In vitro antiviral activities of enzymatic hydrolysates extracted from byproducts of the Atlantic holothurian Cucumaria frondosa

Accepted Manuscript Title: IN VITRO ANTIVIRAL ACTIVITIES OF ENZYMATIC HYDROLYSATES EXTRACTED FROM BYPRODUCTS OF THE ATLANTIC HOLOTHURIAN CUCUMARIA FRONDOSA Author: Ludovic Tripoteau Gilles Bedoux Jacques Gagnon Nathalie Bourgougnon PII: DOI: Reference:

S1359-5113(15)00102-6 http://dx.doi.org/doi:10.1016/j.procbio.2015.02.012 PRBI 10360

To appear in:

Process Biochemistry

Received date: Revised date: Accepted date:

15-12-2014 10-2-2015 23-2-2015

Please cite this article as: Tripoteau L, Bedoux G, Gagnon J, Bourgougnon N, IN VITRO ANTIVIRAL ACTIVITIES OF ENZYMATIC HYDROLYSATES EXTRACTED FROM byproducts OF THE ATLANTIC HOLOTHURIAN CUCUMARIA FRONDOSA, Process Biochemistry (2015), http://dx.doi.org/10.1016/j.procbio.2015.02.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1

IN VITRO ANTIVIRAL ACTIVITIES OF ENZYMATIC HYDROLYSATES EXTRACTED FROM

2

BYPRODUCTS OF THE ATLANTIC HOLOTHURIAN CUCUMARIA FRONDOSA

3

Ludovic TRIPOTEAU

a,b

a

b

*, Gilles Bedoux , Jacques Gagnon , Nathalie Bourgougnon

a,

4 a. Laboratoire de Biotechnologie et Chimie Marines, EA3884, UBS, IUEM, F-56000 Vannes, France.

6

b. Coastal Zones Research Institute Inc., Shippagan, New Brunswick, CANADA

9

Highlights: -

Bioactive compounds were extracted from aquapharyngeal bulb of Cucumaria frondosa

-

Enzymatic extraction permitted the recovery of antiherpetic compounds on our model

-

Antiherpetic activities were highlighted on enzymatic hydrolysates of aquapharyngeal bulb

-

High molecular weight molecules were demonstrated as active against HSV-1

us

8

cr

7

an

10 11 12 13

d te Ac ce p

16

M

14 15

ip t

5

1 Page 1 of 39

16

IN VITRO ANTIVIRAL ACTIVITIES OF ENZYMATIC HYDROLYSATES EXTRACTED FROM

17

BYPRODUCTS OF THE ATLANTIC HOLOTHURIAN CUCUMARIA FRONDOSA

18 19

Ludovic TRIPOTEAU

a,b

a

b

*, Gilles Bedoux , Jacques Gagnon , Nathalie Bourgougnon

a,

ip t

20 a. Laboratoire de Biotechnologie et Chimie Marines, EA3884, UBS, IUEM, F-56000 Vannes, France.

22

b. Coastal Zones Research Institute Inc., Shippagan, New Brunswick, CANADA

cr

21

23 *Corresponding author: Ludovic Tripoteau

25

LBCM, Centre de recherche Yves Coppens. Campus de Tohannic – BP 573, 56017 Vannes Cedex. France.

26

Tel.: +33 2 97 01 71 22

27

E-mail address: [email protected]

an

us

24

28 Pr. Nathalie Bourgougnon.

30

LBCM, Centre de recherche Yves Coppens. Campus de Tohannic – BP 573, 56017 Vannes Cedex. France.

31

Tel.: +33 2 97 01 71 55

32

E-mail address: [email protected]

33

te

d

M

29

Dr. Jacques Gagnon

35

CZRI, PPM. 232B, avenue de l’église, Shippagan, NB, E8S 1J2 Canada

36

Tel.: 1 506 336 6603

37

E-mail address: [email protected]

38

Ac ce p

34

39

Dr. Gilles Bedoux

40

LBCM, Centre de recherche Yves Coppens. Campus de Tohannic – BP 573, 56017 Vannes Cedex. France.

41

Tel.: +33 2 97 01 71 57

42

E-mail address: [email protected]

43

2 Page 2 of 39

43

ABSTRACT

44 45

Herpes Simplex virus 1 (HSV-1), responsible for the common cold sore, can also lead to serious infections in immunocompromised people. Current antiviral chemotherapies face obstacles including

47

the toxicity of therapeutic molecules, interference with normal cellular metabolism, genetic variability

48

and the incurable nature of latent infections. Therefore, the search for new treatments is a public

49

health issue. Marine invertebrates have held great potential for finding novel antiviral compounds.

50

Little is known, about the antiviral activities of compounds isolated from holothurians. In New

51

Brunswick, holothurian is fished for its edible bodywall and muscle, but its processing generates high

52

amounts of byproducts. In vitro evaluation of the anti-HSV-1 activity by cell viability was performed on

53

nine hydrolysates obtained by enzyme-assisted extraction and four solvent extractions from

54

aquapharyngeal bulb and internal organs of Cucumaria frondosa at an MOI of 0.001 ID50/cells. After

55

72 h, four enzymatic hydrolysates from the aquapharyngeal bulb presented effective antiherpetic

56

activities (EC50=7.2–15.2 µg/mL) . After evaluation at a higher MOI (0.01 ID50/cells), the most efficient

57

extract (Papain hydrolysate) was fractionated to identify the active fraction. The fraction superior to

58

100 kDa showed the highest antiherpetic activity (EC50: 18.2 µg/mL). In conclusion, upgrading

59

byproducts of sea cucumber fisheries offers new sources of bioactive molecules.

62

cr

us

an

M

d

te

61

Key words: Cucumaria frondosa; enzymatic hydrolysis; Herpes simplex virus type 1; byproducts.

Ac ce p

60

ip t

46

3 Page 3 of 39

62 63

1. INTRODUCTION

64 Sea cucumbers are benthic marine invertebrates. Of the 1200 species living worldwide, over 60

66

are known to be harvested for human consumption [1]. One of the species that is considered new on

67

the market is the orange-footed sea cucumber Cucumaria frondosa (Gunnerus, 1767), which is

68

approximately 25–30 cm long and can reach 50 cm when relaxed. The Atlantic sea cucumber

69

Cucumaria frondosa is distributed abundantly in the north Atlantic from tide pools of the lower intertidal

70

zone and to down to 300 m depth [2].

us

cr

ip t

65

Cucumaria frondosa is harvested in the Fundy Bay, New Brunswick (NB), Canada to be then

72

transformed for the human consumption. The processing plant generates discards representing more

73

than half of the total amount of raw matter (600 t). The waste is mainly used as fish meal or fish oil.

74

Internal muscle bands and the dried body wall are the products from C. frondosa sold on Asian food

75

markets. The rest of the sea cucumbers, comprising the aquapharyngeal bulb, gut, gonad, and

76

respiratory tree, considered as byproducts, is rejected or underused. This biomass holds, however,

77

considerable potential to generate new biological compounds and can be turned into a commercially

78

viable business [3-4]. The process of enzymatic hydrolysis has been developed in order to transform

79

marine byproducts into marketable forms. By the use of specific enzymes, this process has the ability

80

to enhance the extraction of specific molecules with new properties (functional, biological, aromatic).

81

Consequently, the action of proteases on marine byproducts rich in proteins may lead to the

82

production of peptides, polypeptides and proteins with various biological activities [3].

M

d

te

Ac ce p

83

an

71

Sea cucumbers are a rich source of vitamins, minerals and bioactive components [5]. They are

84

used as healthy foods, traditional medicines and dietary supplements. For example, they have been

85

used for centuries, especially in Asia, as tonic foods and used in folk medicine to treat various ailments

86

[6]. Dried forms of sea cucumber are used as dietary supplements. More recently, sea cucumbers

87

have received increased attention because of their bioactive compounds such as triterpene glycosides

88

[7-9], chondroitin sulfates [10], sulfated polysaccharides [11], sterols [12], phenolics [13], peptides [14],

89

cerebrosides [15], and lectins [16].

90 91

Several studies conducted on holothurians have shown antifungal [17-19] and antibacterial [14; 20-22] activities. Little is known about antiviral activities [23-27], especially regarding Herpes Simplex

4 Page 4 of 39

Virus 1 (HSV-1) [28-31]. Herpes simplex Virus, a DNA enveloped virus, is a common human pathogen

93

with between 60 and up to 95% of certain populations infected with HSV-1, and between 6 and 50%

94

infected with Herpes Simplex Virus type 2 (HSV-2). The frequency of HSV-seropositive males was

95

significantly higher in populations infected with Human Immunodeficiency Virus (HIV). As the HIV

96

disease progresses, cutaneous and mucosal complications become more severe and occur in up to

97

92% of HIV-infected individuals. Medications available for systemic treatment of HSV are acyclovir,

98

famciclovir and valacyclovir. Acyclovir and penciclovir are available for topical use. In clinical practice,

99

treatment of primary HSV infections, while relieving symptoms and reducing the duration of viral

100

shedding, does not prevent recurrences. Moreover, resistance to acyclovir has been reported in

101

immuno-compromised patients. Therefore, there was a need to develop new therapeutic agents for

102

the management of HSV infections. Moreover, the majority of commercially effective molecules are

103

expected to join the public domain in 2015. The search for new treatments is therefore a major public

104

health issue.

cr

us

an

The purpose of this study was to evaluate the in vitro antiherpetic activities of compounds

M

105

ip t

92

obtained after enzyme-assisted extraction and solvent extractions of Cucumaria frondosa

107

(Dendrochirotida, Cucumariidae) byproducts with a bioguided fractionation.

108

te

d

106

2. MATERIALS AND METHODS

110

2.1 Specimen collection

116

Ac ce p

109

117

2.2 Sample processing

111

Specimens of Cucumaria frondosa were collected in Passamaquoddy Bay, Bay of Fundy, New

112

Brunswick, Canada, between January and March 2012. Sea cucumbers (15-30 cm) were placed for

113

acclimation at the Aquaculture Pavilion and Aquaculture Laboratories of the Coastal Zones Research

114

Institute, Shippagan, New Brunswick, in 900L aerated tanks supplied with filtered seawater (4°C)

115

pumped from Shippagan’s bay, prior to experiments.

118

Specimens were sacrificed, then the aquapharyngeal bulb (A.B.) and internal organs (I.O.)

119

were removed from the rest of the sea cucumbers to be separately crushed with a blender (LBC 15,

120

Waring Laboratory Science, USA) and placed at -20°C prior to further treatments. Some of these

5 Page 5 of 39

121

samples were freeze-dried and placed in a labeled glass vial at -20ºC prior to the sequential solvent

122

extraction.

123 Aqueous extraction assisted by enzymatic hydrolysis

125

For each enzymatic hydrolysis, 1.5 kg of ground biomass was placed in a bioreactor (BioFlo

ip t

124

110, New Brunswick Scientific, USA) with 1.5 L of distilled water. Nine different proteases were

127

separately used: Alcalase (0.1%, w/w ; Alcalase® 2.4L FG, Novozymes AS, Denmark) with a

128

declared activity of 2.4 AU/g (Anson Unit), Bromelain (0.1%, w/w ; Bio-Cat, Troy, VA, USA) with a

129

declared activity of 2000 GDU/g (Gelatin-Digesting Unit), Flavourzyme (0.1%, w/w ; Flavourzyme®

130

Novozymes AS, Denmark) with a declared activity of 500 LAPU/g (Leucine Amino Peptidase Unit),

131

Fungal Protease (0.1%, w/w ; Bio-Cat, Troy, VA, USA) with a declared activity of 400 000 HU/g

132

(Hemoglobin Unit), Neutral Protease (0.1%, w/w ; Bio-Cat, Troy, VA, USA) with a declared activity of

133

2 000 000 PC/g (Proteolytic unit), Papain (0.1%, w/w ; Bio-Cat, Troy, VA, USA) with a declared activity

134

of 6500 NFPU/g (Nuclear Floating Power Unit), Peptidase AM (Peptidase Aspergillus Melleus ; 0.1%,

135

w/w ; Bio-Cat, Troy, VA, USA) with a declared activity of 500 LAPU/g, Peptidase AO (Peptidase

136

Aspergillus Oryzae ; 0.1%, w/w ; Bio-Cat, Troy, VA, USA) with a declared activity of 500 LAPU/g and

137

Protamex (0.1%, w/w ; Protamex® Novozymes AS, Denmark) with a declared activity of 1.5 AU/g

138

during 6 h at pH 8.0 and 50°C. After hydrolysis, enzymes were then inactivated at 90°C for 15 min and

139

hydrolysates were centrifuged at 10,000xg for 20 min at 4°C to separate undigested residues and

140

solubilized compounds. The supernatants were sampled, freeze-dried and stored at -20°C prior to

141

cytotoxicity and antiviral evaluation [14]. The enzymatic hydrolysis process is summarized in Figure 1.

143 144

us

an

M

d

te

Ac ce p

142

cr

126

Sequential solvent extraction

Hexane (OmniSolv® Hexanes), acetone (OmniSolv® Acetone) and methanol (OmniSolv®

145

Methanol) are HPLC grade and were provided by Fisher Scientific (Canada). The sequential solvent

146

extraction is summarized in Figure 2.

147

Hexane extraction: 20 g of freeze-dried sample of raw matter were first submitted to hexane

148

extraction into a 500 mL Erlenmeyer flask, and 300 mL (15 mL/g) of hexane was added. The sample

149

was placed on a stir-plate, stirred for 30 min at room temperature and then placed in a sonicator for 15

150

min. After filtration (Filter grade #4; Aldrich, Canada), the soluble part and the first residue (R1) were

6 Page 6 of 39

151

separately collected. The solvent was removed from the filtrate using reduced pressure on the rotary

152

evaporator. The remaining solvent was removed by N2 stream in the heating block (40-50°C). The

153

filtrate was then freeze-dried and placed in a labeled glass vial at -20ºC prior to cytotoxicity and

154

antiviral evaluation.

158 159

ip t

157

using the same procedure according to the sequential solvent extraction described in Figure 2.

For hexane, acetone, and methanol extraction, extractions were repeated twice and the

cr

156

Acetone extraction of R1 and methanol extraction of the second residue (R2) were carried out

fractions obtained were pooled.

Water extraction: The dried filtered residue from methanol extraction (R3) was placed into a

us

155

500 mL Erlenmeyer flask; 200 mL of deionized water (10 mL/g) was added, and the mixture was

161

placed on a heating plate and heated to 80-90°C while being stirred. Once the temperature range was

162

reached, it was kept for 2 h. The mixture was removed from heat, cooled to room temperature, and

163

filtered by vacuum filtration. The filtrate was precipitate using ethanol (3V). After ethanol addition, the

164

flask was placed in an ice bath for 2 h. Once fully precipitated, the solids were separated by

165

centrifugation (2500 rpm /20 min). Solids were then freeze-dried and stored in a labeled glass vial at -

166

20°C prior to cytotoxicity and antiviral evaluation. The final residue was not included in the library of

167

extracts due to its low solubility.

170

Library of extracts

Ac ce p

169

te

168

d

M

an

160

All the extractions carried out on C. frondosa byproducts represented twenty eight different

171

extracts. For one type of byproduct, there were nine enzymatic hydrolysates, four extracts by

172

sequential solvent extraction (hexane, acetone, methanol and water extract) and one byproduct

173

without treatment (control).

174 175 176

2.3 Virus and cell lines

African green monkey kidney cells (Vero cell line n°ATCC CCL81) were grown in Eagle's

177

Minimum Essential Medium (MEM, Laboratory Eurobio, France) supplemented with 8% (v/v) fetal calf

178

serum (FCS, Eurobio, France), to which 1% (v/v) of PCS (penicillin 10 000U, colimycin 25 000 U,

179

streptomycin 10 mg, Sigma, France) was added. Cells were routinely passaged every 3 days.

7 Page 7 of 39

180

Virus stock of Herpes Simplex Virus type 1 was obtained from Pr. Agut, Laboratoire de

181

Dynamique, épidémiologie et traitement des infections virales de la Pitié Salpêtrière (Paris, France).

182

The virus stock was prepared by incubating Vero monolayers (75 cm culture flasks seeded with 350

183

000 cells / mL) at low multiplicity and incubating at 37°C, in a 95% air, 5% CO2 (v/v) atmosphere. Two

184

or three days after infection, the cultures were frozen and thawed twice before clearing the preparation

185

by centrifugation at low speed to remove cell debris. The resulting supernatant aliquot was stored at -

186

80°C until used. Virus titrations were performed by the Reed and Muench dilution method [32], using

187

10 wells on 96-wells microtiter plates per dilution. The virus titer was estimated from cytopathogenicity

188

and expressed as 50% infectious doses / mL (ID50/mL). The virus titer was evaluated at 2.10

189

ID50/mL.

7.57

us

cr

ip t

2

an

190

2.4 Cytotoxicity and antiviral activity of drugs by neutral red dye method

192

Screening for antiviral activities of the library of extracts

193

Dilutions of samples (50 µL) were prepared in Eagle’s MEM supplemented with 8% FCS and

M

191

distributed into the well plates of a 96-well microtest III tissue culture plate (Nunclon, Intermed,

195

France). A series included 10 assays ranging from extreme concentrations of extracts of 0.25 up to

196

250.00 µg/mL (4 wells/concentration). One hundred microliters of cellular suspension (3.5 x 10 Vero

197

cells/mL) in Eagle’s MEM containing 8% FCS were distributed into the wells and infected with HSV-1.

198

Fifty microliters of mock- and virus-infected cell suspensions at multiplicity of infection (MOI) of 0.001

199

ID50/cells were then transferred into each well containing the dilution compound and were incubated

200

for 3 days without change of the medium, at 37°C in 5% CO2. Cells and virus controls were run

201

simultaneously. A MOI of 0.01 ID50/cells was used for the second step of screening on the most

202

promising extracts with a kinetic of 48, 72 and 96 h of incubation.

te

5

Ac ce p

203

d

194

After microscopic examination to check the growth of the virus, 50 µL of neutral red dye (0.15% in

204

saline, pH 5.5) was added to each well, and the cultures were incubated for 45 min at 37°C [33].

205

Excess dye was removed by rinsing with phosphate buffered saline (PBS, pH 7.2; Biomérieux,

206

France), and the neutral red incorporated by the viable cells was eluted into 100 µL/well of citrate

207

ethanol buffer. After shaking the tray for a few minutes, whereby cell monolayers were completely

208

destroyed, the optical density (OD) of the wells was read in a multichannel spectrophotometer

209

(Packard Spectra Count , USA) at 540 nm. The OD was directly related to the percentages of viable

TM

8 Page 8 of 39

cells that were inversely proportional to the cytophathogenecity effect (CPE) ratio. The straight line of

211

the regression was determined for each assay and for each plate on the basis of cell controls (0%

212

CPE) and virus controls (100% CPE) [34]. The 50% cytotoxic concentration (CC50) of the test

213

compound was defined as the concentration that reduced the absorbance of mock-infected cells to

214

50% of that of controls. The 50% antiviral effective concentration (EC50) was expressed as the

215

concentration that achieved a 50% protection of virus-infected cells from the HSV-induced destruction.

216

The following formula was used to calculate the percentage protection:

cr

ip t

210

217 218

us

[ (ODt) HSV - (ODc) HSV ] / [ (ODc) MOCK - (ODc) HSV ] x 100 (%)

219

Where (ODt) HSV is the absorbance of the test sample, (ODc) HSV is the absorbance of the virus-

221

infected control (no compound), and (ODc) MOCK is the absorbance of the mock-infected control. The

222

ratio of (ODc) HSV to (ODc) MOCK is expressed as “% of control“. For all tests, the anti-herpetic

223

compound acyclovir [9-(2-hydroxyethoxymethyl) guanine] (Wellcome Foundation Ltd, U.K.) was used

224

as a reference.

M

an

220

d

225

Antiviral activities and kinetics of the efficient extracts

227

The freeze-dried extracts were tested at 10 different concentrations ranging from 0.25 to 250.00

te

226

µg/mL (4 wells/concentration) at an MOI of 0.01 ID50/cells. Cytotoxicity and antiviral activity of extracts

229

were evaluated by neutral red dye method after 48, 72, and 96 h of treatment.

230

Ac ce p

228

231

Antiviral bioguided fractionation by molecular mass fractionation

232

After fractionation of the most efficient extract with six different membranes (2 kDa, 5 kDa, 10 kDa,

233

30 kDa, 50 kDa and 100 kDa), the freeze-dried extracts were tested at 10 different concentrations

234

ranging from 0.25 to 250.00 µg/mL (4 wells/concentration) at an MOI of 0.01 ID50/cells after 72 h of

235

treatment.

236 237 238 239

2.5 Analysis of the biochemical compositions of the efficient extracts Analysis of biochemical compositions of the efficient extracts and the control were performed. Compositions of freeze-dried samples were determined as described below. Proteins, humidity, and

9 Page 9 of 39

240

ash were determined in accordance with Association of Official Analysts Chemists (A.O.A.C.) official

241

methods, 981.10, 952.08, and 938.08 respectively. Fats were determined according to the Folch

242

method [35] and neutral sugars according to the Dubois method [36].

243

The total free amino acid composition of freeze-dried hydrolysates and the control was determined after hydrolysis in 6 M HCl at 118°C for 18 h. Then, the samples were completely dried

245

under nitrogen atmosphere and diluted by adding 2.5 mL of water. The amino acid analysis was

246

performed according to the EZ faast™ (Phenomenex, France) procedure consisting of a solid phase

247

extraction step followed by derivatization and liquid/liquid extraction. The solid phase extraction was

248

performed via a sorbent packed tip that binds amino acids while allowing interfering compounds to

249

flow through. Amino acids on sorbent were then extracted into the sample and quickly transformed

250

with a reagent at room temperature in an aqueous solution. Derivatized amino acids concomitantly

251

migrated to the organic layer for additional separation from interfering compounds. An aliquot from the

252

organic phase was then analyzed on a GC-MS system. The mass spectrometer was an Agilent 5973

253

series network mass selective detector (Agilent, CA, U.S.A.). The column was a Zebron ZB-AAA GC

254

column (Phenomenex, France) for protein hydrolysates (max. temp. 320/340°C). The amino acids

255

were quantified by their response factor relative to the internal standard Norvaline added at a

256

concentration of 200 μmol/L [37].

te

257

259

2.6 Molecular mass fractionation

Ac ce p

258

d

M

an

us

cr

ip t

244

Molecular mass fractionation was prepared with ultrapure water at 1 mg/mL then fractionated

260

using centrifugal concentrators (Vivaspin 2, Sartorius, France) at different Molecular Weight Cut-Off

261

(MWCO). Centrifugations were carried out for 10 min at 12000 g for 2 kDa, 5 kDa, 10 kDa, 30 kDa and

262

50 kDa PES (Polyethersulfone) membranes and 9000 g for 100 kDa PES membrane. Seven samples

263

were obtained (<2 kDa, 2-5 kDa, 5-10 kDa, 10-30 kDa, 30-50 kDa, 50-100 kDa, >100 kDa) and freeze-

264

dried.

265 266

3. RESULTS AND DISCUSSION The dissection step showed that byproducts represented around 50% of the fresh weight of

267

the whole sea cucumber. The major parts were internal organs (15 ±3% of the fresh weight) and the

268

aquapharyngeal bulb (13 ±2% of the fresh weight)

10 Page 10 of 39

The hydrolysis parameters (ratio enzyme/substrate; pH and temperature) have been selected

270

as the mean of the optimal conditions declared by the manufacturers for all enzymes. Concerning the

271

time of hydrolysis, previous studies showed that the degree of hydrolysis (DH) of Alcalase, Bromelain,

272

Flavourzyme, Protamex, reached a threshold value after 6 h. Therefore, this time value was selected

273

for all enzyme assisted extraction experiments [14; 37-38].

274

ip t

269

3.1 Evaluation of antiviral activities of the library of extracts by cell viability

276

3.1.1

277

Before evaluation of antiviral activity, cytotoxicity of extracts on Vero cells was studied using the

cr

275

us

Evaluation of cytotoxicity

neutral red incorporation in the aim to detect lysosomal functionality. This method is currently used for

279

the screening of anti-HSV-1 molecules from marine organisms [28; 39-40]. An antiviral drug should be

280

active against the virus without inducing significant toxicity on the host cell. Therefore, the

281

concentration range of the extract that did not induce significant toxicity to the host cells was estimated

282

and the 50% cytotoxic concentration (CC50) was determined for each one.

M

283

an

278

Most of the extracts from aquapharyngeal bulb (A.B.) were well tolerated by Vero cells and their CC50 varied in the range of 74.5 and up to more than 500 µg/mL. Only two extracts obtained by

285

solvent extraction were cytotoxic (hexane extract with a CC50 = 81.5 µg/mL and methanol extract with

286

a CC50 = 74.5 µg/mL). None of the hydrolysates evaluated in our conditions induced visible changes in

287

cell morphology and cell density.

te

Ac ce p

288

d

284

The CC50 of the extracts from internal organs (I.O.) varied in the range of 23.3 and up to more

289

than 500 µg/mL. Half of them were cytotoxic in the concentrations tested. The evaluation of

290

cytotoxicity of solvent extracts showed that extracts obtained by highly polar solvent extraction were

291

toxic (methanol extract with a CC50 = 56.7 µg/mL and water extract with a CC50 = 96.3 µg/mL). The

292

majority of the enzymatic hydrolysates were also cytotoxic.

293

The cytotoxic extracts found in the A.B. and especially in I.O. could be explained by the presence

294

of triterpene glycosides [41]. Indeed, these molecules, essentially extracted by alcoholic solvent, have

295

already been described in holothurians species and demonstrated as cytotoxic agents. As an

296

example, the major triterpene glycoside Frondoside A, isolated by solvent extraction from the bodywall

297

of C. frondosa, showed cytotoxicity on THP-1 and HeLa tumor cell lines with IC50 values of 4.5 and

11 Page 11 of 39

298

2.1 μg/mL, respectively [42-43]. Furthermore, a recent study demonstrated the richness of triterpene

299

glycosides in holothurian internal organs [44].

300 301

3.1.2

302

Evaluation of antiviral activity against HSV-1 was examined using CPE inhibitory assay. The

ip t

Evaluation of antiviral activity

reference standard ACV conferred protection (EC50 = 0.2 µg/ml) against HSV-1 with a low percentage

304

of cell destruction. All 28 samples from the library of extracts were evaluated for their antiviral activity

305

at an MOI of 0.001 ID50/cells by cell viability. Results are summarized in Table 1. After 72 h of

306

treatment, seven of nine hydrolysates of the aquapharyngeal exhibited antiherpetic activities with EC50

307

between 7.2 and 170.9 µg/mL. Only the water extract of the aquapharyngeal bulb of C. frondosa

308

demonstrated an antiherpetic activity with an EC50 of 140 µg/mL and the aquapharyngeal bulb without

309

treatment (control) showed an activity (EC50: 75.0 µg/mL). Among the extracts from internal organs,

310

only the control extract showed an antiviral activity (EC50 = 72.5 µg/mL).

us

an

Of 28 extracts evaluated, 10 extracts exhibited antiherpetic activities. The extracts from the

M

311

cr

303

aquapharyngeal bulb showed better antiviral activities in comparison to internal organs extracts. This

313

screening also demonstrated that antiviral compounds seemed to be highly polar. Indeed only the

314

water extract and hydrolysates of the aquapharyngeal bulb possessed antiviral activities without

315

cytotoxicity. In a review, Zhong et al. [31] showed that of 188 plant, animal, or microorganism extracts,

316

162 (86.2%) with inhibitory activity against HSV have been obtained from highly polar solvents such as

317

aqueous solvents, methanol, ethanol, and acetic acid. It appears that most anti-HSV molecules are

318

soluble in polar solvents, including polyphenols and flavones with multiple hydroxyl groups, and this

319

solubility correlates with the reported compound types that have anti-HSV activity. Consequently, it

320

seems relevant to focus on aqueous fractions for the search of antiherpetic compounds. For example,

321

the use of enzymatic hydrolysis with proteases in highly controlled conditions permits the release of

322

bioactive peptides, even though they were inactive when included in the amino sequence of the parent

323

protein [45].

324

Ac ce p

te

d

312

Little is known about antiviral activities from holothurians. Tsushima et al. [23] showed that

325

cucumariaxanthin C from Cucumaria japonica exhibited an inhibitory effect on Epstein-Barr virus

326

(Herpesviridae) activation in a short-term in vitro assay. On the same species, Grishin et al. [24]

327

demonstrated that triterpene glycosides, and especially cucumarioside A2-2, can inhibit the cytopathic

12 Page 12 of 39

effect induced by Vesicular Stomatitis Virus (VSV), Poliovirus, and other viruses in cell culture.

329

According to Rodriguez et al. [25], holothurinosides from Holothuria forskalii can also inhibit the

330

cytopathic effect induced by VSV. Recently, it has been shown that glycosaminoglycan extracted from

331

Thelenota ananas may possess great potential to be further developed as a new Human

332

Immunodeficiency Virus (HIV) entry inhibitor [26-27].

333

ip t

328

Liouvillosides A and B extracted from a whole specimen of Staurocucumis liouvilei [28],

thyonosides A and B from Thyone aurea [29], and a water extract of the body wall of Holothuria sp.

335

[30], have been shown to possess antiviral activities against Herpes simplex virus type 1. In particular,

336

triterpene glycosides from Staurocucumis liouvilei showed a virucidal effect at concentrations below 10

337

µg/mL, and the water extract of Holothuria sp. exhibited an intracellular antiviral activity with an IC50

338

value of 189.9 µg/mL.

an

us

cr

334

In comparison to solvent extraction, enzymatic hydrolysis recovers antiherpetic compounds from

340

the aquapharyngeal bulb. Moreover, this process is a softer technique without the solvent elimination

341

step and can allow a valorization of the entire raw matter, which is useful in a global context of marine

342

biological resources overexploitation [3-4; 46]. The most efficient extracts selected were obtained by

343

using four different proteases which have a common mechanism of action. Indeed, these enzymes

344

have endopeptidase activities resulting mainly in the production of polypeptides or proteins, compared

345

to exopeptidases which mainly promote the production of free amino acids.

d

te

Ac ce p

346

M

339

347

3.2 Biochemical composition of the effective extracts

348

The most effective extracts were selected according to EC50 values. In that way, four hydrolysates

349

from the aquapharyngeal bulb byproducts were chosen to continue the study: Papain hydrolysate

350

(EC50 = 7.2 µg/mL), Protamex hydrolysate (EC50 = 8.3 µg/mL), Neutral Protease hydrolysate (EC50 =

351

12.5 µg/mL) and Alcalase hydrolysate (EC50 = 15.2 µg/mL).

352

Analysis of the biochemical composition was carried out on the four aquapharyngeal bulb

353

hydrolysates and on the control (aquapharyngeal bulb without treatment). Results are presented in

354

Table 2. Results showed no significant differences between the extracts, except for the control.

355

Indeed, a small decrease of the lipid content and increase of the protein content were observed.

356

Compositions of the extracts showed a high protein content with an average of 54.9% (dry weight).

357

These results are in accordance with the previous study of Mamelona et al. [47] on hydrolysis of the

13 Page 13 of 39

358

viscera of the same animal with an average of 55%. Other studies also report that enzymatic

359

hydrolysis does not significantly change the protein percentage, but rather liberates small peptides

360

[48].

361

Total amino acids assays showed no significant differences between the four hydrolysates selected. The mean amino acid profile is presented in three groups of amino acids according to their

363

abundance. Values are expressed as a percentage of the total amino acids assayed: inferior to 4%:

364

Histidine (1.5%), Methionine (1.5%), Phenylalanine (3.5%), Lysine (2.3%), Hydroxyproline (3.5%), and

365

Tyrosine (2.0%); between 4 and 8%: Alanine (7.8%), Threonine (6.2%), Valine (5.1%), Isoleucine

366

(4.7%), Leucine (6.1%), and Serine (7.5%); superior to 8%: Aspartic acid (10.7%), Glutamic acid

367

(12.4%), Glycine (16.5%), and Proline (8.6%). These values are in accordance with the previous study

368

of Zhong et al. [49] on a biochemical comparison of the body wall of C. frondosa with or without

369

internal organs, on fresh or rehydrated matter, where major amino acids were the same as those

370

found in the extracts evaluated in our study. However, the total amino acid profile of the control was

371

slightly different. Indeed, the control was enriched in Leucine (8.2%), Phenylalanine (5.0%), Glycine

372

(25.8%) and Alanine (10.4%) but depleted in Aspartic acid (5.1%), Glutamic acid (5.7%) and Serine

373

(5.5%). In our conditions, the enzymatic hydrolysis process with proteases seems to recover amino

374

acids with acidic side chains and strongly loses the neutral amino acid Glycine.

te

d

M

an

us

cr

ip t

362

Lipid contents (average of 6.6%) are similar between hydrolysates, but twice as high as the

376

control. This difference can be explained by the ability of the enzymatic hydrolysis process to release

377

lipids in the soluble part. Dumay et al. [50] demonstrated on sardine byproducts that enzymatic

378

proteolysis can be used for high lipid recovery.

379

Ac ce p

375

Ash contents are similar with an average of 25.6%. Available data on the ash content of the

380

holothurian aquapharyngeal bulb are quite rare. Results found for C. frondosa are higher than the data

381

found for the viscera content of the same species (7-11%: [47]) and lower than the data found for the

382

body walls of other shallow-water holothuroids with an average of 52% [51]. These variations in ash

383

content can be explained by the structure of their internal anatomy, which is more or less rich in calcite

384

depending on the part of the animal considered [52].

385

Neutral sugar contents are around 1.8% for Alcalase, Papain and Protamex hydrolysates and are

386

similar for those found on the viscera of other shallow-water sea cucumbers [51] but slightly higher for

387

Neutral Protease hydrolysate with 3.1%.

14 Page 14 of 39

388

Studies on natural products with anti-HSV activities reported that most of these compounds targeted the viral attachment and the entry stage. The other major mechanism of action reported was

390

the DNA replication by inhibition of the HSV-encoded DNA polymerase. This latter is the mechanism

391

of action used by the anti-HSV-1 reference drug, acyclovir (ACV). The other mechanisms of action

392

described in natural compounds with antiviral activities target the inhibition of the virion assembly and

393

output, the cellular proteins and the promotion of the immune response [22].

ip t

389

The four selected hydrolysates showed a high protein content. Marine peptides and nucleosides

395

have already shown antiherpetic activities by inhibiting the viral protein synthesis of HSV-1 [53]. The

396

nucleoside spongouridine was used for the synthesis of the marine adenine arabinoside which is

397

rapidly converted into its triphosphate form, leading to the inhibition of the viral DNA polymerase and

398

DNA synthesis of HSV in infected cells [54]. Besides the antiviral activities of these compounds, other

399

chemical classes have also shown such activity. Fucosylated chondroitin sulfate (FuCS) found in sea

400

cucumbers from different species has revealed interesting bioactivities. Among these activities, FuCS

401

extracted from the holothurian T. ananas has shown highly effective antiviral activity against HIV

402

strains. FuCS showed a capacity to block the virus entry and replication on cell models and clinic

403

isolates [10].

us

an

M

d

Sulfated polysaccharides are known to be active on HSV. These compounds are able to inhibit

te

404

cr

394

viral infection by preventing adsorption of the virus into the host cells and/or by inhibiting the

406

production of new viral particles inside the host cells [55]. Natural carrageenans were identified as

407

potent and selective inhibitors of HSV-1 and HSV-2. Studies suggested that the main target for

408

antiviral action of the carrageenans was based on the virus adsorption, whereas no effect on virus

409

internalization or early or later protein synthesis was detected [56]. Alkaloids showed potent anti-HSV-

410

1 activity by impairing the virus penetration and inhibiting immediate early protein synthesis [57].

411

Glycolipids showed antiviral activity against HSV-1 and HSV-2 [58]. The antiviral action might be due

412

to the binding of the virus glycoprotein to the glycolipid leading to an irreversible denaturation that

413

blocks viral infection [59].

414

Ac ce p

405

Many marine compounds of different chemical classes with a broad range of mechanism of action

415

have been described as effective anti-HSV-1 agents. However, according to the biochemical

416

composition of the selected extracts and the differences in activity values modulated by the nature of

417

the enzymes used, the results suggested that the antiviral compounds might be from protein origin.

15 Page 15 of 39

418 419

3.3 Antiviral activities and kinetics of the effective extracts from aquapharyngeal bulb byproducts

420 421

After 48, 72, and 96 h of treatment, viability assay showed no cytotoxic effect of the compounds on Vero cells in the range of concentrations assayed for all four compounds.

422

ip t

The four hydrolysates showed anti-HSV-1 activities for an MOI of 0.01 ID50/cells. Results are summarized in Table 3 in comparison with the reference drug (ACV). After 48 h of incubation, the four

424

extracts exhibited an important antiviral activity: Papain hydrolysate (EC50 = 15.9 µg/mL) > Alcalase

425

hydrolysate (EC50 = 20.4 µg/mL) > Neutral protease hydrolysate (EC50 = 26.9 µg/mL) > Protamex

426

hydrolysate (EC50 = 52.2 µg/mL). After 72 h of incubation, the four extracts still showed an important

427

activity: Papain hydrolysate (EC50 = 25.2 µg/mL) > Neutral protease hydrolysate (EC50 = 36.9 µg/mL) >

428

Alcalase hydrolysate (EC50 = 43.9 µg/mL) > Protamex hydrolysate (EC50 = 102.9 µg/mL). After 96 h of

429

incubation, three of four hydrolysates remained active against HSV-1: Papain hydrolysate (EC50 =

430

44.3 µg/mL) > Protamex hydrolysate (EC50 = 144.9 µg/mL) > Alcalase hydrolysate (EC50 = 150.4

431

µg/mL).

us

an

M

432

cr

423

Extracts seemed to act differently depending on the time of incubation, and the evolution of activities did not follow the same trend. This is well illustrated with a neutral protease hydrolysate

434

activity that, during 48 and 72 h of incubation, possessed interesting antiviral activities but completely

435

lost its activity after 96 h. Antiherpetic compounds from the extracts assayed seemed to act on early

436

cycles of replication but after 96 h faced obstacles with the release of newly formed virions [60].

te

Ac ce p

437

d

433

Results showed that Protamex hydrolysate was the second most efficient extract (EC50 = 8.3

438

µg/mL) at an MOI of 0.001 ID50/cells after 72 h of incubation. At an MOI of 0.01 ID50/cells, it became

439

less active (EC50 = 102.9 µg/mL) with the same concentrations assayed after the same period of

440

incubation. This observation can be explained by the fact that continuous replication of every virus

441

particle will produce an aggressive virus population. A culture infected with high virus multiplicity may

442

therefore need higher concentrations of antiviral compounds for viral inhibition than cells infected with

443

low multiplicity [61].

444

Among all these observations, the Papain hydrolysate remained the more active extract.

445

According to the specificities of each enzyme, a trend seemed to be emerging. Byproducts hydrolysed

446

with Alcalase and mostly with Papain possessed the highest antiviral activities. Papain, and Alcalase

16 Page 16 of 39

447

at a lower rate, have broad specificities to cleave peptide bonds but preferences for amino acids

448

bearing a large hydrophobic side chain compared to Neutral Protease and Protamex enzymes.

449

Ghanbari et al. [14] showed an antibacterial activity of enzymatic hydrolysates from the sea cucumber Actinopyga lecanora. The antibacterial activity was not only correlated to the size, the

451

molecular weight and the degree of hydrolysis, but also on the hydrophobicity of the resulting

452

peptides. Furthermore, Papain hydrolysates have already shown to possess activities such as

453

antioxidant activities [62] and ACE inhibition [63]

cr

ip t

450

454

On the same raw matter, the process of enzymatic hydrolysis with proteases induced different extracts with different activities against HSV-1; therefore, the nature of the enzyme used seems to be

456

a major parameter to modify the antiviral activity. The enzyme specificity is important to peptide

457

functionality because it strongly influences the molecular size and hydrophobicity of the hydrolysate

458

[3].

an

us

455

459

461

3.4 Antiviral activity by bioguided molecular mass fractionation

M

460

After 72 h of treatment, a viability assay showing no cytotoxic effect of the compounds was observed on the Vero cells in the range of concentrations assayed for all seven compounds. Results

463

are presented in Table 4. Out of seven extracts tested, three extracts showed an effective antiviral

464

activity with EC50 of 55.1, 38.1, and 18.2 µg/mL at an MOI of 0.01 ID50/cells for the fractions 30-50

465

kDa, 50-100 kDa and superior to 100 kDa respectively. Other extracts were not active against HSV-1

466

in the range of concentrations tested. Active molecules were present on the last fractions, and activity

467

increased with the size of the cutoff from 30 kDa to over 100 kDa. It is assumed that the compounds

468

responsible for the antiherpetic activity are in an aqueous phase superior to 100 kDa due to the

469

highest activity found for the last fraction. High molecular weight compounds have already been

470

demonstrated as antiherpetic agents [64]. Indeed, sulfated polysaccharides isolated from two red

471

algae with an average molecular weight of 308 and 360 kDa were shown to exert in vitro anti-HSV-1

472

activity. They were capable of inhibiting the in vitro replication of HSV-1 on Vero cells with EC50 values

473

of 4.1 and 17.2 μg/mL [64]. These values have been obtained after an evaluation of 48 h at an MOI of

474

0.001 ID50/cells, ten times lower than the MOI used in the antiviral evaluation by bioguided molecular

475

mass fractionation (0.01 ID50/cells after an evaluation of 72 h). Recently, sulfated polysaccharides

Ac ce p

te

d

462

17 Page 17 of 39

476

isolated from the bodywall of C. frondosa showed biological effects. Indeed, they have demonstrated

477

in vitro the ability to act on the maturation of dendritic cells [11].

478

Generally, due to the poor permeability through membranes, molecular size, physical and chemical instability affect the bioavailability of peptides and more specifically proteins. In vivo, peptides

480

with two or six amino acids reach their target site more easily than a whole protein. However, some

481

marine proteins showed biological activities in animal models. Lectins are carbohydrate-binding

482

proteins found in a wide range of organisms even in holothurians. Bulgakov et al. [65] described a 44-

483

kDa, C-type mannan-binding lectin consisting of two identical subunits isolated from the coelomic fluid

484

of the holothurian Cucumaria japonica. A 400 kDa lectin named SPL-1 and a 68 kDa lectin named

485

SPL-2 were extracted from the holothurian species Stichopus (Apostichopus) japonicus. Inhibition of

486

HSV infections was demonstrated with lectins from plants which are able to recognize and bind to

487

polysaccharides or glycoproteins on cell surface in order to agglomerate the cells. The presence of

488

lectins in holothurians species, the wide range of molecular mass described and their bioactivities

489

might be a possible explanation for the antiviral compounds found in our study. Furthermore, the

490

presence of oligomers with different subunit sizes could also explain the antiviral activities of the two

491

fractions lower than 100 kDa. Glycosylation is essential to the virus for its cellular transport and

492

binding to cellular receptors, in this way, by binding to the glycosylated viral-envelope, lectins could act

493

as entry blockers.

cr

us

an

M

d

te

Protein hydrolysates have been investigated as an alternative for the upgrading of marine

Ac ce p

494

ip t

479

495

byproducts to deal with the significant quantities of waste generated annually by the processing

496

industries [66]. These hydrolysates have broadly contributed to the discovery of new compounds with

497

activities that include the following: antioxidative, anticancer, calcium-binding peptides, appetite

498

suppression, anticoagulant, immunostimulant, hypocholesterolemic, hormone-regulating, and

499

antimicrobial and antiviral [38]. In our study, the aqueous fraction obtained by enzymatic proteolysis

500

from Cucumaria frondosa seems to be a promising extract for the search of active compounds against

501

Herpes Simplex virus.

502 503

In conclusion, this study confirms that, in comparison to solvent extraction, the enzymatic

504

extraction appears to be a promising softer technique for the recovery of industrially important

505

metabolites as antiviral compounds in a time-saving fashion, with less solvent and at lower cost. The

18 Page 18 of 39

specificity of enzymes used was also shown with different levels of response for the activity evaluated.

507

The aquapharyngeal bulb, for a long time considered as a byproduct with low value added, represents

508

a non-negligible part of the animal in terms of proportion but also mainly for its biochemical

509

composition. To our knowledge, no studies have demonstrated the presence of active molecules

510

against HSV-1 from C. frondosa byproducts. The results obtained in this study make the high

511

molecular weight extract from the Papain hydrolysate of the aquapharyngeal bulb from the sea

512

cucumber Cucumaria frondosa a great potential source of antiviral agent against Herpes Simplex virus

513

1. Literature suggested that lectins might be a possible explanation for the nature of the antiviral

514

compounds retrieved in the high molecular weight extract with a potential mechanism of action as

515

entry blockers. This extract could provide high added value to an underestimated biomass with the

516

added bonus of being environmentally friendly through the use of enzymes instead of solvents. These

517

compounds are currently undergoing purification in order to identify the active molecules as well as

518

their mechanism of action.

an

us

cr

ip t

506

M

519 520

d

522

ACKNOWLEDGMENTS This research was supported by the Atlantic Canada Opportunities Agency (Atlantic Innovation

te

521

Fund grant 193594). The authors thank Dr J.P. Bergé and C. Donnay-Moreno from the laboratory

524

BIORAFhe, IFREMER, Nantes, FRANCE for the amino acids analysis.

Ac ce p

523

19 Page 19 of 39

REFERENCES

526

[1] Purcell, S.W. Managing sea cucumber fisheries with an ecosystem approach. Edited/compiled by

527

Lovatelli, A.; M. Vasconcellos and Y. Yimin. FAO Fisheries and Aquaculture Technical Paper. No. 520.

528

Rome, FAO. 2010. 157p

ip t

525

529

[2] Hamel JF, Mercier A. Precautionary management of Cucumaria frondosa in Newfoundland and

531

Labrador, Canada in Sea cucumbers. A global review of fisheries and trade. FAO. Fisheries and

532

Aquaculture Technical Paper 2008;516:pp. 293–306.

us

cr

530

an

533

[3] Kristinsson HG, Rasco BA. Fish Protein Hydrolysates: Production, Biochemical, and Functional

535

Properties. Critical Reviews in Food Science and Nutrition 2000;40:43-81.

M

534

536

[4] Guerard F, Sumaya-Martinez MT, Laroque D, Chabeaud A, Dufossé L. Optimization of free radical

538

scavenging activity by response surface methodology in the hydrolysis of shrimp processing discards.

539

Process Biochem 2007;42:1486-1491.

te

Ac ce p

540

d

537

541

[5] Bordbar S, Anwar F, Saari N. High-Value Components and Bioactives from Sea Cucumbers for

542

Functional Foods-A Review. Mar Drugs 2011;9:1761-1805.

543 544

[6] Kiew PL, Don MM. Jewel of the seabed: sea cucumbers as nutritional and drug candidates.

545

International Journal of Food Sciences and Nutrition. 2012;63:616-36

546 547

[7] Kerr RG, Chen Z. In Vivo and In Vitro Biosynthesis of Saponins in Sea Cucumbers. Journal of

548

Natural Products 1995;58:172-176.

20 Page 20 of 39

549 [8] Kalinin VI, Aminin DL, Avilov SA, Silchenko AS, Stonik VA. Triterpene glycosides from sea

551

cucucmbers (holothurioidea, echinodermata). Biological activities and functions. In: R. Atta ur editor.

552

Studies in Natural Products Chemistry vol. Volume 35: Elsevier; 2008. p. 135-196.

ip t

550

553

[9] Aminin DL, Chaykina EL, Agafonova IG, Avilov SA, Kalinin VI, Stonik VA. Antitumor activity of the

555

immunomodulatory lead Cumaside. International Immunopharmacology 2010; 10:648-654.

us

cr

554

556

[10] Pomin V. Holothurian Fucosylated Chondroitin Sulfate. Mar Drugs 2014;12:232-254.

an

557 558

[11] Kale V, Freysdottir J, Paulsen BS, Friðjónsson ÓH, Óli Hreggviðsson G, Omarsdottir S. Sulphated

560

polysaccharide from the sea cucumber Cucumaria frondosa affect maturation of human dendritic cells

561

and their activation of allogeneic CD4(+) T cells in vitro. Bioactive Carbohydrates and Dietary Fibre.

562

2013;2:108-17

564 565

d

te Ac ce p

563

M

559

[12] Gupta KC, Scheuer PJ. Echinoderm sterols. Tetrahedron 1968;24:5831-5837.

566

[13] Mamelona J, Pelletier E, Girard-Lalancette K, Legault J, Karboune S, Kermasha S. Quantification

567

of phenolic contents and antioxidant capacity of Atlantic sea cucumber, Cucumaria frondosa. Food

568

Chemistry 2007;104:1040-1047.

569 570

[14] Ghanbari R, Ebrahimpour A, Abdul-Hamid A, Ismail A, Saari N. Actinopyga lecanora Hydrolysates

571

as Natural Antibacterial Agents. Int J Mol Sci 2012;13:16796-16811.

572

21 Page 21 of 39

573

[15] La M-P, Shao J-J, Jiao J, Yi Y-H. Three cerebrosides from the sea cucumber Cucumaria

574

frondosa. Chinese Journal of Natural Medicines 2012;10:105-109.

575 [16] Gowda NM, Goswami U, Khan MI. Purification and characterization of a T-antigen specific lectin

577

from the coelomic fluid of a marine invertebrate, sea cucumber (Holothuria scabra). Fish & Shellfish

578

Immunology 2008;24:450-458.

cr

ip t

576

us

579 580

an

581 582

586 587 588 589 590

d te

585

.

Ac ce p

584

M

583

591 592

[17] Yuan W-H, Yi Y, Tang H-F, Liu B-S, Wang Z-L, Sun G-Q, Zhang W, Li L, Sun P. Antifungal

593

Triterpene Glycosides from the Sea Cucumber Bohadschia marmorata. Planta Med 2009;75:168-173.

594

22 Page 22 of 39

595

[18] Mokhlesi A, Saeidnia S, Gohari AR, Shahverdi AR, Nasrolahi A, Farahani F, Khoshnood R,

596

Es'haghi N. Biological Activities of the Sea Cucumber Holothuria leucospilota. Asian Journal of Animal

597

& Veterinary Advances 2012;7:243.

ip t

598 [19] Wang Z, Zhang H, Yuan W, Gong W, Tang H, Liu B, Krohn K, Li L, Yi Y, Zhang W. Antifungal

600

nortriterpene and triterpene glycosides from the sea cucumber Apostichopus japonicus Selenka. Food

601

Chemistry 2012;132:295-300.

cr

599

us

602

an

603 604

M

605 606

[20] Beauregard K, Truong N, Zhang H, Lin W, Beck G. The Detection and Isolation of a Novel

608

Antimicrobial Peptide from the Echinoderm Cucumaria Frondosa. In: G. Beck, M. Sugumaran,E.

609

Cooper editors. Phylogenetic Perspectives on the Vertebrate Immune System vol. 484: Springer US;

610

2001. p. 55-62.

te

Ac ce p

611

d

607

612

[21] Haug T, Kjuul AK, Styrvold OB, Sandsdalen E, Olsen OM, Stensvag K. Antibacterial activity in

613

Strongylocentrotus droebachiensis (Echinoidea), Cucumaria frondosa (Holothuroidea), and Asterias

614

rubens (Asteroidea). Journal of Invertebrate Pathology 2002;81:94-102.

615 616

[22] Schillaci D, Cusimano M, Cunsolo V, Saletti R, Russo D, Vazzana M, Vitale M, Arizza V. Immune

617

mediators of sea-cucumber Holothuria tubulosa (Echinodermata) as source of novel antimicrobial and

618

anti-staphylococcal biofilm agents. AMB Express 2013;3(1):35.

619

23 Page 23 of 39

620

[23] Tsushima M, Fujiwara Y, Matsuno T. Novel marine di-Z-carotenoids: Cucumariaxanthins A, B,

621

and C from the sea cucumber Cucumaria japonica. Journal of Natural Products 1996;59:30-34.

622 [24] Grishin YI, Morozov E, Avilov SA. preliminary studies of antiviral activities of triterpene glycosides

624

from holothurians. 8th conference of young scientist on organic and bioorganic chemistry. 1991. p.

625

181.

cr

ip t

623

us

626

[25] Rodriguez J, Castro R, Riguera R. Holothurinosides: New antitumour non sulphated triterpenoid

628

glycosides from the sea cucumber holothuria forskalii. Tetrahedron 1991;47:4753-4762.

an

627

629

[26] Huang N, Wu M-Y, Zheng C-B, Zhu L, Zhao J-H, Zheng Y-T. The depolymerized fucosylated

631

chondroitin sulfate from sea cucumber potently inhibits HIV replication via interfering with virus entry.

632

Carbohyd Res 2013;380:64-69.

d te

633

M

630

[27] Lian W, Wu M, Huang N, Gao N, Xiao C, Li Z, Zhang Z, Zheng Y, Peng W, Zhao J. Anti-HIV-1

635

activity and structure–activity-relationship study of a fucosylated glycosaminoglycan from an

636

echinoderm by targeting the conserved CD4 induced epitope. Biochimica et Biophysica Acta (BBA) -

637

General Subjects 2013;1830:4681-4691.

638

Ac ce p

634

639

[28] Maier MS, Roccatagliata AJ, Kuriss A, Chludil H, Seldes AM, Pujol CA, Damonte EB. Two new

640

cytotoxic and virucidal trisulfated triterpene glycosides from the antarctic sea cucumber Staurocucumis

641

liouvillei. Journal of Natural Products 2001;64:732-736.

642 643

[29] Bonnard I, Rinehart KL. Thyonosides A and B, two new saponins isolated from the holothurian

644

Thyone aurea. Tetrahedron 2004;60:2987-2992.

24 Page 24 of 39

645 [30] Farshadpour F, Gharibi S, Taherzadeh M, Amirinejad R, Taherkhani R, A. H, K. Z. Antiviral

647

activity of Holothuria sp. a sea cucumber against herpes simplex virus type 1 (HSV-1). Eur Rev Med

648

Pharmacol Sci 2014;Vol. 18 - N. 3:333-337.

ip t

646

649

[31] Zhong M-G, Xiang Y-F, Qiu X-X, Liu Z, Kitazato K, Wang Y-F. Natural products as a source of

651

anti-herpes simplex virus agents. RSC Advances 2013;3:313-328.

us

cr

650

652

[32] Reed LJ, Muench H. A simple method of estimating fifty percent endpoints. American Journal of

654

Epidemiology 1938;27:493-497.

an

653

M

655

[33] McLaren C, Ellis MN, G.A. H. A colorimetric assay for the measurement of the sensitivity of

657

herpes simplex viruses to antiviral agents. Antiviral Research 1983;3:223-234.

te

658

d

656

[34] Langlois M, Allard JP, Nugier F, Aymard M. A rapid and automated colorimetric assay for

660

evaluating the sensitivity of herpes. J Biol Stand 1986;14:201-211.

661

Ac ce p

659

662

[35] Folch J, Lees M, Sloane SG. A simple method for the isolation and purification of total lipides from

663

animal. J Biol Chem 1957 May;226(1):497-509 1957.

664 665

[36] Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric Method for Determination of

666

Sugars and Related Substances. Analytical Chemistry 1956;28:350-356.

667

25 Page 25 of 39

668

[37] Soufi-Kechaou E, Jaouen P, Ben Amar R, Berge J-P. Influence of hydrolysis time on protein

669

recovery and amino acid composition of hydrolysates from Sepia officinalis viscera. Science Research

670

Reporter 2012;2:115-129.

ip t

671 [38] Dumay J. Extraction de lipides en voie aqueuse par bioréacteur enzymatique combine à

673

l'ultrafiltration: application à la valorisation de co-produits de poisson (Sardina pilchardus). Université

674

de Nantes; PhD Thesis; 2006.

cr

672

us

675

[39] Bergé JP, Bourgougnon N, Alban S, Pojer F, Billaudel S, Chermann JC, et al. Antiviral and

677

Anticoagulant Activities of a Water-Soluble Fraction of the Marine Diatom Haslea ostrearia. Planta

678

Med. 1999;65:604-9.

an

676

M

679

[40] Olicard C, Renault T, Torhy C, Benmansour A, Bourgougnon N. Putative antiviral activity in

681

hemolymph from adult Pacific oysters, Crassostrea gigas. Antiviral Research. 2005;66:147-52.

te

682

d

680

[41] Findlay JA, Yayli N, Radics L. Novel Sulfated Oligosaccharides from the Sea Cucumber

684

Cucumaria Frondosa. Journal of Natural Products. 1992;55:93-101.

685

Ac ce p

683

686

[42] Silchenko AS, Avilov SA, Kalinin VI, Kalinovsky AI, Dmitrenok PS, Fedorov SN, et al. Constituents

687

of the sea cucumber Cucumaria okhotensis. Structures of okhotosides B-1-B-3 and cytotoxic activities

688

of some glycosides from this species. Journal of Natural Products. 2008;71:351-6.

689 690

[43] Jin JO, Shastina VV, Shin SW, Xu Q, Park JI, Rasskazov VA, et al. Differential effects of

691

triterpene glycosides, frondoside A and cucumarioside A(2)-2 isolated from sea cucumbers on

692

caspase activation and apoptosis of human leukemia cells. Febs Lett. 2009;583:697-702.

26 Page 26 of 39

693 694

[44] Bahrami Y, Zhang W, Chataway T, Franco C. Structural Elucidation of Novel Saponins in the Sea

695

Cucumber Holothuria lessoni. Marine Drugs. 2014;12:4439-73.

ip t

696 [45] Batista I. Biological Activities of Fish-protein Hydrolysates. Marine Proteins and Peptides: John

698

Wiley & Sons, Ltd; 2013. p. 111-138.

cr

697

us

699 700

an

701

[46] Thorkelsson G, Sigurgisladottir S, Geirsdottir M, Jóhannsson R, Guerard F, Chabeaud A,

703

Bourseau P, Vandanjon L, Jaouen P, Chaplain-Derouiniot M, Fouchereau-Peron M, Martinez Alvarez

704

O, Le Gal Y, Ravallec-Ple R, Picot L, Berge J-P, Delannoy C, Jakobsen G, Johansson I, Batista I. Mild

705

processing techniques and development of functional marine protein and peptide ingredients. In: B. T.

706

editor. Improving seafood products for the consumer; 2008.

te

d

M

702

Ac ce p

707 708

[47] Mamelona J, Saint-Louis R, Pelletier E. Nutritional composition and antioxidant properties of

709

protein hydrolysates prepared from echinoderm byproducts. Int J Food Sci Tech 2010;45:147-154.

710 711

[48] Ravallec-Plé R, Charlot C, Pires C, Braga V, Batista I, Van Wormhoudt A, Le Gal Y, Fouchereau-

712

Péron M. The presence of bioactive peptides in hydrolysates prepared from processing waste of

713

sardine (Sardina pilchardus). J Sci Food Agr 2001;81:1120-1125.

714 715

[49] Zhong Y, Khan MA, Shahidi F. Compositional characteristics and antioxidant properties of fresh

716

and processed sea cucumber (Cucumaria frondosa). Journal of Agricultural and Food Chemistry

717

2007;55:1188-1192.

27 Page 27 of 39

718 [50] Dumay J, Allery M, Donnay-Moreno C, Barnathan G, Jaouen P, Carbonneau ME, Bergé JP.

720

Optimization of hydrolysis of sardine (Sardina pilchardus) heads with Protamex: enhancement of lipid

721

and phospholipid extraction. J Sci Food Agr 2009;89:1599-1606.

ip t

719

722

[51] Sibuet M, Lawrence JM. Organic content and biomass of abyssal holothuroids (Echinodermata)

724

from the Bay of Biscay. Marine Biology 1981;65:143-147.

us

cr

723

725

[52] Lambert P. editor. Sea Cucumbers of British Columbia, Southeast Alaska and Puget Sound. UBC

727

Press; 1997. p. 166.

an

726

M

728

[53] Sagar S, Kaur M, Minneman KP. Antiviral Lead Compounds from Marine Sponges. Marine Drugs.

730

2010;8:2619-38.

te

731

d

729

[54] Vo T-S, Ngo D-H, Ta QV, Kim S-K. Marine organisms as a therapeutic source against herpes

733

simplex virus infection. European Journal of Pharmaceutical Sciences. 2011;44:11-20.

734

Ac ce p

732

735

[55] de Jesus Raposo MF, de Morais RMSC, de Morais AMMB. Bioactivity and Applications of

736

Sulphated Polysaccharides from Marine Microalgae. Marine Drugs. 2013;11:233-52.

737 738

[56] Abad Martinez MJ, Bedoya Del Olmo LM, Bermejo Benito P. Natural marine antiviral products. In:

739

Atta ur R, editor. Studies in Natural Products Chemistry: Elsevier; 2008. p. 101-34.

740

28 Page 28 of 39

741

[57] Souza TML, Abrantes JL, Epifanio RdA, Fontes CFL, Frugulhetti ICPP. The Alkaloid 4-

742

Methylaaptamine Isolated from the Sponge Aaptos aaptos Impairs Herpes simplex Virus Type 1

743

Penetration and Immediate-Early Protein Synthesis. Planta Med. 2007;73:200-5.

ip t

744 [58] de Souza LM, Sassaki GL, Romanos MTV, Barreto-Bergter E. Structural Characterization and

746

Anti-HSV-1 and HSV-2 Activity of Glycolipids from the Marine Algae Osmundaria obtusiloba Isolated

747

from Southeastern Brazilian Coast. Marine Drugs. 2012;10:918-31.

us

748

cr

745

[59] El Baroty GS, El Baz FKA-EI, Abd El-Baky HH, Ali MM, Ibrahim EA. Evaluation of Glycolipids of

750

some Egyptian Marine Algae as A Source of Bioactive Substances. Int Res J Pharmacy. 2011.

an

749

M

751

[60] Mat R, Zalilawati, Lahaye, Elodie, Defer, Diane, Douzenel, Philippe, Perrin, Bertrand,

753

Bourgougnon, Nathalie, Sire, Olivier G. Isolation of a sulphated polysaccharide from a recently

754

discovered sponge species (Celtodoryx girardae) and determination of its anti-herpetic activity vol. 44.

755

Kidlington, ROYAUME-UNI: Elsevier; 2009. p. 8.

te

Ac ce p

756

d

752

757

[61] Harmenberg J, Wahren B, Sundqvist V-A, Levén B. Multiplicity dependence and sensitivity of

758

herpes simplex virus isolates to antiviral compounds. Journal of Antimicrobial Chemotherapy

759

1985;15:567-573.

760 761

[62] You L, Zhao M, Cui C, Zhao H, Yang B. Effect of degree of hydrolysis on the antioxidant activity of

762

loach (Misgurnus anguillicaudatus) protein hydrolysates. Innovative Food Science & Emerging

763

Technologies. 2009;10:235-40.

764

29 Page 29 of 39

765

[63] Barbana C, Boye JI. Angiotensin I-converting enzyme inhibitory activity of chickpea and pea

766

protein hydrolysates. Food Research International. 2010;43:1642-9.

767 [64] Bouhlal R, Haslin C, Chermann J-C, Colliec-Jouault S, Sinquin C, Simon G, et al. Antiviral

769

Activities of Sulfated Polysaccharides Isolated from Sphaerococcus coronopifolius (Rhodophytha,

770

Gigartinales) and Boergeseniella thuyoides (Rhodophyta, Ceramiales). Marine Drugs. 2011;9:1187-

771

209.

cr

ip t

768

us

772

[65] Bulgakov AA, Eliseikina MG, Petrova IY, Nazarenko EL, Kovalchuk SN, Kozhemyako VB, et al.

774

Molecular and Biological Characterization of a Mannan-Binding Lectin from the Holothurian

775

Apostichopus Japonicus. Glycobiology. 2007;17:1284-98.

an

773

M

776

[66] Harnedy PA, Fitzgerald RJ. Bioactive Proteins and Peptides from Macroalgae, Fish, Shellfish and

778

Marine Processing Waste. Marine Proteins and Peptides: John Wiley & Sons, Ltd; 2013. p. 5-39.

te Ac ce p

779

d

777

30 Page 30 of 39

779

Table 1 : Evaluation of anti-HSV activity at an MOI of 0.001 ID50/cells on Vero cell line of the

780

byproducts library of extracts from C. frondosa, by neutral red dye method after 72 h of incubation.

781

Acyclovir (ACV) was used as a reference drug. Treatment

Reagent

CC50 (µg/mL)

Aquapharyngeal bulb

None

None

>500

Enzymatic hydrolysis

Alcalase

>500

Bromelain

>500

cr

>250 160.5 ±8.7

>500

>250

>500

12.5 ±3.1

Papain

>500

7.2 ±1.9

Peptidase AM

>500

45.3 ±5.1

Peptidase AO

>500

170.9 ±7.5

Protamex

>500

8.3 ±2.7

Acetone

>500

>250

Hexane

81.5 ±3.8

7.9 ±3.4

Methanol

74.5 ±4.5

70.9 ±7.4

Water extract

>250

140.0 ±13.8

None

None

>250

72.5 ±9.8

Enzymatic hydrolysis

Alcalase

>500

>250

Bromelain

40.4 ±3.0

60.0 ±3.2

Flavourzyme

>500

>250

Fungal protease

29.0 ±3.0

10.4 ±1.5

Neutral protease

23.3 ±3.1

14.0 ±4.0

Papain

69.6 ±2.8

11.0 ±9.1

Peptidase AM

>500

>250

d

M

Neutral protease

Ac ce p

te

Solvent extraction

Internal organs

15.2 ±2.5

>500

an

Fungal protease

75.0 ±2.1

us

Flavourzyme

EC50 (µg/mL)

ip t

Organ

31 Page 31 of 39

ACV

None

7.8 ±9.5

Protamex

>500

>250

Acetone

>500

>250

Hexane

>500

>250

Methanol

56.7 ±5.8

Water extract

96.3 ±7.3

None

>500

7.9 ±1.8

33.2 ±4.8

0.2 ±0.1

us

Data are shown as mean ± S.D. (n=4).

ip t

74.9 ±4.8

cr

Solvent extraction

Peptidase AO

an

MOI: multiplicity of infection: ratio (virus titer)/(cells number/mL). CC50: the 50% cytotoxic concentration: concentration that reduced the absorbance of mock-infected cells to 50% of that of controls. EC50: the 50% antiviral effective concentration: concentration that achieved 50% protection of virus-infected cells from the HSV-induced destruction

Ac ce p

te

d

M

782 783

32 Page 32 of 39

Table 2: Proximate analysis on freeze-dried aquapharyngeal bulb of C. frondosa hydrolysates and without hydrolysis as a control group; n.d.: Not determined Control

Alcalase hydrolysate

Neutral Protease hydrolysate

Papain hydrolysate

Protamex hydrolysate

Crude protein (%)

58.8

54.5

54.7

55.4

54.9

Humidity (%)

6.5

5.8

5.9

4.7

Ash (%)

23.2

26.2

25.1

26.0

Fats (%)

2.8

6.3

6.5

Neutral sugars (%)

n.d.

1.8

3.1

ip t

Analysis

us

cr

4.3

27.6

7.0

6.4

1.9

1.7

an

783 784 785

786

Ac ce p

te

d

M

787

33 Page 33 of 39

787 Table 3 : Evaluation of anti-HSV activity at an MOI of 0.01 ID50/cells on Vero cell line of the most promising extracts from aquapharyngeal bulb of C. frondosa hydrolysed with: Alcalase (1), Neutral protease (2), Papain (3) and Protamex (4) by neutral red dye method at 48, 72 and 96 h of incubation. Acyclovir (ACV) was used as a reference drug. 96 h

EC50 (µg/mL)

CC50 (µg/mL)

EC50 (µg/mL)

CC50 (µg/mL)

EC50 (µg/mL)

1

>500.0

20.4 ±3.2

>500.0

43.9 ±5.2

>500.0

150.4 ±8.2

2

>500.0

26.9 ±4.4

>500.0

36.9 ±0.5

3

>500.0

15.9 ±2.2

>500.0

25.2 ±4.8

>500.0

44.3 ±3.2

4

>500.0

52.2 ±5.8

>500.0

102.9 ±8.7

>500.0

144.9 ±7.3

0.3 ±0.1

>500.0

0.3 ±0.1

>500.0

1.1 ±0.3

us

>500.0

>500

Ac ce p

te

d

794

an

Data are shown as mean ± S.D. (n=4).

cr

CC50 (µg/mL)

ACV >500.0 792 793

72 h

ip t

48 h

M

788 789 790 791

34 Page 34 of 39

794

801

< 2 kDa

>500.0

>500.0

2-5 kDa

>500.0

>500.0

5-10 kDa

>500.0

>500.0

10-30 kDa

>500.0

>500.0

30-50 KDa

>500.0

50-100 KDa

>500.0

us

> 100 kDa

>500.0

ACV

>500.0

cr

55.1 ±7.2 38.1 ±5.2

an

M

Data are shown as mean ± S.D. (n=4).

ip t

EC50 (µg/mL)

18.2 ±3.6 0.3 ±0.1

d

800

CC50 (µg/mL)

te

798 799

Table 4 : Evaluation of anti-HSV activity at an MOI of 0.01 ID50/cells on Vero cell line by molecular mass fractionation of aquapharyngeal bulb of C. frondosa hydrolysed with Papain after 72 h of incubation, by neutral red dye method. Acyclovir (ACV) was used as a reference drug.

Ac ce p

795 796 797

35 Page 35 of 39

801

Figure 1: Summary of the enzymatic hydrolysis process for the preparation of byproducts

802

hydrolysates from Cucumaria frondosa (A.B.: Aquapharyngeal Bulb and I.O.: Internal Organs); rpm:

803

rotation per minute.

804

806

Figure 2: Summary of the sequential solvent extraction of the freeze-dried byproducts of

ip t

805

Cucumaria frondosa (A.B.: Aquapharyngeal Bulb and I.O.: Internal Organs; RT: Room Temperature.

cr

807

Ac ce p

te

d

M

an

us

808

36 Page 36 of 39

Ac ce p

te

d

M

an

us

cr

ip t

Figure1

Page 37 of 39

Ac ce p

te

d

M

an

us

cr

ip t

Figure2

Page 38 of 39

cr M

an

us

*Graphical Abstract

L.TRIPOTEAU L.TRIPOTEAU

Extraction of the byproducts

L.TRIPOTEAU

ed

Enzymatic hydrolysis Enzymatic and Solvent extraction hydrolysis (variation of proteases nature, pH, temperature, time)

ce

pt

Collection of specimens

Ac

Identification of a potent antiherpetic extract : - > 100 kDa

- Papain hydrolysate - Aquapharyngeal bulb

Selection

Molecular mass fractionation

Antiviral and cytotoxic evaluation of the fractions

Antiviral and cytotoxic evaluation of the selected extracts

Page 39 of 39

Antiviral and cytotoxic evaluation of the library of extracts