Triterpene and Steroid Glycosides from Marine Sponges (Porifera, Demospongiae): Structures, Taxonomical Distribution, Biological Activities

The article is a comprehensive review concerning tetracyclic triterpene and steroid glycosides from sponges (Porifera, Demospongiae). The extensive oxidative transformations of the aglycone and the use of various monosaccharide residues, with up to six possible, are responsible for the significant structural diversity observed in sponge saponins. The saponins are specific for different genera and species but their taxonomic distribution seems to be mosaic in different orders of Demospongiae. Many of the glycosides are membranolytics and possess cytotoxic activity that may be a cause of their anti-predatory activities. All these data reveal the independent origin and parallel evolution of the glycosides in different taxa of the sponges. The information concerning chemical structures, biological activities, biological role, and taxonomic distribution of the sponge glycosides is discussed.


Introduction
Sponges (phylum Porifera) are an oldest living group of Metazoa. Their general organization was so adaptable that they survived during dramatic changes in environment. This group of aquatic organisms (marine and freshwater) are very diverse and includes more than 8500 valid species [1,2]. The unique conditions of the marine environment, such as high pressure and salinity, coupled with the need to defend against predators and microorganisms, have led to a wide range of secondary metabolites in sponges. These metabolites possess medicinal properties that are distinct from those found in terrestrial plants [3]. The number of such metabolites found in sponges by now is more than 5300. Some of them are anticancer agents which have different mechanisms of action including anti-neoplastic efficacy [4,5]. Furthermore, the metabolites obtained from marine sponges may have extensive medicinal applications, including preparations with antiviral and anti-inflammatory properties, as referenced in [6] and [7], respectively.
It is known that many sponge metabolites may be synthesized by symbiotic microorganisms [3]. However, the enzymes responsible for terpene biosynthesis were found recently in sponges but not in symbiotic microorganisms. Hence, these metabolites should be synthesized by sponges directly [8]. The distribution of different groups of secondary metabolites within the class Demospongiae of the phylum Porifera (sponges) was used for chemotaxonomy purposes. The authors of the investigation suggest that the distribution reveals polyphyly of any taxonomical groups and the system of the class should be improved [9].
Tetracyclic steroid and triterpene glycosides are characteristic substances for many terrestrial higher plants. Over the past few decades, a large number of triterpene glycosides as the third monosaccharide, glycosides 4-6, 10-13, as well as 24 having xylose instead of glucose. Nevertheless, glycosides 7-9 are tetraosides with identical carbohydrate chains having xylose as the third monosaccharide residue. This monosaccharide occupies terminal position.
Later, Fouad et al. [30] isolated two related glycosides named as erylosides K and L (26,27), in addition to 25 from the same sponge, which was harvested near Jordan's coast in the Gulf of Aqaba, Red Sea. The absolute stereochemistry of C-23 in eryloside K was established by comparison the NMR data with those published for both possible diastereomers, which exact stereochemistry has been established by Mosher's method. Eryloside L (27) had very uncommon 8α,9α-epoxy-8,9-seco-7,9(11),14-triene fragment in the aglycone (Figure 2) [30].
lecules 2023, 28, x FOR PEER REVIEW 8 of studies began with the study of the sponge Erylus lendenfeldi, which is a habitant of th Indo-West Pacific zoogeographical region. Eryloside A (25), a new bis-nor-triterpene bi side, with 4β,14-di-nor-methyl-lanostane aglycone and β-D-galactopyranose as the fir sugar together with another (terminal) β-D-galactopyranose linked to C-2 of the fir sugar, was found in the Red Sea sample of this sponge species by Carmely et al. in 198 [29]. Glycoside 25 aglycone possesses 8(9)-and 14(15)-double bonds and a hydroxyl at C 23.
Later, Fouad et al. [30] isolated two related glycosides named as erylosides K and (26,27), in addition to 25 from the same sponge, which was harvested near Jordan's coa in the Gulf of Aqaba, Red Sea. The absolute stereochemistry of C-23 in eryloside K w established by comparison the NMR data with those published for both possible diaster omers, which exact stereochemistry has been established by Mosher's method. Erylosid L (27) had very uncommon 8α,9α-epoxy-8,9-seco-7,9(11),14-triene fragment in the agl cone ( Figure 2) [30]. Almost simultaneously, Sandler et al. [31] from Faulkner's laboratory have isolate the same eryloside A (25) with two similar glycosides named erylosides K and L (26, 2 from another Red Sea collection of this species. The structure of eryloside K was identic to that described by Fouad et al. [30]. The second new glycoside 28, also called erylosid L by Sandler et al. [31], differed in structure from the compound described by Fouad et a [30]. Eryloside L1 (28), depicted in Figure 2, contains an aglycone similar to that of eryl sides A and K, as well as a 23-keto group in the side chain. The 23S-configuration of eryl side A (25) was firstly established by Mosher's method. The C-23 absolute configuratio in 26 was assigned by comparison 1 H NMR spectra, HPLC retention times, and optic rotation derivatives of 25 and 26 obtained by hydrogenation using a rhodium catalyst [31 Glycoside 25 reveals antifungal action on Candida albicans (MIC of 15.6 μg/mL) an cytotoxicity on P388 tumor cell line (IC50 of 4.2 μg/mL) [30]. This glycoside is active o Bacillus subtilis, C. albicans, and Escherichia coli (zones of inhibition at 10 μg per disc we of 7, 7, and 6 mm, respectively) [30]. Glycosides 25 and 26 gave the mortality rate of 50 at the concentration of 0.1 μg/mL in the brine shrimp assay. Glycoside 27 was inactive Almost simultaneously, Sandler et al. [31] from Faulkner's laboratory have isolated the same eryloside A (25) with two similar glycosides named erylosides K and L (26, 28) from another Red Sea collection of this species. The structure of eryloside K was identical to that described by Fouad et al. [30]. The second new glycoside 28, also called eryloside L by Sandler et al. [31], differed in structure from the compound described by Fouad et al. [30]. Eryloside L1 (28), depicted in Figure 2, contains an aglycone similar to that of erylosides A and K, as well as a 23-keto group in the side chain. The 23S-configuration of eryloside A (25) was firstly established by Mosher's method. The C-23 absolute configuration in 26 was assigned by comparison 1 H NMR spectra, HPLC retention times, and optical rotation derivatives of 25 and 26 obtained by hydrogenation using a rhodium catalyst [31].
Glycoside 25 reveals antifungal action on Candida albicans (MIC of 15.6 µg/mL) and cytotoxicity on P388 tumor cell line (IC 50 of 4.2 µg/mL) [30]. This glycoside is active on Bacillus subtilis, C. albicans, and Escherichia coli (zones of inhibition at 10 µg per disc were of 7, 7, and 6 mm, respectively) [30]. Glycosides 25 and 26 gave the mortality rate of 50% at the concentration of 0.1 µg/mL in the brine shrimp assay. Glycoside 27 was inactive in this test. Glycosides 25, 26, and 27 are not cytotoxic on THP-1, JURKAT, and MM-1 tumor cell lines [30]. Glycosides 25, 26, and 28 were selectively active on the ∆rad50 budding yeast strain (0.8, 2.0, and 3.4 µg/mL) in comparison with the action on the wild parent yeast strain (IC 50 of 3.5, 6.1, and 11.4 µg/mL, respectively). The selective cytotoxicity against similar mutant yeast is a probable indicator of topoisomerase inhibitors. However, the activities on TOP1oe (IC 50 of 5.7, 7.5, and 10.9 µg/mL) and TOP2oe (10.8, 12.2, and 9.5 µg/mL) were weaker than for camptothecin and idarubicin, which are known inhibitors. The aglycone, obtained from glycoside 25, was inactive against the both yeast strains. This finding confirmed the significant role of the carbohydrate chain in the activity, as reported in reference [31].
The studies on a sponge Erylus sp. collected at a depth of 500 m from New Caledonian waters yielded two new lanostane glycosides: trioside and tetraoside and erylosides C (29) and D (30), respectively [32]. Their aglycones have 8(9)-unsaturation, a carboxyl at C-14, a methylene at C-24 and an additional methyl at C-25. All the sugars are Dgalactose residues in β-pyranose form. One terminal monosaccharide residue is linked to C-2 of the first monosaccharide. Another terminal sugar is attached to C-3 of the first monosaccharide residue. This is characteristic for many triterpene glycosides isolated from sponges belonging to the genus Erylus. Nevertheless, one of the terminal monosaccharide residues in tetraoside 30 is linked to C-4 of the second monosaccharide residue, but not to C-3 position ( Figure 2).
The sponge Erylus goffrilleri is an inhabitant of the tropical shallow waters of the Atlantic. Eryloside E (31), an unusual lanostane glycoside, was isolated by Gulavita et al. It contains a bioside carbohydrate chain consisting of galactose as the first monosaccharide, with N-acetyl-2-deoxy-2-amino-glucose linked to C-2 of the galactose residue. It also has another carbohydrate moiety composed of xylose, attached to a carboxyl at C-14 of the aglycone [33]. The aglycone possesses 8(9)-double unsaturation, oxy-group at C-24, and additional methyls at C-25, as well as C-24 ( Figure 3).
Afiyatullov et al. discovered four lanostane monosides, erylosides R (32), S (33), T (34), and U (35), which are similar glycosides with a β-D-galactopyranose residue serving as a carbohydrate moiety, as depicted in Figure 3 [34]. Glycoside 32 contains a lanostane derivative as an aglycone. It has 8(9)-double unsaturation, carboxyl at C-14, hydroxyl at C-24 and two additional methyls at C-25, as well as C-24 in the side chain. Eryloside S (33) contains a relative aglycone, having an acetate and an additional methyl group at C-24 in the side chain. The aglycone of eryloside T (34) has the same side chain as in 32 but possesses 7(8)-unsaturation and an uncommon lactone linkage between C-14 and C-9. Glycoside 35 is similar to 34 but has an additional 7,8-epoxy group. The configuration of this epoxy-group was indicated by the authors of the present paper as 7α,8α on the basis of the upfield shift of the C-5 signal [34]. Later, Kolesnikova et al. isolated a series of free similar aglycones from a sponge Penares sp. which was collected by dredging in Vietnamese waters. They determined the stereochemistry of these substances using X-ray analysis and CD spectra and found that the aglycones investigated contained a 7β,8β-epoxy group [55]. As a result, the structure of 35 was revised and presented at Figure 3 with a 7β,8β-epoxy group.
Moreover, Afiyatullov et al. found three lanostane biosides: erylosides F 5 -F 7 (36)(37)(38), as well as eryloside V (39), and a trioside ( Figure 3). The carbohydrate chains of glycosides 36 and 37 are very similar, consisting of D-galactose and N-acetyl-2-deoxy-2-amino-Dglucose, and are linked to C-2 of the galactose [34]. The sugars are in β-pyranose forms, and their aglycones have an 8(9)-double bond and a carboxy-group at C-14, which is common for many erylosides. As it also is characteristic for many erylosides, the side chain of the aglycone in glycoside 36 contains a C-24 hydroxyl and two additional methyls at C-25, as well as at C-24. The side chain of 37 contains a 24-OAc-group and only one additional C-24 methyl group. Glycoside 38 has the same aglycone as in 36. However, it has the second monosaccharide residue, β-D-glucopyranose, linked to C-3 of the first sugar. Eryloside V (39) is a trioside having the same aglycon as in 36 and α-L-arabinopyranose as the first Afiyatullov et al. discovered four lanostane monosides, erylosides R (32), S (33), T (34), and U (35), which are similar glycosides with a β-D-galactopyranose residue serving as a carbohydrate moiety, as depicted in Figure 3 [34]. Glycoside 32 contains a lanostane derivative as an aglycone. It has 8(9)-double unsaturation, carboxyl at C-14, hydroxyl at C-24 and two additional methyls at C-25, as well as C-24 in the side chain. Eryloside S (33) contains a relative aglycone, having an acetate and an additional methyl group at C-24 in the side chain. The aglycone of eryloside T (34) has the same side chain as in 32 but possesses 7(8)-unsaturation and an uncommon lactone linkage between С-14 and С-9. Glycoside 35 is similar to 34 but has an additional 7,8-epoxy group. The configuration of this epoxy-group was indicated by the authors of the present paper as 7α,8α on the basis of the upfield shift of the C-5 signal [34]. Later, Kolesnikova et al. isolated a series of free similar aglycones from a sponge Penares sp. which was collected by dredging in Vietnamese waters. They determined the stereochemistry of these substances using X-ray analysis and CD spectra and found that the aglycones investigated contained a 7β,8β-epoxy group [55]. As a result, the structure of 35 was revised and presented at Figure 3 with a 7β,8βepoxy group. Glycoside 31 weakly inhibited the binding of 125 [I]-Bottom Hunter labeled C5a to its receptor (IC 50 > 10 µM). Eryloside E (31) reveals also immunosuppressive activity (EC 50 of 1.3 µg/mL). Its immunosuppressive action was specific and independent from general cytotoxicity [33]. Erylosides R-T (32-34), F 6 (37), F 7 (38), and V (39) were cytotoxic against Ehrlich carcinoma tumor cells (IC 50 of 20-40 µM) [34]. Nevertheless, glycosides 35 and 36 were not active.
In continuation of the investigation of E. goffrillery, which was harvested from the Caribbean Sea near the Arrecife-Seco reef (Cuba), Antonov et al. [35]  Two residues of α-L-arabinopyranose, attached to each other by 1→4 glycosidic link, are linked to C-3 of the first sugar for all the tetraosides 44-46. The fourth (terminal) sugar for 44 and 45 attached to C-2 of the first monosaccharide residue is N-acetyl-2-deoxy-2-aminoβ-D-galactopyranose whereas N-acetyl-2-deoxy-2-amino-β-D-glucopyranose is the fourth sugar for glycoside 46 [35].
Many glycosides of this series have moderate cytotoxicity and hemolytic activities. The activities depend on both aglycone and carbohydrate moieties [35]. Erylosides V 2 (42) and V 3 (43) have the same trioside carbohydrate moieties but different aglycones. They reveal quite different activities. Eryloside V 2 (42), having 24-O-acetyl and having no methyl at C-25, is a moderate hemolytic. Nevertheless, it possesses significant cytotoxic activities. Glycoside 43, having a 24-hydroxyl and a C-25 methyl, has both moderate hemolytic and cytotoxic activities. Eryloside V 2 (42), which is a trioside, is more active in both tests than eryloside W (44), which is a tetraoside that has an additional L-arabinose. The activities of glycoside 44, having D-galactose as the first sugar, are significantly lower than the activities of 45, which possesses a 6-O-acetyl group of the first D-galactose. The activity of glycoside 45 with N-acetyl-D-galactosamine as the fourth sugar are higher than the activities of 46 with an N-acetyl-D-glucosamine at the same position. It is interesting that glycoside 42 possesses strong cytotoxic and moderate hemolytic activities. However, glycoside 43 has moderate cytotoxic activity and low hemolytic activity [35]. This indicates a more complicated character of cytotoxicity for these glycosides than a simple membranolytic action.
The aglycone present in almost all glycosides isolated from E. goffrilleri is a lanostane derivative with a double bond at position 8(9) and a carboxyl group at C-14. This tetracyclic triterpene nucleus was found earlier from Penares sp. as penasterol, a free non-glycosylated triterpene [56]. 3-oxo-and 3-O-acetyl-derivatives of penasterol were also found in the sponge Penares incrustans [57]. Only two types of side chains were found in them: the first one contains a C-24 hydroxyl, along with two additional methyls at C-25 and C-24; the second one contains an acetyl group and one additional C-24 methyl.
All erylosides from E. goffrilleri possess a similar architecture of the carbohydrate chain, and all the sugars are in pyranose form. The first monosaccharide residue attached to C-3 of a 14-carboxylated aglycone is β-D-galactose. Sometimes it may be a 6-O-acetylβ-D-galactose or α-L-arabinose. The sugar number is varied from one to four. Next, monosaccharide residues may be linked to the first sugar C-2 and C-3 positions. Further elongation of the carbohydrate chain may occur only from the sugar that is attached to C-3 of the first sugar. The next (terminal) monosaccharide residue may be attached only to C-4 of this second sugar. A terminal monosaccharide residue attached to the first sugar C-2 position may be D-galactose, N-acetyl-D-galactosamine, or N-acetyl-D-glucosamine. The configurations of the glycosidic centers are α for L-arabinose and β for D-glucose, D-galactose, 6-O-acetyl-D-galactose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, as well as D-xylose.
The sponge Erylus formosus is a typical habitant of shallow waters from the Caribbean Sea and eastern Brazil. It is the most investigated sponge concerning tetracyclic triterpene glycosides. In continuation of investigations into this species, Jaspars and Crews have found formoside (47), a lanostane tetraoside. It was isolated from the sample of this sponge harvested in shallow waters of Bahamas [36] (Figure 4). The aglycone of formoside is a lanostane derivative, with 8(9)-and 24(25)-double bonds and C-14 carboxyl. Such structural features are characteristic for penasterol [56]. All monosaccharide residues (two galactoses and two arabinoses) of 47 are in pyranose forms.   Another glycoside of this type, formoside B (48), was isolated by Kubanek et al. in the extract of E. formosus, also collected near the Bahamas, along with known formoside (47) (Figure 4) [37]. Glycoside 48 differs from 47 because of the presence of terminal N-acetyl-2-deoxy-2-amino-D-galactose instead of a galactose residue. The authors also characterized, using mass-spectrometry without the isolation of individual glycosides, a series of inseparable hexaoside and triosides fractions [37].
Stead et al. discovered eryloside F (49), a similar lanostane bioside, as a result of their investigation into E. formosus. The sponge was harvested near the Bahamas through scuba diving at a depth of 55 feet. It has the same aglycone, also containing arabinose residue as the first monosaccharide residue linked to C-3 of the aglycone and the second sugar (galactose) attached at the arabinose C-2 position [38] (Figure 4).
Erylosides M (54) and N (55), two new triosides, were also found by the present authors in E. formosus [39], as well as eryloside H (the structure is shown below in Figure 5), earlier found in Erylus nobilis [41]. Eryloside N (55) has penasterol as an aglycone. However, eryloside M (54) contains an aglycone with a C-24 methylene group in the side chain. Eryloside O (56), a new tetraoside is similar to formoside (47), but the third terminal sugar was identified as α-L-arabinose but not β-D-galactose. Erylosides P (57) and Q (58), two new hexaosides, have the same carbohydrate chains. They differed by the aglycones side chains. The carbohydrate chains of these glycosides are very closed to that of formoside (47). However, they have an additional glucose residue linked to C-4 of the fourth monosaccharide residue (galactose) and a terminal xylose, attached to this glucose C-2 position [39].
cules 2023, 28, x FOR PEER REVIEW 14 of except arabinose residues, which belong to L-series. The last ones have an α-configurati of glycosidic bonds. Erylosides F1-F4 (50-53) were similar to eryloside F (49) and differi by aglycone side chains [39]. The known eryloside F (49) also was found. Eryloside F1 ( possesses a penasterol such as aglycone having the side chain with a C-24 methyle Penasterol and the discussed relative aglycones are predominant in the Erylus spp. gly sides. Eryloside F2 (51) possesses a 25 (26) (51) and F3 (52) [39]. Erylosides M (54) and N (55), two new triosides, were also found by the present a thors in E. formosus [39], as well as eryloside H (the structure is shown below in Figure  earlier found in Erylus nobilis [41]. Eryloside N (55) has penasterol as an aglycone. Ho ever, eryloside M (54) contains an aglycone with a C-24 methylene group in the side cha Eryloside O (56), a new tetraoside is similar to formoside (47), but the third terminal sug was identified as α-L-arabinose but not β-D-galactose. Erylosides P (57) and Q (58), tw new hexaosides, have the same carbohydrate chains. They differed by the aglycones si chains. The carbohydrate chains of these glycosides are very closed to that of formosi (47). However, they have an additional glucose residue linked to C-4 of the fourth mon saccharide residue (galactose) and a terminal xylose, attached to this glucose C-2 positi [39].  Figure 4). The sugars in all these glycosides were in β-D-pyranose forms except for arabinose residues, which had an α-configuration. Eryloside R1 (59) has penasterol as aglycone. Its terminal galactose is attached to C-3 of the first sugar (arabinose), where another galactose residue is linked to the arabinose C-2 position. Hexaosides 60-62 a contain penasterol as the aglycone. However, hexaosides 63-65 contain an aglycone w a methylene group at C-24 in the side chains which is common in this series [40]. All t hexaosides possess carbohydrate moieties as they have the same architecture (gene structural plan). Their carbohydrate chains are closed to those of erylosides P (57) and (58) [39].  (Figure 4). The sugars in all these glycosides were in β-D-pyranose forms except for L-arabinose residues, which had an α-configuration. Eryloside R 1 (59) has penasterol as its aglycone. Its terminal galactose is attached to C-3 of the first sugar (arabinose), whereas another galactose residue is linked to the arabinose C-2 position. Hexaosides 60-62 also contain penasterol as the aglycone. However, hexaosides 63-65 contain an aglycone with a methylene group at C-24 in the side chains which is common in this series [40]. All the hexaosides possess carbohydrate moieties as they have the same architecture (general structural plan). Their carbohydrate chains are closed to those of erylosides P (57) and Q (58) [39].
It is interesting that all the triterpene glycosides found in E. formosus (totally 20 glycosides) have similar structural features. All the glycosides contain α-L-arabinopyranose as the first sugar linked to an aglycone C-3 position. The number of sugars may vary from two to six.
One of terminal monosaccharides is attached at C-2 of the first sugar (L-arabinose). All the hexaosides also contain another terminal sugar, linked to C-2 of the fourth monosaccharide residue. All other links between the sugars are 1 → 3, except a link between the third and fourth as well as the fourth and fifth sugars, which is 4 → 1 and 2 → 1, respectively. Figure 4 shows that N-acetyl-2-deoxy-2-amino-D-galactose may also serve as a terminal monosaccharide in tetracyclic triterpene glycosides of E. formosus, in addition to arabinose, glucose, and galactose.
There is a possibility that the biosynthesis of carbohydrate chains in E. formosus is initiated by a glycosyltransferase containing L-arabinose. Through the use of other glycosyltransferases, elongation of the carbohydrate chains is possible, which allows for the substitution of different sugars in various triterpene glycosides while maintaining the overall structure of the carbohydrate chains [40]. Eryloside F (49) is a potent thrombin receptor antagonist. It may inhibit human platelet aggregation in vitro. Glycoside 49 facilitates the Ca 2+ mobilization in cells registered by an FLIPR assay [37]. Formoside (47) revealed antiviral effect (IC 50 of 3.5 µg/mL vs. HSV-1) and moderate antibacterial activity (IC 50 of 31.3 µg/mL vs. Corynebacterium xerosis) [36]. Hexaoside fractions have an antifungal effect on Candida albicans resistant to amphotericin B (IC 50 of 3.9 µg/mL) [38]. At comparable concentrations, eryloside F1 (50) and eryloside F (49) both elicit a stimulation of calcium influx into mouse splenocytes (130% of the control), whereas eryloside F3 (52) induces early apoptosis in Ehrlich cells at a concentration of 100 µg/mL, whereas its epimer eryloside F2 (51) does not have this effect [39].
A study [58] has reported the synthesis of two trisaccharides containing D-galactose, L-arabinose, and D-glucosamine hydrochloride, which are structurally similar to saponins found in E. nobilis. Thioglycoside chemistry was used for glycosylation reactions, with activation accomplished using NIS in the presence of La(OTf) 3 .
Okada et al. have found two unique 14-nor-methyl-23,24,25,26,27-pentanorlanostane glycosides in Erylus placenta harvested near the shores of the South Japan (Hachijo Island). An unprecedented sokodoside A (71) aglycone possesses an 8(14)-double bond. However, sokodoside B (72) has an unprecedented conjugated 8(9),14(15),16(17)-triene system [43] ( Figure 6). Sokodoside 71 is a branched tetraoside. The first monosaccharide residue of 71 is β-D-galacturonic acid, the second sugar linked to C-2 of the first monosaccharide residue is also β-D-galacturonic acid, and the terminal (third) one linked to C-2 of the second monosaccharide residue is α-L-fucose. Another terminal monosaccharide is α-L-arabinose residue. Sokodoside B (72) is a branched trioside that has carbohydrate moiety including α-L-arabinose, β-D-galactose and β-D-galacturonic acid. The arabinose residues configuration was erroneously determined as β. However, the presented coupling constant in the 1 H NMR spectra of 71 and 72 demonstrated an α-configuration. A synthesis of the trisaccharide carbohydrate chain of sokodoside B has been realized by thioglycoside activation using sulfuric acid immobilized on silica in conjunction with N-iodosuccinimide [59]. The glycosides 71 and 72 revealed moderate growth-inhibitory activity on the fungus Mortierella ramanniana and several strains of the yeast Saccharomyces cerevisiae with or without mutations (cdc28, erg6 and act1-1). The size of inhibition zones was varied from 8 to 16 mm at 50 µg of the tested glycoside on 6 mm spot on thin paper disk. The glycoside 71 was more active than 72. Glycosides 71 and 72 reveal moderate cytotoxic effects on P388 cells with IC 50 of 100 and 50 µg/mL, respectively. The correlation between their antifungal and cytotoxic activities was observed [43].  erg6 and act1-1). The size of inhibition zones was varied from 8 to 16 mm at 50 μg of the tested glycoside on 6 mm spot on thin paper disk. The glycoside 71 was more active than 72. Glycosides 71 and 72 reveal moderate cytotoxic effects on P388 cells with IC50 of 100 and 50 μg/mL, respectively. The correlation between their antifunga and cytotoxic activities was observed [43]. Thus, all the studied representatives of the genus Erylus contain bioactive tetracyclic triterpene glycosides, preferably having lanostane aglycones with C-14 carboxyl and carbo hydrate chains with predominance of arabinose and galactose monosaccharide residues.  Thus, all the studied representatives of the genus Erylus contain bioactive tetracyclic triterpene glycosides, preferably having lanostane aglycones with C-14 carboxyl and carbohydrate chains with predominance of arabinose and galactose monosaccharide residues. -double bond and 9α-hydroxy and 8(9)-epoxy groups. The aglycone of 77 has 8α,9α-diol group and C-18 methyl shifted to 14β-position. It also has a unique 12(13)-double bond. Most of these glycosides feature a pentasaccharide chain that is very typical of sarasinosides. However, glycoside 73 is unique in that it is a trioside lacking two glucose residues, specifically the terminal and pre-terminal ones [44]. has 8α,9α-diol group and C-18 methyl shifted to 14β-position. It also has a unique 12(13)double bond. Most of these glycosides feature a pentasaccharide chain that is very typical of sarasinosides. However, glycoside 73 is unique in that it is a trioside lacking two glucose residues, specifically the terminal and pre-terminal ones [44].

The Order Bubarida
All the studied glycosides, including known ones, had no antibacterial action on several bacterial strains [44]. However, the most of glycosides possessed moderate cytotoxic activities on K562 leukemia and A549 lung carcinoma cell lines. Nevertheless, glycoside 77 was not active against K562 tumor cell lines whereas glycoside 15 was not active against A549 cells. The author noted that the presence of additional hydroxyls at the nucleus of an aglycone decreased the activity. Sarasinoside A1 (1), its derivative obtained from 1 by glycolysis, and sarasinoside N (73) weakly inhibited Na + /K + -ATPase with IC50 60.0, 59.4, and 54.1 μg/mL, respectively [44].
A group of French investigators has isolated new triterpene bioside, eryloside W (79), from the sponge Dictyonella marsilii (family Dictyonellidae) harvested in the Gibraltar Strait ( Figure 8). This is the first sponge triterpene glycoside found in the Mediterranean region. The glycoside has a lanostane aglycone with an 8(9)-double bond, a carboxyl group at C-14, a methylene group at C-24 in the side chain, and a carbohydrate chain consisting of β-D-glucuronic acid as the first sugar and 2-NAc-β-D-glucopyranose as a terminal monosaccharide residue linked with C-2 of the first glucose residue [46]. = 10.85 μg/mL), and NUGC-3 (IC50 = 10.47 μg/mL) [45].
A group of French investigators has isolated new triterpene bioside, eryloside W (79), from the sponge Dictyonella marsilii (family Dictyonellidae) harvested in the Gibraltar Strait ( Figure 8). This is the first sponge triterpene glycoside found in the Mediterranean region. The glycoside has a lanostane aglycone with an 8(9)-double bond, a carboxyl group at C-14, a methylene group at C-24 in the side chain, and a carbohydrate chain consisting of β-D-glucuronic acid as the first sugar and 2-NAc-β-D-glucopyranose as a terminal monosaccharide residue linked with C-2 of the first glucose residue [46].
A group of French investigators has isolated new triterpene bioside, eryloside W (79), from the sponge Dictyonella marsilii (family Dictyonellidae) harvested in the Gibraltar Strait ( Figure 8). This is the first sponge triterpene glycoside found in the Mediterranean region. The glycoside has a lanostane aglycone with an 8(9)-double bond, a carboxyl group at C-14, a methylene group at C-24 in the side chain, and a carbohydrate chain consisting of β-D-glucuronic acid as the first sugar and 2-NAc-β-D-glucopyranose as a terminal monosaccharide residue linked with C-2 of the first glucose residue [46].
procedures. The absolute configuration of 80 was proposed along with biogenetical reasons. All the isolated triterpene glycosides were not active against two human solid cancer cell lines, Huh-7 (hepatocarcinoma) and A549 (lung carcinoma) [47].
branched at the first sugar tetraosides, having two residues of β-D-glucopyranose and two residues of α-L-rhamnopyranose. Residues of α-L-rhamnose were never discovered in sponge triterpene glycosides before this investigation ( Figure 11). Glycosides 88 and 89 reveal moderate cytotoxic effects in vitro on J774 (murine monocyte-macrophage), WEHI164 (murine fibrosarcoma), and P388 (murine leukemia) cell lines (IC50 from 8.5 to 11.4 μg/mL) [52]. Glycosides 90 and 91 were moderately cytotoxic (IC50 of 19 μg/mL) against the cells of J774 murine monocyte-macrophages [53]. A group of authors from Colombia and France have reported the isolation of three new triterpene glycosides, namely ulososide F (92), urabosides A (93) and B (94) (Figure  11), from a sample of the Caribbean marine sponge Ectyoplasia ferox. The previously known ulososide A (83) was also isolated in this study. The sponge was harvested in the Uraba Gulf (Colombia). All the compounds have an aglycone derived from 14-normethyl A group of authors from Colombia and France have reported the isolation of three new triterpene glycosides, namely ulososide F (92), urabosides A (93) and B (94) (Figure 11), from a sample of the Caribbean marine sponge Ectyoplasia ferox. The previously known ulososide A (83) was also isolated in this study. The sponge was harvested in the Uraba Gulf (Colombia). All the compounds have an aglycone derived from 14-normethyl lanostane with a double bond at the 8(9)-position. The side chain of 92 composed 22α-and 23βhydroxyls and additional 24β-methyl. The carbohydrate chain includes 2-NHAc-β-Dglucopyranosyl as the first monosaccharide residue and β-D-glucuronic acid as the terminal monosaccharide residue attached to C-6 of the first sugar. The aglycones of urabosides A (93) and B (94) are 14-normethyl derivatives with an 8(9)-double bond and a 23-ketogroup. Both aglycones have a 4β-methyl group that is oxidized by a carboxyl group. The aglycone of 93 has a 4α-methyl group that is oxidized by CH 2 OH, whereas the aglycone of 94 has a 4α-methyl group that is oxidized by a carboxyl group.
The first monosaccharide residue of the triosidic carbohydrate chain in glycoside 93 is branched, and it is a β-D-galactopyranose. The terminal monosaccharide residues, β-D-arabinopyranose and β-D-galactopyranose, are linked to C-2 and C-3 of the first monosaccharide residue, respectively. There is no significant cytotoxicity on the two cell lines (Jurkat and CHO cells) or hemolytic action for the isolated compounds [54].
In order to estimate of the diversity of triterpene glycosides in the same collection of E. ferox, the authors used an metabolomic approach and applied the HPLC-ESI-IT-MS/MS method [61]. They have obtained valuable information about the presence of 25 compounds including three that were previously reported and three which are a combination of known aglycones and different carbohydrate chains. The saponins mixture revealed a significant cytotoxic effect on the CHO-k1 and Jurkat cell lines, as was found by the MTT test [61].
It can be noted that most of the glycosides isolated from the sponges of the order Poecilosclerida have aglycones with an 8(9)-double bond and one or two oxidized methyl groups at C-4. The carbohydrate chains of these compounds range from one to four monosaccharide residues. Unusual monosaccharides, as well as α-D-arabinopyranose, β-D-arabinopyranose, and α-L-ramnopyranose, were found.
The isolation of a series of holostane triterpene glycosides from the sponge Ianthella basta (family Iantellidae, order Virongiida) collected in Nha Trang Bay (Vietnam) was reported. These glycosides are very characteristic of sea cucumbers of the family Holothuriidae in terms of both aglycone and carbohydrate chain structures. The isolated compounds include known holothurin A 2 , desulfoechinoside A, echinoside B, holothurin A, holothurin B, and a new glycoside named lanthebastanoside A. However, there is some doubt regarding the isolation procedure as it is possible that the sea cucumber extract belonging to the family Holothuriidae was mixed with the sponge extract. The authors did not provide any explanation or clarification regarding this issue [62].

The Order Tetractinellida
Pachastrelloside A (95) is a steroid bioside obtained from a sponge of the species Pachastrella sp. (belonging to the suborder Astrophorina and family Pachastrellidae). Its sterol aglycone contains a 5(6)-double bond and is oxidized at positions C-2, C-3, C-4, and C-7 in rings A and B. The aglycone is also attached to two sugars, namely D-galactopyranose and 4-O-acetyl-β-D-xylopyranose, at positions C-4 and C-7 (as depicted in Figure 12). [63]. This glycoside inhibits cell division of the fertilized eggs of the starfish Asterina pectinifera. However, it does not affect nuclear divisions to form multinucleated, unicellular embryos. Scrobiculosides A (96) and B (97), two new steroid glycosides, have been found in the deep-sea sponge Pachastrella scrobiculosa which is harvested near the coast of Miura Peninsula, Japan ( Figure 12). The aglycones of scrobiculosides A and B possess a saturated nucleus which is oxidated to carboxyl C-18 and have a unique vinylic cyclopropane and exomethylene group at C-24 in the side chains, respectively. These saponins have bioside carbohydrate chain with β-D-galactopyranosyl as the first sugar and the linked to C-2 of the first monosaccharide residue β-D-glucopyranoside, whereas glycoside 96 has an acetyl group on C-6 of the glucose residue. Glycoside 96 revealed cytotoxicity on P388 and HL-60 tumor cell lines (IC50 = 61 and 52 μM, respectively) [64].
A two-sponge symbiotic association was also studied by Korean authors. This asso- Scrobiculosides A (96) and B (97), two new steroid glycosides, have been found in the deep-sea sponge Pachastrella scrobiculosa which is harvested near the coast of Miura Peninsula, Japan ( Figure 12). The aglycones of scrobiculosides A and B possess a saturated nucleus which is oxidated to carboxyl C-18 and have a unique vinylic cyclopropane and exomethylene group at C-24 in the side chains, respectively. These saponins have bioside carbohydrate chain with β-D-galactopyranosyl as the first sugar and the linked to C-2 of the first monosaccharide residue β-D-glucopyranoside, whereas glycoside 96 has an acetyl group on C-6 of the glucose residue. Glycoside 96 revealed cytotoxicity on P388 and HL-60 tumor cell lines (IC 50 = 61 and 52 µM, respectively) [64].
A two-sponge symbiotic association was also studied by Korean authors. This association included Poecillastra wondoensis (family Vulcanellidae) and Jaspis wondoensis (family Ancorinidae (Jaspis wondoensis is the unaccepted name, the valid name is Rhabdastrella wondoensis)). The sponge association was harvested near Cheju Island, Republic of Korea. Wondosterols A-C (98-100), three steroid glycosides, were found in this association. Their structures including absolute stereochemistry were elucidated by NMR spectroscopy and application of Mosher's method ( Figure 13). The structures of 98-100 are similar to 95, although 98-100 contain only one carbohydrate chain linked to C-4 and hydroxyl at C-7 with β-configuration. The side chains of wondosterols A-C differ in the position of the double bond. Their carbohydrate moieties include D-xylose and D-galactose monosaccharide residues [65]. The deep-water sponge Poecillastra compressa (family Vulcaniidae) harvested near French coasts of Mediterranean Sea was studied by a French group of scientists. They isolated seven new steroid glycosides, poecillastrosides A-G (101-107) ( Figure 13) [66]. All the glycosides are characterized by oxidized C-18 methyl by a primary alcohol or carboxylic acid. Poecillastrosides A-D (101-104) contain a methylene or ethylene group at C-24 of the side chain and the carbohydrate chain consists of the first β-D-galactopyranosyl residue and is linked to C-2 β-D-glucopyranosyl residue. Poecillastrosides E-G (105-107) have aglycones with a 22(23)-E-double bond and a 24(25)-cyclopropane ring in the side chain, which is similar to scrobiculoside A (96) [64]. Additionally, the glycosides have two identical monosaccharide residues linked to each other through C-3 of the first sugar. However, the terminal glucose monosaccharide residues are acetylated by C-6 in the glycosides 105 and 107. It was found that poecillastrosides D and E, having a carboxylic acid at C-18, possess antifungal activity against Aspergillus fumigatus (MIC90 = 6 μg/mL and 24 μg/mL, respectively) [66]. The deep-water sponge Poecillastra compressa (family Vulcaniidae) harvested near French coasts of Mediterranean Sea was studied by a French group of scientists. They isolated seven new steroid glycosides, poecillastrosides A-G (101-107) ( Figure 13) [66]. All the glycosides are characterized by oxidized C-18 methyl by a primary alcohol or carboxylic acid. Poecillastrosides A-D (101-104) contain a methylene or ethylene group at C-24 of the side chain and the carbohydrate chain consists of the first β-D-galactopyranosyl residue and is linked to C-2 β-D-glucopyranosyl residue. Poecillastrosides E-G (105-107) have aglycones with a 22(23)-E-double bond and a 24(25)-cyclopropane ring in the side chain, which is similar to scrobiculoside A (96) [64]. Additionally, the glycosides have two identical monosaccharide residues linked to each other through C-3 of the first sugar. However, the terminal glucose monosaccharide residues are acetylated by C-6 in the glycosides 105 and 107. It was found that poecillastrosides D and E, having a carboxylic acid at C-18, possess antifungal activity against Aspergillus fumigatus (MIC 90 = 6 µg/mL and 24 µg/mL, respectively) [66].

The Order Poecilosclerida
The sample of Caribbean sponge Pandaros acanthifolium belonging to the family Microcionidae was harvested from canyon rock near Martinique Island. A series of new steroid glycosides, named pandarosides, were isolated as result of the investigation into this sponge. Pandarosides A-D (108-111) are glycosides with uncommon sterol aglycones, which have an unusual cis-C/D ring junction that is oxidized in ring D [67]. Their carbohydrate chains include the residues of D-glucose and D-glucuronic acids or D-glucuronic acid. The carbohydrate moiety is attached to C-3 of the aglycone (Figure 14). During the isolation procedure, methyl esters 108a, 110a, and 111a of pandarosides A, C, and D, respectively, were obtained. It is likely that these methyl esters are artefacts of a chemical reaction with methanol using as an extractant. The absolute configuration of the aglycone moiety of 108 was determined by comparing the experimental and TDDFT-calculated CD spectra of the more stable conformer. All the isolated glycosides showed the absence of the activity on three human tumor cell lines (MDA-MB-231 breast cancer cells, HT29 colonic cancer cells, and A549 lung cancer cells) below 10 µg/mL [67].
In continuation, the next series of steroidal glycosides, pandarosides E-J (112-117) and their methyl esters 112a-117a, have been isolated from the same sponge [68] (Figure 14). These compounds were tested for in vitro antiprotozoal activity against four parasitic protozoa and cytotoxic activity on L6 cancer cells. Pandaroside G (114) and its methyl ester 114a were inhibitors of the growth of Leishmania donovani (IC 50 of 1.3 and 0.051 µM, respectively) and Trypanosoma brucei rhodesiense (IC 50 of 0.78 and 0.038 µM, respectively) [68].
Finally, pandarosides K-M (118-120) were isolated as minor components from the same species [69] and their methyl esters 118a-120a were also obtained during the isolation procedure probably as artefacts ( Figure 14). All these metabolites and isolated early compounds 114 and 114a were moderate inhibitors of the growth of four parasitic protozoa and did not indicate cytotoxicity on mammalian cells, except for pandaroside G (114) and its methyl ester (114a), which were inactive on T. b. rhodesiense [69].
Thus, all pandarosides have rare aglycones with 14β-H, a 16(17)-double bond, and 3β,16-diol and 15-keto groups. Pandarosides E and F (112, 113) and pandarosides G and L (114, 119) have additional 8(9)-and 7(8)-double bonds, respectively. All pandarosides have a 23-keto group in their side chains. Differences were observed at C-24, which is either free or contains methyl, ethyl, methylene, or ethylene groups. Four types of carbohydrate chains were found in pandarosides, and all contain β-D-glucuronic acid as the first monosaccharide residue. The obtaining methyl esters of pandarosides, most likely, are artifacts formed during the MeOH extraction steps.
Another series of steroid glycosides from the same species are acanthifoliosides A-F (121-126) ( Figure 15). Acanthifoliosides are closed to pandarosides steroid saponins. However, they have common steroid nuclei with a trans-junction of C and D rings. Similar to aglycones of pandarosides, the aglycones of 121-126 possess an oxidized functionality in the D ring. The configurations of C-23 and C-24 centers in the side chains of 124-126 were not established because the low amounts of substances for using Mosher's method. Although some assumptions about relative stereochemistry of the side chains have been made, the authors noted that confirmation of configurations C-23 and C-24 required more thorough stereochemical analysis. reaction with methanol using as an extractant. The absolute configuration of the aglycone moiety of 108 was determined by comparing the experimental and TDDFT-calculated CD spectra of the more stable conformer. All the isolated glycosides showed the absence of the activity on three human tumor cell lines (MDA-MB-231 breast cancer cells, HT29 colonic cancer cells, and A549 lung cancer cells) below 10 μg/mL [67]. In continuation, the next series of steroidal glycosides, pandarosides E-J (112-117) and their methyl esters 112a-117a, have been isolated from the same sponge [68] (Figure 14). These compounds were tested for in vitro antiprotozoal activity against four parasitic protozoa and cytotoxic activity on L6 cancer cells. Pandaroside G (114) and its methyl ester 114a were inhibitors of the growth of Leishmania donovani (IC50 of 1.3 and 0.051 μM, respectively) and Trypanosoma brucei rhodesiense (IC50 of 0.78 and 0.038 μM, respectively) [68].
Finally, pandarosides K-M (118-120) were isolated as minor components from the same species [69] and their methyl esters 118a-120a were also obtained during the isolation procedure probably as artefacts ( Figure 14). All these metabolites and isolated early compounds 114 and 114a were moderate inhibitors of the growth of four parasitic protozoa and did not indicate cytotoxicity on mammalian cells, except for pandaroside G (114) and its methyl ester (114a), which were inactive on T. b. rhodesiense [69].
The next sample of P. acanthifolium was harvested through scuba diving near the coast of Marathon in the Florida Keys (FL, USA) and was investigated by a group of Canadian chemists and biochemists [71]. They have isolated four new minor steroid glycosides, acanthifoliosides G-J (127-130) ( Figure 15). These glycosides also were characterized by a highly oxygenated D ring. All the isolated glycosides possess β-L-glucopyranose as a first sugar with a terminal α-L-rhamnopyranosyl unit linked to C-2 of the first sugar. This bioside carbohydrate chain attached to C-3 of steroid aglycone whereas another carbohydrate moiety composing of α-L-rhamnopyranosyl residue is attached to C-15 aglycone. The absolute configurations of glucose and rhamnose were found by aldose o-tolylthiocarbamate derivatization followed by comparison with reference substances by LC/HRESIMS. The aglycones of 127, 128 are 5α-cholestane derivatives with 16β-Oacetic group. The aglycone of 128 has a 22(23)E-double bond in the side chain. The aglycones of 129 and 130 are 5α-poriferastan and poriferast-5-en derivatives, respectively. The side chains of the both of aglycones possess a hydroxyl at C-23 and ethyl group at a 24-position, as well as in acanthifoliosides D-F [70]. The hydroxyl configuration was not found because of too low amounts of the substances. The glycosides were tested on antioxidant and cell-protective activities, but only acanthifolioside G (127) exhibited antioxidant and cytoprotective activities [71]. either free or contains methyl, ethyl, methylene, or ethylene groups. Four types of carbohydrate chains were found in pandarosides, and all contain β-D-glucuronic acid as the first monosaccharide residue. The obtaining methyl esters of pandarosides, most likely, are artifacts formed during the MeOH extraction steps.
Another series of steroid glycosides from the same species are acanthifoliosides A-F (121-126) ( Figure 15). Acanthifoliosides are closed to pandarosides steroid saponins. However, they have common steroid nuclei with a trans-junction of C and D rings. Similar to aglycones of pandarosides, the aglycones of 121-126 possess an oxidized functionality in the D ring. The configurations of C-23 and C-24 centers in the side chains of 124-126 were not established because the low amounts of substances for using Mosher's method. Although some assumptions about relative stereochemistry of the side chains have been made, the authors noted that confirmation of configurations C-23 and C-24 required more thorough stereochemical analysis.  (Figure 15). The methyl ester of acanthifolioside F (126a) was also isolated and probably formed during the extraction process as previously observed for pandarosides [67][68][69]. Moderate antiprotozoal activity has been reported for acanthifoliosides A-F (121-126) and the methyl ester of acanthifolioside F (126a) [70]. There is a definite structural closeness of P. acanthifolium glycosides and starfish steroid glycosides, because of oxidation of D-ring in C-15 and C-16 positions [16][17][18][19]. The steroid oligoglycosides, similar to starfish asterosaponins by containing several monosaccharide residues, were first found in a sponge of the genera Mycale (the family Mycalidae) by Russian scientists in 1981 [72]. Unfortunately, the separation of the corresponded glycosidic fractions was impossible because of a low level of development of isolation technologies. Later, steroid oligoglycoside mycaloside A (131) was isolated from the Caribbean sponge Mycale laxissima as individual substance [73]. Then, the structures of related mycalosides B-K (132-141) were reported [74,75] (Figure 16). All the isolated glycosides were tetraosides with the similar carbohydrate parts architecture. They include two D-galactopyranose, one D-glucopyranose, and one D-arabinopyranose residues. In mycalosides D, F, and K (134, 136, 141) the glucopyranose residue has acetate group at C-6. One galactopyranosyl moiety attached to C-4 of glucopyranosyl residue by a β-glycosidic bond, another galactopyranosyl unit attached to C-2 of arabinopyranose by a α-glycosidic bond. Their aglycones are steroid derivatives, as they have oxidized functionalities in rings A, D, and side chains. All mycalosides, with the exception of mycaloside I (139), have an unusual oxidation at C-21, which is characteristic of steroids from ophiuroids [16,17].  (Figure 16). The total fraction of the mycalosides and individual mycalosides A (131) as well as micaloside G (137) have an inhibition action on the fertilization of eggs by sperm of the sea urchin Strongylocentrotus nudus after preincubation [74].

The Order Haplosclerida
The sponge Niphates olemda (formerly known as Cribrochalina olemda), (family Niphatidae) is characteristic for the Indo-Pacific region and was collected at a depth of 40 m in Micronesia. The found in this sponge hapaioside (142) has an uncommon aglycone with a polycyclic nucleus, resembling several steroid hormones. It has a 4-hydroxy-6-oxo-19norpregnane skeleton. The glycoside is a monoside and its monosaccharide residue is 6deoxy-β-L-altropyranose-4-acetate [76] (Figure 17). This sugar was earlier found as a constituent of a lipopolysaccharide isolated from mammalian intestinal microorganisms.

The Order Haplosclerida
The sponge Niphates olemda (formerly known as Cribrochalina olemda), (family Niphatidae) is characteristic for the Indo-Pacific region and was collected at a depth of 40 m in Micronesia. The found in this sponge hapaioside (142) has an uncommon aglycone with a polycyclic nucleus, resembling several steroid hormones. It has a 4-hydroxy-6-oxo-19norpregnane skeleton. The glycoside is a monoside and its monosaccharide residue is 6-deoxy-β-L-altropyranose-4-acetate [76] (Figure 17). This sugar was earlier found as a constituent of a lipopolysaccharide isolated from mammalian intestinal microorganisms.
The sponge Niphates olemda (formerly known as Cribrochalina olemda), (famil phatidae) is characteristic for the Indo-Pacific region and was collected at a depth of in Micronesia. The found in this sponge hapaioside (142) has an uncommon aglycone a polycyclic nucleus, resembling several steroid hormones. It has a 4-hydroxy-6-ox norpregnane skeleton. The glycoside is a monoside and its monosaccharide residue deoxy-β-L-altropyranose-4-acetate [76] (Figure 17). This sugar was earlier found as a stituent of a lipopolysaccharide isolated from mammalian intestinal microorganism

The Order Axinellida
Ptilocaulis spiculifer is a sponge belonging to the family Axinellidae and is found in the tropical waters of the Indo-Pacific region. This sponge is known to contain sulfated polyhydroxysteroids. During the isolation procedure of this sponge specimen, two pregnane glycosides, ptilosaponosides A (143) and B (144), as well as sulfated polyhydroxysteroids were also obtained. The specimen was collected from shallow waters in the Solomon Islands. (Figure 17). The glycosides contain β-D-glucopyranosyl-3-O-sulfate residue as a carbohydrate chain and their rings A are oxidized at C-3, C-4, and C-19. The glycoside 143 also has an additional sulfate in the aglycone. The absolute configuration of glucose was not assigned due to the small amount of isolated compounds. Glycosides 143 and 144 have no cytotoxic action on human tumor KB cells [77].
Thus, steroid glycosides of marine sponge are very interesting class of marine natural products. Some of them also reveal significant antiprotozoal activity and other kinds of biological activity.

Taxonomic Distribution of Glycosides in Sponges
Frequently, the presence of occurring triterpene and steroid glycosides in sponges can result in highly complex mixtures, making their separation a challenging task. Consequently, the difficulty in isolating these glycosides may hinder their utilization as chemotaxonomic markers. Due to the complexity of glycoside fractions, many researchers may only isolate a portion of the components at random, whereas another group may isolate a different portion. Comparing such data may lead to inaccuracies. Furthermore, distinct structural series of triterpene and steroid glycosides are distributed among taxa that are different from each other and widely separated. The most frequently the tetracyclic triterpene and steroid glycosides are presented in two orders-Tetractinellida (suborder Astrophorina) and Poecilosclerida. In total, the glycosides were found in the sponges belonging to five orders (Tables 1 and 2, the taxonomic names corresponded to WoRMS-World Register of Marine Species data base [78]). The distribution of glycosides occurs across multiple families and species and is not strictly dependent on ecological or geographical factors. The major glycoside in Melophlus sarasinorum is sarasinoside A 1 (1), which has been harvested in various geographical locations ranging from Guam to the north-western coast of Australia. Sarasinoside A 3 (3) has been isolated from M. sarasinorum harvested in shallow waters near the north-western coast of Australia, Guam Island, Palau Islands, and Sulawesi Island. Similarly, sarasinoside A 2 (2) was found in specimens harvested from shallow waters near the north-western coast of Australia, Palau Island, and other regions. A very similar character of distribution was found for erylosides of Erylus formosus, from which formoside (47) and eryloside F (49) were isolated from the samples harvested near Puerto Morelos and Bahamas in the Gulf of Mexico. Discoveries of glycosides with identical structures in different species, even those within the same genus, are uncommon. However, an instance of such a finding is eryloside H (67), which has been identified in Erylus formosus harvested near Puerto Morelos and in samples of E. nobilis from the waters of Jaeju Island, Republic of Korea. Nevertheless, some sponge genera may be characterized by the occurrence of closely related glycosides. This may be exemplified by erylosides found in different species of Erylus, which was collected in the shallow waters of three oceans (Pacific, Atlantic and Indian). These glycosides contain 14-carboxy-or 14-nor-methyl-lanostane aglycone. Some of them are additionally dealkylated at C-4 and alkylated in sidechains. General carbohydrate chains architectures for a series of glycosides isolated from Erylus spp. are frequently close, but, as a rule, exact structures are a little different. As we already have noted, a Korean group recently reported on the finding of a series of sarasinosides from the sponge Lipastrotethya sp. belonging to the order Bubarida, the family Dictyonellidae, which was harvested in Micronesian shallow waters [44]. Another representative of this order, Dictyonella marsilii contains eryloside W (79) very similar to other glycosides of eryloside series [46]. A series of sarasinosides was also isolated from Petrosia sp. and Petrosia nigricans, the representative of the order Haplosclerida [47,48]. Very similar steroid glycosides containing unique aglycone side chains having a 23(23)-Edouble bond along with a 24(26)-cyclopropanic ring were isolated from representatives of two different families of the order Tetractinellida (suborder Astrophorina)-Pachastrella scrobiculosa (family Pachastrellidae) and Poecillastra compressa (family Vulcanellidae), which were harvested near Miura Peninsula, Japan [64] and the French coasts of Mediterranean Sea [66], respectively.
The discovery of similar occurrences suggests a parallel origin and evolution of terpenoid and steroid glycosides in Demospongiae.
Therefore, triterpene and steroid glycosides can be regarded as taxonomic markers that are specific to certain species and occasionally even to the level of genus or subgenus. However, the use of them for improvement of sponge taxonomy may be difficult because of the presence of very complicated mixtures of close related substances in glycosidic fractions, and seasonal and ecological variability in the content of some components of glycosidic fractions in the different collection of the same sponge. This was described, for example, by Kubanek et al. [32], who found the presence of formoside in Erylus formosus harvested near Bahamas and its absence in the specimens collected in Floridian waters.

Biological Roles of Sponge Triterpene and Steroid Glycosides
Sponges are very important parts of coral reef ecological systems [79]. Kubanek et al. [80] discovered that triterpene glycosides from Erylus formosus harvested in shallow waters near Bahamas and Southern Florida deters fish-predators Thalassoma bifasciatum. Formoside (47) and formoside B (48), which have four monosaccharide residues in their carbohydrate chains, were found to be less active compared to the non-separated hexaoside fraction, which contains penasterol as the aglycone. The total glycoside sum indicated more activity than any separate subfraction or individual glycoside. Thus, triterpene glycosides of sponges may be a defensive agent against fish-predators [81].
Ecological activities of sponge triterpene glycosides may be moderated even by minor structural differences. Some triterpene glycosides are not excreted in sea water, but their concentration on surface of sponge specimens was high. However, these sponges may deter their enemies and competitors by surface direct contacts with triterpene glycosides. This is more probable than by contacts via sea water [80]. The discovery of the molecular mechanisms of predator deterrence by triterpene glycosides in sponges also suggests their role as signaling substances, indicating their release into the surrounding seawater. It was discovered that zebrafish Danio rerio rejected an artificial diet containing the sponge triterpene glycosides. Transcripts from a zebra fish cDNA library were expressed in oocytes of Xenopus laevis and checked for activation of a chemoreceptor via electrophysiology and electrophysiological responses of ectyoplasides A, B (86, 87), and formoside (47) were revealed [84]. Later, a new RAMP-like triterpene glycoside receptor (RL-TGR) was discovered in predatory fish [84,85]. It was involved in chemical signaling because this receptor responds to triterpene glycosides as chemical defensive substances in the water environment [85]. Hence, sponge triterpene glycosides may have antifouling and allelopathic roles.
Biological activities of sponge steroid glycosides are similar to such of terpenoid ones (see Tables 1 and 2). For example, many steroid glycosides have been found to possess cytotoxic activity, suggesting their potential role as defensive agents against predators, as well as antifouling and allelopathic agents. However, the biological role of sponge steroid glycosides has not been specially studied.

Conclusions
About 144 sponge saponins were isolated by the end of 2022, including 94 tetracyclic triterpenoid glycosides and 50 steroid ones. The presence of triterpene and steroid glycosides is a characteristic feature of a few taxonomic groups (up to date, they were indicated in about 24 species, namely 15 species contain triterpene glycosides and 9 steroid ones). All of them were found within the class Demospongiae, but they are not characteristic for the class as a whole. The fact that terpenoid and steroid glycosides are distributed in a mosaic pattern within the class Demospongiae, and that glycosides with similar structures can be found in representatives of different orders, along with the data on their biological activities and roles, suggests that these substances may have evolved independently as defensive agents in several taxonomic groups of Demospongiae, and may have undergone parallel evolution.
These glycosides are very diverse from a chemical point of view and quite different from the structures of the both aglycone and carbohydrate chains. They are more structurally diverse than other saponins from marine invertebrates or terrestrial plants. The structures of both steroid and triterpene aglycones are the result of intensive oxidative processes that allow high activity of the corresponding cytochrome P-450 family of enzymes in the sponges belonging to saponin-producing taxa. These glycosides may be not only mono-and biosides, but also oligoglycosides. All the monosaccharides are in pyranose form and most of them belong to D-series; however, L-sugars also may be found. The monosaccharides may be acetylated by C-6 or may be in uronic acid form (glucuronic and galacturonic acids); however, they do not contain any OMe groups. Sponge triterpene glycosides demonstrate deterrent and ichthyotoxic properties, and many of them and their steroid analogs have cytotoxic and antiprotozoal activities that may be a cause of their defensive, antifouling, and allelopathic biological functions. It seems probable that new marine expeditionary research and the progress in development of separation and spectral technique may allow for the discovery of new, interesting structural versions of triterpene and steroid glycosides in sponges.