2 pp 167ndash180 2009

[37] F Van De Velde L Pereira and H S Rollema ldquoThe revisedNMR chemical shift data of carrageenansrdquo CarbohydrateResearch vol 339 no 13 pp 2309ndash2313 2004

[38] D D McIntyre H Ceri and H J Vogel ldquoNuclear magneticresonance studies of the heteropolysaccharides alginate Gumarabic and gum xanthanrdquo Starch vol 48 no 7-8 pp 285ndash2911996

[39] H Grasdalen B Larsen and O Smisrod ldquo 13C-nmr studiesof monomeric composition and sequence in alginaterdquo Carbo-hydrate Research vol 89 no 2 pp 179ndash191 1981

[40] S Dinesh T Menon L E Hanna V Suresh M Sathuvanand M Manikannan ldquoIn vitro anti-HIV-1 activity of fucoidanfrom Sargassum swartziirdquo International Journal of BiologicalMacromolecules vol 82 pp 83ndash88 2016

[41] Y Sugiura K Matsuda Y Yamada et al ldquoAnti-allergicphlorotannins from the edible brown alga Eisenia arboreardquoFood Science and Technology Research vol 13 no 1 pp 54ndash602007

[42] J A Zertuche-Gonzalez M Sanchez-Barredo J M Guzman-Calderon and Z Altamirano-Gomez ldquoEisenia arborea JEAreschoug as abalone diet on an IMTA farm in Baja CaliforniaMexicordquo Journal of Applied Phycology vol 26 no 2 pp 957ndash960 2014

[43] Q-T Le Y Li Z-J Qian M-M Kim and S-K KimldquoInhibitory effects of polyphenols isolated from marine algaEcklonia cava on histamine releaserdquo Process Biochemistry vol44 no 2 pp 168ndash176 2009

[44] D L Arvizu Y E Rodrıguez G Hernandez and J I MurilloldquoChemical constituents of Eisenia arboreaAreschoug from BajaCalifornia Sur Mexicordquo Investigaciones Marinas vol 35 no 2pp 63ndash69 2007

[45] K I P J Hidari N Takahashi M Arihara M Nagaoka KMorita and T Suzuki ldquoStructure and anti-dengue virus activityof sulfated polysaccharide from amarine algardquo Biochemical andBiophysical Research Communications vol 376 no 1 pp 91ndash952008

[46] K Hayashi T Nakano M Hashimoto K Kanekiyo and THayashi ldquoDefensive effects of a fucoidan from brown algaUndaria pinnatifida against herpes simplex virus infectionrdquoInternational Immunopharmacology vol 8 no 1 pp 109ndash1162008

[47] KHayashi J-B Lee T Nakano andTHayashi ldquoAnti-influenzaA virus characteristics of a fucoidan from sporophyll ofUndariapinnatifida in mice with normal and compromised immunityrdquoMicrobes and Infection vol 15 no 4 pp 302ndash309 2013

[48] M C Valdez E S Zaragoza D L Belda R Marcos and R ARamırez ldquoEffect of climatic change on the harvest of the kelpMacrocystis Pyrifera on the Mexican Pacific coastrdquo Bulletin ofMarine Science vol 73 no 3 pp 545ndash556 2003

[49] A M S Mayer A Diaz A Pesce M Criscuolo J F Groismanand R M de Lederkremer ldquoBiological activity in Macrocystispyrifera from Argentina sodium alginate fucoidan and lami-naran III Antiviral activityrdquo Hydrobiologia vol 151-152 no 1pp 497ndash500 1987

[50] Z Luo D Tian M Zhou et al ldquo120582-Carrageenan P32 is a potentinhibitor of rabies virus infectionrdquo PLoS ONE vol 10 no 10Article ID e0140586 2015

[51] Q Shao Q Guo W P Xu Z Li and T T Zhao ldquoSpecificinhibitory effect of 120581-carrageenan polysaccharide on swinepandemic 2009 H1N1 influenza virusrdquo PLoS ONE vol 10 no5 Article ID e0126577 2015

[52] I W F De Araujo E D S O Vanderlei J A G Rodrigueset al ldquoEffects of a sulfated polysaccharide isolated from thered seaweed Solieria filiformis on models of nociception andinflammationrdquo Carbohydrate Polymers vol 86 no 3 pp 1207ndash1215 2011

[53] T Ghosh K Chattopadhyay M Marschall P Karmakar PMandal and B Ray ldquoFocus on antivirally active sulfatedpolysaccharides from structure-activity analysis to clinicalevaluationrdquo Glycobiology vol 19 no 1 pp 2ndash15 2009

[54] I Sugawara W Itoh S Kimura S Mori and K ShimadaldquoFurther characterization of sulfated homopolysaccharides asanti-HIV agentsrdquo Experientia vol 45 no 10 pp 996ndash998 1989

[55] T Priengprom T Ekalaksananan B Kongyingyoes S Sueb-sasana C Aromdee and C Pientong ldquoSynergistic effects ofacyclovir and 3 19- isopropylideneandrographolide on herpessimplex virus wild types and drug-resistant strainsrdquo BMCComplementary and Alternative Medicine vol 15 no 1 article56 2015

[56] G Belardo O Cenciarelli S La Frazia J F Rossignol andM G Santoroa ldquoSynergistic effect of nitazoxanide with neu-raminidase inhibitors against influenza A viruses in vitrordquoAntimicrobial Agents and Chemotherapy vol 59 no 2 pp 1061ndash1069 2015

[57] E Vilanova C C Coutinho and P A S Mourao ldquoSul-fated polysaccharides from marine sponges (Porifera) anancestor cell-cell adhesion event based on the carbohydrate-carbohydrate interactionrdquo Glycobiology vol 19 no 8 pp 860ndash867 2009

[58] R Bouhlal C Haslin J-C Chermann et al ldquoAntiviral activ-ities of sulfated polysaccharides isolated from Sphaerococcuscoronopifolius (Rhodophytha Gigartinales) and Boergeseniellathuyoides (Rhodophyta Ceramiales)rdquo Marine Drugs vol 9 no7 pp 1187ndash1209 2011

[59] N N Besednova I D Makarenkova T N Zvyagintseva T IImbs L M Somova and T S Zaporozhets ldquoAntiviral activityand pathogenetic targets for seaweed sulfated polysaccharidesin herpesvirus infectionsrdquo Biochemistry (Moscow) SupplementSeries B Biomedical Chemistry vol 10 no 1 pp 31ndash42 2016

[60] E A Govorkova and R G Webster ldquoCombination chemother-apy for influenzardquo Viruses vol 2 no 8 pp 1510ndash1529 2010


0

20

40

60

80

100

120

Untreated 1 5

sy

ncyt

ia co

unt o

r vira

l RN

A co

pies



(a)

0

20

40

60

80

100

120

Untreated 1 5

sy

ncyt

ia co

unt o

r vira

l RN

A co

pies



(b)

Figure 1 Confirmation of antiviral activity of Eisenia arborea (a) and Solieria filiformis (b) SPs at their best inhibitory concentrations bysyncytia reduction and qPCR assays Syncytia count and viral RNA copies number are given in of the untreated control values

2000 1800 1600 1400 1200 1000 800 600

1210ndash1260 804846929

D

C

B

A

1639

(cmminus1)

(a)

2000 1800 1600 1400 1200 1000 800 600

C

B

124414101627

1039ndash1041

A

(cmminus1)

(b)

Figure 2 (a) Infrared spectra of (A) Solieria filiformis aqueous extract (B) 120580-carrageenan (C) 120581- carrageenan and (D) 120582-carrageenan (b)Infrared spectra of (A) Eisenia arborea aqueous extract (B) fucoidan and (C) alginic acid

Fucoidans have shown a potent antiviral activity againstnumerous enveloped viruses including herpes simplex virustype 1 (HSV-1) [15] human immunodeficiency virus [16]influenza A virus [17] and different kind of paramyxovirusessuch as Newcastle disease virus (NDV) and canine distempervirus (CDV) [18 19] In vitro and in vivo antiretroviral effectsof alginates preventing syncytium formation and reducingthe P24 core antigen level have been demonstrated [20]Antiviral activity of carrageenans has been demonstrated invitro against human papillomavirus (HPV) acting mainly onthe inhibition ofHPV virions binding to cells and also in vivoby preventing infection by different HPV genotypes [21 22]Recently antiviral activity against NDV of ulvan from Ulvaclathrata cultivated in Mexico has been reported [23]

Nowadays combining multiple drugs is a primaryapproach for improving antiviral effects within the antiviraldrug therapy field The advantages of multidrugs combina-tion are the reduction of individual drugs doses a decrease inthe side effects of antiviral agents and the prevention of drug-resistant viruses emergence Drug combination theories pro-vide an ideal tool for this purpose to understand the benefitsof multidrugs combinations therapy [24]

Measles virus (MeV) belongs to the Paramyxoviridaefamily of Mononegavirales is a nonsegmented negative-strand RNA virus and causes a highly contagious disease[25] Although preventable by vaccination measles stillremains one of the causes of death among young childrenworldwide [26] Many new antiviral drugs have been licensed


7

1

4

3

12

6

59

2

11

10 8

O O OO

O

OH

OH 1

234

6

5

78

10

9

12

11

TSP-d4

OSO3minus

HOCH2CH3

250

240

230

220

210

200

190

180

170

160

150

140

130

120

110

100

90 80 70

60

50

40

30 20 10 0

minus10

minus20

105 100 95 90 85 80 75 70 65

(ppm)

O3minusSO

10466

9451

8084

8025

7948

7927

7743

7727

7451

7233

7219

7167

6379

(a)

[MMM][GMM]


250

240

230

220

210

200

190

180

170

160

150

140

130

120

110

100

90 80 70

60

50

40

30 20 10 0

minus10

minus20

(ppm)

TSP-d4

18783

17768

10293

10281

8082

7890

7407

7279

7251

6604

4683

(b)





50(120583gmL)c SId




21 Antiviral Agents


















298 3027 345 44 1059 AntagonismIC75-IC50

298 0985 264 56 308 AntagonismIC75-IC25

298 0011 33 73 147 AntagonismIC50-IC75

0275 3027 284 41 371 AntagonismIC50-IC50

0275 0985 32 59 188 AntagonismIC50-IC25

0275 0011 4 32 0001 SynergismIC25-IC75

001 3027 225 66 185 AntagonismIC25-IC50

001 0985 167 71 031 SynergismIC25-IC25

001 0011 394 25 005 Synergism

2

minus2

minus2

2

SfEa

Comb

log (FaFu)

log (D)

(a)

1

05

00 05 1

A

B

E50-S25E25-S50E25-S25

(b)





25 IC50values




of SO4







0

20

40

60

80

100

120



sy

ncyt

ia co

unt o

r vira

l RN

A co

pies

E50S25 E25S50 E25S25


25and E

50are the SPs


and IC50


25and S



0

20

40

60

80

100

120


sy

ncyt

ia co

unt

Time (minutes)







0

20

40

60

80

100

120

Untreated 1 5

sy

ncyt

ia co

unt


















75 IC50 and IC







2and stained



and IC50


3 Results



500275120583gmL and









25 IC50 and

IC75


75correspond to IC

25 IC50 and

IC75


75




50-S25 E25-S50 and E

25-S25) and


E50-S50 E50-S75 E75-S25 E70-S50 and E






4 Discussion
















5 Conclusions


Competing Interests


Acknowledgments


References
































































7

1

4

3

12

6

59

2

11

10 8

O O OO

O

OH

OH 1

234

6

5

78

10

9

12

11

TSP-d4

OSO3minus

HOCH2CH3

250

240

230

220

210

200

190

180

170

160

150

140

130

120

110

100

90 80 70

60

50

40

30 20 10 0

minus10

minus20

105 100 95 90 85 80 75 70 65

(ppm)

O3minusSO

10466

9451

8084

8025

7948

7927

7743

7727

7451

7233

7219

7167

6379

(a)

[MMM][GMM]


250

240

230

220

210

200

190

180

170

160

150

140

130

120

110

100

90 80 70

60

50

40

30 20 10 0

minus10

minus20

(ppm)

TSP-d4

18783

17768

10293

10281

8082

7890

7407

7279

7251

6604

4683

(b)





50(120583gmL)c SId




21 Antiviral Agents


















298 3027 345 44 1059 AntagonismIC75-IC50

298 0985 264 56 308 AntagonismIC75-IC25

298 0011 33 73 147 AntagonismIC50-IC75

0275 3027 284 41 371 AntagonismIC50-IC50

0275 0985 32 59 188 AntagonismIC50-IC25

0275 0011 4 32 0001 SynergismIC25-IC75

001 3027 225 66 185 AntagonismIC25-IC50

001 0985 167 71 031 SynergismIC25-IC25

001 0011 394 25 005 Synergism

2

minus2

minus2

2

SfEa

Comb

log (FaFu)

log (D)

(a)

1

05

00 05 1

A

B

E50-S25E25-S50E25-S25

(b)





25 IC50values




of SO4







0

20

40

60

80

100

120



sy

ncyt

ia co

unt o

r vira

l RN

A co

pies

E50S25 E25S50 E25S25


25and E

50are the SPs


and IC50


25and S



0

20

40

60

80

100

120


sy

ncyt

ia co

unt

Time (minutes)







0

20

40

60

80

100

120

Untreated 1 5

sy

ncyt

ia co

unt


















75 IC50 and IC







2and stained



and IC50


3 Results



500275120583gmL and









25 IC50 and

IC75


75correspond to IC

25 IC50 and

IC75


75




50-S25 E25-S50 and E

25-S25) and


E50-S50 E50-S75 E75-S25 E70-S50 and E






4 Discussion
















5 Conclusions


Competing Interests


Acknowledgments


References


































































50(120583gmL)c SId




21 Antiviral Agents


















298 3027 345 44 1059 AntagonismIC75-IC50

298 0985 264 56 308 AntagonismIC75-IC25

298 0011 33 73 147 AntagonismIC50-IC75

0275 3027 284 41 371 AntagonismIC50-IC50

0275 0985 32 59 188 AntagonismIC50-IC25

0275 0011 4 32 0001 SynergismIC25-IC75

001 3027 225 66 185 AntagonismIC25-IC50

001 0985 167 71 031 SynergismIC25-IC25

001 0011 394 25 005 Synergism

2

minus2

minus2

2

SfEa

Comb

log (FaFu)

log (D)

(a)

1

05

00 05 1

A

B

E50-S25E25-S50E25-S25

(b)





25 IC50values




of SO4







0

20

40

60

80

100

120



sy

ncyt

ia co

unt o

r vira

l RN

A co

pies

E50S25 E25S50 E25S25


25and E

50are the SPs


and IC50


25and S



0

20

40

60

80

100

120


sy

ncyt

ia co

unt

Time (minutes)







0

20

40

60

80

100

120

Untreated 1 5

sy

ncyt

ia co

unt


















75 IC50 and IC







2and stained



and IC50


3 Results



500275120583gmL and









25 IC50 and

IC75


75correspond to IC

25 IC50 and

IC75


75




50-S25 E25-S50 and E

25-S25) and


E50-S50 E50-S75 E75-S25 E70-S50 and E






4 Discussion
















5 Conclusions


Competing Interests


Acknowledgments


References




































































298 3027 345 44 1059 AntagonismIC75-IC50

298 0985 264 56 308 AntagonismIC75-IC25

298 0011 33 73 147 AntagonismIC50-IC75

0275 3027 284 41 371 AntagonismIC50-IC50

0275 0985 32 59 188 AntagonismIC50-IC25

0275 0011 4 32 0001 SynergismIC25-IC75

001 3027 225 66 185 AntagonismIC25-IC50

001 0985 167 71 031 SynergismIC25-IC25

001 0011 394 25 005 Synergism

2

minus2

minus2

2

SfEa

Comb

log (FaFu)

log (D)

(a)

1

05

00 05 1

A

B

E50-S25E25-S50E25-S25

(b)





25 IC50values




of SO4







0

20

40

60

80

100

120



sy

ncyt

ia co

unt o

r vira

l RN

A co

pies

E50S25 E25S50 E25S25


25and E

50are the SPs


and IC50


25and S



0

20

40

60

80

100

120


sy

ncyt

ia co

unt

Time (minutes)







0

20

40

60

80

100

120

Untreated 1 5

sy

ncyt

ia co

unt


















75 IC50 and IC







2and stained



and IC50


3 Results



500275120583gmL and









25 IC50 and

IC75


75correspond to IC

25 IC50 and

IC75


75




50-S25 E25-S50 and E

25-S25) and


E50-S50 E50-S75 E75-S25 E70-S50 and E






4 Discussion
















5 Conclusions


Competing Interests


Acknowledgments


References
































































0

20

40

60

80

100

120



sy

ncyt

ia co

unt o

r vira

l RN

A co

pies

E50S25 E25S50 E25S25


25and E

50are the SPs


and IC50


25and S



0

20

40

60

80

100

120


sy

ncyt

ia co

unt

Time (minutes)







0

20

40

60

80

100

120

Untreated 1 5

sy

ncyt

ia co

unt


















75 IC50 and IC







2and stained



and IC50


3 Results



500275120583gmL and









25 IC50 and

IC75


75correspond to IC

25 IC50 and

IC75


75




50-S25 E25-S50 and E

25-S25) and


E50-S50 E50-S75 E75-S25 E70-S50 and E






4 Discussion
















5 Conclusions


Competing Interests


Acknowledgments


References


































































75 IC50 and IC







2and stained



and IC50


3 Results



500275120583gmL and









25 IC50 and

IC75


75correspond to IC

25 IC50 and

IC75


75




50-S25 E25-S50 and E

25-S25) and


E50-S50 E50-S75 E75-S25 E70-S50 and E






4 Discussion
















5 Conclusions


Competing Interests


Acknowledgments


References


































































25 IC50 and

IC75


75correspond to IC

25 IC50 and

IC75


75




50-S25 E25-S50 and E

25-S25) and


E50-S50 E50-S75 E75-S25 E70-S50 and E






4 Discussion
















5 Conclusions


Competing Interests


Acknowledgments


References






































































5 Conclusions


Competing Interests


Acknowledgments


References

























































































































































Date post:	03-Dec-2023
Category:	Documents
Upload:	cinvestav
View:	0 times
Download:	0 times

Synergistic Effects of Sulfated Polysaccharides from Mexican Seaweeds against Measles Virus

Documents