In Vitro Anticancer and Cancer-Preventive Activity of New Triterpene Glycosides from the Far Eastern Starfish Solaster pacificus

Sea stars or starfish (class Asteroidea) and holothurians or sea cucumbers (class Holothuroidea), belonging to the phylum Echinodermata (echinoderms), are characterized by different sets of glycosidic metabolites: the steroid type in starfish and the triterpene type in holothurians. However, herein we report the isolation of eight new triterpene glycosides, pacificusosides D–K (1–3, 5–9) along with the known cucumarioside D (4), from the alcoholic extract of the Far Eastern starfish Solaster pacificus. The isolated new compounds are closely related to the metabolites of sea cucumbers, and their structures of 1–3 and 5–9 were determined by extensive NMR and ESIMS techniques. Compounds 2, 5, and 8 have a new type of tetrasaccharide chain with a terminal non-methylated monosaccharide unit. Compounds 3, 6, and 9 contain another new type of tetrasaccharide chain, having 6-O-SO3-Glc as one of the sugar units. The cytotoxic activity of 1–9 against non-cancerous mouse epidermal cells JB6 Cl41 and human melanoma cell lines SK-MEL-2, SK-MEL-28, and RPMI-7951 was determined by MTS assay. Compounds 1, 3, 4, 6, and 9 showed potent cytotoxicity against these cell lines, but the cancer selectivity (SI > 9) was observed only against the SK-MEL-2 cell line. Compounds 1, 3, 4, 6, and 9 at the non-toxic concentration of 0.1 μM significantly inhibited neoplastic cell transformation of JB6 Cl41 cells induced by chemical carcinogens (EGF, TPA) or ionizing radiation (X-rays and UVB). Moreover, compounds 1 and 4 at the non-toxic concentration of 0.1 µM possessed the highest inhibiting activity on colony formation among the investigated compounds and decreased the colonies number of SK-MEL-2 cells by 64% and 70%, respectively. Thus, triterpene glycosides 1 and 4 can be considered as prospective cancer-preventive and anticancer-compound leaders.

The proton and carbon signals of the aglycon side chain of 1 demonstrated the presence of three methyl groups (δ H 1.57 s, 1.61 s, 1.69 s; δ C 30.2, 17.9, 25.6) and the conjugated 22,24-diene system (δ H 5.92 d (J = 15.7), 6.56 dd (J = 15.7, 11.0), 5.86 brd (11.0); δ C 134.0, 122.4, 125. 2, 134.6). The observation of a UV absorption maximum at λ max = 240 nm was consistent with this assignment [30,31] ( Figure S15). The protons sequence from H-22 to H-27 correlated with the corresponding carbon atoms of the side chain of 1 was assigned using the COSY and HSQC experiments (Tables 1 and 2 (Figures 1, S13 and S14). The trans configuration of the 22(23)-double bond followed from J 22,23 = 15.7 Hz. The NMR spectroscopic data of the aglycon part of 1 were coincident with those of the known cucumarioside C 2 from E. fraudatrix, with holostane-type aglycon having 16β-OAc and 7(8)-double bonds in the nucleus and ∆ 22,24 -lanostane side chain [32]. side chain [32]. In addition to the above-mentioned signals, the 1 H NMR spectrum of 1 exhibited five resonances in the deshielded region due to the anomeric protons of monosaccharide units at δH 4.87, 5.20, 4.92, 5.27, and 5.39 that correlated in the HSQC experiment with a carbon signal at δC 105.1, 103.0, 104.7, 105.5, and 105.8, respectively, as well as a resonance due to an O-methyl group at δH 3.87 that correlated in the HSQC experiment with a carbon signal at δC 60.5 (Table 2, Figures S3-S10). In addition to the above-mentioned signals, the 1 H NMR spectrum of 1 exhibited five resonances in the deshielded region due to the anomeric protons of monosaccharide units at δ H 4.87, 5.20, 4.92, 5.27, and 5.39 that correlated in the HSQC experiment with a carbon signal at δ C 105.1, 103.0, 104.7, 105.5, and 105.8, respectively, as well as a resonance due to an O-methyl group at δ H 3.87 that correlated in the HSQC experiment with a carbon signal at δ C 60.5 ( Table 2, Figures S3-S10).
The sequence of monosaccharide units in the carbohydrate chain of 1 was confirmed by ESIMS/MS data. In fact, the (−)ESIMS/MS spectrum of the molecular anion peak  Along with mass spectral information, these data showed the presence of five monosaccharide residues in the oligosaccharide moiety of 1. The existence of a 6-deoxy-sugar unit was supported by one methyl doublet at δ H 1.70. The coupling constants (7.0-8.0 Hz) of anomeric protons were indicative of a β-configuration of all the glycosidic bonds. The chemical shifts and coupling constants of H-1-H-5 or H-6 of monosaccharide units were determined by irradiation of anomeric protons in the 1D TOCSY experiments. 1 H-1 H COSY, HSQC, HMBC, and ROESY experiments led to the assignment of all the proton and carbon signals of the carbohydrate chain of 1 (Table 2, Figures 3, and S11-S14).
the anomeric protons were indicative of a β-configuration of all the glycosidic bonds. The chemical shifts and coupling constants of H-1-H-5, or H-6 of monosaccharide units, were determined by irradiation of the anomeric protons in the 1D TOCSY experiments. 1 H-1 H COSY, HSQC, HMBC, and ROESY experiments led to the assignment of all the proton and carbon signals of the carbohydrate chain of 3 ( Figure 3, Figures S28-S31). The examination of the NMR spectroscopic data of compounds 3 and 1 exhibited that the signals of oligosaccharide moiety of 3 strictly coincided with those of the terminal 3-OMe-β-D-xylopyranosyl residue and internal 4-O-substituted β-D-quinovopyranosyl and 2-O-substituted β-D-xylopyranosyl residues. The signals of H-6, H′-6 at δH 5.25, 4.82, and C-6 at δC 67.6 of the internal 3-O-substituted-β-D-glucopyranosyl residue in the 1 H and 13 C NMR spectra of 3 were deshielded in comparison with the same signals at δH 4.45, 4.18, and δC 61.9 in the 1 H and 13 C NMR spectra of 1, respectively, that revealed the presence of a sulfoxy group at the C-6 position in this monosaccharide unit of 3.     Figure S16). The IR spectrum of compound 2 showed the presence of hydroxy (3414 cm -1 ), γ-lactone (1736 cm -1 ), and olefinic (1619 cm -1 ) groups ( Figure S17). The comparison of the 1 H, 13 C NMR spectra and application of extensive 2D NMR analysis of compounds 1, 2, and 3 exhibited that the triterpene aglycon of 1 is identical to that in compounds 2 and 3, while compounds 1, 2, and 3 differ from each other in oligosaccharide moieties only (Tables 1 and 2 Along with mass-spectra information, these data showed the presence of four monosaccharide residues in the oligosaccharide moiety of 2. The existence of a 6-deoxy-sugar unit was supported by one methyl doublet at δ H 1.71. The coupling constants (6.  Figure S24). The IR spectrum of compound 3 showed the presence of hydroxy (3415 cm -1 ), γ-lactone (1742 cm -1 ), olefinic (1619 cm -1 ), and sulfate (1243, 817 cm -1 ) groups ( Figure S25). The 1 H NMR spectrum of 3 exhibited four resonances in the deshielded region due to the anomeric protons of monosaccharide units at δ H 4.76, 5.06, 4.85, and 5.21 that correlated in the HSQC experiment with a carbon signal at δ C 105.5, 104.9 × 2, and 105.9, respectively, as well as one resonance due to an O-methyl group at δ H 3.85 that correlated in the HSQC experiment with a carbon signal at δ C 60.4 ( Table 2, Figures S26 and S27). Along with mass-spectrometric information, these data showed the presence of four monosaccharide residues in the oligosaccharide moiety of 3. The existence of a 6-deoxy-sugar unit was supported by one methyl doublet at δ H 1.69. The coupling constants (7.   22 Na] + , 1105.5190) in the (+)HRESIMS ( Figure S32). The IR spectrum of compound 5 showed the presence of hydroxy (3419 cm -1 ), γ-lactone (1740 cm -1 ), and olefinic (1618 cm -1 ) groups ( Figure S33). Compounds 5 and 2 have the same molecular weight. The thorough comparison of the 1 H and 13 C NMR data of 5 and 2 showed that they differed from each other only in signals of their side chains (Table 1, Figures S34-S41 [29]. Therefore, the structure of pacificusoside G (5) was established The  Figure S47). The IR spectrum of compound 6 showed the presence of hydroxy (3421 cm -1 ), γ-lactone (1744 cm −1 ), olefinic (1635 cm −1 ), and sulfate (1245, 819 cm −1 ) groups ( Figure S48). Compounds 6 and 3 have the same molecular weight. On the basis of an extensive 2D NMR analysis of glycosides 3 and 6, we suggested that the oligosaccharide moiety of 3 is identical to that of glycoside 6. Moreover, a detailed NMR analysis of the proton and carbon signals of the triterpene aglycon of 6 clearly indicated that glycosides 6 and 5 have the same aglycon. All proton and carbon resonances of 6 were received from the 1 H-1 H COSY, HSQC, HMBC, and ROESY experiments and confirmed the structure of both the aglycon and carbohydrate moieties (Tables 1 and 2 Table 1, Figures S57 and S58). The signals of the OAc group in the 1 H and 13 C NMR spectra of compound 7 were absent. The 1 H-1 H COSY and HSQC correlations attributable to the triterpene nucleus revealed the corresponding sequences of protons from C-1 to C-3, C-5 to C-7, C-9 to C-12 through C-11, and C-15 to C-17 (  (Figures 2A and S61). The key ROESY cross-peaks showed the common 5α/9β/10β/13β,14α stereochemistry of the triterpene nucleus and 3β-configuration of the oxygenated substituent in 7 ( Figures 2B and S62) (Figure 1, Figures S61 and S62). The NMR spectroscopic data of the aglycon part of 7 were coincident with those of the known cucumarioside A 10 from E. fraudatrix and pacificusoside B from S. pacificus with 23,24,25,26,27-pentanor-lanosta-7,20(22)-diene-18(16)-lactone-3β-ol aglycon [28,33].
On the basis of extensive 2D NMR and MS analysis of glycosides 1, 4, and 7, we suggested that the oligosaccharide moiety of 7 is identical to those of glycosides 1 and 4. Thus, the structure of pacificusoside I (7)  The comparison of 1 H, 13 C NMR and MS spectra and the application of extensive 2D NMR analysis for compounds 8, 9, and 7 exhibited that they have the identical triterpene aglycon and differ from each other only in the oligosaccharide moieties (Tables 1 and 2 Figure S63).The IR spectrum of compound 8 showed the presence of hydroxy (3415 cm −1 ), γ-lactone (1766 cm −1 ), and olefinic (1638 cm −1 ) groups ( Figure S64). The examination of the 1 H, 13 C NMR and MS spectra of compounds 8, 2, and 5 clearly revealed that glycoside 8 has the same oligosaccharide chain ( Table 2, Figures S65-S70). Therefore, the structure of pacificusoside J (8) Figure S71). The IR spectrum of compound 9 showed the presence of hydroxy (3415 cm −1 ), γ-lactone (1744 cm −1 ), olefinic (1638 cm −1 ), and sulfate (1241, 815 cm -1 ) groups ( Figure S72). The detailed comparison of the 1 H, 13 C and 2D NMR and MS spectra of glycosides 9, 3, and 6 exhibited that the oligosaccharide moiety of 9 is identical to that of 3 and 6 ( Table 2, Figures S73-S84). Accordantly, the structure of pacificusoside K Previously, only three triterpene glycosides were isolated from two species of starfish, A. rollestoni and C. granulatus [26,27], and we also reported about six triterpene glycosides from S. pacificus [28]. Now, we have isolated nine additional compounds of this type. To the best of our knowledge, triterpene glycosides with this type of carbohydrate chain are very rare triterpene glycosides. In addition, a tertrasaccharide carbohydrate chain containing a 6-O-SO 3 -glucopyranose residue, in glycosides 3, 6, and 9, was not earlier found in echinoderms.
Previously, we investigated the metabolite profiling of triterpene glycosides of the Far Eastern sea cucumber E. fraudatrix using LC-ESI QTOF-MS [34]. Totally, 54 triterpene glycosides were detected by this method, including compounds 1, 2, and 5. However, even such a sensitive method as LC-ESI QTOF-MS did not allow detecting and predicting the structures of compounds 3 and 6-9. This fact may also indicate that the starfish S. pacificus can partially metabolize the triterpene glycosides of sea cucumbers using their own enzyme systems. It is worth noting that at the present time, in the starfish S. pacificus, we did not find polar steroid compounds-typical secondary metabolites of sea stars. At the same time, triterpene glycosides from starfish A. rollestoni and C. granulatus were isolated together with steroid glycosides that make the detection of triterpene glycosides in the starfish S. pacificus unique [26,27]. Probably, dietary glycosides play the same protective biological role against predators and pathogens in this starfish as they play in holothurians.

The Effect of the Triterpene Glycosides on Cell Viability
To determine the cytotoxic activities of triterpene glycosides 1-9 from the starfish S. pacificus, non-cancerous mouse epidermal cells JB6 Cl41 and the panel of human melanoma cell lines SK-MEL-2, SK-MEL-28, and RPMI-7951 were treated by the investigated compounds at a concentrations range of 0.1-62.5 µM for 24 h, and cell viability was estimated by MTS assay. As shown in Table 3, Table 3). The triterpene glycosides 2, 5, 7, and 8 were found to be less effective against the tested cells ( Table 3). The RPMI-7951 cell line seemed to be resistant to the action of the investigated compounds (Table 3). In this work, mouse non-cancerous epidermal cells JB6 Cl41 were used as a reference to check compounds 1-9 for cancer selectivity. The selectivity indexes of triterpene glycosides 1-9 were calculated as the ratio of respective IC 50 values against cancer cell lines SK-MEL-2, SK-MEL-28, and RPMI-7951 and that against JB6 Cl41 normal cells (Table 3). Ideally, the potential drug should kill the cancer cells, but it should not affect the normal cells, so the higher the magnitude of the selectivity index, the greater is its cancer selectivity [35]. Thus, compounds 1, 3, 4, 6, and 9 showed a high selectivity (more than nine) only against the SK-MEL-2 cell line. Since the most known triterpene glycosides belong to a class of saponins and possess membranolytic and hemolytic activities, the hemolytic activity of investigated compounds 1-9 was studied. It was found that compounds 1, 3, 4, and 6 have high cytotoxic activity and were able to lyse the erythrocytes with ED 50 of 2.03, 0.72, 2.48, and 1.68 µM, respectively (Table 4). Based on obtained results, the triterpene glycosides 1-9 from S. pacificus were used at low non-toxic concentrations of 0.1 µM for further study of their biological activity. 1.68 ± 0.14 7 4.07 ± 0.10 8 6.16 ± 0.05 9 2.85 ± 0.14 a ED 50 -The effective dose causing 50% of erythrocytes hemolysis was calculated using the computer program SigmaPlot 10.0. All experiments were made in triplicate, p < 0.01.

The Effect of the Triterpene Glycosides on Neoplastic Cell Transformation Induced by Carcinogenic Factors
The initial stage of cancer development is the neoplastic transformation of noncancerous cells into cancer ones induced by chemical (growth factors: epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), transforming growth factor β (TGF-β), formaldehydes, nitrites, peroxides, etc.), physical (ionizing radiation: UV and X-ray), and biological (oncogenic viruses, some bacteria) carcinogens [36,37]. In light of this, the investigation of effective and non-toxic cancer-preventive compounds could be a winning weapon to manage cancer initiation. The promotion-sensitive mouse epidermal cells JB6 Cl41 are known to respond irreversibly to carcinogens such as EGF, 12-Otetradecanoilphorbol 13-acetate (TPA), or UV irradiation with the induction of anchorageindependent growth in soft agar [38]. That is why this well-established culture system was used to identify the effect of the triterpene glycosides 1-9 from starfish S. pacificus on EGF-, TPA-, X-ray-, or UVB-induced neoplastic cell transformation. JB6 Cl41 cells without EGF or TPA treatment did not form colonies in soft agar (negative control), while under the action of EGF (1 ng/mL) or TPA (10 ng/mL), they were transformed and formed colonies (positive control) ( Figure 4A,B). Compounds 1, 3, 4, 6, and 9 at a non-toxic concentration of 0.1 µM significantly inhibited EGF-induced neoplastic transformation of JB6 Cl41 cells by 94%, 70%, 92%, 44%, and 34%, respectively, ( Figure 4A) or TPA-induced neoplastic transformation by 98%, 36%, 99%, 61%, and 42%, respectively, compared with the positive control ( Figure 4B).
The percentage of inhibition of neoplastic cell transformation of JB6 Cl41 cells of compounds 2, 5, 7, and 8 was 25%, 16%, 20%, and 10% for EGF-induced cell transformation and 18%, 17%, 33%, and 20% for TPA-induced cell transformation compared to the positive control ( Figure 4A,B). People are exposed to both natural sources of ionizing radiation (UV, soil, water, plants) and artificial sources (X-rays and medical devices). As the use of ionizing radiation increases, the potential for health hazards also increases. It was shown that low doses of ionizing radiation (X-rays and UV) can increase the risk of longer-term effects such as cancer, especially skin cancer.
or TPA treatment did not form colonies in soft agar (negative control), while under the action of EGF (1 ng/mL) or TPA (10 ng/mL), they were transformed and formed colonies (positive control) ( Figure 4A,B). Compounds 1, 3, 4, 6, and 9 at a non-toxic concentration of 0.1 μM significantly inhibited EGF-induced neoplastic transformation of JB6 Cl41 cells by 94%, 70%, 92%, 44%, and 34%, respectively, ( Figure 4A) or TPA-induced neoplastic transformation by 98%, 36%, 99%, 61%, and 42%, respectively, compared with the positive control ( Figure 4B). The culture was maintained at 37 °C in a 5% CO2 atmosphere for 2 weeks. The colonies were counted under a microscope with the aid of the ImageJ software program (n = 9 for control and each compound; n-quantity of photos). The magnification of representative photos of colonies is ×10. The asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001) indicate a significant decrease of colony formation in cells treated with compound compared with the EGF-or TPAtreated cells.
The percentage of inhibition of neoplastic cell transformation of JB6 Cl41 cells of compounds 2, 5, 7, and 8 was 25%, 16%, 20%, and 10% for EGF-induced cell transformation and 18%, 17%, 33%, and 20% for TPA-induced cell transformation compared to the positive control ( Figure 4A,B). People are exposed to both natural sources of ionizing radiation (UV, soil, water, plants) and artificial sources (X-rays and medical devices). As the use of ionizing radiation increases, the potential for health hazards also increases. It was shown that low doses of  1 µM) in agar mix. The culture was maintained at 37 • C in a 5% CO 2 atmosphere for 2 weeks. The colonies were counted under a microscope with the aid of the ImageJ software program (n = 9 for control and each compound; n-quantity of photos). The magnification of representative photos of colonies is ×10. The asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001) indicate a significant decrease of colony formation in cells treated with compound compared with the EGF-or TPA-treated cells.
In this work, we hypothesized that the triterpene glycosides from starfish S. pacificus were able to prevent the neoplastic cell transformation induced by low doses of X-ray and UVB radiation. The optimal doses and time of cells treatment were experimentally chosen, so in a soft agar the formation and growth of colonies of JB6 Cl41 cells were observed under the irradiation of cells by 0.5 Gy of X-rays or by 0.3 J/cm 2 of UVB (wavelength 312 nm) three times in week for three weeks ( Figure 5A,B). The triterpene glycosides 1 and 4 at 0.1 µM were found to almost completely suppress X-ray-induced cell transformation by 94% and 97%, respectively. Compounds 3, 6, and 9 at the same concentration inhibited JB6 Cl41 colonies formation by 26%, 23%, and 28%, respectively ( Figure 5A). Other investigated compounds, 2, 5, 7, and 8, possessed slight cancer-preventive activity; the percentage of inhibition was less than 15% ( Figure 5A). It was shown that the inhibiting effect of compounds 1-9 on UVB-induced neoplastic cell transformation was weaker than on X-rayinduced cell transformation. The most effective compounds, 1 and 4 (0.1 µM), decreased the colonies number by 96% and 86%, respectively, while compounds 3, 6, and 9 inhibited JB6 Cl41 cell transformation induced by UVB by 13%, 10%, and 11%, respectively. Triterpene glycosides 2, 5, 7, and 8 at the same concentration were not effective in the prevention of UVB-induced neoplastic cell transformation. tigated compounds, 2, 5, 7, and 8, possessed slight cancer-preventive activity; the percentage of inhibition was less than 15% ( Figure 5A). It was shown that the inhibiting effect of compounds 1-9 on UVB-induced neoplastic cell transformation was weaker than on Xray-induced cell transformation. The most effective compounds, 1 and 4 (0.1 µ M), decreased the colonies number by 96% and 86%, respectively, while compounds 3, 6, and 9 inhibited JB6 Cl41 cell transformation induced by UVB by 13%, 10%, and 11%, respectively. Triterpene glycosides 2, 5, 7, and 8 at the same concentration were not effective in the prevention of UVB-induced neoplastic cell transformation. 3% basal medium Eagle (BME's) agar containing 10% FBS and overlaid with 3.5 mL of 0.5% BME's agar containing 10% FBS. The culture was maintained at 37 °C in a 5% CO2 atmosphere for 2 weeks. The colonies were counted under a microscope with the aid of the ImageJ software program (n = 9 for control and each compound; n-quantity of photos). The magnification of representative photos of colonies is ×10. The asterisks (* p < 0.05, *** p < 0.001) Figure 5. The effect of the triterpene glycosides 1-9 from S. pacificus on neoplastic cells transformation of JB6 Cl41 induced by ionizing radiation. JB6 Cl41 cells (2.4 × 10 4 /mL) treated with/without (A) X-rays (0.5 Gy/9 times) and investigated compound (0.1 µM) or (B) UVB (0.3 mJ/cm 2 ) or investigated compound (0.1 µM) in 1 mL of 0.3% basal medium Eagle (BME's) agar containing 10% FBS and overlaid with 3.5 mL of 0.5% BME's agar containing 10% FBS. The culture was maintained at 37 • C in a 5% CO 2 atmosphere for 2 weeks. The colonies were counted under a microscope with the aid of the ImageJ software program (n = 9 for control and each compound; n-quantity of photos). The magnification of representative photos of colonies is ×10. The asterisks (* p < 0.05, *** p < 0.001) indicate a significant decrease of colony formation in cells treated with compound compared with the X-ray-or UVB-treated cells.
To the best of our knowledge, an investigation of the inhibition of the EGF-, TPA-, X-ray-, and UVB-induced neoplastic cell transformation of JB6 Cl41 cells by triterpene glycosides has been described here for the first time.
Next, we determined the influence of the investigated triterpene glycosides on the colony formation of human melanoma cells SK-MEL-2. As in the experiments described above, compounds 1 and 4 at a non-toxic concentration of 0.1 µM possessed the highest activity among the investigated compounds and decreased the colonies number of SK-MEL-2 cells by 64% and 70%, respectively; compounds 3 and 6 decreased the number at the same concentration by 34% and 40%, respectively, while compounds 2, 5, 7, and 8 inhibited colony formation by 3%, 13%, 15%, and 3%, respectively ( Figure 6A,B).
Next, we determined the influence of the investigated triterpene glycosides on the colony formation of human melanoma cells SK-MEL-2. As in the experiments described above, compounds 1 and 4 at a non-toxic concentration of 0.1 µ M possessed the highest activity among the investigated compounds and decreased the colonies number of SK-MEL-2 cells by 64% and 70%, respectively; compounds 3 and 6 decreased the number at the same concentration by 34% and 40%, respectively, while compounds 2, 5, 7, and 8 inhibited colony formation by 3%, 13%, 15%, and 3%, respectively ( Figure 6A,B). The culture was maintained at 37 °C in a 5% CO2 atmosphere for 2 weeks. The colonies were counted under a microscope with the aid of the ImageJ software program (n = 9 for control and each compound; n-quantity of photos). The magnification of representative photos of colonies is ×10. The asterisks (* p < 0.05, *** p < 0.001) indicate a significant decrease of colony formation in cells treated with compound compared with the X-ray-or UVB-treated cells.
We were able to isolate nine compounds (1-9), which consist of three types of triterpene aglycons and three types of oligosaccharide chains. It was found that compounds 1 and 4 were the most active compounds in all test systems, compounds 3, 6, and 9 possessed moderate activities, and compounds 2, 5, 7, and 8 are not active. This indicates that the biological activity of triterpene glycosides depends both on the structure of the triterpene aglycon (in particular, the side chain) and on the structure of the carbohydrate chain. Indeed, the most active compounds, 1 and 4, have trans-and cis-Δ 22,24 -lanostane side chains in triterpene aglycon and a pentasaccharide chain with terminal 3-O-methyl-D-glucose. The least active compounds, 2, 5, 7, and 8, have a tetrasaccharide chain without a terminal 3-O-methyl-monosaccharide (2 and 5) or non-holostane type of aglycon (23,24,25,26,27pentanorlanosta-7,20(22)-diene-18(16)-lactone-3β-ol (7 and 8)), respectively. It is obvious that such structural changes lead to an almost complete loss of biological activity. While  1 µM) in agar mix. The culture was maintained at 37 • C in a 5% CO 2 atmosphere for 2 weeks. The colonies were counted under a microscope with the aid of the ImageJ software program (n = 9 for control and each compound; n-quantity of photos). The magnification of representative photos of colonies is ×10. The asterisks (* p < 0.05, *** p < 0.001) indicate a significant decrease of colony formation in cells treated with compound compared with the X-ray-or UVB-treated cells.
We were able to isolate nine compounds (1-9), which consist of three types of triterpene aglycons and three types of oligosaccharide chains. It was found that compounds 1 and 4 were the most active compounds in all test systems, compounds 3, 6, and 9 possessed moderate activities, and compounds 2, 5, 7, and 8 are not active. This indicates that the biological activity of triterpene glycosides depends both on the structure of the triterpene aglycon (in particular, the side chain) and on the structure of the carbohydrate chain. Indeed, the most active compounds, 1 and 4, have transand cis-∆ 22,24 -lanostane side chains in triterpene aglycon and a pentasaccharide chain with terminal 3-O-methyl-D-glucose. The least active compounds, 2, 5, 7, and 8, have a tetrasaccharide chain without a terminal 3-O-methyl-monosaccharide (2 and 5) or non-holostane type of aglycon (23,24,25,26,27-pentanorlanosta-7,20(22)-diene-18(16)-lactone-3β-ol (7 and 8)), respectively. It is obvious that such structural changes lead to an almost complete loss of biological activity. While compounds 3, 6, and 9 consisted of a tetrasaccharide chain with terminal 3-O-methyl-D-xylose, they demonstrated moderate activity regardless of the structure of the triterpene aglycon.

Colony Formation of Cancer Cells
SK-MEL-2 cells (2.4 × 10 4 ) were treated either with DMSO (control) or the triterpene glycosides 1-9 from S. pacificus (0.1 µM) in 1 mL of agar mix. After 14 days, colonies of cancer cells were scored as described above.

Statistical Analysis
All of the assays were performed in at least three independent experiments. Results are expressed as the mean ± standard deviation (SD). The Student's t-test was used to evaluate the data with the following significance levels: * p < 0.05, ** p < 0.01, *** p < 0.001.

Conclusions
Thus, nine triterpene glycosides, including eight new pacificusosides D-K, were isolated from the Far Eastern starfish S. pacificus, and their chemical structures were established. The glycosides with a tetrasaccharide carbohydrate chain containing a 6-O-SO 3 -glucopyranose residue, as in pacificusosides F, H, and K, were not earlier found in echinoderms. Pacificusosides E, G, and J have a very rare tetrasaccharide carbohydrate chain without 3-O-methyl-D-glucose or 3-O-methyl-D-xylose as a terminal monosaccharide unit.
The isolation of a series of new triterpene glycosides from starfish is a rare case. Pacificusosides D-K have a structural similarity to the triterpene glycosides obtained from the sea cucumber E. fraudatrix earlier. Most of these glycosides contain 3-O-methyl-Dxylose or 3-O-methyl-D-glucose as a terminal monosaccharide unit. These monosaccharide residues are characteristic for the triterpene glycosides from sea cucumber E. fraudatrix [34]. Previously, we suggested that the triterpene glycosides from the starfish S. pacificus are food markers because they can be obtained by starfish through diet [28]. The discovery of a series of nine triterpene glycosides confirmed our assumptions that the starfish S. pacificus feeds mainly on sea cucumbers E. fraudatrix or related species. At the same time, these glycosides from S. pacificus contain some structural features, differing them from the triterpene glycosides from these sea cucumbers, which suggests the participation of the enzymes of this starfish in the metabolism of dietary compounds.
The anticancer activity against human melanoma cells and the prevention of the EGF-, TPA-, X-ray-, and UVB-induced neoplastic cell transformation of JB6 Cl41 cells of triterpene glycosides were investigated for the first time. It was found that compounds 1, 3, 4, 6, and 9 possessed the highest cytotoxic effects against SK-MEL-2 cell lines with IC 50 less than 1 µM with high cancer selectivity (more than nine) . Compounds 1, 3, 4, 6, and 9 at a non-toxic concentration of 0.1 µM significantly inhibited EGF-or TPA-induced neoplastic transformation compared with positive control. Moreover, the triterpene glycosides 1 and 4 at 0.1 µM were shown to almost completely suppress X-ray-or UVB-induced cell transformation. It should be noted that the most active triterpene glycosides, 1 and 4, possessed cancer preventive and anticancer activities at concentrations much lower than their effective dose of hemolysis.
The investigated compounds (1-9) consist of three types of triterpene aglycons and three types of oligosaccharide chains, and analysis of their activity allows for revealing the structure-activity relationship. This indicates that the biological activity of triterpene glycosides depends both on the structure of the triterpene aglycon and on the structure of the carbohydrate chain. Indeed, the most active compounds, 1 and 4, have transand cis-∆ 22,24 -lanostane side chains in triterpene aglycon and a pentasaccharide chain with terminal 3-O-methyl-D-glucose. The least active compounds, 2, 5, and 7, in these rows have a tetrasaccharide chain without a terminal 3-O-methyl-monosaccharide (2 and 5) or non-holostane type of aglycon (7), respectively. As a result, our studies of triterpene glycosides from S. pacificus not only revealed the compound leaders (1 and 4), which have potent cancer-preventive and anticancer activities, but also brought to light their structure-activity relationships.
Institutional Review Board Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding authors.