Putative mechanism for apoptosis-inducing properties of crude saponin isolated from sea cucumber (Holothuria leucospilota) as an antioxidant compound

Document Type : Original Article


1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran

2 Department of Biology, Mashhad Branch, Islamic Azad University, Mashhad, Iran

3 Medical Genetics Research Center, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

4 Department of Pharmacognosy, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran


Objective(s):Marine organisms are known as a potential source of natural products, which contain bioactive substances with therapeutic properties. Sea cucumbers are prominent among marine organisms because of their dietary and therapeutic applications. In addition, they have capacity of synthesizing saponins molecules and other metabolites with therapeutic properties such as antitumor, antimicrobial, anti-inflammatory and antioxidant activities. The aim of this study was to evaluate the antioxidant and pro-apoptotic effects of sea cucumber saponins (SCS) isolated from Holothuria leucospilota species.
Materials and Methods: Evaluation of antioxidant activity of SCS was carried out by DPPH (1, 1-diphenyl-2-picrylhydrazyl), ABTS (azino-bis-3-ethylbenzothiazoline-6-sulfonic acid), power reducing and total antioxidant assays. The anti-proliferative effect was studied by МТТ (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. Mechanisms leading to apoptosis were also evaluated byfluorescence microscopy, flow cytometry and real time PCR.
Results: The results showed that the DPPH and ABTS activities increased in a dose dependent manner. The reducing capacity enhanced with increasing concentration of the saponin extract (0 to 2 mg/ml). The SCS exhibited moderate total antioxidant activity. Evaluation of anti-proliferative effect revealed that SCS with IC50 of about 6 μg/ml, can display a good cytotoxic activity in a dose dependent manner. Further apoptosis induction was confirmed by fluorescence microscopy and flow cytometry. Sea cucumber saponin was also found to exert a pro-apoptotic effect by increasing the expression of Bax and decreasing the expression of Bcl2.
Conclusion: These results indicate that the SCS may act as a natural antioxidant and antitumor agent.


1. Althunibat O, Ridzwan B, Taher M, Daud J, Jauhari Arief Ichwan S, Qaralleh H. Antioxidant and cytotoxic properties of two sea cucumbers, Holothuria edulis Lesson and Stichopus horrens Selenka. Acta Biol Hung 2013; 64:10-20.
2. Patel-Rajesh M, Patel-Natvar J. In vitro antioxidant activity of coumarin compounds by DPPH, Super oxide and nitric oxide free radical scavenging methods. JAPER. 2011;1:52-68.
3. da Silva Gomes ECB, Jimenez GC, da Silva LCN, de Sá FB, de Souza KPC, Paiva GS, et al. Evaluation of antioxidant and antiangiogenic properties of caesalpinia echinata extracts. J Cancer 2014; 5:143-150.
4. Seifried HE, McDonald SS, Anderson DE, Greenwald P, Milner JA. The antioxidant conundrum in cancer. Cancer Res 2003; 63:4295-4298.
5. Yang HL, Chen CS, Chang WH, Lu FJ, Lai YC, Chen CC, et al. Growth inhibition and induction of apoptosis in MCF-7 breast cancer cells by Antrodia camphorata. Cancer Lett 2006; 231:215-227.
6. Yang B, Johnson TS, Thomas GL, Watson PF, Wagner B, Skill NJ, et al. Expression of apoptosis-related genes and proteins in experimental chronic renal scarring. J Am Soc Nephrol 2001; 12:275-288.
7. Sarfaraj HM, Sheeba F, Saba A, Mohd S. Marine natural products: A lead for Anti-cancer. Indian J Mar Sci 2012; 41:27-39.
8. Soltani M, Baharara J. Antioxidant and antiprolifereative capacity of dichloromethane extract of holoturia leucospilota sea cucumber. Int J Data Min Bioinform 2014; 1-9.
9. Bordbar S, Anwar F, Saari N. High-value components and bioactives from sea cucumbers for functional foods—a review. Mar Drugs 2011; 9:1761-1805.
10. Li R, Zhou Y, Wu Z, Ding L. ESI‐QqTOF‐MS/MS and APCI‐IT‐MS/MS analysis of steroid saponins from the rhizomes of Dioscorea panthaica. J Mass Spectrom 2006; 41:1-22.
11. Podolak I, Galanty A, Sobolewska D. Saponins as cytotoxic agents: a review. Phytochem Rev 2010; 9:425-474.
12. Sun H, Chen L, Wang J, Wang K, Zhou J. Structure–function relationship of the saponins from the roots of Platycodon grandiflorum for hemolytic and adjuvant activity. Int Immunopharmacol 2011; 11:2047-2056.
13. Arabski M, Węgierek-Ciuk A, Czerwonka G, Lankoff A, Kaca W. Effects of saponins against clinical E. coli strains and eukaryotic cell line. J Biomed  Biotechnol 2012; 2012.
14. Patra J, Dhal N, Thatoi H. In vitro bioactivity and phytochemical screening of Suaeda maritima (Dumort): A mangrove associate from Bhitarkanika, India. Asian Pac J Trop Med 2011; 4:727-734.
15. Moghimipour E, Sadaghi-nejad B, Handali S, Ameri A, Azemi ME. In vitro screening of anti-candida activity of saponins extracted from Glycyrrhiza glabra and Quillaja saponaria. Asian J Pharm Clin Res 2014; 7:7-9.
16. Prabakaran R, Kalimuthu K, Vani C, Brindha C. Angiogenesis and antioxidant activity of in vitro and in vivo tuber of ceropegia pusilla wight and arn. Br J Pharm Res 2014; 4: 608–616.
17. Tian F, Zhu C-h, Zhang X-w, Xie X, Xin X-l, Yi Y-h,  et al. Philinopside E, a new sulfated saponin from sea cucumber, blocks the interaction between kinase insert domain-containing receptor (KDR) and αvβ3 integrin via binding to the extracellular domain of KDR. Mol Pharmacol 2007; 72:545-552.
18. Hu X-Q, Wang Y-M, Wang J-F, Xue Y, Li Z-J, Nagao K, et al. Dietary saponins of sea cucumber alleviate orotic acid-induced fatty liver in rats via PPARa and SREBP-1c signaling. Lipids Health Dis 2010; 9:25.
19. Rajamanikandan S, Sindhu T, Durgapriya D, Sophia D, Ragavendran P, Gopalakrishnan V. Radical scavenging and antioxidant activity of ethanolic extract of mollugo nudicaulis by In vitro assays. IJPER 2011; 45:310-316.
20. Li P, Huo L, Su W, Lu R, Deng C, Liu L, et al. Free radical-scavenging capacity, antioxidant activity and phenolic content of Pouzolzia zeylanica. J Serb Chem Soc 2011; 76:709-717.
21. Chang HY, Ho YL, Sheu MJ, Lin YH, Tseng MC, Wu SH, et al. Antioxidant and free radical scavenging activities of Phellinus merrillii extracts. Bot Stud 2007; 48:407-417.
22. Shajiselvin C, Muthu AK. In-vitro antioxidant studies of various extracts of whole plant of Borreria hispida (Linn). Res J Pharm Biol Chem Sci 2010; 1:14-20.
23. Namvar F, Rahman HS, Mohamad R, Baharara J, Mahdavi M, Amini E, et al. Cytotoxic effect of magnetic iron oxide nanoparticles synthesized via seaweed aqueous extract. Int J Nanomed 2014; 9: 2479–2488.
24. Pan SK, Yao DR, Zhou MQ, Wu SJ. Hydroxyl radical scavenging activity of peptide from sea cucumber using enzyme complex isolated from the digestive tract of sea cucumber. Afr J Biotechnol 2014; 11:1214-1219.
25. Philip JP, Madhumitha G, Mary SA. Free radical scavenging and reducing power of Lawsonia inermis L. seeds. Asian Pac J Trop Med 2011; 4:457-461.
26. Althunibat OY, Hashim RB, Taher M, Daud JM, Ikeda M-A, Zali B. In vitro antioxidant and antiproliferative activities of three Malaysian sea cucumber species. Eur J Sci Res 2009; 37:376-387.
27. Zhong Y, Khan MA, Shahidi F. Compositional characteristics and antioxidant properties of fresh and processed sea cucumber (Cucumaria frondosa). J Agric Food Chem 2007; 55:1188-1192.
28. Chenghui L, Beiwei Z, Xiuping D, Liguo C. Study on the separation and antioxidant activity of enzymatic hydrolysates from sea cucumber. Food Ferment Ind  2007; 33:50–53.
29. Chen Y, Miao Y, Huang L, Li J, Sun H, Zhao Y, Yang J. Antioxidant activities of saponins extracted from Radix Trichosanthis: an in vivo and in vitro evaluation. BMC
Complement Altern Med 2014; 14:1-8.
30. Ashraf MF, Abd Aziz M, Stanslas J, Ismail I, Abdul Kadir M. Assessment of antioxidant and cytotoxicity activities of saponin and crude extracts of chlorophytum borivilianum. Scientific WorldJournal 2013; 2013: 216894.
31. Arslan I, Çelik A. Saponin rich fractions (SRPs) from soapwort show antioxidant and hemolytic activity. APCBEE Procedia 2013; 7:103-108.
32. Hu JL, Nie SP, Huang DF, Li C, Xie MY. Extraction of saponin from Camellia oleifera cake and evaluation of its antioxidant activity. Int  J Food Sci Technol 2012; 47:1676-1687.
33. Lu C, Li C, Wu D, Lu J, Tu F, Wang L. Induction of apoptosis by Rhizoma Paridis saponins in MCF-7 human breast cancer cells. Afr J Pharm Pharmaco 2011; 5:1086-1091.
34. Xu K, Shu Z, Xu Q-M, Liu Y-L, Li X-R, Wang Y-L, Yang SL. Cytotoxic activity of Pulsatilla chinensis saponins and their structure–activity relationship. J Asian Nat Prod Res 2013; 15:680-686.
35. Kaswandi M, Hing H, Sahalan A, Farah F, Ridzwan B, Samsudin M, et al. Saponin from sea cucumber Stichopus badionotus sluiter as potential cytotoxic agent on CEM-SS T-lymphoblastic cell. J Microsc Soc Thailand 2004; 18:79-84.
36. Yang P, Collin P, Madden T, Chan D, Sweeney‐Gotsch B, McConkey D, et al. Inhibition of proliferation of PC3 cells by the branched‐chain fatty acid, 12‐methyltetradecanoic acid, is associated with inhibition of 5‐lipoxygenase. Prostate 2003; 55:281-291.
37. Huang W, Zou K. Cytotoxicity of a plant steroidal saponin on human lung cancer cells. Asian Pac J Cancer Prev 2011; 12:513-517.
38. Wijesinghe W, Jeon YJ, Ramasamy P, Wahid MEA, Vairappan CS. Anticancer activity and mediation of apoptosis in human HL-60 leukaemia cells by edible sea cucumber (Holothuria edulis) extract. Food Chem 2013; 139:326-331.
39. Hsu MJ, Cheng JS, Huang HC. Effect of saikosaponin, a triterpene saponin, on apoptosis in lymphocytes: association with c‐myc, p53, and bcl‐2 mRNA. Br J Pharmacol 2000; 131:1285-1293.
40. Du L, Li Z-J, Xu J, Wang J-F, Xue Y, Xue C-H, et al. The anti-tumor activities of cerebrosides derived from sea cucumber Acaudina molpadioides and starfish Asterias amurensis in vitro and in vivo. J Oleo Sci 2012; 61:321-330.