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Use of triterpene glycosides for resolving taxonomic problems in the sea cucumber genus Cucumaria (Holothurioidea, Echinodermata)
Biochemical Systematics and Ecology 32 (2004) 715–733 www.elsevier.com/locate/biochemsyseco
Use of triterpene glycosides for resolving taxonomic problems in the sea cucumber genus Cucumaria (Holothurioidea, Echinodermata) Sergey A. Avilov a, Vladimir I. Kalinin a,, Alexey V. Smirnov b a
Pacific Institute of Bioorganic Chemistry, Far-Eastern Division of the Russian Academy of Sciences, 690022, Vladivostok, Russia b Zoological Institute of the Russian Academy of Sciences, 199164, Saint Petersburg, Russia Received 30 January 2003; accepted 30 December 2003
Abstract This article presents a review of the data on the structures of glycosides from five species of sea cucumbers belonging to the genus Cucumaria (Holothurioidea, Echinodermata). A set of aglycones is specific for glycosides from each species but carbohydrate chains are similar for all five species. These sea cucumbers contain glycosides with three-sulphated branched pentasaccharide carbohydrate chains which are characteristic for the genus Cucumaria. The glycosides from all the Cucumaria species studied are different from the glycosides of Aslia lefevrei and Pseudoocnus echinata that confirms the earlier exclusion of these species from the genus Cucumaria. They are also significantly different from the glycosides of Staurocucumis liouvillei and Hemoiedema spectabilis, the other representatives of the family Cucumariidae. # 2004 Elsevier Ltd. All rights reserved. Keywords: Sea cucumbers; Cucumaria; Cucumariidae; Taxonomy; Triterpene glycosides
1. Introduction The distribution of different triterpene glycosides was successfully applied to the taxonomy of sea cucumbers belonging to the order Aspidochirotida. (Kalinin et al., 1994). Such data were used to revise the genus Bohadschia with the establishment
Corresponding author. Fax: +7-4232-31-40-50. E-mail address: [email protected] (V.I. Kalinin).
0305-1978/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2003.12.008
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of a new genus Pearsonothuria (Levin et al., 1984). A taxonomic relation has been found among the North Pacific representatives of the family Stichopodidae by simultaneous use of the information on the glycoside distribution and morphological data (Levin et al., 1986). The phylogeny of the order Aspidochirotida (Levin et al., 1985) has also been improved by a similar approach. It was confirmed that the triterpene glycosides are specific for genera and groups of genera within this order. However, the glycosides of sea cucumbers belonging to the order Dendrochirotida are much more structurally diverse than those of Aspidochirotida and are species-specific in some cases (Stonik et al., 1999). In this paper we consider the distribution of glycosides in representatives of the genus Cucumaria (Cucumariidae, Dendrochirotida) to clarify the relation of the species.
2. Systematics of the genus Cucumaria and non-resolved taxonomical problems The referrence species of the genus Cucumaria Blainville is Cucumaria frondosa (Gunner, 1767; Baranova, 1976). During different periods this genus included about 90 species of the family Cucumariidae. However, Panning (1949, 1955) showed that only some of these species may really belong to the genus Cucumaria according to data on the structure of throat calcareous ring and sclerites (ossicles) of the body walls. Indeed, among all the sea cucumbers inhabiting the Northern part of Pacific and Atlantic Oceans, only C. frondosa, C. japonica, C. koraeensis, C. djakonovi, C. savelijevae (Baranova, 1976, 1980), and the recently described species C. conicospermium (Levin and Stepanov, 2002), are unquestionably members of this genus. For example, the Atlantic sea cucumber Aslia (=Cucumaria) lefevrei, was assigned by Panning to the genus Ludwigia along with C. lactea and C. planci (Panning, 1949). Rowe (1970) assigned it to the genus Aslia. Though Panning (Panning, 1971) placed it in the genus Thyonella, now it is generally accepted as belonging to the genus Aslia. The Pacific sea cucumber Cucumaria echinata was assigned by Panning (1949) to the genus Pseudoocnus though this species was not listed in this genus in his later work (Panning, 1949, 1955, 1971). Comparison of the set of glycosides of the sea cucumbers definitely included in the genus Cucumaria with those species excluded from the genus is also of interest. There are serious problems in the identification of different representatives of the genus Cucumaria, including even the reference species, caused by the great degree of variability in the ossicles of the body walls (which are the main characteristic of the species) (Levin and Gudimova, 1997). As an example, Cucumaria japonica inhabiting the Sea of Japan and the southern part of the Sea of Okhotsk was erroneously classified as C. frondosa (Levin and Gudimova, 1997). Moreover, four new North Pacific species, C. djakonovi, C. savelijevae (Baranova, 1980), C. conicospermium (Levin and Stepanov, 2002) and C. okhotensis (Levin, in press), were earlier classified as Cucumaria japonica. The method of quantitative analysis of the form of the ossicles proposed for identification of different species of Cucumaria is
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very complicated (Gudimova, 1991). Hence, finding new approaches to the taxonomy of the genus Cucumaria is essential.
3. Triterepene glycosides The glycosides of the North Pacific sea cucumber Cucumaria japonica is best documented among representatives of the genus. The structures of 13 glycosides from this species are known. This species contains a very complicated mixture of glycosides distinguished from each other both by their aglycone structures and carbohydrate chains. The general features of these glycosides are, (a) the presence of a pentasaccharide carbohydrate chain branched at the second monosaccharide unit (quinovose), (b) the presence of a sulphate group at C-4 position of the first xylose residue and (c) the presence of a 7(8)-double bond in the aglycon. Cucumariosides belonging to the series A0 (1, 2, 3), A1 (4), and A7 (11, 12, 13) are mostly minor components; cucumarioside A4-2 (8) is present in slightly larger amounts and cucumarioside A2-2 (5) is the major component of the glycoside content. However, structurally and chromatographycally similar cucumariosides A2-3 (6) and A2-4 (7) are minor glycosides. Cucumariosides A3 (9) and A6-2 (10) are also minor glycosides.
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An interesting peculiarity of cucumarioside A0-1 (1) is the presence of both a 23keto group and 16b-O-Ac in the aglycone (Drozdova et al., 1993a). This glycoside, as well as other representatives of the group A0, are distinguished from most other glycosides by the presence of xylose and not glucose as the third monosaccharide unit. Cucumarioside A0-2 (2) has no keto group in the side chain of the aglycone but has a terminal double bond (Drozdova et al., 1992b). Cucumarioside A0-3 (3) is distinguished from (2) by the presence of a 16-keto group, which is characteristic for most of the glycosides from Cucumaria japonica (Drozdova et al., 1993a). Cucumarioside A1-2 (4) has a 6-OAc group on the terminal glucose residue. It is unique for sea cucumber glycosides (Drozdova et al., 1992a). Cucumarioside A2-2 (5), a major component of the glycoside fraction, contains a 16-keto group and 25(26)-double bond in the aglycone. Cucumariosides A2-3 (6) and A2-4 (7) are 25,26-dihydro- and 16-de-oxo analogs of the glycoside (5). Cucumarioside A4-2 (8) has glucose as the terminal monosaccharide residue in the carbohydrate chain and is distinguished from other glycosides of this fraction which contain a terminal 3-O-methylglucose as in most other triterpene glycosides from sea cucumber (Avilov et al., 1990). Cucumariosides A3 (9) and A6-2 (10) are structurally close to cucumarioside A2-2 (5). The only difference between them is the presence of an additional sulphate group at 3-O-methylglucose in (9) and at C-6 of glucose residue in (10) (Drozdova et al., 1997). Cucumariosides A7-1 (11), A7-2 (12) and A7-3 (13) are structurally similar to the glycosides (5), (6) and (7), respectively, but in addition to the sulphate group at C-4 of the first xylose residue, they have two more sulphate groups at C-6 of glucose and 3-O-methylglucose residues (Drozdova et al., 1993b).
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In the Northern Pacific sea cucumber Cucumaria miniata, collected near Bering Island (Komandor Islands), cucumarioside A7-3 (13) was identified as the major component of the glycoside fraction (Drozdova et al., 1997). The North Atlantic sea cucumber C. frondosa has been the subject of extensive chemical investigations. A major component of the glycoside fraction of C. frondosa, is frondoside A (14). It has five monosaccharide units like most of the other glycosides from species belonging to the genus Cucumaria (Girard et al., 1990). It is very similar to the minor glycoside cucumarioside A0-2 (2) from C. japonica because it contains xylose as the third monosaccharide unit, a 16b-acetate group and 7(8)-double bond in the aglycone. However, it is distinguished by the absence of the terminal double bond. A minor glycoside, frondoside A1 (15), which was isolated along with frondoside A from the glycoside fraction of C. frondosa collected in the Barents Sea near the shore of Kolsky Peninsula is a sulphated tetraoside. It is distinguished from the major component of the fraction only by the absence of a terminal xylose residue attached to quinovose (Avilov et al., 1993). It is probable that this glycoside is a so-called ‘‘hot metabolite’’, a biosynthetic precursor of frondoside A. Frondoside D (16) is structurally similar to frondoside A, but is distinguished by the presence of a hydroxy group at 23-position in the side chain of the aglycone (Yayli and Findlay, 1999).
Two minor monosulphated isomeric pentaosides, frondosides E1 (5) and E2 (17), distinguished only by the position of the double bond in the cyclic system of the aglycone and double bond in the side chain, have been isolated as a non-separable mixture in studies of minor components of the glycoside fraction of C. frondosa (Yayli, 2001). Frondoside E1 (5) is structurally identical to cucumarioside A2-2 from C. japonica.
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Disulphated glycosides are presented in C. frondosa by frondoside B (18) having holostane aglycone with 7(8)- and 24(25)-double bonds and common pentasaccharide carbohydrate chain with glucose as third monosaccharide unit and two sulphate groups at C-4 of first xylose residue and at C-6 of the glucose (Findlay et al., 1992).
A fraction containing trisulphated pentaosides from C. frondosa is an almost inseparable and complicated mixture of many glycosides having the same carbohydrate chain but different aglycons. An isolate called ‘‘frondecaside’’ (19) has been
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reported as hexasulphated decaoside (Findlay et al., 1992). However, peculiarities in the method of isolation of this compound, namely its adsorption on hydrophobic adsorbent Amberlit XAD-2 followed by elution with methanol absolutely excludes the proposed structure. The authors did not present any decisive spectral data which confirmed the proposed structure (19) (Findlay et al., 1992). Our own experience in studying trisulphated pentaosides of C. frondosa (Avilov et al., 1998) suggests ‘‘frondecaside’’ was a very complicated mixture of glycosides having the same carbohydrate chains which induced accumulation of well resolved signals of the pentasaccharide trisulphated carbohydrate chain in the 13C NMR spectrum but not of corresponding aglycones because of low concentrations of each different aglycone.
A new trisulfated pentaoside frondoside F (20), having a 18(22)-lactone instead of 18(20)-lactone and a saturated polycyclic nucleus in the aglycone was isolated (Yayli, 2001). These structural features are unprecedented for sea cucumber glycosides. Although the author mentions the use of the most modern physicochemical methods of structural elucidation, the experimental data raise many questions. Indeed, there is no peak of quasi-molecular ion in the FAB mass spectrum, no correlation between protons at C-8 and C-9 in the 1H-1H-COSY spectrum, no aglycone conformation formula in the interpretation of the NOESY data and no information on the equipment used for obtaining the IR spectrum. A simple explanation for these experimental data could be that, as in the case of ‘‘frondecaside’’, the author was working with a very complicated mixture of glycosides having aglycones with both 7(8)-, and 9(11)-double bonds whose signals were not accumulated in the NMR spectra. Moreover, the author states that frondoside A (14) and A1 (15) are isomers, based on the structure of the side chain in the aglycone. This is not correct since there is no double bond in the side chain and hence no possibility of isomers (Yayli, 2001). These glycosides contain different numbers of monosaccharide units and are not isomers.
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Another structure reliably elucidated is trisulphated pentaoside frondoside C (21), isolated from C. frondosa from the Barents Sea. Its aglycone has 9(11)- and 24(25)-double bonds, acetate group at position C-22 and a hydroxyl at C-20. Its aglycone does not have a lactone and consequently is not a holostane derivative, in contrast to the most sea cucumber glycosides (Avilov et al., 1998).
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Cucumaria frondosa contains a very complicated mixture of glycosides that requires further investigation. However, even at this stage it may be concluded that individuals of this species living either near the Atlantic shore of North America or in Barents Sea have the same glycosides. Cucumaria miniata collected in the Bering Sea near the shore of Komandor Islands contains cucumarioside A7-3 (13) as a major component. This has also been isolated from C. japonica. (Drozdova et al., 1997) Koreoside A (22), is a major glycoside from C. koraeensis, collected near the middle Kurile Islands. It is a trisulphated pentaoside with 7(8)-unsaturated, nonholostane aglycone with a shortened side chain and no lactone ring (Avilov et al., 1997).
Cucumaria conicospermium, collected in the northern part of the Sea of Japan, contains a complicated set of glycosides. (Avilov et al., 2003). Cucumarioside A2-5 (23) isolated from this species is structurally similar to cucumarioside A0-1 (1) from C. japonica, and distinguished only by the third monosaccharide unit in the carbohydrate chain (glucose residue instead of xylose residue). Its monosulphated pentasaccharide carbohydrate chain is identical to that of cucumarioside A2-2 (5).
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Disulphated glycosides are represented in C. conicospermium by cucumariosides A3-2 (24) and A3-3 (25), nonholostane pentaosides with carbohydrate chains identical to that of cucumarioside A3 (9). The aglycone of cucumarioside A3-2 (24) is identical to that of koreoside A (21) from C. koraeensis, while the aglycone of cucumarioside A3-3 (25) is its D9(11)-isomer.
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Trisulphated glycosides from C. conicospermium (22) and (26) are also isomers by position of double bond in the cyclic system of the aglycone. Glycoside (22) is koreoside A, isolated from C. koraeensis. Its isomer glycoside (26) with 9(11)-double bond was named isokoreoside A. The Atlantic species Cucumaria (=Aslia) lefevrei contains four linear tetrasaccharide oligoglycosides, lefevreiosides A1 (27), A2 (15), B (28) and C (29) (Rodriguez and Riguera, 1989). These substances contain xylose as the third monosaccharide, a 7(8)-double bond and 16b-acetoxy group in the aglycone. Side chains in the aglycone of lefevreiosides A1 and A2 are saturated. Lefevreioside A2 contains a sulphate group in the carbohydrate chain but lefevreioside A1 has no sulphate group. Lefevreiosides B and C are sulphated at C-4 of the first xylose residue. They are distinguished only by their unsaturation, having 24(25)- and 25(26)double bonds respectively. In a study on levevreiosides A1, A2, B and C (Rodriguez and Riguera, 1989), an a-configuration of 16-O-acetate group in the aglycone was proposed by the comparison of spectral characteristics with the major aglycone obtained from the glycosides of Cucumaria frondosa. However, since the configuration of 16-O-acetate group in this aglycone was revised as b- (Girard et al., 1990) it is necessary to revise the corresponding configuration for levefreiosides also. Here we present the revised configuration. As a result, the structure of lefevreioside A2 is identical to that of frondoside A1 (15) from C. frondosa.
Six polar triterpene glycosides, cucumechinosides A, B, C, D, E and F (30–35) have been isolated from Cucumaria (=Pseudoocnus) echinata (Miamoto et al., 1990). All these substances have linear tetrasacchatide carbohydrate chains containing both glucose and xylose residues as a third monosaccharide unit, 7(8)-double bond and 23-ketogroup in the aglycone and also a sulphate at C-4 of the first xylose residue. Cucumechinosides A–C (30–32) contain two sulphate groups at the
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first and third monosaccharide units. Cucumechinoside B (31), containing xylose as the third monosaccharide unit, has a sulphate group attached to C-2 of the xylose residue. This is unique for sea cucumber glycosides. 26-keto group is characteristic for cucumechinosides A (30) and B (31), while cucumechinoside C (32) is a 16-desoxoderivative of the substance (30). Cucumechinoside D (33), E (34) and F (35) have an additional sulphate group at C-6 of the terminal residue 3-O-methylglucose, in comparison with substances (30), (31) and (32), and consequently are trisulphated glycosides.
Two polar glycosides liovillosides A (36) and B (37) have been isolated from the Antarctic Staurocucumis liouvillei (Maier et al., 2001). These substances have linear carbohydrate chains, which is common for sea cucumber glycosides, but they contain three sulphate groups attached to C-4 of xylose, C-6 of glucose and 3-Omethylglucose residues. The aglycone of liovilloside B (37) contains 7(8)-double bond and 16b-OAc and is identical to the aglycone of frondoside A (14) from C. frondosa. The aglycone of liovilloside A (36) is distinguished from that of liovilloside B (37) only by the presence of 24(25)-double bond in the side chain. This aglycone was identified earlier in the glycosides from Eupentacta fraudatrix (Stonik et al., 1999).
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The Patagonian Hemoideima spectabilis has two polar glycosides, chemoidesmosides A (38) and B (39) (Chludil et al., 2002). These glycosides have holotoxinogenin, a genin containing 9(11)- and 25(26)-double bonds and 16-keto group in the aglycone. This aglycone is common for glycosides from many different sea cucumbers (Stonik et al., 1999). Glycosides (38) and (39) have common linear tetrasaccharide carbohydrate chains and are distinguished from each other by the number of sulphate groups. Glycoside (38) contains two sulphate groups in the carbohydrate chain while glycoside (39) has three sulphate groups.
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4. Triterpene glycosides and taxonomy of the genus Cucumaria Known distribution of triterpene glycosides in sea cucumbers belonging to in the family Cucumariidae is presented in Table 1. A set of triterpene glycosides is distinct for each species, even within the same genus Cucumaria, and is significantly distinguished from each other. Overlapping structures for different species belonging to this genus are very rare. Indeed, in the glycoside fraction of C. japonica, where 13 glycosides were identified, only one component, cucumarioside A2-2 (5), is also present in the taxonomically related species C. frondosa. Additionally,
Table 1 Distribution of triterpene glycosides in the sea cucumber belonging to the family Cucumariidae Taxon
Place of collection
Glycosides
Reference
North Western shore of the Sea of Japan Atlantic shore of Canada
22, 23, 24, 25, 26
Avilov et al., 2003
5, 14, 16, 17, 18
22
Girard et al., 1990; Findlay et al., 1992; Yayli and Findlay, 1999; Yayli, 2001 Avilov et al., 1993; Avilov et al., 1998 Avilov et al., 1997
13
Drozdova et al., 1997
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13
Drozdova et al., 1997
Genus Cucumaria C. conicospermium C. frondosa
C. frondosa C. koraeensis C. miniata C. japonica
Kolsky shore of Barents Sea Catch Rocks (Kurile Islands) Bering Island (Komandor Islands) Gulf of Posiet (Sea of Japan)
14, 15, 21
Genus Aslia A. lefevrei
Galicia (North 27, 15, 28, 29 Western shore of Spain)
Rodriguez and Riguera, 1989
Inland Sea (Japan)
20, 31, 32, 33, 34, 35
Miamoto et al., 1990
South Georgia (Antarctic)
36, 37
Maier et al., 2001
Patagonia (South Atlantic)
38, 39
Chludil et al., 2002
Genus Pseudoocnus P. echinata
Genus Staurocucumis S. liouvillei
Genus Hemoidema H. spectabilis
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glycoside (5) is a major component in C. japonica but a minor one in C. frondosa. It was identified in trace amounts and could not be isolated as a pure substance. Cucumarioside A7-3 (13) is a major glycoside in C. miniata and a minor one in C. japonica. A major component of the glycoside fraction from C. koraeensis, koreoside A (22), containing nonholostane aglycone with 7(8)-double bond was also isolated as a mixture with isokoreoside A (26) from C. conicospermium. In other known cases the structures of the glycosides are quite different for different species belonging to the genus Cucumaria. Structural differences of the glycosides from the family Cucumariidae may be very great both inter- and intraspecifically. The glycosides may contain both holostane aglycones, i.e. containing 18(20)-lactone characteristic for most of the known sea cucumber glycosides, and nonholostane aglycons having no such lactone. These substances may contain both aglycones with oxidized position at C-16, (acetate or ketogroup) and those having no oxygen in this position. Glycosides may have either 7(8)- or 9(11)-double bond in the nucleus of the aglycone. Aglycones may have both saturated and nonoxidized side chains and side chains having double bonds at positions 24(25) and 25(26), and also oxygen functions at C-23 and C-22. Hence the glycosides from each species in the genus Cucumaria have their own set of complex aglycones, very complicated in composition and characteristic for certain species. The situation with carbohydrate chains of the glycosides is quite different. As a rule, all the glycosides from species belonging to the genus Cucumaria are pentaosides, sulphated at C-4 of the first xylose residue. The only exception is frondoside A1 (15) from C. frondosa, a tetraoside, sulphated at the first xylose residue. However, the amount of this glycoside is low and, as mentioned earlier, it probably is a ‘‘hot’’ metabolite, a precursor of pentaosides. Monosulphated pentaosides such as cucumarioside A2-2 (5) from C. japonica usually have a carbohydrate chain containing glucose as the third monosaccharide unit. In some cases the third monosaccharide unit may be xylose as, for example, in frondoside A (14) from C. frondosa. In monosulphated pentaosides there may be variations in the terminal monosaccharide residue. Instead of the usual 3-O-methylglycose, which is common for most sea cucumber glycosides, there may be either glucose as in cucumarioside A4-2 (8), or 6-O-acetylglucose as in cucumarioside A1 (4) from C. japonica. Monosulphated pentaosides were isolated from C. japonica, C. frondosa and C. conicospermium. All other Cucumaria species contain such glycosides (along with data of TLC) in trace amounts and were not identified by physico-chemical methods. The next two groups of carbohydrate chains of glycosides from Cucumaria contain glucose as the third monosaccharide unit and 3-O-methylglucose as the fourth or terminal monosaccharide unit. Pentaosides containing two sulphate groups have one sulphate at C-4 of the first xylose residue and another one at C-6 of the third glucose residue as, in cucumarioside A3 (9), or at C-6 of 3-O-methylglucose residue as, in cucumarioside A6-2 (10), respectively. Both glycosides were isolated from C. japonica. Disulphated pentaosides were identified in C. japonica, C. frondosa and C. conicospermium.
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Pentaosides containing three sulphate groups, namely one at C-4 of the first xylose residue and two additional ones at the third glucose residue and terminal residue of 3-O-methylglucose have been identified in all species of Cucumaria studied: C. japonica, C. miniata, C. frondosa, C. koraeensis and C. conicospermium. The carbohydrate chain structures of glycosides of Cucumaria species studied differ greatly from those of other genera of the family Cucumariidae. Indeed, lefevreiosides from Aslia (=Cucumaria) lefevrei, cucumechinosides from Pseudoocnus (=Cucumaria) echinata, liovillosides from Staurocucumis liouvillei, and also hemoidemosides from Hemoidema spectabilis are tetraosides and do not contain fifth terminal monosaccharide unit attached to C-2 of quinovose residue.
5. Conclusions Species belonging to the genus Cucumaria may be clearly distinguished by the species-specific set of aglycones in the glycosides. Structures of carbohydrate chains branched at the quinovose residue, sulphated at the first xylose residue and containing, as a rule, five monosaccharide units and having xylose, 3-O-methylglucose, glucose and 6-O-acetylglucose as terminal monosaccharide residues are quite similar for all the glycosides isolated from species of this genus. Such a set of carbohydrate chains is correspondingly characteristic for all species of this genus studied. Glycosides with trisulphated pentaoside carbohydrate chains were isolated from all species studied. Hence, the presence of such a carbohydrate chain may serve as a reliable taxonomical character of the genus. The structure of the carbohydrate chains of glycosides from all other studied species of other genera of the family Cucumariidae are different from those of species belonging to the genus Cucumaria. This validates their earlier elimination from this genus. The sets of glycosides of the species belonging to the genus Cucumaria (Cucumariidae, Dendrochirotida) are species-specific for each studied species. They are significantly different from the glycosides of species belonging to the order Aspidochirotida, where the structures are specific for taxa above the genus level. The decrease in level of taxonomic specificity for triterpene glycoside structures at the transition from species belonging to the order Dendrochrirotida (including the genus Cucumaria) to those belonging to the order Aspidochriotida and the much greater structural diversity of glycosides in the Dendrochirotida may indicate the relatively more phylogenetic primitiveness of Dendrochirotida. Indeed, as well known to paleontologists, the closer a taxon is to the evolutionary stage where any morphofunctional system is formed, the greater the structural diversity of the system in this taxon. In this and similar cases, a level of taxonomical specificity of such structures ought to be decreased during phylogeny (Mamkaev, 1991). The taxonomic distribution of biochemical characteristics in species of the genus Cucumaria confirm the correctness of this.
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Acknowledgements The authors are very grateful to Mrs. Greta Moraes (Industrial Research Limited, New Zealand) and Professor John M. Lawrence (University of South Florida, USA) for discussion of the manuscript and correction of the English text. Vladimir I. Kalinin thanks the Science Support Foundation for Grant for Talented Young Researches. This work was partially supported by the Russian Foundation for Basic Researches (RFBR 00-15-97806, RFBR 01-04-96907 and 01-03-96903). The authors are grateful to Professor Valentin A. Stonik (Pacific Institue of Bioorganic Chemistry, Russia) for discussion of the manuscript and correction of the text.
References Avilov, S.A., Stonik, V.A., Kalinovsky, A.I., 1990. Structures of four new triterpene glycosides from the sea cucumber Cucumaria japonica. Khim. Prirod. Soedin. 6, 787–792. Avilov, S.A., Kalinin, V.I., Drozdova, O.A., Kalinovsky, A.I., 1993. Triterpene glycosides of the sea cucumber Cucumaria frondosa. Khim. Prirod. Soedin. 2, 49–52. Avilov, S.A., Kalinovsky, A.I., Kalinin, V.I., Stonik, V.A., Riguera, R., Jimenes, C., 1997. Koreoside A, a new nonholostane triterpene glycoside from the sea cucumber Cucumaria koraiensis. J. Nat. Prod. 60, 808–810. Avilov, S.A., Drozdova, O.A., Kalinin, V.I., Kalinovsky, A.I., Stonik, V.A., 1998. Frondoside C, a new nonholostane triterpene glycoside from the sea cucumber Cucumaria frondosa: structure and cytotoxicity of its desulphated derivative. Can. J. Chem. 76, 137–141. Avilov, S.A., Antonov, A.S., Silchenko, A.S., Kalinin, V.I., Kalinovsky, A.I., Dmitrenok, P.S., Stonik, V.A., Riguera, R., Jimenes, C., 2003. Triterpene glycosides from the Far Eastern sea cucumber Cucumaria conicospermium Levin et Stepanov. J. Nat. Prod. 66, 910–916. Baranova, Z.I., 1976. Sea cucumbers belonging to the genus Cucumaria of the seas of the USSR. Problemy Zoologii. Nauka, Leningrad, pp. 5–7. Baranova, Z.I., 1980. New species of the sea cucumber belonging to the genus Cucumaria. Novoe v sistematike morskikh besposvonochnych, Issledovanija fauny morej XXV (XXXIII). ZIN AN SSSR, Leningrad, pp. 109–120. Chludil, H.D., Munian, C.C., Seldes, A.M., Maier, M.S., 2002. Cytotoxic and antifungal triterpene glycosides from the Patagonian sea cucumber Hemoidema spectabilis. J. Nat. Prod. 65, 860–865. Drozdova, O.A., Avilov, S.A., Kalinovsky, A.I., Stonik, V.A., 1992a. New acetylated glycoside from the sea cucumber Cucumaria japonica. Khim. Prirod. Soedin. 5, 590–591. Drozdova, O.A., Avilov, S.A., Kalinovsky, A.I., Stonik, V.A., 1992b. A minor glycoside from the sea cucumber Cucumaria japonica. Khim. Pririod. Soedin. 5, 593. Drozdova, O.A., Avilov, S.A., Kalinovsky, A.I., Stonik, V.A., Milgrom, Y.M., Rashkes, J.W., 1993a. New glycosides from the sea cucumber Cucumaria japonica. Khim. Prirod. Soedin. 2, 242–248. Drozdova, O.A., Avilov, S.A., Kalinovsky, A.I., Stonik, V.A., Milgrom, Y.M., Rashkes, J.W., 1993b. Trisulphated glycosides from the sea cucumber Cucumaria japonica. Khim. Prirod. Soedin. 3, 369–374. Drozdova, O.A., Avilov, S.A., Kalinin, V.I., Kalinovsky, A.I., Stonik, V.A., Riguera, R., Jimenes, C., 1997. Cytotoxic triterpene glycosides from far-eastern sea cucumbers belonging to the genus Cucumaria. Liebigs Ann. 12, 2351–2356. Findlay, J., Yayli, N., Radics, L., 1992. Novel sulphated oligosaccharides from the sea cucumber Cucumaria frondosa. J. Nat. Prod. 55, 93–101. Girard, M., Belanger, J., ApSimon, J.W., Garneau, F.-X., Harvey, C., Brisson, J.-R., 1990. Frondoside A. A novel triterpene glycoside from the holothurian Cucumaria frondosa. Can. J. Chem. 68, 11–18.
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Gudimova, E.N., 1991. Methods of quantative analysis of the sclerite shapes of the sea cucumbers belonging to the genus Cucumaria. Biologia Morya. 6, 80–87. Gunner, J.E., 1767. Beskrifning pa trenne Norska Sjo-Krak, Sjo-Pungar kallade. Stockholm Vetensk for Ar 28, 114–124. Kalinin, V.I., Levin, V.S., Stonik, V.A., 1994. Chemical morphology: Triterpene glycosides from sea cucumbers. Dalnauka, Vladivostok. Levin, V.S., Kalinin, V.I., Stonik, V.A., 1984. An attempt to apply chemical characters at revise of taxonimical status of the sea cucumber Bohadschia graeffei with erection of new genus. Biol. Morya. 2, 33–38. Levin, V.S., Kalinin, V.I., Maltsev, I.I., Stonik, V.A., 1985. Structure of triterpene glycosides and taxonomy of aspidochirotian sea cucumbers. Biol. Morya. 2, 3–11. Levin, V.S., Kalinin, V.I., Fedorov, S.N., Smiley, S., 1986. Structure of triterpene glycosides and taxonomical position of two species of the family Stichopodidae. Biol. Morya. 4, 72–77. Levin, V.S., Gudimova, E.N., 1997. On taxonomical relationships of the sea cucumbers Cucumaria frondosa and C. japonica (Dendrochirotida, Cucumariidae). Zool. Zhurn. 76, 575–584. Levin, V.S., Stepanov, V.G., 2002. Cucumaria conicospermium sp. n. (Dendrochirotida, Cucumariidae)— new sea cucumber from the Sea of Japan. Biol. Morya. 28, 66–69. Maier, M.S., Roccatagliata, A., Kuriss, A., Chludil, H., Seldes, A.M., Pujiol, C.A., Damonte, E.B., 2001. Two new cytotoxic and virucidal trisulphated triterpene glycosides from the Antarctic sea cucumber Staurocucumis liouvillei. J. Nat. Prod. 64, 732–736. Mamkaev, Y.V., 1991. Methods and trends of evolutionary morphology. Sovremwennaja Evolutsionnaja Morfologia. Nauka, Moskva, pp. 33–55. Miamoto, T., Togawa, K., Higuchi, R., Komori, T., Sasaki, T., 1990. Constituents of Holothurioidea. II. Six newly identified biologically active triterpenoid glycoside sulphates from the sea cucumber Cucumaria echinata. Liebigs Ann. 5, 453–460. Panning, A., 1949. Versuch einer Neuordnung der Familie Cucumariidae (Holothurioidea, Dendrochirota). Zool. Jarb. Abt. System. Okol. Geogr. Tiere. 78, 404–470. Panning, A., 1955. Bemerkungen uber die Holothurien—Familie Cucumariidae (Ordnung Dendrochirotida). Mitt. Hamburg Zool. Muz. Inst. 53, 33–47. Panning, A., 1971. Bemerkungen uber die Holothurien-Familie Cucumariidae (Ordnung Dendrochirota). 6. Die Gattungen um Ocnus forbes 1841 und um Pentacte goldfuss 1820. Mitt. Hamburg Zool. Muz. Inst. 67, 29–51. Rodriguez, J., Riguera, R., 1989. Lefevriosides: four novel triterpenoid glycosides from the sea cucumber Cucumaria lefevrei. J. Chem. Res. M, 2620–2630. Rowe, F.W.E., 1970. A note on the British species of cucumarians, involving the erection of two new nominal genera. J. Mar. Biol. Assoc. UK 50, 683–687. Stonik, V.A., Kalinin, V.I., Avilov, S.A., 1999. Toxins from sea cucumbers (Holothuroids): chemical structures, properties, taxonomic distribution, biosynthesis and evolution. J. Nat. Toxins. 8, 235–248. Yayli, N., Findlay, J., 1999. A triterpenoid saponin from Cucumaria frondosa. Phytochemistry 50, 135–138. Yayli, N., 2001. Minor saponins from the sea cucumber Cucumaria frondosa. Indian J Chem 40B, 399–404.
Use of triterpene glycosides for resolving taxonomic problems in the sea cucumber genus Cucumaria (Holothurioidea, Echinodermata) Sergey A. Avilov a, Vladimir I. Kalinin a,, Alexey V. Smirnov b a
Pacific Institute of Bioorganic Chemistry, Far-Eastern Division of the Russian Academy of Sciences, 690022, Vladivostok, Russia b Zoological Institute of the Russian Academy of Sciences, 199164, Saint Petersburg, Russia Received 30 January 2003; accepted 30 December 2003
Abstract This article presents a review of the data on the structures of glycosides from five species of sea cucumbers belonging to the genus Cucumaria (Holothurioidea, Echinodermata). A set of aglycones is specific for glycosides from each species but carbohydrate chains are similar for all five species. These sea cucumbers contain glycosides with three-sulphated branched pentasaccharide carbohydrate chains which are characteristic for the genus Cucumaria. The glycosides from all the Cucumaria species studied are different from the glycosides of Aslia lefevrei and Pseudoocnus echinata that confirms the earlier exclusion of these species from the genus Cucumaria. They are also significantly different from the glycosides of Staurocucumis liouvillei and Hemoiedema spectabilis, the other representatives of the family Cucumariidae. # 2004 Elsevier Ltd. All rights reserved. Keywords: Sea cucumbers; Cucumaria; Cucumariidae; Taxonomy; Triterpene glycosides
1. Introduction The distribution of different triterpene glycosides was successfully applied to the taxonomy of sea cucumbers belonging to the order Aspidochirotida. (Kalinin et al., 1994). Such data were used to revise the genus Bohadschia with the establishment
Corresponding author. Fax: +7-4232-31-40-50. E-mail address: [email protected] (V.I. Kalinin).
0305-1978/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2003.12.008
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of a new genus Pearsonothuria (Levin et al., 1984). A taxonomic relation has been found among the North Pacific representatives of the family Stichopodidae by simultaneous use of the information on the glycoside distribution and morphological data (Levin et al., 1986). The phylogeny of the order Aspidochirotida (Levin et al., 1985) has also been improved by a similar approach. It was confirmed that the triterpene glycosides are specific for genera and groups of genera within this order. However, the glycosides of sea cucumbers belonging to the order Dendrochirotida are much more structurally diverse than those of Aspidochirotida and are species-specific in some cases (Stonik et al., 1999). In this paper we consider the distribution of glycosides in representatives of the genus Cucumaria (Cucumariidae, Dendrochirotida) to clarify the relation of the species.
2. Systematics of the genus Cucumaria and non-resolved taxonomical problems The referrence species of the genus Cucumaria Blainville is Cucumaria frondosa (Gunner, 1767; Baranova, 1976). During different periods this genus included about 90 species of the family Cucumariidae. However, Panning (1949, 1955) showed that only some of these species may really belong to the genus Cucumaria according to data on the structure of throat calcareous ring and sclerites (ossicles) of the body walls. Indeed, among all the sea cucumbers inhabiting the Northern part of Pacific and Atlantic Oceans, only C. frondosa, C. japonica, C. koraeensis, C. djakonovi, C. savelijevae (Baranova, 1976, 1980), and the recently described species C. conicospermium (Levin and Stepanov, 2002), are unquestionably members of this genus. For example, the Atlantic sea cucumber Aslia (=Cucumaria) lefevrei, was assigned by Panning to the genus Ludwigia along with C. lactea and C. planci (Panning, 1949). Rowe (1970) assigned it to the genus Aslia. Though Panning (Panning, 1971) placed it in the genus Thyonella, now it is generally accepted as belonging to the genus Aslia. The Pacific sea cucumber Cucumaria echinata was assigned by Panning (1949) to the genus Pseudoocnus though this species was not listed in this genus in his later work (Panning, 1949, 1955, 1971). Comparison of the set of glycosides of the sea cucumbers definitely included in the genus Cucumaria with those species excluded from the genus is also of interest. There are serious problems in the identification of different representatives of the genus Cucumaria, including even the reference species, caused by the great degree of variability in the ossicles of the body walls (which are the main characteristic of the species) (Levin and Gudimova, 1997). As an example, Cucumaria japonica inhabiting the Sea of Japan and the southern part of the Sea of Okhotsk was erroneously classified as C. frondosa (Levin and Gudimova, 1997). Moreover, four new North Pacific species, C. djakonovi, C. savelijevae (Baranova, 1980), C. conicospermium (Levin and Stepanov, 2002) and C. okhotensis (Levin, in press), were earlier classified as Cucumaria japonica. The method of quantitative analysis of the form of the ossicles proposed for identification of different species of Cucumaria is
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very complicated (Gudimova, 1991). Hence, finding new approaches to the taxonomy of the genus Cucumaria is essential.
3. Triterepene glycosides The glycosides of the North Pacific sea cucumber Cucumaria japonica is best documented among representatives of the genus. The structures of 13 glycosides from this species are known. This species contains a very complicated mixture of glycosides distinguished from each other both by their aglycone structures and carbohydrate chains. The general features of these glycosides are, (a) the presence of a pentasaccharide carbohydrate chain branched at the second monosaccharide unit (quinovose), (b) the presence of a sulphate group at C-4 position of the first xylose residue and (c) the presence of a 7(8)-double bond in the aglycon. Cucumariosides belonging to the series A0 (1, 2, 3), A1 (4), and A7 (11, 12, 13) are mostly minor components; cucumarioside A4-2 (8) is present in slightly larger amounts and cucumarioside A2-2 (5) is the major component of the glycoside content. However, structurally and chromatographycally similar cucumariosides A2-3 (6) and A2-4 (7) are minor glycosides. Cucumariosides A3 (9) and A6-2 (10) are also minor glycosides.
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An interesting peculiarity of cucumarioside A0-1 (1) is the presence of both a 23keto group and 16b-O-Ac in the aglycone (Drozdova et al., 1993a). This glycoside, as well as other representatives of the group A0, are distinguished from most other glycosides by the presence of xylose and not glucose as the third monosaccharide unit. Cucumarioside A0-2 (2) has no keto group in the side chain of the aglycone but has a terminal double bond (Drozdova et al., 1992b). Cucumarioside A0-3 (3) is distinguished from (2) by the presence of a 16-keto group, which is characteristic for most of the glycosides from Cucumaria japonica (Drozdova et al., 1993a). Cucumarioside A1-2 (4) has a 6-OAc group on the terminal glucose residue. It is unique for sea cucumber glycosides (Drozdova et al., 1992a). Cucumarioside A2-2 (5), a major component of the glycoside fraction, contains a 16-keto group and 25(26)-double bond in the aglycone. Cucumariosides A2-3 (6) and A2-4 (7) are 25,26-dihydro- and 16-de-oxo analogs of the glycoside (5). Cucumarioside A4-2 (8) has glucose as the terminal monosaccharide residue in the carbohydrate chain and is distinguished from other glycosides of this fraction which contain a terminal 3-O-methylglucose as in most other triterpene glycosides from sea cucumber (Avilov et al., 1990). Cucumariosides A3 (9) and A6-2 (10) are structurally close to cucumarioside A2-2 (5). The only difference between them is the presence of an additional sulphate group at 3-O-methylglucose in (9) and at C-6 of glucose residue in (10) (Drozdova et al., 1997). Cucumariosides A7-1 (11), A7-2 (12) and A7-3 (13) are structurally similar to the glycosides (5), (6) and (7), respectively, but in addition to the sulphate group at C-4 of the first xylose residue, they have two more sulphate groups at C-6 of glucose and 3-O-methylglucose residues (Drozdova et al., 1993b).
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In the Northern Pacific sea cucumber Cucumaria miniata, collected near Bering Island (Komandor Islands), cucumarioside A7-3 (13) was identified as the major component of the glycoside fraction (Drozdova et al., 1997). The North Atlantic sea cucumber C. frondosa has been the subject of extensive chemical investigations. A major component of the glycoside fraction of C. frondosa, is frondoside A (14). It has five monosaccharide units like most of the other glycosides from species belonging to the genus Cucumaria (Girard et al., 1990). It is very similar to the minor glycoside cucumarioside A0-2 (2) from C. japonica because it contains xylose as the third monosaccharide unit, a 16b-acetate group and 7(8)-double bond in the aglycone. However, it is distinguished by the absence of the terminal double bond. A minor glycoside, frondoside A1 (15), which was isolated along with frondoside A from the glycoside fraction of C. frondosa collected in the Barents Sea near the shore of Kolsky Peninsula is a sulphated tetraoside. It is distinguished from the major component of the fraction only by the absence of a terminal xylose residue attached to quinovose (Avilov et al., 1993). It is probable that this glycoside is a so-called ‘‘hot metabolite’’, a biosynthetic precursor of frondoside A. Frondoside D (16) is structurally similar to frondoside A, but is distinguished by the presence of a hydroxy group at 23-position in the side chain of the aglycone (Yayli and Findlay, 1999).
Two minor monosulphated isomeric pentaosides, frondosides E1 (5) and E2 (17), distinguished only by the position of the double bond in the cyclic system of the aglycone and double bond in the side chain, have been isolated as a non-separable mixture in studies of minor components of the glycoside fraction of C. frondosa (Yayli, 2001). Frondoside E1 (5) is structurally identical to cucumarioside A2-2 from C. japonica.
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Disulphated glycosides are presented in C. frondosa by frondoside B (18) having holostane aglycone with 7(8)- and 24(25)-double bonds and common pentasaccharide carbohydrate chain with glucose as third monosaccharide unit and two sulphate groups at C-4 of first xylose residue and at C-6 of the glucose (Findlay et al., 1992).
A fraction containing trisulphated pentaosides from C. frondosa is an almost inseparable and complicated mixture of many glycosides having the same carbohydrate chain but different aglycons. An isolate called ‘‘frondecaside’’ (19) has been
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reported as hexasulphated decaoside (Findlay et al., 1992). However, peculiarities in the method of isolation of this compound, namely its adsorption on hydrophobic adsorbent Amberlit XAD-2 followed by elution with methanol absolutely excludes the proposed structure. The authors did not present any decisive spectral data which confirmed the proposed structure (19) (Findlay et al., 1992). Our own experience in studying trisulphated pentaosides of C. frondosa (Avilov et al., 1998) suggests ‘‘frondecaside’’ was a very complicated mixture of glycosides having the same carbohydrate chains which induced accumulation of well resolved signals of the pentasaccharide trisulphated carbohydrate chain in the 13C NMR spectrum but not of corresponding aglycones because of low concentrations of each different aglycone.
A new trisulfated pentaoside frondoside F (20), having a 18(22)-lactone instead of 18(20)-lactone and a saturated polycyclic nucleus in the aglycone was isolated (Yayli, 2001). These structural features are unprecedented for sea cucumber glycosides. Although the author mentions the use of the most modern physicochemical methods of structural elucidation, the experimental data raise many questions. Indeed, there is no peak of quasi-molecular ion in the FAB mass spectrum, no correlation between protons at C-8 and C-9 in the 1H-1H-COSY spectrum, no aglycone conformation formula in the interpretation of the NOESY data and no information on the equipment used for obtaining the IR spectrum. A simple explanation for these experimental data could be that, as in the case of ‘‘frondecaside’’, the author was working with a very complicated mixture of glycosides having aglycones with both 7(8)-, and 9(11)-double bonds whose signals were not accumulated in the NMR spectra. Moreover, the author states that frondoside A (14) and A1 (15) are isomers, based on the structure of the side chain in the aglycone. This is not correct since there is no double bond in the side chain and hence no possibility of isomers (Yayli, 2001). These glycosides contain different numbers of monosaccharide units and are not isomers.
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Another structure reliably elucidated is trisulphated pentaoside frondoside C (21), isolated from C. frondosa from the Barents Sea. Its aglycone has 9(11)- and 24(25)-double bonds, acetate group at position C-22 and a hydroxyl at C-20. Its aglycone does not have a lactone and consequently is not a holostane derivative, in contrast to the most sea cucumber glycosides (Avilov et al., 1998).
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Cucumaria frondosa contains a very complicated mixture of glycosides that requires further investigation. However, even at this stage it may be concluded that individuals of this species living either near the Atlantic shore of North America or in Barents Sea have the same glycosides. Cucumaria miniata collected in the Bering Sea near the shore of Komandor Islands contains cucumarioside A7-3 (13) as a major component. This has also been isolated from C. japonica. (Drozdova et al., 1997) Koreoside A (22), is a major glycoside from C. koraeensis, collected near the middle Kurile Islands. It is a trisulphated pentaoside with 7(8)-unsaturated, nonholostane aglycone with a shortened side chain and no lactone ring (Avilov et al., 1997).
Cucumaria conicospermium, collected in the northern part of the Sea of Japan, contains a complicated set of glycosides. (Avilov et al., 2003). Cucumarioside A2-5 (23) isolated from this species is structurally similar to cucumarioside A0-1 (1) from C. japonica, and distinguished only by the third monosaccharide unit in the carbohydrate chain (glucose residue instead of xylose residue). Its monosulphated pentasaccharide carbohydrate chain is identical to that of cucumarioside A2-2 (5).
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Disulphated glycosides are represented in C. conicospermium by cucumariosides A3-2 (24) and A3-3 (25), nonholostane pentaosides with carbohydrate chains identical to that of cucumarioside A3 (9). The aglycone of cucumarioside A3-2 (24) is identical to that of koreoside A (21) from C. koraeensis, while the aglycone of cucumarioside A3-3 (25) is its D9(11)-isomer.
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Trisulphated glycosides from C. conicospermium (22) and (26) are also isomers by position of double bond in the cyclic system of the aglycone. Glycoside (22) is koreoside A, isolated from C. koraeensis. Its isomer glycoside (26) with 9(11)-double bond was named isokoreoside A. The Atlantic species Cucumaria (=Aslia) lefevrei contains four linear tetrasaccharide oligoglycosides, lefevreiosides A1 (27), A2 (15), B (28) and C (29) (Rodriguez and Riguera, 1989). These substances contain xylose as the third monosaccharide, a 7(8)-double bond and 16b-acetoxy group in the aglycone. Side chains in the aglycone of lefevreiosides A1 and A2 are saturated. Lefevreioside A2 contains a sulphate group in the carbohydrate chain but lefevreioside A1 has no sulphate group. Lefevreiosides B and C are sulphated at C-4 of the first xylose residue. They are distinguished only by their unsaturation, having 24(25)- and 25(26)double bonds respectively. In a study on levevreiosides A1, A2, B and C (Rodriguez and Riguera, 1989), an a-configuration of 16-O-acetate group in the aglycone was proposed by the comparison of spectral characteristics with the major aglycone obtained from the glycosides of Cucumaria frondosa. However, since the configuration of 16-O-acetate group in this aglycone was revised as b- (Girard et al., 1990) it is necessary to revise the corresponding configuration for levefreiosides also. Here we present the revised configuration. As a result, the structure of lefevreioside A2 is identical to that of frondoside A1 (15) from C. frondosa.
Six polar triterpene glycosides, cucumechinosides A, B, C, D, E and F (30–35) have been isolated from Cucumaria (=Pseudoocnus) echinata (Miamoto et al., 1990). All these substances have linear tetrasacchatide carbohydrate chains containing both glucose and xylose residues as a third monosaccharide unit, 7(8)-double bond and 23-ketogroup in the aglycone and also a sulphate at C-4 of the first xylose residue. Cucumechinosides A–C (30–32) contain two sulphate groups at the
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first and third monosaccharide units. Cucumechinoside B (31), containing xylose as the third monosaccharide unit, has a sulphate group attached to C-2 of the xylose residue. This is unique for sea cucumber glycosides. 26-keto group is characteristic for cucumechinosides A (30) and B (31), while cucumechinoside C (32) is a 16-desoxoderivative of the substance (30). Cucumechinoside D (33), E (34) and F (35) have an additional sulphate group at C-6 of the terminal residue 3-O-methylglucose, in comparison with substances (30), (31) and (32), and consequently are trisulphated glycosides.
Two polar glycosides liovillosides A (36) and B (37) have been isolated from the Antarctic Staurocucumis liouvillei (Maier et al., 2001). These substances have linear carbohydrate chains, which is common for sea cucumber glycosides, but they contain three sulphate groups attached to C-4 of xylose, C-6 of glucose and 3-Omethylglucose residues. The aglycone of liovilloside B (37) contains 7(8)-double bond and 16b-OAc and is identical to the aglycone of frondoside A (14) from C. frondosa. The aglycone of liovilloside A (36) is distinguished from that of liovilloside B (37) only by the presence of 24(25)-double bond in the side chain. This aglycone was identified earlier in the glycosides from Eupentacta fraudatrix (Stonik et al., 1999).
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The Patagonian Hemoideima spectabilis has two polar glycosides, chemoidesmosides A (38) and B (39) (Chludil et al., 2002). These glycosides have holotoxinogenin, a genin containing 9(11)- and 25(26)-double bonds and 16-keto group in the aglycone. This aglycone is common for glycosides from many different sea cucumbers (Stonik et al., 1999). Glycosides (38) and (39) have common linear tetrasaccharide carbohydrate chains and are distinguished from each other by the number of sulphate groups. Glycoside (38) contains two sulphate groups in the carbohydrate chain while glycoside (39) has three sulphate groups.
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4. Triterpene glycosides and taxonomy of the genus Cucumaria Known distribution of triterpene glycosides in sea cucumbers belonging to in the family Cucumariidae is presented in Table 1. A set of triterpene glycosides is distinct for each species, even within the same genus Cucumaria, and is significantly distinguished from each other. Overlapping structures for different species belonging to this genus are very rare. Indeed, in the glycoside fraction of C. japonica, where 13 glycosides were identified, only one component, cucumarioside A2-2 (5), is also present in the taxonomically related species C. frondosa. Additionally,
Table 1 Distribution of triterpene glycosides in the sea cucumber belonging to the family Cucumariidae Taxon
Place of collection
Glycosides
Reference
North Western shore of the Sea of Japan Atlantic shore of Canada
22, 23, 24, 25, 26
Avilov et al., 2003
5, 14, 16, 17, 18
22
Girard et al., 1990; Findlay et al., 1992; Yayli and Findlay, 1999; Yayli, 2001 Avilov et al., 1993; Avilov et al., 1998 Avilov et al., 1997
13
Drozdova et al., 1997
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13
Drozdova et al., 1997
Genus Cucumaria C. conicospermium C. frondosa
C. frondosa C. koraeensis C. miniata C. japonica
Kolsky shore of Barents Sea Catch Rocks (Kurile Islands) Bering Island (Komandor Islands) Gulf of Posiet (Sea of Japan)
14, 15, 21
Genus Aslia A. lefevrei
Galicia (North 27, 15, 28, 29 Western shore of Spain)
Rodriguez and Riguera, 1989
Inland Sea (Japan)
20, 31, 32, 33, 34, 35
Miamoto et al., 1990
South Georgia (Antarctic)
36, 37
Maier et al., 2001
Patagonia (South Atlantic)
38, 39
Chludil et al., 2002
Genus Pseudoocnus P. echinata
Genus Staurocucumis S. liouvillei
Genus Hemoidema H. spectabilis
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glycoside (5) is a major component in C. japonica but a minor one in C. frondosa. It was identified in trace amounts and could not be isolated as a pure substance. Cucumarioside A7-3 (13) is a major glycoside in C. miniata and a minor one in C. japonica. A major component of the glycoside fraction from C. koraeensis, koreoside A (22), containing nonholostane aglycone with 7(8)-double bond was also isolated as a mixture with isokoreoside A (26) from C. conicospermium. In other known cases the structures of the glycosides are quite different for different species belonging to the genus Cucumaria. Structural differences of the glycosides from the family Cucumariidae may be very great both inter- and intraspecifically. The glycosides may contain both holostane aglycones, i.e. containing 18(20)-lactone characteristic for most of the known sea cucumber glycosides, and nonholostane aglycons having no such lactone. These substances may contain both aglycones with oxidized position at C-16, (acetate or ketogroup) and those having no oxygen in this position. Glycosides may have either 7(8)- or 9(11)-double bond in the nucleus of the aglycone. Aglycones may have both saturated and nonoxidized side chains and side chains having double bonds at positions 24(25) and 25(26), and also oxygen functions at C-23 and C-22. Hence the glycosides from each species in the genus Cucumaria have their own set of complex aglycones, very complicated in composition and characteristic for certain species. The situation with carbohydrate chains of the glycosides is quite different. As a rule, all the glycosides from species belonging to the genus Cucumaria are pentaosides, sulphated at C-4 of the first xylose residue. The only exception is frondoside A1 (15) from C. frondosa, a tetraoside, sulphated at the first xylose residue. However, the amount of this glycoside is low and, as mentioned earlier, it probably is a ‘‘hot’’ metabolite, a precursor of pentaosides. Monosulphated pentaosides such as cucumarioside A2-2 (5) from C. japonica usually have a carbohydrate chain containing glucose as the third monosaccharide unit. In some cases the third monosaccharide unit may be xylose as, for example, in frondoside A (14) from C. frondosa. In monosulphated pentaosides there may be variations in the terminal monosaccharide residue. Instead of the usual 3-O-methylglycose, which is common for most sea cucumber glycosides, there may be either glucose as in cucumarioside A4-2 (8), or 6-O-acetylglucose as in cucumarioside A1 (4) from C. japonica. Monosulphated pentaosides were isolated from C. japonica, C. frondosa and C. conicospermium. All other Cucumaria species contain such glycosides (along with data of TLC) in trace amounts and were not identified by physico-chemical methods. The next two groups of carbohydrate chains of glycosides from Cucumaria contain glucose as the third monosaccharide unit and 3-O-methylglucose as the fourth or terminal monosaccharide unit. Pentaosides containing two sulphate groups have one sulphate at C-4 of the first xylose residue and another one at C-6 of the third glucose residue as, in cucumarioside A3 (9), or at C-6 of 3-O-methylglucose residue as, in cucumarioside A6-2 (10), respectively. Both glycosides were isolated from C. japonica. Disulphated pentaosides were identified in C. japonica, C. frondosa and C. conicospermium.
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Pentaosides containing three sulphate groups, namely one at C-4 of the first xylose residue and two additional ones at the third glucose residue and terminal residue of 3-O-methylglucose have been identified in all species of Cucumaria studied: C. japonica, C. miniata, C. frondosa, C. koraeensis and C. conicospermium. The carbohydrate chain structures of glycosides of Cucumaria species studied differ greatly from those of other genera of the family Cucumariidae. Indeed, lefevreiosides from Aslia (=Cucumaria) lefevrei, cucumechinosides from Pseudoocnus (=Cucumaria) echinata, liovillosides from Staurocucumis liouvillei, and also hemoidemosides from Hemoidema spectabilis are tetraosides and do not contain fifth terminal monosaccharide unit attached to C-2 of quinovose residue.
5. Conclusions Species belonging to the genus Cucumaria may be clearly distinguished by the species-specific set of aglycones in the glycosides. Structures of carbohydrate chains branched at the quinovose residue, sulphated at the first xylose residue and containing, as a rule, five monosaccharide units and having xylose, 3-O-methylglucose, glucose and 6-O-acetylglucose as terminal monosaccharide residues are quite similar for all the glycosides isolated from species of this genus. Such a set of carbohydrate chains is correspondingly characteristic for all species of this genus studied. Glycosides with trisulphated pentaoside carbohydrate chains were isolated from all species studied. Hence, the presence of such a carbohydrate chain may serve as a reliable taxonomical character of the genus. The structure of the carbohydrate chains of glycosides from all other studied species of other genera of the family Cucumariidae are different from those of species belonging to the genus Cucumaria. This validates their earlier elimination from this genus. The sets of glycosides of the species belonging to the genus Cucumaria (Cucumariidae, Dendrochirotida) are species-specific for each studied species. They are significantly different from the glycosides of species belonging to the order Aspidochirotida, where the structures are specific for taxa above the genus level. The decrease in level of taxonomic specificity for triterpene glycoside structures at the transition from species belonging to the order Dendrochrirotida (including the genus Cucumaria) to those belonging to the order Aspidochriotida and the much greater structural diversity of glycosides in the Dendrochirotida may indicate the relatively more phylogenetic primitiveness of Dendrochirotida. Indeed, as well known to paleontologists, the closer a taxon is to the evolutionary stage where any morphofunctional system is formed, the greater the structural diversity of the system in this taxon. In this and similar cases, a level of taxonomical specificity of such structures ought to be decreased during phylogeny (Mamkaev, 1991). The taxonomic distribution of biochemical characteristics in species of the genus Cucumaria confirm the correctness of this.
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Acknowledgements The authors are very grateful to Mrs. Greta Moraes (Industrial Research Limited, New Zealand) and Professor John M. Lawrence (University of South Florida, USA) for discussion of the manuscript and correction of the English text. Vladimir I. Kalinin thanks the Science Support Foundation for Grant for Talented Young Researches. This work was partially supported by the Russian Foundation for Basic Researches (RFBR 00-15-97806, RFBR 01-04-96907 and 01-03-96903). The authors are grateful to Professor Valentin A. Stonik (Pacific Institue of Bioorganic Chemistry, Russia) for discussion of the manuscript and correction of the text.
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