The Isolation, Structure Elucidation and Bioactivity Study of Chilensosides A, A1, B, C, and D, Holostane Triterpene Di-, Tri- and Tetrasulfated Pentaosides from the Sea Cucumber Paracaudina chilensis (Caudinidae, Molpadida)

Five new triterpene (4,4,14-trimethylsterol) di-, tri- and tetrasulfated pentaosides, chilensosides A (1), A1 (2), B (3), C (4), and D (5) were isolated from the Far-Eastern sea cucumber Paracaudina chilensis. The structures were established on the basis of extensive analysis of 1D and 2D NMR spectra and confirmed by HR-ESI-MS data. The structural variability of the glycosides concerned the pentasaccharide chains. Their architecture was characterized by the upper semi-chain consisting of three sugar units and the bottom semi-chain of two sugars. Carbohydrate chains of compounds 2–5 differed in the quantity and positions of sulfate groups. The interesting structural features of the glycosides were: the presence of two sulfate groups at C-4 and C-6 of the same glucose residue in the upper semi-chain of 1, 2, 4, and 5 and the sulfation at C-3 of terminal glucose residue in the bottom semi-chain of 4 that makes its further elongation impossible. Chilensoside D (5) was the sixth tetrasulfated glycoside found in sea cucumbers. The architecture of the sugar chains of chilensosides A–D (1–5), the positions of sulfation, the quantity of sulfate groups, as well as the aglycone structures, demonstrate their similarity to the glycosides of the representatives of the order Dendrochirotida, confirming the phylogenetic closeness of the orders Molpadida and Dendrochirotida. The cytotoxic activities of the compounds 1–5 against human erythrocytes and some cancer cell lines are presented. Disulfated chilensosides A1 (2) and B (3) and trisulfated chilensoside C (4) showed significant cytotoxic activity against human cancer cells.


Introduction
Despite triterpene glycosides from sea cucumbers having a rather long history of investigations, there are some systematic groups, including the order Molpadida, comprising the studied species Paracaudina chilensis, which are poorly studied or unexplored chemically. The majority of recent research concerning the sea cucumber triterpene glycosides deals with the structure elucidation of the compounds isolated from representatives of the orders Dendrochirotida, Synallactida, and Holothuriida [1][2][3][4][5][6][7][8][9]. The use of mass-spectrometrybased metabolomics for the solving of diverse chemical and biological issues concerning secondary metabolites has become very popular and has provided some significant results in the exploration of triterpene glycoside chemical diversity, their content and composition in different body parts [10][11][12][13], and their chemotaxonomy [14,15]. The application of this approach in combination with molecular phylogenetic analysis allowed to clarify the evolution of the Holothuroidea taxons [16]. Different investigations have also confirmed the defensive role of glycosides [17,18]. The biosynthetic studies of triterpene glycosides 2. Results and Discussion

Structural Elucidation of the Glycosides
The crude glycosidic fraction of the sea cucumber Paracaudina chilensis was obtained as a result of hydrophobic chromatography of the concentrated ethanolic extract on a Polychrom-1 column (powdered Teflon, Biolar, Latvia). Its subsequent separation by chromatography on Si gel columns with the stepped gradient of the system of eluents CHCl 3 /EtOH/H 2 O used in ratios (100:100:17), (100:125: 25), and (100:150:50) gave the fractions I-III. Each of the obtained fractions was additionally purified on a Si gel column with the solvent system CHCl 3 /EtOH/H 2 O (100:125: 25), which resulted in the isolation of five subfractions I.0, I.1, II, III.1 and III.2. The individual compounds 1-5 ( Figure 1) were isolated through HPLC of these subfractions on the silica-based column Supelcosil LC-Si (4.6 × 150 mm), and reversed-phase columns Supelco Discovery HS F5-5 (10 × 250 mm) and Diasfer 110 C-8 (4.6 × 250 mm). The configurations of the monosaccharide residues in the glycosides 1-5 were assigned as D based on the biogenetic analogies with the monosaccharides from all other known sea cucumber triterpene glycosides.
The molecular formula of chilensoside A (1) was determined to be C60H92O34S2Na2 from the [M2Na-Na] − Table 1). The same aglycone was found only once earlier in cladoloside A5 from the sea cucumber Cladolabes schmeltzii [42]. The configurations of the monosaccharide residues in the glycosides 1-5 were assigned as D based on the biogenetic analogies with the monosaccharides from all other known sea cucumber triterpene glycosides.
The molecular formula of chilensoside A (1) was determined to be C 60 H 92 O 34 S 2 Na 2 from the [M 2Na -Na] − ion peak at m/z 1443.4800 (calc. 1443.4815), and [M 2Na -2Na] 2− ion peak at m/z 710.2466 (calc. 710.2461) in the (−) HR-ESI-MS ( Figure S8). The 13 C NMR spectrum of the aglycone part of chilensoside A (1) demonstrated the signals of quaternary oxygen-bearing carbons at δ C 176.8 (C-18) and 82.8 (C-20), corresponding to 18(20)-lactone and the signals of olefinic carbons at δ C 151.1 (C-9), 111.2 (C-11) ( Table 1, Figures S1-S7), indicating the presence of 9(11)-double bond, typical of many sea cucumber glycosides. An additional deshielded signal at δ C 214.6 was assigned to C-16 oxo-group in the holostane nucleus, confirmed by the singlet signal of H-17 at δ H 2.89 with a corresponding carbon signal at δ C 61.2 (C-17). The protons of the side chain H-22/H-23/H-24 formed the isolated spin system deduced by the COSY spectrum, indicating the presence of an additional 23Z(24)-double bond (δ H-23 5.90 (dd, J = 6.3; 11.8 Hz), δ H-24 5.90 (d, J = 11.8 Hz)). The presence of the signal of quaternary oxygen-bearing carbon at δ C 81.3 along with the coincidence of the signals of 26, 27-methyl groups to each other (δ C 24.  Table 1). The same aglycone was found only once earlier in cladoloside A 5 from the sea cucumber Cladolabes schmeltzii [42].   Noticeably, such architecture of carbohydrate chains is not common for the holothuroid glycosides, but similar sugar moieties have been found in some glycosides of recently studied species of sea cucumbers: Thyonidium kurilensis [43] and Psolus chitonoides [6] (order Dendrochirotida).
The availability of two sulfate groups in the sugar moiety of 1 was deduced on the basis of shifting effects observed in its 13 C NMR spectrum. These were the signals of two hydroxy methylene groups of glucopyranose residues at δ C 61.8 (C-6 Glc3) and 62.0 (C-6 MeGlc5), indicating the absence of sulfate groups in these positions and one signal at δ C 68.2 (deshielded due to α-shifting effect of sulfate group) corresponding to sulfated at C-6 Glc4 residue. Additional shifting effects of the sulfate group became evident when the 13 C NMR spectrum of 1 was compared with the spectrum of the carbohydrate part of kuriloside A 1 [43]. The signals of all monosaccharides in the spectra of these glycosides were close to each other, with the exception of the signals of glucose residue in the upper semi-chain (Glc4). The signals of C-3 Glc4 and C-5 Glc4 were shielded in the spectrum of 1 (to δ C 82.9 and 74.5, correspondingly) in comparison with the same signals in the spectrum of kuriloside A 1 (δ C 86.9 and 75.7, correspondingly) due to the β-shifting effect of the sulfate group, which was attached to C-4 Glc4 of 1. This was confirmed by an α-shifting effect: the signal of C-4 Glc4 in the spectrum of chilensoside A (1) was deshielded (δ C 75.6) when compared with the signal of C-4 Glc4 in the spectrum of kuriloside A 1 (δ C 69.6). Therefore, two sulfate groups were attached to one monosaccharide unit (Glc4) in the sugar chain of 1. Such a structural feature was also recently found in psolusoside P from Psolus fabricii [44]. These data indicate that chilensoside The aglycones of chilensosides A 1 (2), B (3), C (4) and D (5) (Tables 3 and S2-S4, Figures S9-S14, S17-S22, S25-S30 and S33-S38) were identical to each other and to those of cladoloside A 4 [42] and psolusoside D 1 [45]. This holostane aglycone has the same polycyclic system as 1 and differs in the side chain structure with a 24(25)-double bond.
The molecular formula of chilensoside A 1 (2) was determined to be C 60 H 92 O 33  All these data indicate that chilensoside  (Table 4, Figures S17-S23) of 3 indicated the same monosaccharide composition, positions of glycosidic linkages, and architecture established for the glycosides 1, 2. The differences in the chemical shifts of carbon signals of chilensosides A (1) and B (3) were attributed to the diverse positions of sulfate groups. The signal of C-4 Glc4 in the 13 C NMR spectrum of 3 was shielded to δ C 68.9 instead of δ C 75.6 in 1 due to the absence of a sulfate group in this position of 3. Additionally, the signal of C-3 Glc4 was deshielded to 85.9 due to the glycosylation effect and the absence of the β-shifting effect of sulfate group. The signal of C-6 Glc4 at δ C 67.2 was characteristic for the sulfated hydroxy methylene group of the glucopyranose unit. Therefore, the glucose residue attached to C-4 Xyl1 of the carbohydrate chain of 3 bears one sulfate group at C-6. The comparison of the signals assigned to carbons of the 3-O-methylglucose unit of the compounds 3 (Table 4) and 1 ( Table 2) showed that the signal of C-4 MeGlc5 of 3 was deshielded by 6.1 ppm (to δ C 76.1) and the signals of C-3 and C-5 MeGlc5 were shielded by 1.7 and 1.0 ppm, corresponding to the shifting effects of the sulfate group attached to C-4 MeGlc5 of chilensoside B (3). Thus, the glycoside 3 is a new disulfated pentaoside having sulfate groups at C-6 Glc4 and C-4 MeGlc5. The compound with identical positions of sulfates but differing in the terminal xylose residue in the bottom semi-chain was chitonoidoside H, found recently in the sea cucumber Psolus chitonoides [6].    The extensive analysis of the 1 H, 1 H-COSY, 1D TOCSY, HSQC, ROESY, and HMBC spectra of 4 indicated the same monosaccharide composition, glycosidic bond locations and architecture of carbohydrate chains as in the previously discussed glycosides 1−3. Differences were found in the quantity of sulfate groups, which was also confirmed by MS data, where three-charged ions were registered, indicating the presence of three sulfate groups.
The comparison of the 13 C NMR spectra of sugar moieties of 4 and 1 showed the coincidence of all the signals except the signals of glucose residue in the bottom semi-chain. The signal of C-3 Glc3 was deshielded to δ C 84.3 in the spectrum of 4, which could be explained by the α-shifting effect of the sulfate group as well as by the glycosylation effect. However, the latter was excluded due to the absence of the ROE-and HMBC correlations of H-3 Glc3 with any protons or carbons of neighboring monosaccharide residues (Table 5). Moreover, the signals of C-2 Glc3 and C-4 Glc3 in the spectrum of 4 were shielded to δ C 73.1 and 69.8, respectively, in comparison with the corresponding signals in the spectrum of 1 due to β-shifting effect of sulfate group at C-3 Glc3. Therefore, the third sulfate group in chilensoside C (4) Figure S40). Chilensoside D (5), analogously to compounds 1-4, has a pentasaccharide branched by C-4 Xyl1 chain consisting of xylose, quinovose, two glucose and 3-O-methylglucose residues deduced from thorough analysis of its 1D and 2D NMR spectra ( Table 6, Figures S33-S39). The availability of four-charged ion peaks in the ESI-MS spectra of 5 indicated four sulfate groups are present in its carbohydrate chain. The analysis of 1D TOCSY spectrum corresponding to Glc3 showed strongly deshielded signals of protons of the hydroxy methylene group at δ H 4.61 (m) and 5.00 (d, J = 11.9 Hz), which were assigned to the corresponding carbon signal at δ C 67.6. These data indicate that the glucose residue in the bottom semichain was sulfated by C-6. The glucose unit (Glc4) attached to C-4 Xyl1 in chilensoside D (5) had two sulfate groups at C-4 Glc4 and C-6 Glc4, deduced from the deshielding of its signals to δ C 75.1 and 68.5, respectively. The fourth sulfate group was positioned at C-6 MeGlc5 because of the deshielding of the signals of hydroxy methylene group to δ C 67.0 and δ H 4.99 (brd, J = 11.9 Hz); 4.78 (dd, J = 5.1; 11.9 Hz). Therefore, chilensoside D (5) is a new, sixth tetrasulfated glycoside found in sea cucumbers [7,44].  The structural peculiarities of the glycosides of P. chilensis showed similarity to the compounds of the representatives of the order Dendrochirotida, i.e., sea cucumbers of the species Thyonidium kurilensis and Psolus chitonoides (the same architecture of the carbohydrate chains), Psolus fabricii (attachment of sulfates to C-4 Glc4 and C-6 Glc4) and Cladolabes schmeltzii (the same aglycones). All these data significantly support the phylogenetic closeness of the order Molpadida to the order Dendrochirotida, rather than to the order Aspidochirotida (in accordance with the system of Pawson and Fell). This order is absent in the last revision of the system of the class Holothuroidea, and the families, which were part of it, are now included in the orders Holothuriida, Persiculida and Synallactida [46]. The obtained structural data are in good agreement with the phylogenetic study of Holothuroidea using a multi-gene approach, which showed poor support of Molpadida as a sister group to Synallactida but demonstrated the close relationship of Molpadida to Dendrochirotida [46].

Bioactivity of the Glycosides
Cytotoxic activity of chilensosides A-D (1-5) against human cells, including erythrocytes and cancer cell lines SH-SY5Y, HeLa, DLD-1, HL-60, and THP-1, was studied. The earlier tested chitonoidoside L [7] was used as the positive control (Table 7). Table 7. The cytotoxic activities of glycosides 1-5, and chitonoidoside L (positive control) against human erythrocytes, and SH-SY5Y, HeLa, DLD-1, HL-60, THP-1 human cell lines. The less active compounds in the series were chilensosides A (1) and D (5). The first of these substances has a hydroxyl group in the aglycone side chain, which is the cause of the decrease in its membranolytic activity [3]. In fact, its structural analog chilensoside A 1 (2), with the same carbohydrate chain and aglycone without the OH-group, demonstrated high hemolytic and cytotoxic effects against all tested cell lines. Chilensoside D (5) is a tetrasulfated glycoside that itself is not the cause of activity depletion, because it is known that tetrasulfated hexaosides from P. chitonoides, chitonoidosides K and L, were significantly active [7]. The combination of sulfate group positions in 5, especially at C-6 Glc3 and C-6 MeGlc5, probably negatively affected the activity. Disulfated chilensosides A 1 (2), B (3) and trisulfated chilensoside C (4) displayed similar cytotoxicity. The differing sulfate groups in these glycosides were attached to C-4 or C-3 of glucopyranose units while C-6 positions of terminal sugar residues were free from sulfation.

Glycosides
The differential sensitivity of the cell lines in relation to the cytotoxic action of sea cucumber glycosides depended both on the glycoside's chemical structures and the composition of cellular membranes [47]. In the current tests, erythrocytes were, as usual, more sensitive than cancer cells to the action of the glycosides, but leukemia cells (promyeloblast HL-60 and monocytic THP-1) displayed increased sensitivity compared to the other cancer cells.
Therefore, three of the five glycosides isolated from P. chilensis demonstrated high hemolytic and moderate cytotoxic activities against cancer cells. These data, along with the previous investigations of highly polar tri-and tetrasulfated glycosides [7], indicate the possible potential of these water-soluble compounds to be used as anticancer drugs.  All the studied substances (including chitonoidoside L used as positive control) were tested in concentrations between 0.1 µM to 100 µM using 2-fold dilution in d-H2O. The cell suspension (180 µL) and solutions (20 µL) of tested compounds in different concentrations were injected in wells of 96-well plates (SH-SY5Y, 1 × 104 cells/well, HeLa and DLD-1, 6 × 10 3 /200 µL) and incubated at 37 • C for 24 h in atmosphere with 5% CO 2 . Then, 100 µL of fresh medium was added instead of the tested substances in the same volume of medium. After that, 10 µL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich, St. Louis, MO, USA) stock solution (5 mg/mL) was added to each well, and the microplate was incubated for 4 h. Next, each well was additionally incubated for 18 h with 100 µL of SDS-HCl solution (1 g SDS/10 mL d-H2O/17 µL 6 N HCl). Multiskan FC microplate photometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to measure the absorbance of the converted dye formazan at 570 nm. Cytotoxic activity of the tested compounds was calculated as the concentration that caused 50% cell metabolic activity inhibition (IC50). The experiments were carried out in triplicate, p < 0.05.

Cytotoxic Activity (MTS Assay) (for HL-60 and THP-1 Cells)
The cells of HL-60 line (10 × 10 3 /200 µL) and THP-1 (6 × 10 3 /200 µL) were placed in 96-well plates at 37 • C for 24 h in a 5% CO 2 incubator. The cells were treated with tested substances and chitonoidoside L as positive control at concentrations from 0 to 100 µM for an additional 24 h incubation. Then, the cells were incubated with 10 µL MTS ([3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) for 4 h, and the absorbance in each well was measured at 490/630 nm with plate reader PHERA star FS (BMG Labtech, Ortenberg, Germany). The experiments were carried out in triplicate and the mean absorbance values were calculated. The results were presented as the percentage of inhibition that produced a reduction in absorbance after tested compounds treatment compared to the non-treated cells (negative control), p < 0.01.

Hemolytic Activity
Erythrocytes were isolated from human blood (AB(IV) Rh+) by centrifugation with phosphate-buffered saline (PBS) (pH 7.4) at 4 • C for 5 min by 450 g on centrifuge LABOFUGE 400R (Heraeus, Hanau, Germany) three times. Then, the residue of erythrocytes was resuspended in ice cold phosphate saline buffer (pH 7.4) to a final optical density of 1.5 at 700 nm, and kept on ice. For the hemolytic assay, 180 µL of erythrocyte suspension was mixed with 20 µL of test compound solution (including chitonoidoside L used as positive control) in V-bottom 96-well plates. After 1 h of incubation at 37 • C, plates were exposed to centrifugation for 10 min at 900 g on laboratory centrifuge LMC-3000 (Biosan, Riga, Latvia). Then, 100 µL of supernatant was carefully selected and transferred in new flat-plates, respectively. Lysis of erythrocytes was determined by measuring the concentration of hemoglobin in the supernatant with microplate photometer Multiskan FC (Thermo Fisher Scientific, Waltham, MA, USA), λ = 570 nm. The effective dose causing 50% hemolysis of erythrocytes (ED50) was calculated using the computer program SigmaPlot 10.0. All the experiments were carried out in triplicate, p < 0.01.

Conclusions
As a result of investigation of the glycosidic composition of the sea cucumber Paracaudina chilensis, the structures of five new glycosides, chilensosides A-D (1-5), were established and their cytotoxic activities were studied. Two different aglycones were found and one of them was a part of four compounds. Four diverse carbohydrate chains were detected in the studied glycosides. They differed in the quantity of sulfate groups: two in chilensosides of groups A (1, 2) and B (3), three in chilensoside C (4), and four in chilensoside D (5). The positions of sulfation were also variable: two sulfates were attached to C-4 and C-6 of Glc4 residue in the glycosides 1, 2; additional third sulfate group bonded C-3 Glc3 in chilensoside C (4); two sulfates bonded different monosaccharide residues, C-6 Glc4 and C-4 Meglc5, in chilensoside B (3); and, finally, two positions of sulfation at C-6 Glc3 and C-6 MeGlc5, additional to those observed in 1, 2, were detected in chilensoside D (5).
Such diversity in sulfate group quantity and positions indicates the high enzymatic activity of sulfatases. They have low specificity to attach a sulfate group to different positions of the same or several monosaccharide residues in glycosides 1-5. Especially interesting was the observation that the sulfatase could compete with the glycosidase, bonding the sulfate group to C-3 Glc3 in chilensoside C (4) instead of potential glycosylation of this position. Additionally, it is interesting to note that only the aglycones with intranuclear 9(11)-double were found. This indicates that one oxidosqualene cyclase (OSC), forming the parkeol (precursor of the glycosides with 9(11)-double bond), is expressed in P. chilensis.
The structures of the glycosides of P. chilensis were similar to those found in some representatives of the order Dendochirotida, confirming phylogenetic closeness of the order Molpadida to the order Dendrochirotida.
Rather high hemolytic and cytotoxic activity of three out of five isolated glycosides along with the previous investigations of highly polar tri-and tetrasulfated glycosides indicate the possible potential of these water-soluble compounds to be used as anticancer drugs.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/molecules27217655/s1, The original spectral data ( Figures S1-S40 and Tables S1-S4).  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.