Sea Cucumber Glycosides: Chemical Structures, Producing Species and Important Biological Properties

Sea cucumbers belonging to echinoderm are traditionally used as tonic food in China and other Asian countries. They produce abundant biologically active triterpene glycosides. More than 300 triterpene glycosides have been isolated and characterized from various species of sea cucumbers, which are classified as holostane and nonholostane depending on the presence or absence of a specific structural unit γ(18,20)-lactone in the aglycone. Triterpene glycosides contain a carbohydrate chain up to six monosaccharide units mainly consisting of d-xylose, 3-O-methy-d-xylose, d-glucose, 3-O-methyl-d-glucose, and d-quinovose. Cytotoxicity is the common biological property of triterpene glycosides isolated from sea cucumbers. Besides cytotoxicity, triterpene glycosides also exhibit antifungal, antiviral and hemolytic activities. This review updates and summarizes our understanding on diverse chemical structures of triterpene glycosides from various species of sea cucumbers and their important biological activities. Mechanisms of action and structural–activity relationships (SARs) of sea cucumber glycosides are also discussed briefly.


Introduction
Nature is the largest source of pharmaceutical lead drugs for the remedies of many diseases. Earlier scientists mainly focused on terrestrial samples (plants and microorganisms) for the discovery of lead bioactive compounds. With the passage of time, the search for new drugs or agrochemicals has been switching from land to ocean due to re-isolation of known natural products from terrestrial samples. Marine organisms produce diversified bioactive compounds because of large species biodiversities and living in extremely harsh environment.
Among so many sources, numerous bioactive metabolites have been isolated from marine invertebrates such as echinoderms with a broad spectrum of biological activities [1]. The echinoderms are divided into five classes, i.e., Holothuroidea (sea cucumbers), Asteroidea (starfishes), Echinoidea (sea urchins), Crinoidea (sea lilies), and Ophiuroidea (brittle stars and basket stars), which live exclusively in the marine habitat, distributed in almost all depths and latitudes, as well as reef
The glycone parts may contain up to six monosaccharide units covalently connected to C-3 of the aglycone. The sugar moieties mainly consist of D-xylose (Xyl), D Sixty percent of the triterpene glycosides isolated so far from sea cucumbers have sulfate groups linked to the monosaccharide units of the carbohydrate chain. Most of them are monosulfsated, but many di-and trisulfated glycosides have also been isolated. Most tetrasaccharides and pentasaccharides are sulfated at C-4 of xylose unit. In both the cases, additional sulfate groups at C-6 of the 3-O-methylglucose and glucose units have also been found. The term "Ds" stands for desulfated. Sea cucumber triterpene glycosides are chemotaxonomic markers specific for groups of genera within each family.
Sixty percent of the triterpene glycosides isolated so far from sea cucumbers have sulfate groups linked to the monosaccharide units of the carbohydrate chain. Most of them are monosulfsated, but many di-and trisulfated glycosides have also been isolated. Most tetrasaccharides and pentasaccharides are sulfated at C-4 of xylose unit. In both the cases, additional sulfate groups at C-6 of the 3-O-methylglucose and glucose units have also been found. The term "Ds" stands for desulfated. Sea cucumber triterpene glycosides are chemotaxonomic markers specific for groups of genera within each family.  Triterpene glycosides can be classified as holostane type having 3β-hydroxy-5α-lanostanoγ (18,20)-lactone structural feature and nonholostane type do not have a γ (18,20)-lactone but have other structural features like holostane type glycosides.

Holostane Type Triterpene Glycosides
Depending on the position of double bond in the B and C ring of the aglycone (Figure 1), holostane type glycosides can be further subdivided into three groups: glycosides with 3β-hydroxyholost-7(8)-ene, 3β-hydroxyholost-9(11)-ene, and 3β-hydroxyholost-8(9)-ene aglycone skeletons. There are eight pentacyclic triterpene and 30 alkane side chain aglycone architectures commonly found in holostane type glycosides ( Figure 4). In these architectures, certain functional groups are generally attached to the specific carbons: keto and β-acetoxy groups at C-16, and α-hydroxy group at C-12 and C-17.    Substantial number of triterpene glycosides in this category is produced by sea cucumbers. The species Eupentacta fraudatrix, Holothuria lessoni, Bohadschia marmorata, Stichopus chloronotus and Staurocucumis liouvillei produce most of the compounds in this group. For convenience, the large number of compounds in this category can be further subdivided into four groups depending on the number of sugar units.

Holostane Glycosides with 3β-Hydroxyholost-7(8)-ene Skeleton and Six Sugar Units
The name of the compounds in this group, their producing species, chemical structures and references are summarized in Table 1 and Figure 5. The most common features of glycosides in this category are the presence of α-acetoxy group at C-23, double bond at C-25(C-26) and terminal 3-O-methyl-D-glucose in carbohydrate chain. An interesting point to be noted in here is that the sulfate group is totally absent in this group of compounds. Table 1. Name and producing species of glycosides with 3β-hydroxyholost-7(8)-ene and sixs ugar units.
Holostane Glycosides with 3β-Hydroxyholost-7(8)-ene Skeleton and Six Sugar Units The name of the compounds in this group, their producing species, chemical structures and references are summarized in Table 1 and Figure 5. The most common features of glycosides in this category are the presence of α-acetoxy group at C-23, double bond at C-25(C-26) and terminal 3-O-methyl-D-glucose in carbohydrate chain. An interesting point to be noted in here is that the sulfate group is totally absent in this group of compounds.  The name of the compounds in this group, their producing species, chemical structures and references are summarized in Table 2 and Figure 6. The most common structural features in this group are the sulfate groups at C-4 of xylose and C-6 of glucose and methylglucose with either β-acetoxy or keto group at C-16 and C-25(26) double bond. A quite number of compounds contain a keto group at C-23.The rare structural features of triterpene glycoside are the presence of 16,22-epoxy group (33), ethoxy group (29) and methylglucuronic acid (51). Cucumarioside A1-2 (17) is the only example of triterpene glycosides containing an acetate group at C-6 of the terminal sugar unit. Carbohydrate chain can be one branched (14-48 and52-54) or straight (49)(50)(51). 3-O-methyl-D-xylose as a terminal monosaccharide unit that is a characteristic feature of all the glycosides isolated from Eupentacta fraudatrix. Table 2. Name and producing species of glycosides with 3β-hydroxyholost-7(8)-ene and five sugar units.

The Important Biological Properties of Sea Cucumber Glycosides
Triterpene glycosides are the prime bioactive metabolites of sea cucumbers, and are commonly known as toxins of sea cucumbers to eukaryotic cells. These glycosides showed a wide range of biological activities including cytotoxic, antifungal, antiviral, hemolytic, antiprotozoal and immunomodulatory activities. Sea cucumbers produce some major glycosides in sufficient amount to carry out a wide range of biological activity tests [37,94]. Besides major glycosides, they also produce minor glycosides insufficient to test a range of biological activities [66,67]. The point to be noted here is that sea cucumber glycosides are able to exhibit biological activities in both in vitro and in vivo models [5]. The remarkable biological properties showed by some triterpene glycosides are summarized in Table 11. Triterpene glycosides do not exhibit antibacterial activity, indicating that these glycosides are probably produced by sea cucumbers for defence against eukaryotic predators.

The Important Biological Properties of Sea Cucumber Glycosides
Triterpene glycosides are the prime bioactive metabolites of sea cucumbers, and are commonly known as toxins of sea cucumbers to eukaryotic cells. These glycosides showed a wide range of biological activities including cytotoxic, antifungal, antiviral, hemolytic, antiprotozoal and immunomodulatory activities. Sea cucumbers produce some major glycosides in sufficient amount to carry out a wide range of biological activity tests [37,94]. Besides major glycosides, they also produce minor glycosides insufficient to test a range of biological activities [66,67]. The point to be noted here is that sea cucumber glycosides are able to exhibit biological activities in both in vitro and in vivo models [5]. The remarkable biological properties showed by some triterpene glycosides are summarized in Table 11. Triterpene glycosides do not exhibit antibacterial activity, indicating that these glycosides are probably produced by sea cucumbers for defence against eukaryotic predators.

Mechanisms of Action
Natural products derived from marine organisms have incredible structural and functional diversity. The mechanism by which triterpene glycosides exhibit anticancer activity primarily involve induction of tumor cell apoptosis through the activation of intracellular caspase cell death pathways, arrest of the cell cycle at S or G 2 /M phases and increase of the sub-G 0 /G 1 cell population; regulation of nuclear factor NF-κB expression; reduction in cancer cell adhesion; suppression of cell migration and tube formation; suppression of angiogenesis; inhibition of cell proliferation, colony formation, and tumor invasion [141]. However, the detailed mechanism(s) of the anticancer activities of these glycosides remains largely unclear.
Marked membranolytic effects such as increased membrane permeability, loss of barrier function, and the rupture of cell membrane are considered the basic mechanisms underlying a variety of biological activities exerted by triterpene glycosides of sea cucumbers. The glycosides form complex with ∆ 5(6) -sterols of cellular membrane especially cholesterol. This interaction induces significant changes in the physicochemical properties of cell membranes, such as variations in their stability, microviscosity, and permeability. Saponins form complexes with membrane sterols, leading to cell disruption by the formation of pores. Due to this irreversible interaction, the selective permeability of cell membranes is impaired and cell compounds are transferred into the extracellular matrix, ultimately resulting in cell death [142,143].

Structure-Activity Relationships (SARs)
Both glycone and aglycone parts are important for biological activities of sea cucumber glycosides. The structure-activity relationships among sea cucumber glycosides are presumably more complicated. The most important structural characteristics of glycosides that probably contribute in biological activities are mentioned below.
The cytotoxicity not only depends on the chemical structures of the glycosides but also cell types [144]. The presence of 12α-hydroxy and 9(11)-ene structural units in holostane aglycone play key role in cytotoxicity [144]. Number of monosaccharide units in sugar chains and the substitution in side chain of aglycone could affect cytotoxicity. The presence of hydroxy groups in the side chains of glycosides significantly reduces cytotoxicity of the glycosides with increasing distance of hydroxy group from the 18(20)-lactone [30,31]. Linear tetraoside unit plays important role in different biological activities of sea cucumber glycosides [144]. Hexaoside chain containing glycosides show stronger cytotoxic activity than pentaoside chain containing glycosides. Glycosides with hexaosides residue with xylose or quinovose in the fifth position are the most active cytotoxins [144]. Different activities test result indicates that the number of sulfate groups and their position in the carbohydrate chains affect cytotoxicity [144]. It has been shown that the sulfate group attached to C-6 of terminal 3-O-methylglucose unit greatly decrease and attached to C-6 of glucose (the third monosaccharide unit) generally increase membranotropic activity [145].

Conclusions
Sea cucumbers (or holothurians), a class of marine invertebrates, are used as human food and traditional medicine, especially in some parts of Asia. The majority of the sea cucumbers synthesize glycosides with a polycyclic aglycone that contain either 7(8)-or 9(11)-double bond with up to six monosaccharide residues containing carbohydrate chain. A few of them are known to synthesize aglycone with 8(9)-ene. Sea cucumber glycosides are cytotoxic to eukaryotes; probably produce for escaping from predation by marine eukaryotic organisms. These cucumber metabolites have shown profound cytotoxic and hemolytic activities against eukaryotic organisms but not prokaryotic organisms. Due to significant cytotoxic and antifungal activities, extensive differential SAR studies of these glycosides can be helpful to develop new drugs and agrochemicals.