Open Access

Bioactive compounds from Holothuria atra of Indian ocean

SpringerPlus20143:673

https://doi.org/10.1186/2193-1801-3-673

Received: 30 July 2014

Accepted: 10 October 2014

Published: 14 November 2014

Abstract

The sea cucumber (Holothuria atra) extracts have been evaluated for the presence of bioactive compounds and various biological activities. The methanol extracts showed anti proliferative activities against the Hela and MCF-7 cell lines. Similarly the inhibitory effects of Herpes simplex virus 1 and 2 cells were detected using the plaque reduction assay. The extracts of H. atra were purified using the silica gel column chromatography. The active fractions collected were observed for antimicrobial activity. The GC-MS analysis showed the availability of 59 compounds. The active bioactive compounds found in the H. atra were analyzed and their structure was identified using the 1HNMR and 13C NMR experiments.

Keywords

Holothuria atra Bioactive compoundsCell lines Herpes simplex virus MCF-7

Introduction

The marine holothurians are spiny skinned invertebrates, which form important commercial group among the echinoderms. In India, about 200 species of holothurians are known of which 75 species are from shallow waters within 20 m depths (Cuvillier 2002). (James 1994) suggests that great potential exists for the extraction of valuable bioactive compounds from the sea cucumbers in the Indian coast. A new immunomodulatory lead Cumaside a complex of monosulfated triterpene glycosides from the sea cucumber Cucumaria japonica possesses cytotoxic activity against Ehrlich carcinoma cells (Aminin et al.2004). Sphingoid base composition of cerebrosides from sea cucumber Stichopus variegatus exhibited cytotoxicity against human colon cancer cell and induced apoptosis. They are major constituents of c-17, c-19 alkyl chain and 1-3 double bonds (Sugwara et al. 2006). Triterpene glycosides are the predominant secondary metabolites of the sea cucumber Hemoiedema spectabilis which exhibited wide spectra of biological activities, including antifungal, cytotoxic, hemolytic, cytostatic and immunomodulatory functions Chludil et al. (2002). A new lanostane-type triterpene glycoside, impatienside A and bivittoside D were isolated from the sea cucumber Holothuria impatiens Sun et al. (2007). The potential angiogenesis inhibitors, a novel sulfated saponin philinopside A, isolated from the sea cucumber Pentacta quandrangulari, possessed dual antiangiogenic and antitumour effects (Tong et al. 2005).

Fuscocineroside C bioactive compound obtained from sea cucumber Holothuria fuscocinerea a triterpene glycoside showed cytotoxic nature against human cancer cells Zhang et al. (2006). Hillaside C a triterpene derived from sea cucumber Holothuria hilla inhibited the growth of human leukemia, breast and colon cancer cells in vitro in a dose and time-dependent manner by a mechanism that required induction of apoptosis and the concomitant reduction of the apoptosis-suppressing protein Bcl-effect Wu et al. (2006). Intercedenside D–I iso lated a cytotoxic triterpene glycoside from the sea cucumber Mensamaria intercedens a marine natural product inhibited proliferation of several human cancer cell lines Zou et al. (2005). Steroid glycosides are a class of wide-spread natural products having marine origins. Spirostan and furostan steroid saponins, pregnane glycosides have a potential to be used as cancer therapies. Structurally, these glycosides exhibit a moderate cytotoxicity against human leukemia cell lines (Prassas and Diamandis 2008). Linhardt et al. (1990) found that low molecular weight sulphated polysaccharides are noted from sea cucumbers with efficient anticoagulant activities and several pharmacological properties. The chondroiton and glucosamine components of holothuria were reported to be important cartilage building blocks and other bioactivities including anti-inflammatory and anti tumor activity properties (Herecia and Ubeda 1998).

The extract LPS obtained from Stichopus japonicus induced inflammatory response via blocks the MAPK signaling pathway in murine macrophages, showed in vitro with anti-inflammatory potential Himayaa et al. (2010). The sea cucumber Telenata ananas derived bioactive compounds were reported to act as the chemokine receptor subtype-5 (CCR5) with possible anti-HIV activity Hegde et al. (2002). Potential use of sea cucumber S. liouvillei isolated compound chondroitin sulfate (the polysaccharides) are reported to exhibit antiviral activity to inhibit human immunodeficiency virus (HIV) infection (Chen 2003). Considering this as an evidence in the present study, attempts were made to find out the bioactive compounds from marine invertebrates such as the Holothuria.

Materials and methods

Sample collection and extract preparation

Holothuria atra specimens with a size range of 10 to 30 cm in length and 30 to 180 g weight were collected from fishing nets operated off Kanyakumari (8° 03′ and 8° 35′ of the north latitudes and 77° 15′ and 77° 36′ of the east longitudes) in the Indian Ocean. Immediately upon collection, they were dissected to remove the internal organs and packed using ice prior and kept at –80°C or extraction. The skin portion was peeled off and stored in methanol in separate containers. The biologically active compounds were extracted as a function of their polarity using water and organic solvents. About 200 g of frozen samples were homogenized with deionized water and methanol. The mixture was continuously stirred in the dark at 4°C for 24 h. Then it was centrifuged at 5000 rpm for 15 min. The supernatant was collected and filtrated. The collected organic extracts were freeze-dried and kept at -80°C, while the insoluble solid materials were re-extracted with methanol (100%) (Chen 2003).

MTT assay using Hela cell lines and MCF-7 cancer cell lines

The cells were preincubated at a concentration of 1 × 106 cells/ml in culture medium for 3 h at 37°C and 6.5% CO2. Then, the cells were seeded at a concentration of 5 × 10 4 cells/well in 100 μl culture medium and at various concentrations of extracts (dissolved in 2% DMSO dimethylsulphoxide solution) into microplates (tissue culture grade, 96 wells, flat bottom) and incubated for 24h at 37°C and 6.5% CO 2. Then, 10 μl MTT labelling mixture was added and incubated for 4 h at 37°C and 6.5% CO 2. Each experiment was conducted as triplicates sets. Then 100 μl of solub ilization solution was added into each well and incubated for overnight. The spectrophotometric absorbance of the samples was measured using a microplate (ELISA) reader. The wavelength to measure absorbance of the formazan product in 570 nm according to the filters available for the ELISA reader was used. The reference wavelength was more than 650 nm. IC50 values were calculated Percentage inhibition of novel compounds against all cell lines was calculated using the following formula:
A t Ab % cell survival = × 100 A c Ab

Trypan blue dye exclusion test

Being an essential dye, Tryphan blue was used in estimating the number of viable cells present in a population. The culture sample was mixed to resuspend cells. 20 μl of cell culture sample was taken and filled into sterile microfuge tube. To this 20 μl of 0.4% Trypan blue solution was added and mixed well by gently aspirating and dispensing the solution with the help of micropipette. The coverslip was fixed on the centre top of the hemocytometer. To the 10 μl mixture of the cell culture t he Trypan Blue mixture taken from the microfuge tube was added and kept in the hemocytometer assembly on microscope stage using 100 X magnification. The number of live and dead cells were recorded.
% viability = live cell count / total cell count × 100

Plaque reduction assay

Vero monolayer cells grown in 24 well tissue culture plates were infected with HSV -1 and HSV-2. Virus dilutions were made from 101 to 107 using 0.1 ml of viral suspension. Virus adsorption was carried out for 1h at 37°C in the presence of test extract. Virus dilutions were prepared in Eagles minimum essential medium. Prior to incubation, an overlay medium comprising of 0.8% carboxy methyl cellulose with 2% FBS was added. It was done to avoid formation of secondary plaques. Infected cell cultures were incubated at 37°C at 5.0% CO2 incubator for 2 to 3 days. The infected cells were stained and observed for plaque reduction. The infectivity titers were expressed as the number of plaque forming units per ml (pfu ml−1). After incubation, cultures were stained with 1% (w/v) crystal violet solution. The plaques were counted by visual examination and the percentage of plaque inhibition was calculated. The Pfu = Plaque number × reciprocal of dilution × reciprocal of volume in ml.

The antiviral activity was defined as the percentage of plaque inhibition as follows:
% Plaque inhibition = [ 1 ( Number of plaque in test ÷ Number of plaque in control ) × 100 ]

Column chromatography

Silica (230-400 mesh) gel slurry was prepared using methanol. The column was packed with silica gel. After washing the column with same solvents, the sample (3 to 5 ml) was poured and then eluted with methanol: water on different percentages (10 to 100% methanol). The active fractions were collected and used for NMR analysis.

Antibacterial activity

Disc diffusion method was employed to test the active fractions of H. atra obtained from the column chromatography Bauer et al. (1966). Antibacterial activity was determined using Muller Hinton agar (Hi Media).

The bacterial cultures were obtained from the Microbial type culture collection and gene bank (MTCC), Institute for Microbial Technology, Chandigarh, India. They were Staphylococcus aureus MTCC 737, E.coli MTCC 443, Klebsiella pneumonia MTCC 109, Listeria monocytogenes MTCC 1143, and Serratia liquefaciens MTCC 3039. The plates were aseptically streaked with the test microorganism using a sterile swab and allowed to dry for a few minutes. Sterilized filter paper discs (Whatman no.1; 6 mm diameter) were used. The fractions were collected from 10 to 100% levels of methanolic extracts and were evaluated at 100 μl concentration. The plates were then incubated for 24 h at 37°C. Controls were blank discs impregnated with solvent. The diameter of the inhibition zone formed around the disc was measured.

GC-MS analysis

The methanol extract of the sea cucumber H. atra was analyzed by GC-MS (Make: Fisons GC8000 series and MS: md800). The GC column dimension was: 30 mm, 0.25 mm, 0.5 mm AB-35MS fused silica capillary column. The GC conditions were as follows: injector temperature 250°C column temp isothermal at 100°C then programmed to rise up to 250°C at 6°C/min and held at this temperature for 10 minutes. The ion source temperature was 200°C and the interface temperature was 250°C. Helium gas was engaged for carrier gas at the rate of 1ml/min. Spectra was obtained in the EI mode with 70eV ionization energy. The compounds were identified by comparison with the standards. If not available, the mass spectra was matched with inbuilt library like wileys, NIST (Stonik et al. 1998).

NMR analysis

The active fractions obtained from column chromatography were analysed for Nuclear magnetic resonance spectroscopy (NMR) analysis. Optical rotations were measured on a Perkin- Elmer Model 341 LC polarimeter. 1H NMR and 13C NMR experiments were performed on Bruker Unity 400 and 600 MHz spectrometers. NMR spectra were referenced to the CD3OD solvent signals at δ 3.30 (1H) and 49.00 (13C), respectively. The spectra were obtained using the standard Bruker software. The samples were dissolved in different solvents (i.e. DMSO-d 6, CDCl3, and CD3OD), the choice of which was dependent on the solubility of the samples. The observed chemical shift (δ) values were given in ppm and the coupling constants (J) in Hz.

Results and discussion

The results in Table 1 show that the amount of sea cucumber H. atra extract required to inhibit 50% of the antitumor activity against the Hela cell lines in 96 well plates could be determined. Antitumor activity was measured by using the IC50 values. The anti proliferative effect (IC50 value) exhibited by the Holothuria atra was 468.0 against the cervical cancer cell line (Hela). The cell inhibition was determined from the extract concentration ranged from 0.078 mg/ml to 10 mg/ml. The absorbance values were measured at 570 nm. Percentage of growth inhibition was identified at different concentration of the extracts. The gradual decrease in absorbance values showed increase in inhibition effect of the extracts against the Hela cell lines. The findings suggest that H. atra showed cell inhibition to the tune of 90% to the maximum in Hela cells and 75% of cell inhibition in MCF-7 cells. Five cerebrosides, PA-0-1, PA-0-5, PA-2-5, PA-2-6 and CE-2c were reported from the Japanese sea cucumber Pentacta australis Higuchi et al. (1994). A ganglioside molecular species SJG-1, isolated from the sea cucumber Stichopus japonicus. SJG-1 possessed a sialic acid, nonhydroxy fatty acids and phytosphingosine-type long chain bases as major ceramide components. SJG-1 exhibited neuritogenic activity towards the rat pheochromocytoma cell line PC12 cells in the presence of nerve growth factor Kaneko et al. (1999). Additionally, a cerebroside isolated from the sea cucumber Stichopus japonicas showed effective antitumor activity (Hayashi et al. 1990). The data presented in the Table 2 suggested the antitumor activity of the methanol extracts of H. atra against the breast cancer cell lines MCF-7 in 96 well microtitre plates. The susceptibility of cells to the extract exposure was characterized by IC50 values. Results indicated that the anti proliferative effect increased with the increase in concentration of the extracts. The IC50 value for the sea cucumber Holothuria atra was 352.0 A decrease in number of viable cells with the increase in concentration of the extracts was noted. From Table 3 the results of cell counting and viability of cells using tryphan blue staining were indicators for the influence of extracts. The percentage cell inhibition of Hela was 81.81% and MCF-7was 72%. The cell proliferation and inhibition measurment of the sea cucumber Holothuria atra showed that it can be developed as an antitumor agent. The cytotoxic effects of extracts against the Hela and MCF-7 cell lines are observed through the inverted microscope (Figure 1). Promising in vitro cytotoxic compounds such as the Calcigeroside B, C1 and C2 identified from the holothurians included triterpene glycosides. They showed antiproliferative action against the human and murine tumour cell lines (Alejandro and Gustafson 2003). In the present study, the tumor growth was inhibited by Holothuria atra extract and dosage was an important criteria which influenced the efficacy of the cell death in Hela, and MCF-7 cell lines. These suggest the possibility of apoptotic cell death through the activation of Bax a proapoptotic protein.
Table 1

Cytotoxicity analysis of Holothuria atra extracts against Hela cell lines using MTT assay

Concentration (mg/ml)

Absorbance (570 nm)

% inhibition

IC50

0.078

0.125

40.56

468.0

0.156

0.103

52.12

0.3125

0.10

60.42

0.625

0.093

68.30

1.25

0.085

75.34

2.5

0.070

83.26

5

0.069

88.32

10

0.032

90.56

Cell control

0.45

100

Table 2

Cytotoxicity analysis of Holothuria atra extracts against MCF-7 cell lines using MTT assay

Concentration (mg/ml)

Absorbance (570 nm)

% inhibition

IC50

0.078

0.120

30.22

352.0

0.156

0.102

35.64

0.3125

0.10

48.72

0.625

0.088

55.60

1.25

0.085

60.62

2.5

0.075

65.20

5

0.062

70.22

10

0.052

75.60

Cell control

0.45

100

Table 3

Percentage cell inhibition and characterization of cell line exposed to Holothuria atra using tryphan blue

Cell line

% cell inhibition

Dead cell count

Total cell count

pH

HeLa

81.81

1.80 × 105

2.20 × 10 5

6.9

MCF-7

72.72

1.76 × 105

2.42 × 10 5

7.2

Figure 1

Cytotoxic effects of H.atra extracts on A) Hela and B) MCF-7 cell lines.

Angiogenesis inhibitors and aromatase inhibitors present in sea cucumbers play a major role in reducing the growth of breast cancer and prostate cancers, especially the solid tumors. Research results showed that angiogenesis inhibitors effectively block the growth of tumors by cutting off their nutrient and blood supply. The mechanism by which they block tumor growth is driven by the inhibition of receptor tyrosine kinases (RTKs) that are over expressed by cancer cells (Chi 2006). Using MTT assay it could be inferred that H. atra extracts can block the growth of breast cancer cells (MCF-7) by inducing apoptosis. In H. atra extract there is a possibility of blocking the receptors such as the tyrosine kinases (RTKs) in cancer cells of Hela and MCF-7. The methanol extracts of Holothuria atra showed maximum inhibition of antitumor cells and it was observed by cell inhibition using tryphan blue.

The concentration of extracts for H. atra was from 10 μg/ml to 70 μg/ml, respectively. The tested viruses were affected with the increase in concentration of extracts. The H. atra exhibited significant antiviral activity, and suggested the potential role of extracts. The effect of inhibition in plaque formation was evaluated based on the 101 to 107 dilutions of HSV-1. and HSV-2. In H. atra the highest plaque inhibition rate was as at 75% with 2.4 × 103 pfu ml−1. Less inhibition rate was observed at 33% for 6.0 × 109 pfu ml−1. The results of (Figure 2) suggest the effects of H. atra extracts on the inhibition of virus replication after attachment of HSV-1and 2 on Vero cells. In H. atra, the plaque inhibition obtained was high at 74% with 2.3 × 109 pfu ml−1 whereas less effect was seen in H .atra was 27% with 6.4 × 103 pfu ml−1 units (Table 4). Saponins the secondary metabolites which are triterpene glycosides present in sea cucumbers like H. forskali are reported to have antiviral property by in vitro and in vivo methods (Kerr and Chen 1995). It was observed that the H. atra extracts exhibited antiviral activity on plaque reduction assay in which maximum effect was seen against the HSV-1 to the tune of 74%. Thus Holothuria atra extracts have the ability to arrest the multiplication of virus and suppress its growth by influencing the growth factors. This could have resulted in appearance plaques in the plaque reduction assay. Bioactive peptides and hemolytic lectins have been reported from sea cucumber as a source of antiviral activity. Among Holothuroidea genera, Cucumaria echinata and C. frondosa contained lectin and peptide, respectively. They have been found in the body wall mucus Hisamatsu et al. (2008). Fucoidans, polysaccharides containing substantial percentages of L-fucose and sulfate ester groups are generally considered as constituents of sea cucumbers. Fucoidan can inhibit the development of cytopathic effect (CPE) and protect cultural cells from infection caused by viruses (Hemmingson et al. 2006). This can induce the antiviral effects against the Herpes simplex viruses.
Figure 2

In vitro antiviral activity of Holothuria atra extracts against A) HSV-1and B) HSV-2 using plaque reduction assay.

Table 4

Inhibitory action of Holothuria atra extracts against HSV strains (- ?)

Dilution of virus

Concentration of H. atra extracts

Plaque forming units pfu/ml

% Plaque inhibition [HSV-1 and HSV-2]

  

HSV-1

HSV-2

H. atra

Control

Nil

9.2 × 101

8.7 × 10 1

-

-

10−1

10 μg

6.0 × 109

6.4 × 10 3

33

27

10−2

20 μg

5.6 × 10 8

5.6 × 10 4

40

36

10−3

30 μg

5.2 × 107

4.7 × 105

44

46

10−4

40 μg

4.4 × 10 6

4.3 × 10 6

53

51

10−5

50 μg

3.9 × 10 5

2.3 × 109

58

60

10−6

60 μg

3.3 × 10 4

2.8 × 10 8

65

68

Figure 3 shows the antibacterial activity for the active fractions obtained from column chromatography of H. atra against various Gram positive and Gram negative bacteria viz., Klebsiella pneumonia, Serratia liquefaciens, Staphylococcus aureus, Listeria monocytogenes, and Escherichia coli. The fractions collected from 50 to 100% showed maximum effect whereas the fractions from 10 to 40% did not show any activity. In H. atra the E. coli had less effect at 50 and 60% of fractions which showed the zone diameter of 2 and 3mm range. Figure 4 shows the GC-MS graphical representation of the extracts of sea cucumber. The interpretation on mass spectrum GC-MS was carried out and the spectra of the unknown component were compared to the spectrum of the known components stored in the NIST library. The name, molecular weight of the components was ascertained. A total of 59 natural compounds were identified from the extracts of sea cucumber (H. atra). The active principles with their retention time (RT), molecular formula, molecular weight (MW) were ascertained (Table 5).
Figure 3

Anti bacterial activity of the column purified fractions of sea cucumber, Holothuria atra against A) Serratia liquefaciens B) Escherichia coli C) Klebsiella pneumoniae D) Staphylococcus aureus.

Figure 4

GC-MS Analysis of the methanolic extracts of the sea cucumber Holothuria atra.

Table 5

Compounds identified in the crude methanolic extracts of sea cucumber ( Holothuria atra ) using GC-MS Analysis

No

Name of the compound

Molecular formula

M.W

1

Ethane 11dichloro(CAS)11dichloro ethane.

C2H4Cl2

98

2

Carbamic acid,hydroxyl, ethyl ester

C3H7O3N

105

3

Ethane sulphonyl chloride,2 chloro

C2H4O2Cl2

162

4

Methyl 40-methyl-beta-D-xylophyanoside

C7H1405

178

5

Propylhexedrine

C10H21N

155

6

2-methyl-1,3-dithiacyclopentane

C4H8S2

120

7

Ethane dithioic acid

C2H4S2

92

8

4 ketopinelic

C7H10O5

174

9

2-ethylomethyl-1,3-dithiacyclopentane

C8H16S3

208

10

Carbomic chloride,methoxymethyl

C3H6O2NCl

123

11

Xylane,ethyldimethyl

C4H12SL

88

12

D-pseudo ephedrine or ephedrine or pholedrine

C10H15ON

165

13

Ethanol,1-methoxy-benzoate

C10H12O3

180

14

Methanomine hippurate artefact

H40

0

15

2,6,10,14 tetra methylpentadecane-2-ol

C1940O

284

16.

2,5,8-heptadecatrien-1-ol

C17H30O

250

17

Methyl 19-hydroxyeicosan-5(z),8(z),11(z),14(z)-tetrae.

C21H34O3

334

18

3,4epoxy-6,9-octadecadiene

C18H32O

264

19

(14z,17z)-3,20-dibromo-21-ethyl-2,6-epoxy-1-oxacyclo

C22HO3B12

496

20

(-)-elema-1,3,11(13)-trien-12-ol or Beta –costol

C15H24O

220

21

Phenylpropanolamine

C9H13ON

187

22

3,3-dibromotricyclo(2,4) undecane

C11H16B12

306

23

Transcaryophyllene or Alpha far nesene or epsilon cadinene.

C15H24

204

24

2,3-2poxy-5,8-hectadecadien-1-ol

C17H30O2

266

25.

(+-)-3-methylidenetricyclo(2,9)undecan-8-one

C12H16O

176

26

7-methylbicyclo oct-7-ene

C9H14

122

27

Methylareachidonate 5,8,11,14 eicosatetraenoic

C21H34O2

318

28

2 –naphthalenemethanol-decahydro-5-methylene-8

C 14H22O

206

29

Tricyclo decanedimethanol

C12H20O2

196

30

2-3-29Methoxycarbonyl) ethyl bicyclo 1-1-1pent-1

C13H18O4

238

31

Transcaryophyllene.

C15H24

204

32

2xo,2endo:3endo,3exo-bis (epoxymethane) bicyclo

C9H12O2

152

33

2-pyrrolidinnarbocylic acid.5-methyl,phenylne

C13H17O2N

219

34

Cyclopropane,1-etenyl-2-hexenyl,1apha,1 e Bet

C11H18

150

35

Propene,3-phenyl-3-ol

C9H10O

134

36

1-bromo-12cyclohexylidene-3-buen-2ol-$$3-buten

C10H15Obr

230

37

2-4 nonadien-6-yn -1-0l,(e,e)

C9H12O

136

38

Tricyclo3,2,1,02,4 octane 8-one,3,3-dimethyl,(1 alp)

C10H14O

150

39

Trans-ocimene 1,3,7-octatriene,3,7- dimethyl

C10H16

136

40

Cycloprpane,{(1-propenyloxy) methyl}- (CAS) CY

C7H12O

112

41

3T-Bromo-1-ethynyl-1c,2r-cyclohexanediol

C8H11O2Br

218

42

Didodecyl phthalate

C32H54O4

502

43

Phendimetrazine

C12H17ON

191

44

Methyl 2-ethylhexyl phthalate

C17H24O4

292

45

Di 2 ethylhexyl phthalate

C24H38O4

390

46

1,2-benxenedicarboxylic acid, dioctyl ester (cas)

C22H34O4

362

47

5,8-methanospiro(4,5) deccane-1,4 dione

C11H14O2

178

48

3-octyl acetate

C10H20O2

172

49

Bicyclo[3.2.1]octan-2-one, 1-(1-propenyl)- a(cas) 1-p

C11H16O

164

50

Ethylphenyl-n-ethylamine

C10H15N

177

51

Phthalic acid, didecyl ister 1,2-benzenedicarbo

C28H46O4

446

52

Bupranolol (betadrenol)

C13H2102N

271

53

Ethosuxinide (sematin)

C7H11O2N

141

54

1,2-Benzenedicarbozylicacid,diethy ester (CAS)

C12H14O4

222

55

2-acetylbenzoic acid

C18H26O5

164

56

1,2-Benzenedicarboxylic acid dipropyl ester(CAS)

C14H18O4

250

57

Nitrofurantion

C8H6O5N4

238

58

Benzoic acid-4 forml

C8H6O3

150

59

1,2-Benzenedicarbozylicacid dibutyl ester(CAS)

C16H22O4

278

Fucoidan of sea cucumber Laminaria japonica has anti RNA and DNA virus functions. The antivirus effects of fucoidan on infection was against poliovirus III, adenovirus III, ECHO6 virus, coxsackie B3 virus and coxsackie A16. Fucoidan inhibited the development of cytopathic effect (CPE) and protected the cultural cells from infection caused by the viruses Li et al. (1995). Sulfated polysaccharides from sea cucumbers such as the Cucumaria japonica, Holothuria impatiens are reported to exhibit antiviral activity. Based on this fact, Japanese scientists have patented their scientific findings regarding the potential use of sea cucumber chondroitin sulfate to inhibit human immunodeficiency virus (HIV) infection Beutler et al. (1993). Triterpene glycosides, namely holothurinosides A, B, C and D as well as desholothurin A from sea cucumber (Holothuria forskali), have considerable antitumour activity against P388 cell lines. The saponins isolated from the aqueous and methanolic extract of sea cucumber (Holothuria forskali) have showed considerable antiviral activities Mulloy et al. (2000). Considering these as well as the results of present investigations, the methanolic extracts of Holothuria atra could form effective antitumour and antiviral agents. Previous work showed that sulphated polysaccharides such as glycosaminoglycans an inhibitor of human immunodeficiency virus binds to T lymphocytes and showed antiviral activity Toido et al. (2003). In low concentrations, the extract showed potent inhibitory effect towards Herpes simplex virus and thus has got significant drug value. The antiviral activity using the sea cucumber Ludwigothurea grisea and Thelenota ananas derived fucosylated chondroitin sulfates (FCS), was recognized as the sulfated polysaccharides. It inhibited human immunodeficiency virus (HIV) infection Mc Clure et al. (1992). The present work suggested that Holothuria atra extracts showed virucidal action through reduction in number of plaques formed during plaque reduction assay against the HSV-1 and HSV-2. The strong growth inhibitory activity found in the extract of Holothuria atra might be the source for the development of antiherpetic compound.

The structure of bioactive compounds was elucidated by using NMR spectra from the active fractions. In addition, the methyl groups were observed in the 1H NMR spectra including singlets and doublets which were integrated relatively for olefinic proton at δ position. The 13C NMR spectrum showed the presence of a carbon–carb on double and indicated the presence of two conjugated carbonyls. It also showed the appearance of two carbon signals. Figure 5 represent the 1H and 13C NMR data of Holothuria atra. Some of the bioactive compounds identified with their structures are given below. Sea cucumber derived fucosylated chondroitin sulfates (FCS) which inhibited the growth of human immunodeficiency virus and also acted as a cytotoxic agent was initially obtained from Stichopus badionotus Kaswandi et al. (2004). It could be predicted that high molecular weight compounds present in Holothuria atra detected by NMR analysis could form a potent antiviral sources (Additional file 1).
Figure 5

NMR analysis of active fractions obtained from Column chromatography (A) 1 H NMR Spectrum of Holothuria atra (B) 1 H NMR Spectrum of Holothuria atra (C) 13 C NMR Spectrum of Holothuria atra.

Conclusions

The H. atra extract had various compounds such as the flavonoids, phenolic components, terpenoids, saponins, alkaloids etc. The GC-MS analysis revealed the presence of 59 compounds. It was found that H. atra extracts showed anti proliferative activities against the Hela and MCF-7 cell lines. Similarly the inhibitory action of extracts were found against the HSV-1 and HSV-2 strains was analyzed by plaque reduction assay. From NMR analysis the structural elucidation of the active compounds were studied. These results will direct future efforts to optimize the anti proliferative activity of these bioactive compounds.

Declarations

Acknowledgments

The authors are grateful and thankful to Dr. S. Joseph Selvin the Co-ordinator, and Head, Department of Microbiology, Pondicherry University for his help during the work.

Authors’ Affiliations

(1)
Centre for Marine Science and Technology, Manonmaniam Sundaranar University, Rajakkamangalam
(2)
Marine Biotechnology Laboratory, Central Marine Fisheries Research Institute

References

  1. Alejandro MS, Gustafson RK: Marine pharmacology in 2000: antitumor and cytotoxic compounds. Int J Cancer 2003, 105: 291-299. 10.1002/ijc.11080View ArticleGoogle Scholar
  2. Aminin DL, Agafonova IG, Berdyshev EV, Isachenko EG, Avilov SA, Stonik VA: Immunomodulatory properties of cucumariosides from the edible Far-eastern holothurian cucumaria japonica. J Med Food 2004, 4(3):127-135.View ArticleGoogle Scholar
  3. Bauer AW, Kirby W, Sherris J, Truck M: Antibiotic susceptibility testing by a standardized single disc diffusion method. Am J Clin Path 1966, 45: 493-496.Google Scholar
  4. Beutler JA, McKee TC, Fuller RW, Tischler MV, Cardellina JH, Snader KM, McCloud TG, Boyd MR: Frequent occurrence of HIV-inhibitory sulphated polysaccharides in marine invertebrates. Antivir Chem Chemother 1993, 4: 167-172.View ArticleGoogle Scholar
  5. Chen J: Overview of sea cucumber farming and sea ranching practices in China. SPC Beche-de-mer Inf Bull 2003, 18: 18-23.Google Scholar
  6. Chi TT: Benefits of a sea cucumber extract in anti-angiogenic therapy and RTK inhibition for cancer. Townsend Lett 2006, 8: 91-95.Google Scholar
  7. Chludil HD, Muniain CC, Seldes AM: Cytotoxic and antifungal triterpene glycosides from the Patagonian sea cucumber Hemoiedema spectabilis. J Nat Prod 2002, 65: 860-865. 10.1021/np0106236View ArticleGoogle Scholar
  8. Cuvillier O: Sphingosine in apoptosis signaling. Biochim Biophys Acta 2002, 1585: 153-162. 10.1016/S1388-1981(02)00336-0View ArticleGoogle Scholar
  9. Hayashi A, Chen HC, Shiono H: Studies on sterol sulfate, sterol glycoside and cerebroside of sea cucumber Stichopus japonicus. Kinki Daigaku Rikogakubu Kenkya Hokoku 1990, 26: 73-85.Google Scholar
  10. Hegde VR, Chan TM, Pu H, Gullo VP, Patel MG, Das P, Wagner N, Parameswaran PS, Naik CG: Two selective novel triterpene glycosides from sea cucumber, Telenota Ananas: inhibitors of chemokine receptor-5. Bioorg Med Chem Lett 2002, 12: 3203-3205. 10.1016/S0960-894X(02)00599-1View ArticleGoogle Scholar
  11. Hemmingson JA, Falshaw R, Furneaux RH, Thompson K: Structure and antiviral activity of the galactofucan sulfates extracted from Undaria pinnatifida (Phaeophyta). J Appl Phycol 2006, 18: 185-193. 10.1007/s10811-006-9096-9View ArticleGoogle Scholar
  12. Herecia F, Ubeda A: Anti-inflammatory activity in mice of extracts from Mediterranean marine invertebrates. Life Sci 1998, 62(9):115-120.Google Scholar
  13. Higuchi R, Inagaki M, Tokogawa K, Miyamoto T, Komori T: Constituents of Holothuroideae. V Isolation and structure of cerebrosides from the sea cucumber Pentacta australis Liebigs. Ann Chem 1994, 7(12):653-658.Google Scholar
  14. Himayaa SWA, Ryua B, Qian ZJ, Kwon KS: Sea cucumber, Stichopus japonicus ethyl acetate fraction modulates the lipopolysaccharide induced iNOS and COX-2 via MAPK signaling pathway in murine macrophages. Environ Toxicol Phar 2010, 10: 1016.Google Scholar
  15. Hisamatsu K, Tsuda N, Goda S, Hatakeyama T: Characterization of the {alpha}-helix region in domain 3 of the haemolytic lectin CEL-III: Implications for selfoligomerization and haemolytic processes. J Biochem 2008, 143: 79-86.View ArticleGoogle Scholar
  16. James DB: Zoogeography and systematic of holothurians used for Beche-de-mer in India, Proc. Natl. Workshop on Beche-de-mer, (Eds) K. Rengarajan and D.B. James. Bull Cent Mar Fish Res Inst 1994, 46: 34-36.Google Scholar
  17. Kaneko M, Kisa F, Yamada K, Constituents of Holothuroidea, 8: Structure of neuritogenic active gangliosides from the sea cucumber Stichopus japonicas. Eur J Org Chem 1999, 3171-3174.Google Scholar
  18. Kaswandi MA, Hing HL, Sahalan AZ, Farah F, Ridzwan BH, Samsudin MW, Yasin MS, Ali A: Saponin from sea cucumber Stichopus badionotus Sluiter as potential cytotoxic agent on CEM-SS T-lymphoblastic cell. JMST 2004, 18: 79-84.Google Scholar
  19. Kerr RG, Chen Z: In vivo and in vitro biosynthesis of saponins in sea cucumbers. J Nat Prod 1995, 58: 172-176. 10.1021/np50116a002View ArticleGoogle Scholar
  20. Li F, Tian TC, Shi YC: Study on antivirus effect of fucoidan in vitro . J N Bethune Univ Med Sci 1995, 21: 255-257.Google Scholar
  21. Linhardt RJ, Loganathan D, Al-Hakim A: Oligosaccharide mapping of low molecular weight heparins: structure and activity differences. J Med Chem 1990, 33: 1639-1645. 10.1021/jm00168a017View ArticleGoogle Scholar
  22. Mc Clure MO, Moore JP, Blanc DF: Investigations into the mechanism by which sulfated polysaccharides inhibit HIV infection in vitro . AIDS Res Hum Retrovir 1992, 8: 19-26. 10.1089/aid.1992.8.19View ArticleGoogle Scholar
  23. Mulloy B, Mourao PAS, Gray E: Structure/function studies of anticoagulant sulphated polysaccharides using NMR. J Biotechnol 2000, 77: 123-135. 10.1016/S0168-1656(99)00211-4View ArticleGoogle Scholar
  24. Prassas I, Diamandis EP: Novel therapeutic applications of cardiac glycosides. Nat Rev Drug Discov 2008, 7: 926-935. 10.1038/nrd2682View ArticleGoogle Scholar
  25. Stonik VA, Ponomarenko LP, Makarieva TN, Boguslavsky VM, Dmitrenok AS, Fedorov SN, Strobikin SA: Free sterol compositions from the sea cucumbers Pseudostichopus trachus, Holothuria (Microtele) nobilis, Holothuria scabra, Trochostoma orientale and Bathyplotes natans. Comp Biochem Physiol 1998, 120: 337-347. 10.1016/S0305-0491(98)10023-8View ArticleGoogle Scholar
  26. Sugwara T, Zamia N, Yamamoto T, Sakai S, Noguchi R, Hiraia T: Isolation of shignoid bases of sea cucumber cerebrosides and their cytotoxic activity against colon cancer cells. Biosci Biotechnol and Biochem 2006, 70: 2906-2912. 10.1271/bbb.60318View ArticleGoogle Scholar
  27. Sun P, Liu BS, Yi YH: A new cytotoxic lanostane-type triterpene glycoside from the sea cucumber Holothuria impatiens. Chem Biodivers 2007, 4: 450-457. 10.1002/cbdv.200790037View ArticleGoogle Scholar
  28. Toido T, Gumjorn C, Mornrut A: Structure and Bioactivity of sulphated polysaccharides. Trends Glycosci Glycotechnol 2003, 15(81):29-46. 10.4052/tigg.15.29View ArticleGoogle Scholar
  29. Tong Y, Zhang X, Tian F: Philinopside A, a novel marine-derived compound possessing dual anti-angiogenic and anti-tumor effects. Int J Cancer 2005, 114(6):843-853. 10.1002/ijc.20804View ArticleGoogle Scholar
  30. Wu J, Yi YH, Tang HF: Structure and cytotoxicity of a new lanostane-type triterpene glycoside from the sea cucumber Holothuria hilla. Chem Biodivers 2006, 3(11):1249-1254. 10.1002/cbdv.200690126View ArticleGoogle Scholar
  31. Zhang SY, Yi YH, Tang HF: Bioactive triterpene glycosides from the sea cucumber Holothuria fuscocinerea. J Nat Prod 2006, 69(10):1492-1495. 10.1021/np060106tView ArticleGoogle Scholar
  32. Zou Z, Yi Y, Wu H, Yao L, Jiuhong LD: Intercedensides D-I, cytotoxic triterpene glycosides from the sea cucumber Mensamaria intercedens Lampert. J Nat Prod 2005, 68(4):540-546. 10.1021/np040205bView ArticleGoogle Scholar

Copyright

© Dhinakaran and Lipton; licensee Springer. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.