Nine New Triterpene Glycosides, Magnumosides A1–A4, B1, B2, C1, C2 and C4, from the Vietnamese Sea Cucumber Neothyonidium (=Massinium) magnum: Structures and Activities against Tumor Cells Independently and in Synergy with Radioactive Irradiation

Nine new sulfated triterpene glycosides, magnumosides A1 (1), A2 (2), A3 (3), A4 (4), B1 (5), B2 (6), C1 (7), C2 (8) and C4 (9) as well as a known colochiroside B2 (10) have been isolated from the tropical Indo-West Pacific sea cucumber Neothynidium (=Massinium) magnum (Phyllophoridae, Dendrochirotida) collected in the Vietnamese shallow waters. The structures of new glycosides were elucidated by 2D NMR spectroscopy and mass-spectrometry. All the isolated new glycosides were characterized by the non-holostane type lanostane aglycones having 18(16)-lactone and 7(8)-double bond and differed from each other by the side chains and carbohydrate moieties structures. Magnumoside A1 (1) has unprecedented 20(24)-epoxy-group in the aglycone side chain. Magnumosides of the group A (1–4) contained disaccharide monosulfated carbohydrate moieties, of the group B (5, 6)—tetrasaccharide monosulfated carbohydrate moieties and, finally, of the group C (7–9)—tetrasaccharide disulfated carbohydrate moieties. The cytotoxic activities of the compounds 1–9 against mouse spleen lymphocytes, the ascites form of mouse Ehrlich carcinoma cells, human colorectal carcinoma DLD-1 cells as well as their hemolytic effects have been studied. Interestingly, the erythrocytes were more sensitive to the glycosides action than spleenocytes and cancer cells tested. The compounds 3 and 7 significantly inhibited the colony formation and decreased the size of colonies of DLD-1 cancer cells at non-cytotoxic concentrations. Moreover, the synergism of effects of radioactive irradiation and compounds 3 and 7–9 at subtoxic doses on proliferation of DLD-1 cells was demonstrated.

The glycosides from the sea cucumber Neothyonidium (=Massinium) magnum (Phyllophoridae, Dendrochirotida) have been previously investigated. The first studied sample of N. magnum was collected near the shores of New Caledonia [15]. The main component of glycosidic fraction, monosulfated tetraoside, "neothyonidioside", had the holostane-type aglycone with 9(11)-and 25 (26)-double bonds and a 16-keto-group. Another sample of N. magnum was collected near Vietnam's shore [16]. The main component of its glycosidic fraction, neothyonidioside C, was different from "neothyonidioside" and characterized by the C-16-acetylated holostane-type aglycone having 7(8)and 25(26)-double bonds. The carbohydrate chain of neothyonidioside C had the same set of monosaccharide residues as "neothyonidioside" but was disulfated.
Herein we report the results of investigation of N. magnum also collected in Vietnamese shallow waters but having the glycosides, magnumosides A 1 -A 4 (1-4), B 1 (5), B 2 (6), C 1 (7), C 2 (8) and C 4 (9), which is significantly different from the compounds isolated previously. The structures of the glycosides were established based on 1 H and 13 C NMR spectra and 2D NMR ( 1 H, 1 H-COSY, HMBC, HSQC, ROESY) and confirmed by HR-ESI mass spectrometry. The cytotoxic activities of 1-9 against mouse spleen lymphocytes, the ascites form of mouse Ehrlich carcinoma cells, mouse erythrocytes, and human colorectal adenocarcinoma DLD-1 cells were tested. The effects of compounds 1-9 on proliferation, colony formation of DLD-1 cells as well as the synergism of radioactive irradiation and compounds effects have been studied.
The molecular formula of magnumoside A1 (1) was determined to be C41H63O16SNa from the [MNa − Na] − (18) in the HMBC spectrum ( Figure  2) and H(16)/H (21) in the ROESY spectrum ( Figure 3). The signals of olefinic methine group H-C(7) (δ(C) 122.4; δ(H) 5.61 (dt, J = 2.3, 7.4 Hz)) and quaternary carbon C(8) (δ(C) 147.6) in the 13 C-and 1 H NMR spectra were indicative of 7(8)-double bond in the aglycone nucleus. The signal of oxygenated tertiary asymmetric carbon C (20) assigned by the characteristic for the aglycones of sea cucumber glycosides HMBC correlation H(21)/C (20) was observed at δ(C) 81.9. Its δ(C) value was similar to those values in onekotanogenin [21] and in colochiroside E [22] having the 18(16)-lactone and acetylated C (20) position. However, the presence of acetoxy-group in the aglycone of 1 was excluded by ESI mass spectrometry. The 1 H and 13 C NMR spectra of carbohydrate parts of magnumosides A 1 -A 4 (1-4) were coincident to each other, indicating the identity of carbohydrate chains of these glycosides. The presence of two characteristic doublets at δ(H) 4.66 (J = 7.0 Hz) and 5.00 (J = 7.6 Hz) in the 1 H NMR spectra of the carbohydrate chains of 1-4 correlated by the HSQC spectra with the signals of anomeric carbons at δ(C) 104.8 and 105.2, correspondingly, were indicative of a disaccharide chain and β-configuration of glycosidic bonds. The 1 H, 1 H-COSY and 1D TOCSY spectra of 1-4 showed the signals of two isolated spin systems assigned to the xylose and quinovose residues. The positions of interglycosidic linkages were confirmed by the ROESY and HMBC spectra of 1-4 (SM , Table 1) where the cross-peaks between H(1) of the xylose and H(3) (C(3)) of an aglycone and H(1) of the quinovose and H(2) (C(2)) of the xylose were observed. Thus, the carbohydrate chains of magnumosides of the group A (1-4) were identical to those of holothurins of the group B, that are characteristic glycosides for the representatives of the genus Holothuria and Actinopyga (Holothuriidae, Aspidochirotida) [1,[18][19][20].
The molecular formula of magnumoside A 1 (1) was determined to be C 41 H 63 O 16 SNa from the [M Na − Na] − ion peak at m/z 843.3838 (calc. 843.3842) in the (−)HR-ESI-MS. Analysis of the 1 H and 13 C NMR spectra (Tables 2 and 3) of the aglycone part of magnumoside A 1 (1) suggested the presence of an 18(16)-lactone that was deduced from the characteristic signals of carbons C(18) (δ(C) 180.9) and C(20) (δ(C) 81.9) and characteristic signals of oxygen-bearing methine CH-O(16) (δ(C) 79.6; δ(H) 4.84 (s)). The availability of an 18(16)-lactone and (S)-configuration of C(16) asymmetric center was confirmed by the absence of coupling constant J 17/16 for H(17) signal (δ(H) 2.48 (s)) in the 1 H NMR spectrum of 1 as well as by the presence of correlations H(16)/C (18) in the HMBC spectrum ( Figure 2) and H(16)/H (21) in the ROESY spectrum ( Figure 3). The signals of olefinic methine group H-C(7) (δ(C) 122.4; δ(H) 5.61 (dt, J = 2.3, 7.4 Hz)) and quaternary carbon C(8) (δ(C) 147.6) in the 13 C-and 1 H NMR spectra were indicative of 7(8)-double bond in the aglycone nucleus. The signal of oxygenated tertiary asymmetric carbon C(20) assigned by the characteristic for the aglycones of sea cucumber glycosides HMBC correlation H(21)/C (20) was observed at δ(C) 81.9. Its δ(C) value was similar to those values in onekotanogenin [21] and in colochiroside E [22] having the 18(16)-lactone and acetylated C(20) position. However, the presence of acetoxy-group in the aglycone of 1 was excluded by ESI mass spectrometry. Additionally, there was another downshifted resonance in the 13 C NMR spectrum of 1 at δ(C) 86.7 corresponding to the oxygen bearing methine carbon located at α-position to hydroxylated C-25. Its position as C(24) was deduced from the 1 H, 1 H-COSY spectrum where the signals of isolated spin system from H (22) to H(24) were observed ( Figure 2). Based on the NMR and ESI-MS data the presence of 20(24)-epoxy-group was suggested. The correlations from H-C(24) (δ(H) 3.97 (dd, J = 5.6; 9.3) to C (22), C(23), Me (26), Me (27); from H-C (22) to C (20), Me (21) and from Me(26) (27) to C (24) in the HMBC spectrum were in good agreement with this suggestion. Another downshifted signal (δ(C) 70.1) was assigned to the oxygen-bearing tertiary carbon positioned as C(25) that was deduced from the HMBC correlations Me(26)/C (25) and Me(27)/C (25). The multiplicity of the proton H(24), neighboring it, which was observed as doublet of doublets, confirmed this. The presence in the 1 H-1 H-COSY spectrum of the signals of four isolated spin systems confirmed the sequences of protons from H(1) to H(3); from H(5) to H (12); from H (15) to H(17) and finally from H (22) to H (24). The HMBC correlations Me(30)/C(4), Me(31)/C(4), Me(19)/C(10), Me(32)/C(13), C (14) and H(17)/C(20) allowed to elucidate the positions of quaternary carbons in the aglycone polycyclic nucleus.   The ROESY correlations (        Based on these results, the structure of magnumoside A1 (1) was determined as 3β The molecular formula of magnumoside A2 (2) was determined to be C41H63O16SNa from the [MNa − Na] − molecular ion peak at m/z 843.3838 (calc. 843.3842) in the (−)HR-ESI-MS. Extensive analysis of the 1 H-, 13 C- (Tables 2 and 3) and 2D NMR spectra of the aglycone part of magnumoside A2 (2) showed similarity of its polycyclic system to that of 1. Indeed, the signals at δ(C) 182.6 (C(18)), 80.1 (C(16)), 62.2 (C(17)) as well as at δ(H) 5.12 (brs, H(16)) and 2.72 (s, H(17)) confirmed the presence of an 18(16)-lactone moiety, the signals at δ(C) 122.3 (C(7)) and 147.6 (C(8)) with corresponding proton signal at δ(H) 5.55-5.57 (m, H(7)) demonstrated the presence of 7(8)-double bond. However the signal of C(20) was high-shifted to δ(C) 71.3 when compared with that of 1 and was close to those of progenins obtained from cladoloside C by its alkaline treatment and having the identical to each other aglycone nuclei with hydroxylated C(20) position and non-shortened side chains [25]. The signals corresponding to the side chain of 2 formed the isolated spin system in the 1 H, 1 H-COSY spectrum ( Figure 2) from H (22) to H(24), indicating the latter signal was down-shifted to δ(H) 4.33 (brt, J = 6.0 Hz, H(24)) due to hydroxylation of this position and the multiplicity of the signal showed its vicinity to quaternary carbon C (25). The characteristic signals at δ(C) 148.4 (C(25)) and 110.6 (C(26)) with corresponding signals of olefinic protons at δ(H) 5.16 and 4.87 (each brs, H2 (26)    Based on these results, the structure of magnumoside A 1 (1) was determined as The molecular formula of magnumoside A 2 (2) was determined to be C 41 H 63 O 16 SNa from the [M Na − Na] − molecular ion peak at m/z 843.3838 (calc. 843.3842) in the (−)HR-ESI-MS. Extensive analysis of the 1 H-, 13 C- (Tables 2 and 3) and 2D NMR spectra of the aglycone part of magnumoside A 2 (2) showed similarity of its polycyclic system to that of 1. Indeed, the signals at δ(C) 182.6 (C(18)), 80.1 (C(16)), 62.2 (C(17)) as well as at δ(H) 5.12 (brs, H(16)) and 2.72 (s, H(17)) confirmed the presence of an 18(16)-lactone moiety, the signals at δ(C) 122.3 (C(7)) and 147.6 (C(8)) with corresponding proton signal at δ(H) 5.55-5.57 (m, H (7)) demonstrated the presence of 7(8)-double bond. However the signal of C(20) was high-shifted to δ(C) 71.3 when compared with that of 1 and was close to those of progenins obtained from cladoloside C by its alkaline treatment and having the identical to each other aglycone nuclei with hydroxylated C(20) position and non-shortened side chains [25]. The signals corresponding to the side chain of 2 formed the isolated spin system in the 1 H, 1 H-COSY spectrum ( Figure 2) from H (22) to H(24), indicating the latter signal was down-shifted to δ(H) 4.33 (brt, J = 6.0 Hz, H(24)) due to hydroxylation of this position and the multiplicity of the signal showed its vicinity to quaternary carbon C (25). The characteristic signals at δ(C) 148.4 (C(25)) and 110.6 (C (26) 3)) of the third (xylose) residue were observed. The signals of C(4) and C(5) of the first (xylose) residue were observed at δ(C) 76.0 and 63.9, correspondingly, indicating the presence of a sulfate group at C(4) of the sugar unit, analogically to the carbohydrate chains of magnumosides of the group A (1-4). The linear tetrasaccharide monosulfated carbohydrate chain of magnumosides of the group B (5, 6), having the xylose as third monosaccharide unit, were found earlier in "neothyonidioside" [15] isolated from another collection of N. magnum, also coincided with the sugar moiety of colochiroside B 2 (9) [17] found in the investigated sample of N. magnum and are widely distributed in the triterpene glycosides of sea cucumbers of the orders Aspidochirotida [2] and Dendrochirotida [2,8,[26][27][28].
The molecular formula of magnumoside B 1 (5)  The 1 H and 13 C NMR spectra of carbohydrate parts of magnumosides C 1 (7), C 2 (8) and C 4 (9) were coincident to each other and to those of neothyonidioside C, isolated earlier from this species [16], that indicated the identity of carbohydrate chains of these glycosides. The presence of four characteristic doublets at δ(H) 4.65-5.12 (J = 7.0-7.9 Hz) in the 1 H NMR spectra of the carbohydrate chains of 7-9 correlated by the HSQC spectra with the signals of anomeric carbons at δ(C) 104.3-104.8 were indicative of a tetrasaccharide chain and β-configuration of glycosidic bonds. The positions of interglycosidic linkages were elucidated based on the ROESY and HMBC spectra (SM , Table 1), where the cross-peaks analogical to 5, and 6 were observed. The differences between the 13 C NMR spectra of magnumosides of the groups C (7-9) and B (5, 6) were in the signals of C(5) and C(6) of terminal monosaccharide residue that were observed in the 13 C NMR spectra of 7-9 at δ(C) 75.5 and 67.1, correspondingly, due to the shifting effects of a sulfate group attached to C(6) of 3-O-methylglucose unit. Actually, magnumosides of the group C (7-8) are characterized by the linear tetrasaccharide disulfated carbohydrate chain, which have been found earlier in the glycosides of Mensamaria intercedens [29], Pseudocolochirus violaceus [30] and Colochirus robustus [17], representatives of the family Cucumariidae, order Dendrochirotida.
The molecular formula of magnumoside C 1 (7) was determined to be C 53 H 82 O 28 S 2 Na 2 from the [M 2Na − Na] − molecular ion peak at m/z 1253.4328 (calc. 1253.4337) in the (−)HR-ESI-MS. The aglycones of magnumoside C 1 (7) and B 1 (5) were identical to each other due to the coincidence of their 13 C NMR spectra (Tables 2 and 3  The molecular formula of magnumoside C 2 (8) coincided with that of 7 (C 53 H 82 O 28 S 2 Na 2 ) that was deduced from the [M 2Na − Na] − molecular ion peak at m/z 1253.4341 (calc. 1253.4337) in the (−)HR-ESI-MS. The aglycone of magnumoside C 2 (8) was identical to those of the magnumosides A 2 (2) and B 2 (6) that was suggested based on the coincidence of their 13 C NMR spectra (Tables 2 and 3). So, magnumoside C 2 (8) is additionally sulfated, by C(6) of 3-O-methylglucose residue, analog of magnumoside B 2 (6). The (−)ESI-MS/MS of 8 corroborated its isomerism to the magnumoside C 1 (7) since their fragmentation patterns were the same and the ion-peaks were characterized by the identical m/z values.
The molecular formula of magnumoside C 4 (9) was determined to be C
These aglycones are strongly differing from the holostane-type aglycones found in isolated earlier glycosides, "neothyonidioside" and neothyonidioside C [15,16], from New-Caledonian and Vietnamese collections of N. magnum, correspondingly. In contrast, the aglycone of colochiroside B 2 (10) found in the investigated sample of N. magnum is biogenetically very close to that of neothyonidioside C which also has the holostane-type aglycone with 16-acetoxygroup and differs in the side chain structure. Furthermore, the monosulfated tetrasaccharide carbohydrate chain of 10 was identical to those of "neothyonidioside" and magnumosides of the group B (5, 6). The disulfated tetrasaccharide chain of magnumosides of the group C (7-9) was identical to that of neothyonidioside C. The finding of compound 10 in recent Vietnamese collection of N. magnum structurally close to found earlier glycosides from this species confirms the correctness of biological identification of the all studied samples. The predominance of non-holostane-type glycosides and the fact that neither "neothyonidioside" nor neothyonidioside C have been found in the studying sample of the animal could be explained by the changes in the quantitative and qualitative composition of different components of glycosidic fraction in the samples of one species collected in different places and seasons. The representative example of such changes demonstrated the components of glycosidic fraction of Psolus fabricii. Psolusoside A, a holostane glycoside was predominant in P. fabricii collected near the shore of the Onekotan Island of Kuril Ridge and non-holostane psolusoside B having a 18(16)-lactone was the minor one [35]. The opposite situation was observed in samples of P. fabricii collected in the Kraternaya Bay of Ushishir Islands of the Kuril Archipelago, where psolusoside A was found only in trace amount and psolusoside B was the predominant component [36].
The sets of magnumosides A 2 →B 2 →C 2 and B 1 →C 1 , in which the glycosides were characterized by the same aglycones and different carbohydrate chains within the set, demonstrated the biosynthetic transformations of the carbohydrate chains resulting in their elongation and additional sulfation. The biosynthetic modifications of the aglycones are mainly concerned with side chains transformations, when the double bonds and hydroxyls are introduced in different positions. The aglycone of magnumosides B 1 (5) and C 1 (7) with 23(24)-double bond and 20,25-hydroxyls is obviously could be the precursor of the unusual aglycone of magnumoside A 1 (1) with 20(24)-epoxy-25-hydroxy-fragment. The similar conversion of the aglycone having a linear side chain with a double bond and hydroxyl group to the aglycone having epoxy-group was observed in the process of obtaining the genins with 18(16)-lactone moieties by chemical transformations of the holostane aglycone of cladoloside C [25]. It was considered as a biomimetic reaction, modeling the biosynthetic process in the glycoside aglycones. The aglycone structure of colochiroside B 2 (10) is biogenetically related with the aglycones of 1, 5 and 7. It is known that the formation of 18(16)-lactone biosynthetically occurs in C(18)-carboxylated derivatives having both hydroxylated C (16) and C (20) positions. However, when the further oxidation (acetylation or carboxylation) of C(16) precedes the carboxylation of C(18) the holostane-type aglycones with functionality at C(16) are biosynthesized [37] (Figure 5). In the case of 10, this process took place. Interestingly, the oxidative transformations of the side chain in biosynthesis of 10 may precede the lactonization.

Hemolytic and Cytotoxic Activities of the Glycosides 1-9 against Mouse Spleenocites and the Ascites Form of Mouse Ehrlich Carcinoma Cells
The cytotoxic action of the compounds 1-9 against mouse spleenocytes and the ascites form of mouse Ehrlich carcinoma cells as well as hemolytic action against mouse erythrocytes have been studied (Table 4). Magnumoside A1 (1) was the only glycoside inactive in all tests that was caused by unusual aglycone structure having 18(16)-lactone in combination with 20(24)-epoxy-25-hydroxyfragment. Magnumosides A2 (2), B1 (5) and B2 (6) demonstrated moderate hemolytic activity and were non-cytotoxic up to the ultimate investigated concentration of 100 μM, that was explained by the presence of hydroxyls in their side chains [17,32,33]. However, magnumosides C1 (7) and C2 (8) that had aglycones identical to those of magnumosides B1 (5) and B2 (6) and differed from the latter compounds by the presence of the additional sulfate group were the exclusions from this relation and demonstrated significant effects in all tests. Moreover, magnumosides C1 (7) and C4 (9) turned out to be the most active compounds in this series. So, the activity-decreasing influence of hydroxyl-group in the side chain of 7 and 8 was counterbalanced by the additional sulfate group attached to C-6 of terminal monosaccharide residue of these compounds. On the whole, the erythrocytes were more sensitive to the glycosides action than the spleenocytes and cancer cells studied. Table 4. Hemolytic activity of the glycosides 1-9 against mouse erythrocytes and cytotoxic activity against mouse spleenocytes and the ascites form of mouse Ehrlich carcinoma cells.

Hemolytic and Cytotoxic Activities of the Glycosides 1-9 against Mouse Spleenocites and the Ascites Form of Mouse Ehrlich Carcinoma Cells
The cytotoxic action of the compounds 1-9 against mouse spleenocytes and the ascites form of mouse Ehrlich carcinoma cells as well as hemolytic action against mouse erythrocytes have been studied (Table 4). Magnumoside A 1 (1) was the only glycoside inactive in all tests that was caused by unusual aglycone structure having 18(16)-lactone in combination with 20(24)-epoxy-25-hydroxy-fragment. Magnumosides A 2 (2), B 1 (5) and B 2 (6) demonstrated moderate hemolytic activity and were non-cytotoxic up to the ultimate investigated concentration of 100 µM, that was explained by the presence of hydroxyls in their side chains [17,32,33]. However, magnumosides C 1 (7) and C 2 (8) that had aglycones identical to those of magnumosides B 1 (5) and B 2 (6) and differed from the latter compounds by the presence of the additional sulfate group were the exclusions from this relation and demonstrated significant effects in all tests. Moreover, magnumosides C 1 (7) and C 4 (9) turned out to be the most active compounds in this series. So, the activity-decreasing influence of hydroxyl-group in the side chain of 7 and 8 was counterbalanced by the additional sulfate group attached to C-6 of terminal monosaccharide residue of these compounds. On the whole, the erythrocytes were more sensitive to the glycosides action than the spleenocytes and cancer cells studied. Table 4. Hemolytic activity of the glycosides 1-9 against mouse erythrocytes and cytotoxic activity against mouse spleenocytes and the ascites form of mouse Ehrlich carcinoma cells.

The Effect of the Glycosides on Cell Viability of Human Colorectal Adenocarcinoma DLD-1 Cells
To determine cytotoxic effect of the compounds against human colorectal adenocarcinoma, DLD-1 cells were treated with various concentrations of 1-9 (0-100 µM) for 24 h and then cell viability was assessed by the MTS assay. It was showed that 1, 2, 4-6 did not possessed cytotoxic effect against DLD-1 cells that is in good correlation with the data on Echrlich carcinoma cells. The magnumoside A 3 (3), magnumoside C 1 (7), magnumoside C 2 (8) and magnumoside C 4 (9) decreased cell viability with IC 50 value of 30.3, 34.3, 32.9, 37.1, and 33.9 µM, respectively (Table 5). Thus, for further experiments we chose the concentrations of investigated compounds lower than IC 50 , at which no significant cytotoxic effect on DLD-1 cells was observed.  (4) >100 magnumoside C 4 (9) 33.9 magnumoside B 1 (5) >100 --* IC 50 is a concentration of substance caused 50% reduction in cell viability.

The Effect of the Glycosides on Formation and Growth of Colonies of Human Colorectal Adenocarcinoma DLD-1 Cells
The effect of 3, 7-9 on the colony formation of DLD-1 cells using soft agar assay has been studied. The magnumoside A 3 (3) and magnumoside C 1 (7) inhibited spontaneous colony formation by 22% and 26%, respectively, ( Figure 6A) and, at the same time, they reduced colonies size of DLD-1 cancer cells by 49% and 43%, respectively, ( Figure 6B). On the other hand, magnumosides C 2 (8) and C 4 (9) possessed slight inhibitory activity in this experiment (the percentages of inhibition were less than 20%) ( Figure 6A,B).

The Effect of the Glycosides on Cell Viability of Human Colorectal Adenocarcinoma DLD-1 Cells
To determine cytotoxic effect of the compounds against human colorectal adenocarcinoma, DLD-1 cells were treated with various concentrations of 1-9 (0-100 μM) for 24 h and then cell viability was assessed by the MTS assay. It was showed that 1, 2, 4-6 did not possessed cytotoxic effect against DLD-1 cells that is in good correlation with the data on Echrlich carcinoma cells. The magnumoside A3 (3), magnumoside C1 (7), magnumoside C2 (8) and magnumoside C4 (9) decreased cell viability with IC50 value of 30.3, 34.3, 32.9, 37.1, and 33.9 μM, respectively (Table 5). Thus, for further experiments we chose the concentrations of investigated compounds lower than IC50, at which no significant cytotoxic effect on DLD-1 cells was observed. Table 5. The cytotoxic activity of the glycosides 1-9 against DLD-1 cells.

The Effect of the Glycosides on Formation and Growth of Colonies of Human Colorectal Adenocarcinoma DLD-1 Cells
The effect of 3, 7-9 on the colony formation of DLD-1 cells using soft agar assay has been studied. The magnumoside A3 (3) and magnumoside C1 (7) inhibited spontaneous colony formation by 22% and 26%, respectively, ( Figure 6A) and, at the same time, they reduced colonies size of DLD-1 cancer cells by 49% and 43%, respectively, ( Figure 6B). On the other hand, magnumosides C2 (8) and C4 (9) possessed slight inhibitory activity in this experiment (the percentages of inhibition were less than 20%) ( Figure 6A,B).

The Synergism of Radioactive Irradiation and the Compounds Effects on Proliferation and Colony Formation of Human Colorectal Adenocarcinoma Cells
At first, the individual effect of radiation or the compounds at concentration 2 µM on colony formation of DLD-1 cells was checked. The tested compounds did not influence the process of colonies formation at the dose of 2 µM. The number and size of colonies of DLD-1 cells were found to be decreased by 7% and 67%, respectively, after radiation exposure at dose of 4 Gy. Moreover, the synergism of effects of radioactive irradiation (4 Gy) and the compounds 3, 7-9 (2 µM) was not observed (data not shown).
Nevertheless, the investigated compounds (2 µM) enhanced the antiproliferative effect from radioactive irradiation (4 Gy). Magnumoside C 4 (9) possessed the highest activity in this experiment; it increased the inhibitory effect from radiation on proliferation of DLD-1 cancer cells by 45%. Magnumoside A 3 (3), magnumoside C 1 (7) and magnumoside C 2 (8) enhanced the effect from radiation by more than 30% (Figure 7). Recently, it was reported that ginsenoside Rg3 isolated from the roots of Panax ginseng sensitized human lung carcinoma A549 and H1299 cells to γ-radiation and significantly enhanced the efficacy of radiation therapy in C57BL/6 mice bearing a Lewis lung carcinoma cell xenograft tumor [38]. Nevertheless, our finding of a synergism of the antiproliferative effect of the radioactive irradiation and a series of glycosides from Neothyonidium magnum on human tumor cells is the first study on sea cucumber triterpene glycosides. At first, the individual effect of radiation or the compounds at concentration 2 μM on colony formation of DLD-1 cells was checked. The tested compounds did not influence the process of colonies formation at the dose of 2 μM. The number and size of colonies of DLD-1 cells were found to be decreased by 7% and 67%, respectively, after radiation exposure at dose of 4 Gy. Moreover, the synergism of effects of radioactive irradiation (4 Gy) and the compounds 3, 7-9 (2 μM) was not observed (data not shown).
Nevertheless, the investigated compounds (2 μM) enhanced the antiproliferative effect from radioactive irradiation (4 Gy). Magnumoside C4 (9) possessed the highest activity in this experiment; it increased the inhibitory effect from radiation on proliferation of DLD-1 cancer cells by 45%. Magnumoside A3 (3), magnumoside C1 (7) and magnumoside C2 (8) enhanced the effect from radiation by more than 30% (Figure 7). Recently, it was reported that ginsenoside Rg3 isolated from the roots of Panax ginseng sensitized human lung carcinoma A549 and H1299 cells to γ-radiation and significantly enhanced the efficacy of radiation therapy in C57BL/6 mice bearing a Lewis lung carcinoma cell xenograft tumor [38]. Nevertheless, our finding of a synergism of the antiproliferative effect of the radioactive irradiation and a series of glycosides from Neothyonidium magnum on human tumor cells is the first study on sea cucumber triterpene glycosides. Figure 7. The effect of radioactive irradiation and a combination of radioactive irradiation and the compounds 3, 7-9 on DLD-1 cancer cell proliferation. DLD-1 cells (8.0 × 10 3 ) were treated with radiation 4 Gy and the compounds 3, 7-9 (2 μM) for 96 h. Cell viability was estimated using the MTS assay. Data are represented as the mean ± SD as determined from triplicate experiments. A Student's t-test was used to evaluate the data with the following significance levels: * p < 0.05, ** p < 0.01.

General Experimental Procedures
Specific rotations were measured on Perkin-Elmer 343 polarimeter. NMR spectra were obtained on an AVANCE III-700 Bruker spectrometer (Bruker BioSpin, Fällanden, Switzerland) at 700.13 ( 1 H) and 176.04 ( 13 C) MHz, δ in ppm rel. to Me4Si, J in Hz. ESI-MS/MS and HR-ESI-MS were run on an Agilent 6510 Q-TOF apparatus (Agilent Technologies, Santa Clara, CA, USA), sample concentration 0.01 mg/mL, in m/z. HPLC was carried out on an Agilent 1100 chromatograph equipped with a differential refractometer using Supelcosil LC-Si  The effect of radioactive irradiation and a combination of radioactive irradiation and the compounds 3, 7-9 on DLD-1 cancer cell proliferation. DLD-1 cells (8.0 × 10 3 ) were treated with radiation 4 Gy and the compounds 3, 7-9 (2 µM) for 96 h. Cell viability was estimated using the MTS assay. Data are represented as the mean ± SD as determined from triplicate experiments. A Student's t-test was used to evaluate the data with the following significance levels: * p < 0.05, ** p < 0.01.

Animal Material
Specimens of the sea cucumber Neothyonidium (=Massinium) magnum (family Phyllophoridae; order Dendrochirotida) were collected during the expedition of Joint Russia

Cell Culture
The spleenocytes from CD-1 line mice were used. The spleen was isolated from mice and homogenized. The spleenocytes were washed thrice and resuspended with RPMI-1640 medium contained gentamicine 8 µg/mL (Biolot, Saint Petersburg, Russia). The museum tetraploid strain of murine ascites Ehrlich carcinoma cells from the All-Russian cancer center RAMS (Moscow, Russia) was used. The cells of the ascites Ehrlich carcinoma were separated from ascites, which were collected on day 7 after inoculation in mouse CD-1 line. The cells were washed of ascites thrice and then resuspended in liquid media DMEM with L-Glutamine (Biolot, Saint Petersburg, Russia).

Cytotoxic Activity
The solutions of tested substances in different concentrations (20 µL) and cell suspension (200 µL) were added in wells of 96-well plate and incubated over night at 37 • C and 5% CO 2 . After incubation the cells were sedimented by centrifugation, 200 µL of medium from each well were collected and 100 µL of pure medium were added. Then 10 µL of MTT solution 5 µg/mL (Sigma, St. Louis, MO, USA) were added in each well. Plate was incubated 4 h, after that 100 µL SDS-HCl were added to each well and plate was incubated at 37 • C 4-18 h. Optical density was measured at 570 nm and 630-690 nm. The activity of the substances was calculated as the ratio of the dead cells to general cells amount (ED 50 ). Typicoside A 1 [28] and cucumarioside A 2 -2 [11] were used as positive controls.

Hemolytic Activity
Blood was taken from a CD-1 mouse. The erythrocytes were washed thrice with 0.9% NaCl, centrifuged (450 g) on a centrifuge LABOFUGE 400R (Heraeus, Hanau, Germany) for 5 min followed by re-suspending in phosphate-buffered saline (PBS), pH 7.2-7.4. Erythrocytes were used at a concentration provided an optical density of 1.5 at 700 nm for a non-hemolyzed sample. 20 µL of a water solution of test substance with a fixed concentration (0.12-100.00 µM) were added to a well 3.5.9. Cell Irradiation DLD-1 cells (5.0 × 10 5 ) were plated at 60 mm dishes and incubated for 24 h. After the incubation, the cells were cultured in the presence or absence of 2 µM 3, 7-9 for additional 24 h before irradiation at the dose of 4 Gy. Immediately after irradiation, cells were returned to the incubator for recovery. Three hour later, the cells were harvested and used for soft agar assay or proliferation assay to establish the synergism of radioactive irradiation and investigated compounds effects on colony formation or proliferation of tested cells.

Statistical Analysis
All assays were performed in triplicate. The results are expressed as the means ± standard deviation (SD). A Student's t-test was used to evaluate the data with the significance level of p < 0.05. The mean and standard deviation were calculated and plotted using SigmaPlot 3.02 Software (Jandel Scientific, San Rafael, CA, USA).

Conclusions
Summarizing the obtained data, three types of previously known carbohydrate chains, five new aglycones as well as one known aglycone have been found in the isolated glycosides 1-9. Magnumoside A 1 (1) has uncommon non-holostane aglycone with a unique for sea cucumber triterpene glycosides 20(24)-epoxy-25-hydroxy-fragment in the side chain. The biosythesis of the glycosides found in N. magnum has mosaic character, i.e., transformation in cyclic systems of aglycones and in their side chains as well as in carbohydrate chains (elongation and sulfation) proceed parallel and independently and they have the characteristics of a biosynthetic network. Disulfated glycosides 7 and 8 showed surprisingly high hemolytic and cytotoxic actions in spite of the presence of hydroxyl-groups in their side chains. The data concerning cytotoxic activities on DLD-1 human colorectal adenocarcinoma cells good correlated with the data on mouse ascites Echrlich carcinoma cells that confirmed the usefulness of the last model tumor for screening of substances that are cytotoxic against human tumor cells. The data concerning synergy of the activities of the glycosides 3 and 7-9 in subcytotoxic doses and subtoxic doses of radiation were obtained for the first time where magnumoside C 4 (9) revealed the highest increase of the inhibitory effect of radiation on cell proliferation of 45%. The substances having such effects allow a decrease in the effective doses of radiation that may be used for radiation therapy of human tumors. The search for substances with a similar mode of action among sea cucumber triterpene glycosides should be continued.