Devaraj Isaac DHINAKARAN, and Aaron PremnathLIPTON



Antitumor and Antifungal Activities of Organic Extracts of Seacucumberfrom the Southeast Coast of India

Devaraj Isaac DHINAKARAN1), *, and Aaron PremnathLIPTON2)

1),,629502,,,2),,695521,

In phylum Echinodermata, the family Holothuridae is distinguished by its capacity of bioactive compounds. Seacucumberis commonly known as the lollyfish. Theantifungalactivitywasdetected using agarwelldiffusionmethodagainstthevariousfungalstrainssuchas,,,and. Relatively high antifungal activity was seen againstat 100µL−1concentration of extracts. Zone of inhibition was measured at 18mm of diameter. Theanti-tumoractivitiesweredetectedagainsttheVeroandHep2celllines using MTTassay. The cells were treated withextract at concentrations 0.078-10mgmL−1. The extract showed high proliferative activity against the Hep2 cells. The body wall extracts ofseacucumber(.showedeffectiveantifungalandantitumoractivities. All these findings suggest that the extracts could be used for the development of drugs.

;MTTassay; Verocelllines;Hep2celllines;antifungalactivity;antitumor activity

1 Introduction

Seacucumbers, belonging to the class, are marine invertebrates, habitually found in the benthic areas and deep seas across the world. They have high commercial value coupled with increasing global production and trade (Bordbar., 2011). In a recent study, the smallest peptide fragment of a beta-thymosin detected in the coelomocytes of Mediterranean sea-urchinshowed the common chemical-physical characteristics of AMPs (Schillaci., 2012).The characterization of a purified extract obtained from(Holothuria) by chromatographic and mass spectrometric techniques was identified as holothurinoside D. The biological activity of the purified extract showed antibacterial, antifungal and cytotoxic activity on a neuroblastoma cell line (Sottorff., 2013).

Sea cucumbers are one of the marine animals which are important as human food source, particularly in some parts of Asia. They are usually soft-bodied echinoderms comprising a diverse group of flexible, elongated, worm- like organisms, with a leathery skin and gelatinous body, looking like a cucumber (Taiyeb., 2003). Sea cu-cumbers such as,,extracts collected from the Malaysian coastal waters act as the appropriate source of wound healing promoter, anti-microbial, anticancer and immunomodulatory activities (Hawa., 1999). Triterpenoid glycosides (saponins) are the major and most abundant type of compounds isolated from sea cucumbers. In Sea cucumber, namely holothurin A3, is found to be strongly cytotoxic to cancer cell lines, human epidermoid carcinoma and human hepatocellular carcinoma cells (Zhang., 2006).

Arguside, a bioactive compound isolated from sea cucumberexhibites significant cyto- toxFicity against different human tumor cell lines while showing the highest activity towards human colorectal carcinoma (HCT-116) cells (IC50=0.14μmolL−1).They have also shown potent cytotoxicity against human tumor cell lines, adenocarcinomic human alveolar basal epithelial cells (A549), HCT-116, HepG2, and human breast adenocarcinoma (MCF-7) cell lines (Liu., 2008). The southeastern Gulf of Mannar and the southwestern Palk Bay are rich in echinoderms like sea cucumber, star fishes and sea urchins. Sea cucumbers,,.and.contain saponins (glycosidic steroids) and holothurin compounds are ichthyotoxic in nature. This property has been studied on fish fingerlings, mice and erythrocytes using haemolytic activity (Rajendran, 2000).,a processed form of sea cucumber, has the curative power for ailments like high blood pressure and muscular disorders with pharmacological effects (Ogushi., 2005). Several studies have shown multiple biological activities of sea cucumber species. These include wound healing promoters and antinociceptive, antibacterial, antifungal and antioxidant properties (Himaya., 2010). The medicinal benefits and health functions of sea cucumbers can be attributed to the presence of appre- ciable amounts of bioactive compounds, especially the triterpene glycosides, saponins, chondroitin sulfates, glycosaminoglycan, sulfated polysaccharides, sterols, phenolics, peptides, cerberosides and lectins (Sugawara., 2006).The immune mediators inact as a source of novel antimicrobial peptides for the deve- lopment of new agents against biofilm bacterial communities that are often intrinsically resistant to conventional antibiotics (Schillaci., 2013). Therefore, the present study was to investigate the antifungal and antitumor activities of.extract from species found along the Indian coast. This could be used to identify its potentials in the development of pharmaceuticals in drug industry.

2 Materials and Methods

2.1 Sample Collection and Extract Preparation

.specimens with a size range of 10 to 30cm in length and 30 to 180g in 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). Immediately upon collection, they were dissected to remove the internal organs and packed using ice and kept at −80℃ prior to 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 following the method given by Rashid., (2001) with slight modifications. About 200g of frozen samples were homogenized with deionized water and methanol. The mixture was continuously stirred in the dark at 4℃ for 24h. Then it was centrifuged at 5000rmin−1for 15min. The supernatant was collected and filtrated. The collected organic extracts were freeze-dried and kept at −80℃, while the insoluble solid materials were re-extracted with methanol (100%). The organic extracts were combined and the solvents were removed by rotary evaporation at 40℃ under low pressure to avoid degradation of compounds.

2.2 Antifungal Activity

The antifungal activity was determined by well diffusion method using Potato dextrose agar. Fungal isolates were obtained from the microbial type culture collection and gene bank (MTCC) at Institute for Microbial Technology, Chandigarh, India. Isolates such asMTCC 2047,MTCC 1344,MTCC 183,MTCC 5108 andMTCC 873 were used for the experiment.To each well,100μL of methanol extract (5μg) of seacucumber was added. For the positive control, methanol was used. All the plates were incubated at room temperature 30℃ for 5d.

2.3 Cytotoxicity Studies (MTT Assay)

HEp2 cell line and Vero cell lines were obtained from King Institute, Chennai. They were maintained in MEM medium (with high glucose and glutamine) supplemented with 10% heat inactivated FBS and 1% penicillin/strepto- mycin, at 37℃ in a humidified atmosphere containing 5% CO2. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-te- trazolium bromide (MTT) assay was used to evaluate the anti-proliferative activities of the sea cucumber extracts against the cancer cell lines in twenty four well plates. Each well was washed with MEM (w/o) FCS twice or thrice in which 200μL of MTT (5mgmL−1) was added and incubated for 6 to 7h in 5% CO2incubator. After incubation, 1.0mL of DMSO was added in each well, mixed thoroughly by pipette and left for 45sec. The presence of viable cells was detected with purple colour due to the formazan crystals after adding DMSO. The suspension was transferred into the cuvette of spectrophotometer and an O. D values were read at 595nm by keeping DMSO as a blank. Values were plotted as concentration of the sample onaxis and relative cell viability onaxis. The cytotoxic activities of.extracts against the Hep2 and Vero cell lines were observed as images using the inverted microscope at 25%, 50%, 75% and 100% levels. They were also compared with that of normal cell lines.

3 Results

Maximum antifungal activity was recorded towards,andwith a diameter of 16, 14 and 12mm respectively. Then inandthe inhibitory zone diameter was 18 and 14mm at 100μL concentration of extract (Fig.1).From the MTT assay, it could be noted that the sea cucumber extracts reduced the viability of the Hep2 and Vero cell lines. The concen- tration of the extract ranged from 0.078 to 10mgmL−1and at these lower concentrations, the survival rate of the cells was high whereas at higher concentrations the survival rate was less. It is possible that cell death induction could be due to the presence of bioactive compounds. The cell viability was also seen in Vero cell lines and when compared to Hep2 cell lines the effect was less (Fig.2 and Fig.3). The monolayer of Hep2 and Vero cell lines and the cytotoxic effects of the extracts in Hep2 and Vero cell lines were observed as 25%, 50%, 75% and 100% dead cells. It was observed using inverted micro- scope (Fig.4 and Fig.5). These results suggest that sea cucumber.has antitumor property.

Fig.1 Antifungal activities of the methanol extracts of sea cucumber.

Fig.2 MTT assay of Hep 2 cell lines using H. atra extracts.

Fig.3 MTT assay of Vero cell lines using H. atra extracts.

Fig.4 Cytotoxicity images of sea cucumber extracts against Hep2 cell lines using inverted microscope. 1+, for 25% dead cells; 2+, for 50% dead cells; 3+, for 75% dead cells; 4+, for 100% dead cells.

Fig.5 Cytotoxicity images of sea cucumber extract against Vero cells using inverted microscope.

4 Discussion

In the present study the antitumor activities of.extracts against the Hep2 and Vero cell lines were observed. An example of this is Frondoside A, isolated from sea cucumber which demonstrates an important antimetastatic activity (Ma. 2012). A similar study revealed that the solvent extracts and the compounds isolated from sea cucumbers showed a variety of biological activities such as antifungal, anticancer, hemolytic, cytostatic, antioxidant and immunomodulatory effects (Dang., 2007). The antifungal activity of the.could be due to the presence of compounds such as terpenes, alkaloids and saponins. Patagonicoside, a bioactive novel antifungal disulphated triterpene glycoside from the sea cucumber,was found to be active against the pathogenic fungus(Ana.,2001). It was also noted that the antifungal activity was maximum againstand. Similarly the extracts ofandhave the capacity to inhibit the growth of(an amphotericin B and nystatin resistant strain) as well as other bacteria (Rifai., 2005). Triterpene glycosides from the sea cucumber,a bioactive compound, showed effective anifungal activity against,,(Kumar., 2007).

Inhibition effect of the extracts from the sea cucumber.on the growth of two cancer cell lines,.., Hep2 and Vero cell lines was demonstrated by using MTT assay. The results on activity of the extracts showed that sea cucumber (contains bioactive compounds that exhibit antitumor activities. High molecular fraction of hot water extracts from sea cucumber,, inhibits the growth of human colon adenocarcinoma and induction of apoptosis (Ogushi., 2006). In the present study the antitumor activity was detected by using the methanolic extracts of. In the propanol extracts fromandthe bioactive components like the glycosides and the desulfated derivatives of calcigerosides have been seen (Avilov., 2000).

The antitumor property in.could be due to the presence of compounds such as the lectins, terpenes, sterols, terpenoids, alkaloids,. So Liouvillosides from,hemoiedemosides fromand a lectin from brown sea cucumbers show antitumor activities. Other components in sea cucumbers also show cytotoxic activities including echinosides, holothurins, holotoxin and cucumarioside (Maier.,2005). It is observed that Hep2 cell line was more sensitive to the active extracts than Vero cells. This is in agreement with Han. (2009) who reported that different cell lines exhibit different responses to the cytotoxic compounds of seacucumbers. The morphological changes and changes in membrane permeability could be easily inferred from the images observed in our experiment through the inverted microscope among the cell lines of Vero and Hep2. These results suggest the cytotoxicity activity of the extracts leading to the death of tumor cells.

5 Conclusion

The present study shows thatextracts have effective antifungal activities against various fungal strains such as,,,and. The.extracts also exhibit antitumor effects against Vero and Hep2 cell lines. Hence it is observed that thecould be used in the development of marine derived drugs. Further studies are needed to identify its bioactive compounds.

Acknowledgements

The authors gratefully thank the Centre for Marine Fisheries and Research Institute, Vizhinjam, Thiruvan- anthapuram. We are also thankful to Dr. S. Joseph Selvin, Professor and Head, Department of Microbiology, Pon- dicherry University for his help and cooperation.

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(Edited by Ji Dechun)

DOI 10.1007/s11802-015-2528-x

ISSN 1672-5182, 2015 14 (1): 185-189

© Ocean University of China, Science Press and Springer-Verlag Berlin Heidelberg 2015

(November 1, 2013; revised March 28, 2014; accepted April 13, 2014)

* Corresponding author. E-mail:isaacdhina@yahoo.co.in