48 SPC Beche-de-mer Information Bulletin #31 – January 2011

Review of saponin diversity in sea belonging to the family Guillaume Caulier,1* Séverine Van Dyck,1 Pascal Gerbaux,2 Igor Eeckhaut1 and Patrick Flammang1

Abstract

Saponins are secondary metabolites produced by holothurians. Structurally, they are triterpene glycosides that play an important role in chemical defense and possess a wide spectrum of pharmacological effects. This review highlights the very high diversity of saponins detected in different of the family Hol- othuriidae. No less than 59 triterpene glycosides are reported. Several saponins are shared by many species but others are very specific. Overall, most species appear to possess a specific congener mixture. The most evident inter-specific differences that can be highlighted among Holothuriidae are based on the presence or absence of a sulfate group attached to the carbohydrate chain of their saponins. Within a single , saponin mixtures also present different concentrations and compositions depending on the organ, with Cuvierian tubules showing the highest saponin concentrations. All of the data combined indicate a complex chemical defence mechanism with different sets of saponins originating from different body compartments and presenting different properties in relation to their ecological role(s).

Introduction ecology, saponins are deleterious for most organ- isms and probably function as a chemical defense Saponins are an important class of natural prod- to deter (Kalinin et al. 1996a, b; Van Dyck ucts first discovered in higher plants where they et al. unpubl. obs.). are widely spread (Li et al. 2006). In the search for new pharmacologically active substances, saponins Saponin diversity in Holothuriidae have also been isolated from marine organisms such as holothurians (Nigrelli 1952; Yamanouchi 1955), This article reviews the diversity of saponins seastars (Mackie and Turner 1970) and sponges detected in different species of the family Holothuri- (Thompson et al. 1985). Structurally, holothurian idae. Table 1 presents all the saponins extracted and saponins are described as triterpene glycosides characterized from holothurians of the genera Actin- composed of an oligosaccharide chain and an agly- opyga, , and dur- cone based on holostane-3b-ol (Fig.1A) (Kornprobst ing the last 40 years. Saponins detailed in this table 2005). Saponins of Holothuriidae (Fig. 1B,C) con- have been purified by different methods including tain a D9(11) double bound in the aglycone and the liquid-liquid extractions with different solvents, carbohydrate chain encloses up to 6 sugars units, solid phase extraction or chromatography (silica including xylose, glucose, 3-O-methylglucose and gel or resins), and high performance liquid chroma- quinovose, and can be branched only once (Kalinin tography. Mass spectrometry-based techniques and et al. 2005). Some of these saponins can be sulfated nuclear magnetic resonance combined with chemi- at the level of the sole xylose (Fig. 1C). cal reactions and chemical evidence were used to highlight the chemical structure of these saponins. Holothurian triterpene glycosides present a high Table 1 emphasizes the very high diversity of scientific interest in pharmacology and ecology. saponins in holothuriids. Indeed, no less than 59 Indeed, these secondary metabolites have been triterpene glycosides are reported. When these data reported to possess a wide spectrum of pharma- are gathered from the literature, some nomencla- cological effects including hemolytic, antitumoral, ture problems can be identified. It happens that anti-inflammatory, antifungal, anti-bacterial, anti- two names have been given independently to the viral, ichthyotoxic, cytostatic and antineoplastic same molecule. For instance, the structure of nobi- activities (Kerr and Chen 1995; Kalinin et al. 1996a, liside 2a detailed by Wu et al. (2006c) corresponds 1996b; Prokofieva et al. 2003). Many of these activi- exactly to desholothurin A described by Rodrigez ties are the result of their tensioactive properties. In et al. (1991). Also, authors should homogenize the

1. University of Mons, Marine Biology Laboratory, 7000 Mons, Belgium. 2. University of Mons, Organic Chemistry Laboratory, Interfaculty Center for Mass Spectrometry (CISMa), 7000 Mons, Belgium. * Corresponding author: [email protected] SPC Beche-de-mer Information Bulletin #31 – January 2011 49

Figure 1. Molecular structure of (A) a hypothetic saponin composed of an aglycone of holostanol (according to the Comparative Toxicogenomic Database) and a linear glycosidic chain constituted by the four most frequent monosaccharides found in holothurian saponins; (B) holothurinoside A, a non-sulfated saponin; and (C) holothurin A, a sulfated saponin. saponin nomenclature by giving logical names to (Elyakov et al. 1973, 1975). In the future, one should new molecules, based on the structure of known expect that new studies, using contemporary tech- congeners, rather than names based on the specific niques, will detect additional saponins in these spe- origin of the molecules. cies. Indeed, Table 1 clearly shows that many new congeners have been described only recently. Most The most evident inter-specific differences that can species therefore appear to possess a specific conge- be highlighted among Holothuriidae are based on ner mixture, a valuable chemotaxonomic character the presence or absence of sulfate group attached allowing the assignment of a holothuriid species to to the carbohydrate chain of their saponins (Koba- a specific taxa according to its chemical signature. yashi et al. 1991). The contains For example, the taxonomic position of Bohadschia only sulfated saponins (in green in Table 1), the graeffei was revised, following the isolation and genus Bohadschia encloses only non-sulfated ones characterization of its saponins, to a newly estab- (in red in Table 1), and the genera Pearsonothuria and lished genus Pearsonothuria graeffei (Kalinin et al. Holothuria present both saponin types. In this last 2005). genus, the situation is even more complex with sev- eral species containing only sulfated saponins, some Among the numerous studies dealing with sapon- others presenting the two types of congeners, and ins from holothuroids, very few make the distinc- finally one species, H. forskali, enclosing exclusively tion between their different body compartments non-sulfated saponin. Several saponins are shared (Table 1) although, within an animal, saponins by a lot of species, holothurins A and B for example, may present different concentrations and composi- but others are very specific like griseaside A or argu- tions according to the organ considered. Matsuno sides A-E. It must be noted that holothurins A and and Ishida (1969) reported about the distribution B were the first to be discovered (Yamanouchi 1955; of saponins in sea body compartments. Kitagawa et al. 1978, 1979) and were subsequently Saponins were found in the digestive organs, the detected in many species of the genus Holothuria longitudinal retractor muscles, the epidermis, the 50 SPC Beche-de-mer Information Bulletin #31 – January 2011 (1991) . (1991) (1991) . . Radhika et al. (2002) Radhika et al. (1973) et al. Elyakov (1975) et al. Elyakov (1991) et al. Kobayashi (1991) et al. Kobayashi 2 Chapter (2008) et al. Yuan (1991) et al. Kobayashi (1991) et al. Kobayashi (2008) et al. Yuan (1973) et al. Elyakov (1973) et al. Elyakov (1973) et al. Elyakov (1975) et al. Elyakov (1975) et al. Elyakov (1973) et al. Elyakov (1973) et al. Elyakov (1986) Stonik (1986) Stonik (1986) Stonik (1986) Stonik (1982) et al. Elyakov x 1 Chapter x 1 Chapter x 1 Chapter (2002) Radhika et al. xxx xx 2 Chapter x 2 Chapter x x 2 Chapter x x 2 Chapter x x 2 Chapter x 2 Chapter x 2 Chapter (2002) Radhika et al. (2002) Radhika et al. xxx (1991) et al. Kobayashi x et al Kobayashi 2 Chapter 2 Chapter x (1991) et al. Kobayashi x xx (1991) Rodrigez et al. xx (1991) Rodrigez et al. x x x Rodrigez et al x Rodrigez et al x (1991) Rodrigez et al. 1 1 1 1 1 1 (Arguside E)** (Arguside 1 * 4 2 2 2 1 2 1 2 3 1 Holothurinoside F B* Holothurinoside H/Marmoratoside Holothurinoside H Holothurinoside I Holothurinoside I Holothurinoside J Holothurinoside K C Bivittoside D Bivittoside C Bivittoside D Bivittoside Holothurin A Holothurin B Holothurin A Holothurin A Holothurin B Holothurin B/B Holothurin B Holothurin B Holothurin B Axilogoside A Holothurin A Holothurin A Holothurin B Holothurin A Holothurin A Holothurin B Holothurin A Holothurin B Holothurin A Holothurin B Holothurin A Holothurin A Holothurin B Holothurin A Holothurin A Holothurin B Desholothurin A (Nobiliside 2a)** Desholothurin A Holothurinoside A Holothurinoside A Holothurinoside B Holothurinoside C Holothurinoside C Holothurinoside D Bohadschia subrubra Holothuria cubana Holothruia edulis Bohadschia vitiensis Holothuria coluber cilis Holothuria diffi oridana Holothuria fl Bohadschia tenuissima Holothuria arenicola Holothuria axiloga (Microthele) Kitagawa et al. (1980) Kitagawa et al. (1975) et al. Elyakov (1980) Kitagawa et al. (1973) et al. Elyakov 2 Chapter (1985) Bhatnagar et al. (1985) Bhatnagar et al. (1973) et al. Elyakov (1973) et al. Elyakov (1973) et al. Elyakov (1973) et al. Elyakov (2007) Liu et al. (2008a) Liu et al. (2008b) Liu et al. (2009) et al. Yuan (2009) et al. Yuan (2009) et al. Yuan (2009) et al. Yuan (2009) et al. Yuan (2009) et al. Yuan xx (1982) Kitagawa et al. (1982) Kitagawa et al. (Continued) x (2008a) Liu et al. xx Ohta and Hikino (1981) x Ohta and Hikino (1981) x Ohta and Hikino (1981) Ohta and Hikino (1981) xx 2 Chapter 2 Chapter x 2 Chapter (1973) et al. Elyakov xx xx x 2 Chapter (1973) et al. Elyakov x (1980) Kitagawa et al. xx (1980) Kitagawa et al. 2 Chapter 2 Chapter xxx (1991) et al. Kobayashi x (1991) et al. Kobayashi x (1991) et al. Kobayashi (1973) et al. Elyakov (1991) et al. Kobayashi x x (2008b) Liu et al. x xx 2 Chapter 2 Chapter )** 1 2 2 1 * 4 2 2 2 1 1 2 3 1 24-dehydroholothurin A 24-dehydroholothurin Holothurin A Holothurin A Holothurin B Holothurin B B/C* Fuscocineroside Holothurin A Holothurin A Holothurin B Holothurin B/B Holothurin B Holothurin B Holothurin B Holothurin A Holothurin B Holothurin A Holothurin B A 24-dehydroholothurin B 24-dehydroholothurin Holothurin A Holothurin A Holothurin B Holothurin B Holothurin A Holothurin B A Arguside B Arguside C Arguside D Arguside E (Desholothurin A Arguside A Bivittoside B Bivittoside C Bivittoside D Bivittoside 17 impatienside A -hydroxy D bivittoside 25-acetoxy C Bivttoside D Bivittoside A (Impatienside A)** Marmoratoside B/Holothurinoside H* Marmoratoside C Arguside B Bivittoside C Bivittoside D Bivittoside A)** Impatienside A (Marmoratoside Saponins of Holothuriidae by species. Species agassizi Actinopyga Saponins BW CT References Species Saponins BW CT References Actinopyga fl ammea fl Actinopyga mauritana Actinopyga Bohadschia bivittata Actinopyga Bohadschia subrubra Table 1. Table SPC Beche-de-mer Information Bulletin #31 – January 2011 51 Yasumoto et al. (1967) et al. Yasumoto (1967) et al. Yasumoto (1975) et al. Elyakov (1975) et al. Elyakov Matsuno and Iba (1966) Matsuno and Iba (1966) (1973) et al. Elyakov (2006a) et al. Wu (2006a) et al. Wu (2006b) et al. Wu (2006b) et al. Wu (2006b) et al. Wu (1973) et al. Elyakov (1973) et al. Elyakov (1973) et al. Elyakov (1973) et al. Elyakov (2007) Dang et al. (2007) Dang et al. (1973) et al. Elyakov (1986) Stonik (1975) et al. Elyakov (1975) et al. Elyakov (2005) Silchenko et al. 2 Chapter x 2 Chapter (2005) Silchenko et al. x (2002) Radhika et al. xx xx (1989) Kitagawa et al. xx (1989) Kitagawa et al. x (1989) Kitagawa et al. x (2005) et al. Silchenko x (2005) et al. Silchenko (2005) et al. Silchenko (2005) et al. Silchenko x (2005) et al. Silchenko xx (1991) et al. Kobayashi (2007) et al. Dang (2007) et al. Dang xx xx x 2 Chapter x x 2 Chapter x x (1973) et al. Elyakov x (1986) Stonik x (1973) et al. Elyakov 2 Chapter 2 * 4 2 3 4 2 2 3 4 Holothurin A Holothurin B Holothurin A Holothurin B Holothurin A Holothurin A Holothurin A Holothurin B Nobiliside 1a Nobiliside 2a (Desholothurin A)** Nobiliside A Nobiliside B Nobiliside C Holothurin A Holothurin B A Pervicoside B Pervicoside C Pervicoside Holothurin A Holothurin B Holothurin B Holothurin B Holothurin B Holothurin A Holothurin B A 24-dehydroholothuin Holothurin A Holothurin A Holothurin A Holothurin A Holothurin B Holothurin A Holothurin A Holothurin B Holothurin A Holothurin B D Bivittoside Desholothurin A (Nobiliside 2a)** B/C* Fuscocineroside Holothurin A Holothurin A Holothurin B Holothurin B/B Holothurinoside C Holothuria polii Holothuria pulla Holothuria lubrica Holothuria moebi Holothuria surina- mensis graeffei Pearsonothuria Holothuria monacaria Holothuria squamifera Chapter 4 Chapter 4 Chapter (2006) Zhang et al. (1973) et al. Elyakov (2006) Zhang et al. (2006) Zhang et al. (2006) Zhang et al. (2006) Zhang et al. (1973) et al. Elyakov (1973) et al. Elyakov (2008) Sun et al. (2008) Sun et al. (1975) et al. Elyakov (1975) et al. Elyakov (1975) et al. Elyakov (2006c) et al. Wu (1973) et al. Elyakov (1973) et al. Elyakov (2007) Sun et al. (1973) et al. Elyakov (2007) Sun et al. 2 Chapter (2007) Han et al. (2008) Han et al. x 1 Chapter x 1 Chapter x 1 Chapter x 1 Chapter x 1 Chapter x 2 Chapter xx 2 Chapter x 2 Chapter (1979) Kitagawa et al. x 2 Chapter x xx 1 Chapter xx 1 Chapter xx 1 Chapter xx 1 Chapter x 1 Chapter x x 2 Chapter x xx (1978) Kitagawa et al. xx 2 Chapter 2 Chapter 2 Chapter 1 1 1 1 1 1 1 1 2 3 Holothurinoside E Holothurinoside E Holothurinoside F Holothurinoside F Holothurinoside G Holothurinoside G B* Holothurinoside H/Marmoratoside Holothurinoside H Holothurinoside I Holothurinoside I Holothurinoside L*** Holothurinoside M*** Holothurin A Holothurin B A Fuscocineroside B Fuscocineroside C Fuscocineroside C Pervicoside Holothurin A Holothurin B A 17-dehydroxyholothurinoside Griseaside A Holothurin A Holothurin A Holothurin B Hillaside C Holothurin A Holothurin B D Bivittoside Holothurin A A)** Impatienside A (Marmoratoside C Bivittoside D Bivittoside Desholothurin A (Nobiliside 2a)** B/C* Fuscocineroside Holothurin A Holothurin B Holothurin B/B4* Holothurin B Holothurin B Holothurin B Holothurinoside E A Leucospilotaside C Leucospilotaside (continued)

Holothuria gracilis Holothuria impatiens Table 1 Table Species Holothuria forskali Saponins BW CT References Species Saponins BW CT References BW = body wall; CT = Cuvierian tubules; sulfated saponins in green; non-sulfated saponins in red. non-sulfated saponins in green; sulfated tubules; CT = Cuvierian BW = body wall; * Isomeric saponins. the same structure. names for ** Different in which the animal stayed. *** Extracted seawater from 52 SPC Beche-de-mer Information Bulletin #31 – January 2011 intestinal hemal vessels, the ovaries, the testes indicate a complex chemical defence mechanism and the Cuvierian tubules. Amounts of saponins with, for a single species, different sets of saponins (expressed by the hemolytic index) were different originating from different body compartments and between body compartments and the body wall presenting different responses to stress. This pre- and Cuvierian tubules showed the highest values sumably finely tunes the saponin properties accord- (ovaries also presented high saponin concentrations ing to their ecological role(s). but they varied with the reproductive cycle of the animal; Matsuno and Ishida 1969). Van Dyck et al. Acknowledgements (2010) also highlighted a variation of saponin quan- tities between the Cuvierian tubules and the body SVD and GC benefited from a FRIA doctoral grant wall in several species of Holothuriidae. Triterpene (Belgium). PF and PG are respectively Senior glycosides appear to be particularly concentrated in Research Associate and Research Associate of the the Cuvierian tubules, a specialized defense system Fund for Scientific Research of Belgium (F.R.S.- developed by some species within the family Hol- FNRS). PG thanks the F.R.S.-FNRS for financial sup- othuriidae (Matsuno and Ishida 1969; Elyakov et al. port in the acquisition of the Waters Q-ToF Premier 1973; Kobayashi et al. 1991). This organ located in Mass Spectrometer and for continuing support. the posterior part of the animal consists of multi- Work supported by a FRFC Grant n° 2.4535.10. This ple tubules that can, in some species, be expelled study is a contribution of the “Centre Interuniversi- by the individual after stimulation (Bingham and taire de Biologie Marine” (CIBIM). Braithwaite 1986; Hamel and Mercier 2000; Becker and Flammang in press). References

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