New triterpene glycosides from the far eastern starfish solaster pacificus and their biological activity

biomolecules

Article

New Triterpene Glycosides from the Far Eastern Starﬁsh

Solaster paciﬁcus and Their Biological Activity

Timofey V. Malyarenko ^1,2,*, Alla A. Kicha ¹, Anatoly I. Kalinovsky ¹, Pavel S. Dmitrenok ¹

,

Olesya S. Malyarenko ¹, Alexandra S. Kuzmich ¹, Valentin A. Stonik ^1,2and Natalia V. Ivanchina ¹

1

G.B. Elyakov Paciﬁc Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of

Sciences, Pr. 100-let Vladivostoku 159, 690022 Vladivostok, Russia; kicha@piboc.dvo.ru (A.A.K.);

kaaniw@piboc.dvo.ru (A.I.K.); paveldmt@piboc.dvo.ru (P.S.D.); malyarenko.os@gmail.com (O.S.M.);

assavina@mail.ru (A.S.K.); stonik@piboc.dvo.ru (V.A.S.); ivanchina@piboc.dvo.ru (N.V.I.)

2

Department of Bioorganic Chemistry and Biotechnology, School of Natural Sciences,

Far Eastern Federal University, Sukhanova str. 8, 690000 Vladivostok, Russia

*

Correspondence: malyarenko-tv@mail.ru; Tel.: +7-423-2312-360; Fax: +7-423-2314-050

Abstract: Three new triterpene glycosides, paciﬁcusosides A–C (

triterpene glycosides, cucumariosides C₁( ), C₂( ), and A₁₀), were isolated from the alcoholic

extract of the Far Eastern starﬁsh Solaster paciﬁcus. The structures of were elucidated by extensive

NMR and ESIMS techniques and chemical transformations. Compound has a novel, unique

1–3), and three previously known

4

5

(6

1–3

1

structure, containing an aldehyde group of side chains in its triterpene aglycon. This structural

fragment has not previously been found in the sea cucumber triterpene glycosides or starﬁsh steroidal

ꢀꢁꢂꢀꢃꢄꢅꢆꢇ

glycosides. Probably, paciﬁcusoside A (

through dietary means from a sea cucumber in the starﬁsh. Another two new triterpene glycosides

) have closely related characteristics to sea cucumber glycosides. The cytotoxicity of compounds

was tested against human embryonic kidney HEK 293 cells, colorectal carcinoma HT-29 cells,

melanoma RPMI-7951 cells, and breast cancer MDA-MB-231 cells using MTS assay. Compounds

revealed the highest cytotoxic activity against the tested cell lines, while the other investigated

compounds had moderate or slight cytotoxicity. The cytotoxic effects of were reduced by

cholesterol like the similar effects of the previously investigated individual triterpene glycosides.

Compounds , and almost completely suppressed the colony formation of the HT-29, RPMI-7951,

1) is a product of the metabolism of the glycoside obtained

ꢀꢁꢂꢃꢄꢅꢆ

Citation: Malyarenko, T.V.; Kicha,

A.A.; Kalinovsky, A.I.; Dmitrenok,

P.S.; Malyarenko, O.S.; Kuzmich, A.S.;

Stonik, V.A.; Ivanchina, N.V. New

Triterpene Glycosides from the Far

Eastern Starﬁsh Solaster paciﬁcus and

Their Biological Activity. Biomolecules

2021, 11, 427. https://doi.org/

10.3390/biom11030427

(2, 3

1–6

4–6

2–6

3,

4

5

and MDA-MB-231 cells at a nontoxic concentration of 0.5 µM.

Academic Editor: Elena Leychenko

Keywords: triterpene glycosides; starﬁsh; Solaster paciﬁcus; biosynthesis; food markers; cytotoxic

activity; colony formation

Received: 16 February 2021

Accepted: 11 March 2021

Published: 14 March 2021

1. Introduction

Publisher’s Note: MDPI stays neutral

with regard to jurisdictional claims in

published maps and institutional afﬁl-

iations.

The phylum Echinodermata includes ﬁve classes: Holothuroidea (sea cucumbers),

Asteroidea (starﬁsh), Echinoidea (sea urchins), Crinoidea (sea lilies), and Ophiuroidea

(brittle stars and basket stars). These invertebrates are a rich source of a variety of low

molecular metabolites with potent biological activities: triterpene glycosides, peptides, fatty

acids, polar steroids and their glycosides, carotenoids, quinones, spinochromes pigments,

and also sphingolipids and their derivatives [1].

Triterpene glycosides are considered to be characteristic secondary metabolites and chemo-

taxonomic markers of sea cucumbers. They are amphiphilic compounds consisting of lipophilic

Copyright:

Licensee MDPI, Basel, Switzerland.

This article is an open access article

distributed under the terms and

conditions of the Creative Commons

Attribution (CC BY) license (https://

creativecommons.org/licenses/by/

4.0/).

aglycon and a hydrophilic oligosaccharide chain [

type of aglycons having lanostane-3 -ol with (18,20)-lactone in the E-ring of the pentacyclic

triterpene core. Some triterpene glycosides contain nonholostane aglycons [ ]. As a rule, their

2 ]. Usually, these glycosides contain holostane

β

3

carbohydrate chains include five or six monosaccharides such as D-xylose, D-quinovose, and

D-glucose or their methylated or sulfated derivatives attached to C-3 of the aglycon. In the car-

bohydrate chains, the first sugar unit is always xylose, whereas 3-O-methyl-D-glucose and/or

Biomolecules 2021, 11, 427. https://doi.org/10.3390/biom11030427

https://ww w .mdpi.com/journal/biomolecules

Biomolecules 2021, 11, 427

2 of 15

3-O-methyl-D-xylose are predominantly the terminal monosaccharide units. These glycosides

have been reported to exhibit a wide spectrum of biological activities such as cytotoxic [

tifungal [ ], bactericidal, hemolytic, antiviral, and antiparasitic properties [ ]. Some triterpene

glycosides were found to induce apoptosis and inhibit the growth of cancer cells [ ], and show

immunomodulatory properties [10 ].

4 ,5 ], an-

6 ,7

8

9

At the same time, polar steroids and their glycosides are characteristic secondary metabo-

lites of starfish (sea stars). The starfish steroidal glycosides may be divided into three major

classes: glycosides of polyhydroxysteroids, cyclic glycosides, and asterosaponins [11 –19 ]. Poly-

hydroxysteroidal glycosides from starfish have highly oxygenated steroidal aglycon. In most

cases, monosaccharides of these compounds are represented by D-xylose and L-arabinose

or their methylated or sulfated derivatives, forming monoside, bioside and/or sometimes

trioside carbohydrate chains. Cyclic glycosides have several unusual structural features in-

cluding a trisaccharide carbohydrate chain cyclized between C-3 and C-6 of the

dihydroxysteroidal aglycon as well as the presence of a glucuronic acid unit in the carbohydrate

moiety. The majority of asterosaponins contain 3-O-sulfonated ,6 -dihydroxysteroidal

⁹⁽¹¹⁾-3

∆ β,6β-

⁷-3

∆

β α

aglycon and carbohydrate chains, comprising usually five or six monosaccharide residues

attached at the C-6 position of an aglycon.

It is worth noting that triterpene glycosides are not typical secondary metabolites of starfish.

Earlier only two new triterpene glycosides, rollentosides A and B were isolated from the starfish

Asterias rollestoni Bell, collected in the Yellow Sea near the shore of Jiangsu Province [20 ]. In

addition, one previously known triterpene glycoside, holothurin A₂(= echinoside A), was

isolated from the tropical starfish Choriaster granulatus [21 ]. In all other starfish triterpene

glycosides were not found.

Herein, we report the results of our studies on triterpene glycosides from the ethanolic

extract of the Far Eastern carnivorous starfish Solaster pacificus (order Valvatida, family Solas-

teridae) collected in the Sea of Okhotsk near Iturup Island. As a result, we have established

the structures of three new triterpene glycosides, pacificusosides A–C (

diet of this starfish. Moreover, we examined the cytotoxic activity of the individual compounds

or in combination with cholesterol in human embryonic kidney, colorectal carcinoma,

1–3), derived from the

1–6

melanoma, and breast cancer cells. The effects of these compounds on the colony formation of

the tested cancer cells were investigated using soft agar assay.

2. Materials and Methods

2.1. General Methods

Optical rotations were determined on a PerkinElmer 343 polarimeter (PerkinElmer, Waltham,

MA, USA). UV spectra were recorded on a Shimadzu UV-1601 PC spectrophotometer (Shi-

madzu, Kioto, Japan). IR spectra were recorded using a Bruker Equinox 55 spectrophotometer

(Bruker, Göttingen, Germany). The ¹H and ¹³C NMR spectra were otained on Bruker Avance III

700 spectrometer (Bruker BioSpin, Bremen, Germany) at 700.13 and 176.04 MHz, respectively,

chemical shifts were referenced to the corresponding residual solvent signals (δ_H3.30/δ_C49.0 for

CD₃OD). The HRESIMS spectra were recorded on a Bruker Impact II Q-TOF mass spectrometer

(Bruker, Bremen, Germany); the samples were dissolved in MeOH (c 0.001 mg/mL). HPLC

separations were carried out on an Agilent 1100 Series chromatograph (Agilent Technologies,

Santa Clara, CA, USA) equipped with a differential refractometer; Diasfer-110-C18 (10

250 × 15 mm, Biochemmack, Moscow, Russia) and Discovery C₁₈(5 m, 250 4 mm, Supelco,

North Harrison, PA, USA) columns were used. Low-pressure liquid column chromatography

was carried out with Polychrom-1 (powdered Teflon, 0.25 0.50 mm; Biolar, Olaine, Latvia), Si

gel KSK (50–160 µm, Sorbpolimer, Krasnodar, Russia), and Florisil (60–100 m, Sigma Aldrich,

m, Sorbpolimer, Krasnodar, Russia)

µm,

µ

×

−

µ

St. Louis, MO, USA). Sorbfil Si gel plates (4.5

were used for thin-layer chromatography.

×

6.0 cm, 5–17 µ

2.2. Animal Material

Specimens of S. paciﬁcus Djakonov, 1938 (order Valvatida, family Solasteridae) were

collected at a depth of 10 m–20 m by scuba diving in the Sea of Okhotsk near Iturup Island

Biomolecules 2021, 11, 427

3 of 15

during the 42nd scientiﬁc cruise of the research vessel Akademik Oparin in August 2012.

Species identiﬁcation was carried out by Mr. B.B. Grebnev (G.B. Elyakov Paciﬁc Institute

of Bioorganic Chemistry of the FEB RAS, Vladivostok, Russia). A voucher specimen (no.

042–112) is on deposit at the marine specimen collection of the G.B. Elyakov Paciﬁc Institute

of Bioorganic Chemistry of the FEB RAS, Vladivostok, Russia.

2.3. Extraction and Isolation

◦

The fresh animals (1.7 kg) were chopped and extracted twice with EtOH at 20 C.

The H₂O/EtOH layer was evaporated, and the residue was dissolved in H₂O (0.5 L). The

H₂O-soluble materials were passed through a Polychrom-1 column (6.5

with distilled H₂O until a negative chloride ion reaction was obtained, and eluted with

EtOH. The combined EtOH eluate was evaporated to give a brownish residue (48 g). These

×

21 cm), eluted

materials were chromatographed over a Si gel column (6

×

22 cm) using CHCl₃/EtOH

(stepwise gradient, 8:1 to 1:1), EtOH, and EtOH/H₂O (4:1, 2:1, and 1:2) to yield ﬁfteen

fractions, 1–15, that were then analyzed by TLC on Si gel plates in the eluent system

BuOH/EtOH/H₂O (4:1:2). Fractions 4 (11.49 g) and 5 (22.79 g) were additionally chro-

matographed over a Florisil column (4.5

5:1 to 1:1) to yield three fractions, 41 (1.459 g), 42 (1.13 g), and 51 (652 mg). HPLC separation

of fractions 41, 42, and 51 on a Diasfer-110-C18 column (10 m, 250 15 mm, 2.5 mL/min)

with EtOH/H₂O (55:45) as an eluent system yielded pure 2 (31 mg, t_R25.8 min), 3 (13.0 mg

×

12 cm) using CHCl₃/EtOH (stepwise gradient,

µ

×

,

t_R91.5 min), 4 (89.0 mg, t_R57.8 min), 5 (59 mg, t_R71.2 min), and subfractions 41-3 (30 mg)

and 42-1 (94.5 mg). HPLC separation of subfractions 41-3 and 42-1 on a Discovery C18

column (5

µ

m, 250

×

10 mm, 2.5 mL/min) with EtOH/H₂O (50:50) as an eluent system

yielded pure 1 (12.5 mg, t_R6.6 min) and 6 (3.0 mg, t_R16.3 min).

2.4. Spectral Data of New Compounds

Pacificusoside A (1), C₅₇H₈₆O₂₇, white amorphous powder; [α]_D

²⁵—27.7 (c 0.23, MeOH);

UV (MeOH) λ_max199, 224 nm; IR (KBr): ν_max= 3438, 2927, 1745, 1688, 1633, 1422, 1384, 1243,

1160, 1113, 1059, 894 cm⁻¹; ¹H and ¹³C NMR data are listed in Tables 1 and 2 ; (+)ESIMS/MS of

the ion [M+Na]⁺at m/z 1225: 1093 [(M+Na)–C₅H₈O₄]⁺; 947 [(M+Na)–C₆H₁₀O₄–C₅H₈O₄]⁺; 759

[(carbohydrate chain+Na)]⁺; 609 [(carbohydrate chain+Na)–C₅H₈O₄–H₂O]⁺; 477 [(carbohydrate

chain+Na)–2

×

C₅H₈O₄–H₂O]⁺; 331 [(carbohydrate chain+Na)–2 C₅H₈O₄–H₂O–C₆H₁₀O₄]⁺;

×

185 [C₆H₁₀O₅+Na]⁺; 169 [C₆H₁₀O₄+Na]⁺; (+)HRESIMS m/z 1225.5273 [M+Na]⁺(calcd for

[C₅₇H₈₆O₂₇Na]⁺, 1225.5249); (–)ESIMS/M₋S of the ion [M–H]⁻at m/z 1201: 1069 [(M–H)–

−

C₅H₈O₄] ; 923 [(M–H)–C₆H₁₀O₄–C₅H₈O₄] ; 761 [(M–H)–C₆H₁₀O₅–C₆H₁₀O₄–C₅H₈O₄] ; 615

[(M–H)–C₆H₁₀O₅–2

(–)HRESIMS m/z 1201.5318 [M–H]⁻(calcd for [C₅₇H₈₅O₂₇]⁻, 1201.5284).

Paciﬁcusoside B ( ), C₅₃H₈₂O₂₄, white amorphous powder; [

²⁵—28.0 (c 0.1, MeOH);

×

C₆H₁₀O₄–C₅H₈O₄]⁻; 483 [(M–H)–C₆H₁₀O₅–2

×

C₆H₁₀O₄–2

×

C₅H₈O₄]⁻;

2

α]

D

IR (KBr): ν_max= 3427, 2926, 2360; 1772, 1635, 1558, 1541, 1384, 1507, 1457, 1384, 1180,

1

1074 cm⁻¹; H and ¹³C NMR data are listed in Tables 1 and 2; (+)ESIMS/MS of the ion

[M+Na]⁺at m/z 1125: 993 [(M+Na)–C₅H₈O₄]⁺; 979 [(M+Na)–C₆H₁₀O₄]⁺; 847 [(M+Na)–

C₆H₁₀O₄–C₅H₈O₄]⁺; 817 [(M+Na)–C₆H₁₀O₄–C₆H₁₀O₅]⁺; 759 [(carbohydrate chain+Na]⁺;

609 [(carbohydrate chain+Na)–C₅H₈O₄–H₂O]⁺; 477 [(carbohydrate chain+Na)–2

×

C₅H₈O₄–

H₂O]⁺; 331 [(carbohydrate chain+Na)–2

×

C₅H₈O₄–H₂O–C₆H₁₀O₄]⁺; 185 [C₆H₁₀O₅+Na]⁺;

169 [C₆H₁₀O₄+Na]⁺; (+)HRESIMS m/z 1125.5091 [M+Na]⁺(calcd for [C₅₃H₈₂O₂₄Na]⁺,

1125.5088); (–)ESIMS/MS of the ion [M–H]⁻at m/z 1101: 969 [(M–H)–C₅H₈O₄]⁻; 955 [(M–

H)–C₆H₁₀O₄]⁻; 823 [(M–H)–C₆H₁₀O₄–C₅H₈O₄]⁻; 793 [(M–H)–C₆H₁₀O₄–C₆H₁₀O₅]⁻; 661

[(M–H)–C₆H₁₀O₅–C₆H₁₀O₄–C₅H₈O₄]⁻; 515 [(M–H)–C₆H₁₀O₅–2 C₆H₁₀O₄–C₅H₈O₄]⁻;

×

(–)HRESIMS m/z 1101.5119 [M–H]⁻(calcd for [C₅₃H₈₁O₂₄]⁻, 1101.5123).

Pacificusoside C (3), C₆₀H₉₄O₂₆, white amorphous powder; [α]_D

²⁵—9.0 (c 0.23, MeOH); IR

(KBr): ν_max= 3427, 2927, 2854; 1745, 1635, 1457, 1384, 1238, 1180, 1065 cm⁻¹; ¹H and ¹³C NMR

data are listed in Tables 1 and 2 ; (+)ESIMS/MS of the ion [M+Na]⁺at m/z 1253: 1121 [(M+Na)–

C₅H₈O₄]⁺; 759 [(carbohydrate chain+Na]⁺; 609 [(carbohydrate chain+Na)–C₅H₈O₄–H₂O]⁺;

477 [(carbohydrate chain+Na)–2

×

C₅H₈O₄–H₂O]⁺; 331 [(carbohydrate chain+Na)–2

C₅H₈O₄–

×

Biomolecules 2021, 11, 427

4 of 15

H₂O–C₆H₁₀O₄]⁺; 185 [C₆H₁₀O₅+Na]⁺; 169 [C₆H₁₀O₄+Na]⁺; (+)HRESIMS m/z 1253.5920

−

[M+Na]⁺(calcd for [C₆₀H₉₄O₂₆Na]⁺, 1253.5926); (–)ESIMS/MS of the ion [M–H] at m/z 1229:

−

1097 [(M–H)–C₅H₈O₄] ; 1083 [(M–H)–C₆H₁₀O₄] ; 951 [(M–H)–C₆H₁₀O₄–C₅H₈O₄] ; 921 [(M–

−

H)–C₆H₁₀O₄–C₆H₁₀O₅] ; 789 [(M–H)–C₆H₁₀O₅–C₆H₁₀O₄–C₅H₈O₄] ; 643 [(M–H)–C₆H₁₀O₅–

C₆H₁₀O₄–C₅H₈O₄]⁻; 511 [(M–H)–C₆H₁₀O₅–2 C₅H₈O₄]⁻; (–)HRESIMS m/z

C₆H₁₀O₄–2

1229.5952 [M–H]⁻(calcd for [C₆₀H₉₃O₂₆]⁻, 1229.5961).

2×

×

1

◦

Table 1. H and ¹³C NMR data of aglycon part of compounds 1–3 (35 C, C₅D₅N, J in Hz) ^a.

Position

1

2

3

δ_H

δ_C

δ_H

δ_C

δ_H

δ_C

1

2

1.49 m

2.21 m

1.96 m

3.35 dd

(11.6, 4.1)

-

36.3

27.2

1.48 m

2.16 m

1.89 m

3.31 dd

(11.7, 3.8)

-

1.00 dd

(11.9, 3.6)

2.07 m

1.97 m

5.64 brd

(7.1)

35.9

26.9

1.48 m

2.19 m

1.94 m

3.34 dd

(11.5, 3.9)

-

36.4

27.3

3

4

5

89.1

39.7

48.1

88.9

39.4

47.5

89.3

39.8

48.3

1.06 m

2.08 m

5.68 m

1.06 m

2.06 m

1.93 m

6

23.4

23.3

23.6

7

8

121.1

145.3

47.4

122.6

147.4

46.4

5.68 brs

120.7

145.9

47.4

-

3.44 brd

(14.2)

-

1.88 m

1.60 m

2.15 m

2.02 m

-

3.48 brd

(13.5)

-

1.81 m

1.55 m

2.16 m

2.00 m

-

3.00 brd

(14.6)

-

2.00 m

1.47 m

2.37 m

1.88 m

-

9

10

11

35.8

35.5

35.8

22.5

21.7

22.8

12

30.7

20.0

31.6

13

14

59.1

48.2

56.7

46.0

59.5

47.7

-

2.16 d

(13.3)

1.97 dd

(13.3, 2.2)

4.74 brd

(2.2)

2.94 s

-

1.02 s

-

2.58 dd

(12.5, 7.2)

1.70 m

2.63 dd

(12.4, 7.4)

1.75 m

15

43.3

43.8

43.9

16

5.81 q (8.3)

73.6

80.4

5.96 q (8.7)

75.2

17

18

19

20

21

3.01 d (8.3)

56.8

178.5

24.1

82.4

28.9

59.1

180.7

23.9

139.9

23.0

2.66 d (9.5)

-

54.8

179.7

24.2

85.2

28.5

-

1.24 s

-

1.56 s

1.23 s

-

1.75 s

1.53 s

2.57 td

(12.0, 4.4)

1.93 m

2.07 m2.01

m

7.28 d

(16.0)

5.06 s

4.98 s

22

159.4

113.9

38.9

6.46 dd

(16.0, 7.6)

23

127.6

-

23.6

24

25

26

27

30

31

32

9.78 d (7.6)

193.1

-

5.12 m

-

124.4

132.1

17.9

25.7

17.7

29.0

32.4

170.0

21.2

-

1.59 s

1.65 s

1.15 s

1.31 s

1.17 s

-

1.17 s

1.32 s

1.15 s

-

17.6

28.7

32.3

169.7

21.1

1.12 s

1.30 s

1.35 s

-

17.3

28.6

33.9

-

CO

CH₃-CO

1.86 s

-

2.01 s

a

Assignments from 700 MHz ¹H-¹H COSY, HSQC, HMBC (8 Hz), and ROESY (270 msec) data.

Biomolecules 2021, 11, 427

5 of 15

◦

Table 2. ¹H, ¹³C, HMBC, and ROESY NMR data of oligosaccharide chains of 1–3 (35 C, C₅D₅N,

J in Hz) ^a.

1–3

Position

δ_H

δ_C

HMBC

ROESY

Xyl-I

H3, H30-Agl;

H3, H5-Xyl-I

1

2

4.84 d (7.1)

105.0

83.2

C3-Agl; C5-Xyl-I

C1, C3-Xyl-I;

C1-Qui

3.91 dd (8.7, 7.1)

H4-Xyl-I

3

4

4.19 t (8.7)

4.11 m

4.32 dd (11.6, 5.4)

3.69 dd (11.6, 9.3)

77.8

70.2

C2, C4-Xyl-I

C3-Xyl-I

C1, C3-Xyl-I

H1-Xyl-I

H2-Xyl-I

5

66.5

H1-Xyl-I

Qui

H3, H5-Qui;

H2-Xyl-I

1

5.20 d (7.3)

103.0

C2-Xyl-I

C1, C3-Qui;

C1-Xyl-II

C2, C4-Qui

C3, C5, C6-Qui;

C1-Glc

2

3

4

4.11 m

4.09 m

3.61 m

82.7

75.6

86.5

H1-Qui

H1-Glc; H6-Qui

5

6

3.67 m

1.70 d (6.5)

71.0

17.9

C4-Qui

C4, C5-Qui

H1-Qui

H4-Qui

Glc

H4-Qui; H3,

H5-Glc

1

2

3

4.94 d (7.9)

4.00 m

104.7

73.8

87.1

C4-Qui

C1, C3-Glc

C2, C4-Glc

C3, C5, C6-Glc

H1-3-OMe-Xyl;

H1, H5-Glc

4.21 t (9.0)

4

5

4.05 t (9.0)

3.99 m

69.3

77.8

H1, H3-Glc

4.47 dd (11.5, 2.6)

4.20 m

6

61.7

3-OMe-Xyl

H3,

C3-Glc;

C5-3-OMe-Xyl

1

2

5.21 d (7.6)

3.94 t (8.4)

106.0

74.6

H5-3-OMeXyl;

H3-Glc

C1,

C3-3-OMe-Xyl

C2,

C4-3-OMe-Xyl;

OMe

H4-3-OMe-Xyl

H1-3-OMe-Xyl;

OMe

3

4

3.60 t (8.8)

4.08 m

87.7

69.9

H2-3-OMe-Xyl

H1-3-OMe-Xyl

H3-3-OMe-Xyl

C1,

4.21 dd (11.3, 5.5)

3.63 t (11.3)

C3-3-OMe-Xyl

C1, C3,

C4-3-OMe-Xyl

C3-3-OMe-Xyl

5

3-OMe

1

67.0

60.4

3.85 s

Xyl-II

C2-Qui;

C5-Xyl-II

H3, H5-Xyl-II;

H2-Qui

5.36 d (7.0)

105.8

2

3

4

4.04 m

4.10 t (8.1)

4.12 m

75.5

77.0

70.4

C1, C3-Xyl-II

C2, C4-Xyl-II

C3-Xyl-II

C1, C3, C4-Xyl-II

C1, C4, C5-Xyl-II

H1-Xyl-II

4.33 dd (11.7, 4.6)

3.65 dd (11.7, 9.6)

5

66.9

H1-Xyl-II

a

Assignments from 700 MHz ¹H-¹H COSY, HSQC, HMBC (8 Hz), and ROESY (270 msec) data.

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2.5. Acid Hydrolysis and Determination of Absolute Conﬁgurations of Monosaccharides

The acid hydrolysis of

acid (TFA) (1 mL) in a sealed vial on a H₂O bath at 100 C for 2 h. The H₂O layer was

1

(3.5 mg) was carried out in a solution of 2 M triﬂuoroacetic

◦

washed with CHCl₃(3

TFA and 0.5 mL of L-(–)-2-octanol (Sigma Aldrich) were added to the sugar mixture, and

the sealed vial was heated on a glycerol bath at 130 C for 6 h. The solution was evaporated

×

1.0 mL) and concentrated in vacuo. One drop of concentrated

◦

in vacuo and treated with a mixture of pyridine/acetic anhydride (1:1, 0.6 mL) for 24 h

at room temperature. The acetylated 2-octylglycosides were analyzed by GC using the

corresponding authentic samples prepared by the same procedure. The following peaks

were detected in the hydrolysate of 1: D-quinovose (t_R20.32, 20.55, 21.91, and 21.13 min),

D-xylose (t_R20.75, 20.91, and 21.19 min), D-glucose (t_R24.24, 24.86, 25.10, and 25.38 min),

and 3-OMe-D-xylose (t_R19.33, 19.57, 19.84, and 20.00 min). The retention times of the

authentic samples were as follows: D-quinovose (t_R20.30, 20.50, 20.86, and 21.13 min),

D-xylose (t_R20.70, 20.89, and 21.15 min), D-glucose (t_R24.23, 24.83, 25.06, and 25.37 min),

3-OMe-D-xylose (t_R19.32, 19.57, 19.86, and 20.02 min), L-quinovose (t_R20.12, 20.60, and

21.17 min), L-xylose (t_R20.57, 21.03, and 21.34 min), L-glucose (t_R24.39, 24.63, 24.83, and

25.06 min), and 3-OMe-L-xylose (t_R18.92, 19.50, and 20.10 min).

2.6. Anticancer Activity

2.6.1. Reagents

Phosphate buffered saline (PBS), L-glutamine, penicillin-streptomycin solution

(

10,000 U/mL, 10 µg/mL), dimethyl sulfoxide (DMSO), BuOH, 99.9% (pure p.a.), and

cholesterol, 99.5% were from Sigma-Aldrich. MTS reagent (3-(4,5-dimethylthiazol-2-

yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) was purchased

from Promega (Madison, WI, USA). The Basal Medium Eagle (BME), Minimum Essen-

tial Medium Eagle (MEM), Dulbecco’s Modified Eagle’s Medium (DMEM), McCoy’s

5A Modified Medium (McCoy’s 5A), trypsin, fetal bovine serum (FBS), and agar were

purchased from Thermo Fisher Scientific (Waltham, MA, USA). Organic solvents,

inorganic acids, and salts were commercial products (Laverna, Moscow, Russia).

2.6.2. Cell Lines and Culture Conditions

Human embryonic kidney HEK 293 cells (ATCC^®no. CRL-1573

noma HT-29 cells (ATCC^®no. HTB-38 ), melanoma RPMI-7951 cells (ATCC^®no. HTB-66),

and breast cancer MDA-MB-231 cells (ATCC^®HTB-26

) were obtained from the Amer-

™), colorectal carci-

™

ican Type Culture Collection (Manassas, VA, USA). HEK 293, HT-29, RPMI-7951, and

MDA-MB-231cells were cultured in complete MEM/10% FBS, McCoy’s 5A/10% FBS, and

DMEM/10% FBS, respectively, contained 1% of penicillin-streptomycin solution. The

◦

cell cultures were maintained at 37 C in humidiﬁed atmosphere containing 5% CO₂.

Every 3–4 days cells were rinsed with PBS, detached from the tissue culture ﬂask by

0.25% trypsin/0.05 M EDTA, and 10–20% of the harvested cells were transferred to a

new ﬂask containing fresh culture media. After three passages the cells were used for

the experiments.

2.6.3. The Preparation of Solutions of Compounds

Compounds

1

–

6

were dissolved in sterile DMSO to prepare stock concentrations of

(culture medium used as

20 mM. Cells were treated with serially diluted compounds

1–6

diluent) to give the intended ﬁnal concentrations. The vehicle control is the cells treated

with equivalent volume of DMSO (less than 0.5%) for all presented experiments.

Cholesterol (10 mg/mL) was dissolved in 99.5 % BuOH and added to compounds 1–6

to get their stock concentrations of 20 mM and incubated for 12 h at room temperature. Then

samples were vacuum evaporated and dissolved in DMSO to their stock concentrations of

20 mM.

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2.6.4. Cell Viability Assay

Cells (1.0

cultured in 200

The cell monolayer was treated with compounds

at concentrations of 5, 10, 20, and 40 M in fresh appropriate culture medium for 24 h.

Cells were also treated by cisplatin (positive control) at 1, 5, 10, 50, 100 M for 24 and 48 h.

Subsequently, the cells were incubated with 15 L MTS reagent for 3 h, and the absorbance

×

10⁴) were seeded in 96-well plates (Jet Bioﬁl, Guangzhou, China) and

◦

µL of complete culture medium for 24 h at 37 C in 5% CO₂incubator.

1–6 or 1–6 combined with cholesterol

µ

of each well was measured at 490/630 nm using Power Wave XS microplate reader (BioTek,

Wynusky, VT, USA).

2.6.5. Colony Formation Assay

Human cancer cells (2.4

compounds (0.1, 0.5, and 1

×

10⁴/mL) were seeded into 6-well plate and treated with

M) or cisplatin (3 M) in 1 mL of 0.3% BME agar containing

g/mL gentamicin. The cultures were maintained

1

–6

µ

10% FBS, 2 mM L-glutamine, and 25

µ

in a 37 ^◦C, 5% CO₂incubator for 14 days, and the cell colonies were scored using a Motic

microscope AE 20 (XiangAn, Xiamen, China) and ImageJ software bundled with 64-bit

Java 1.8.0_112 (NIH, Bethesda, MD, USA).

2.6.6. Statistical Analysis

All assays were performed in at least three independent experiments. Results are

expressed as the mean

data with the following signiﬁcance levels: * p < 0.05, ** p < 0.01, *** p < 0.001.

±

standard deviation (SD). Student’s t test was used to evaluate the

3. Results

3.1. The Isolation and Structure Elucidation of Compounds 1–6 from S. paciﬁcus

The concentrated ethanol extract of S. paciﬁcus was subjected to sequential separation

by chromatography on columns with Polichrom-1, Si gel, and Florisil followed by HPLC on

semipreparative Diasfer-110-C18 and Discovery C₁₈columns to yield three new triterpene

glycosides, named as paciﬁcusosides A–C (

glycosides (Figure 1). Compounds

were identiﬁed by comparison of their ¹H and

¹³C NMR, and MS spectra with those reported for cucumariosides C₁(

), C₂( ), and A₁₀

(6), triterpene glycosides from the holothurian Eupentacta fraudatrix [22,23].

1–3), and three previously known triterpene

4–

6

4–6

4

5

Figure 1. The structures of compounds 1–6 isolated from S. paciﬁcus.

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The molecular formula of

1

was determined to be C₅₇H₈₆O₂₇from the [M+Na]⁺

sodiated molecular ion peak at m/z 1225.5273 (calcd for [C₅₇H₈₆O₂₇Na]⁺, 1225.5249) in

the (+)HRESIMS and from the [M–H]⁻ion peak at m/z 1201.5318 (calcd for [C₅₇H₈₅O₂₇]⁻,

1201.5284) in the (–)HRESIMS (Figure S1). The IR spectrum of compound

the presence of hydroxy group (3438 cm⁻¹), lactone carbonyl (1745 cm⁻¹) and oleﬁnic

1633 cm⁻¹) groups, as well as one characteristic absorption band at ν_max= 1688 cm⁻¹of

-unsaturated aldehyde group (Figure S2). The ¹H and ¹³C NMR spectroscopic data of

1 showed

(

α,β

the pentacyclic moiety of the aglycon of 1 showed the resonances of protons and carbons of

four methyl groups (δ_H1.15 s, 1.17 s, 1.24 s, 1.32 s; δ_C32.3, 17.6, 24.1, 28.7), the 7(8)-double

bond (δ_H5.68 m; δ_C121.1, 145.3), two oxygenated methine groups [δ_H3.35 dd (J = 11.6,

4.1), 5.81 q (J = 8.3); δ_C89.1, 73.6], one OAc-group (δ_H1.86 s, δ_C21.1, 169.7), and one

1

lactone carbonyl (δ_C178.5) (Table 1, Figures S4 and S5). The H-¹H COSY and HSQC

correlations attributable to 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 (Figure 2A

,

Figures S6 and S7). Key HMBC cross-peaks, such as H-3/C-30; H-5/C-10, C-19; H-6/C-10;

H-7/C-9; H-12/C-13, C-14, C-18; H-15/C-13, C-14, C-17, C-32; H-16/CO(OAc); H-17/C-18,

C-21; H₃-19/C-1, C-5, C-9, C-10; H₃-30/C-3, C-4, C-5, C-31; H₃-31/C-3, C-4, C-5, C-30; H₃-

32/C-8, C-14, C-15; H₃C(OAc)/CO(OAc) conﬁrmed the overall structure of the pentacyclic

triterpene moiety of 1 (Figure 2A, Figure S8). The key ROESY cross-peaks H-1

H-2 /H₃-19, H₃-30; H-3/H-5 ; H-5 /H₃-31; H-6 /H₃-19; H-7/H-15 ; H-9

12 /H₃-21, H₃-32; H-15 /H₃-32; H-16 /H₃-32; H-17 /H₃-21, H₃-32 showed the common

/9 /10 /13 ,14 stereochemistry of the triterpene nucleus and 3

α

/H-3, H-5

α;

β

α

β

α

β/H₃-19; H-

α

5α

β

α

β,16β-conﬁgurations

of oxygenated substituents in 1 (Figure 2B, Figure S9).

O

H

O

C

H

H₃C

H

CH₃

H

CH₃

H

OAc

H

O

H

O

RO

H₃C

H

H₃C

H

RO

1

O

H₃C

C

H

CH₂

H

CH₃

H

CH₃

H

H₃C

RO

H

H₃C

H

RO

H

2

H

O

H₃C

O

H

C

H

CH₃

H

CH₃

H

OAc

CH₃

H

O

H

H₃C

H

O

RO

H₃C

H

H₃C

H

RO

H

3

B

A

Figure 2.

aglycon part of compounds 1–3.

(A 1–3. (B) Key ROESY correlations for

) ¹H-¹H COSY and key HMBC correlations for aglycon part of compounds

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Table 1 demonstrated the presence of one methyl group (δ_H1.56 s; δ_C28.9) and

unsaturated aldehyde group [δ_H7.28 d (J = 16.0), 6.46 dd (J = 16.0, 7.6), 9.78 d (7.6); δ_C159.4,

127.6, 193.1] (Table 1, Figures S4 and S5). The existence of the -unsaturated aldehyde

group was conﬁrmed by the UV spectrum ( = 224 nm in MeOH) (Figure S3). The

α,β-

α,β

λ

max

protons sequence from H-22 to H-24, correlated with the corresponding carbon atoms

of the side chain of , was assigned using the COSY and HSQC experiments (Table 1,

1

Figures S6 and S7). The key HMBC correlations H₃-21/C-17, C-20, C-22; H-22/C-20, C-

23, C-24; H-23/C-20; and H-24/C-23 and ROESY correlation H-22/H-24 supported the

total structure of the

conﬁguration of 22(23)-double bond followed from J_22,23= 16.0 Hz. To the best of our

knowledge, compound has a novel type of triterpene aglycon, containing

²²-25,26,27-

∆

²²-24-aldo-25,26,27-trinorlanostane side chain (Figure 1 ). The trans

1

∆

trinorlanostane side chain with unique C-24 aldehyde group, never found before in the sea

cucumber triterpene glycosides or starﬁsh steroidal glycosides.

In addition to the above-mentioned signals, the ¹H NMR spectrum of

1 exhibited ﬁve

resonances in the deshielded region due to the anomeric protons of monosaccharide units

at δ_H4.84, 5.20, 4.94, 5.21, and 5.36 that correlated in the HSQC experiment with a carbon

signals at δ_C105.0, 103.0, 104.7, 106.0, and 105.8, respectively, as well as one resonance due

to an O-methyl group at δ_H3.85 that correlated in the HSQC experiment with a carbon

signal at δ_C60.4 (Table 2, Figures S4 and S5).

Along with mass spectral information, these data showed the presence of ﬁve monosac-

charide residues in the oligosaccharide moiety of 1. The existence of 6-deoxy-sugar unit

was supported by methyl doublet at δ_H1.70. The coupling constants (7.0–8.0 Hz) of the

anomeric protons were indicative of β-conﬁgurations of all glycosidic bonds. The chemical

shifts and coupling constants of H-1⁰–H-6⁰of the monosaccharide units were determined

by irradiation of anomeric protons in the 1D TOCSY experiment. The ¹H-¹H 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 S4–S9). The spectral data of the oligosaccha-

ride moiety strictly coincided with those of terminal 3-O-methyl-

-D-xylopyranosyl residues, and internal 3-O-substituted -D-glucopyranosyl, 2,4-di-O-

substituted -D-quinovopyranosyl, and 2-O-substituted -D-xylopyranosyl residues as in

β-D-xylopyranosyl and

β

the earlier reported spectra of known cucumarioside C₁from E. fraudatrix [22].

On the basis of all the above-mentioned data, the structure of pacificusoside A (1) was

determined as 3

xylopyranosyl-(1

β

-O-{3-O-methyl-

β

-D-xylopyranosyl-(1

→

3)-

β

-D-glucopyranosyl-(1

→

4)-[β-D-

→

2)]- -D-quinovopyranosyl-(1

β

→

2)- -D-xylopyranosyl}-16β

β

-acetoxy-25,26,27-

trinorholosta-7,22-dien-24-al.

The molecular formula of

2

was determined to be C₅₃H₈₂O₂₄from the [M+Na]⁺

sodiated-molecular ion peak at m/z 1125.5091 (calcd for [C₅₃H₈₂O₂₄Na]⁺, 1125.5088) in

the (+)HRESIMS and from the [M–H]⁻ion peak at m/z 1101.5119 (calcd for [C₅₃H₈₁O₂₄]⁻,

1101.5123) in the (–)HRESIMS (Figure S10). The IR spectrum of compound

2 showed

the presence of hydroxy group (3427 cm⁻¹), lactone carbonyl (1772 cm⁻¹), and oleﬁnic

1

(1635 cm⁻¹) groups (Figure S11). The H and ¹³C NMR spectroscopic data belonging

to the pentacyclic moiety of the aglycon of

2 showed the resonances of the protons and

carbons of four methyl groups (δ_H1.02 s, 1.12 s, 1.30 s, 1.35 s; δ_C23.9, 17.3, 28.6, 33.9),

a 7(8)-double bond [δ_H5.64 brd (7.1); δ_C122.6, 147.4), two oxygenated methine groups

[

δ_H3.31 dd (J = 11.7, 3.8), 4.74 brd (J = 2.2); δ_C88.9, 80.4], and one lactone carbonyl (δ_C

180.7) (Table 1, Figures S12 and S13). The proton and carbon signals of the OAc-group in

the ¹H and ¹³C NMR spectra of compound were absent. The ¹H-¹H COSY and HSQC

correlations attributable to triterpene nucleus revealed the corresponding sequences of

2

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 (Figure 2A

Figures S14 and S15). Key HMBC cross-peaks, such as H-2/C-10; H-3/C-4, C-30, C-31;

H-5/C-4, C-19; H-7/C-9; H-12/C-13, C-14, C-18; H-15/C-8, C-32; H-16/C-18; H-17/C-

13, C-14, C-20, C21, C-22; H₃-19/C-1, C-5, C-9, C-10; H₃-30/C-3, C-4, C-31; H₃-31/C-4,

C-5, C-30; H₃-32/C-8, C-13, C-14, C-15 conﬁrmed the overall structure of the pentacyclic

,

triterpene moiety of 2 (Figure 2A, Figure S16). The key ROESY cross-peaks H-1α/H-3,

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H-5

32; H-9

/9 /10

α

; H-2

/H₃-19; H-12

/13 ,14 stereochemistry of the triterpene nucleus and 3

β

/H₃-19, H₃-30; H-3/H-5

α

, H₃-31; H-5

α

/H₃-31; H-6

/H₃-32 showed the common

,16 -conﬁgurations

β/H₃-19; H-7/H-15α, H₃-

β

α

/H₃-32; H-15

α

/H-17 , H₃-32; H-17

α

5

α

β

α

β

of oxygenated substituents in 2 (Figure 2B, Figure S17). The proton and carbon signals

of the aglycon side chain of 2 demonstrated the presence of only one methyl group (δ_H

1.75 s; δ_C23.0) and one 20,22-double bond (δ_H5.06 s, 4.98 s; δ_C139.9, 113.9). The key

HMBC correlations H₃-21/C-17, C-20, C-22 and H-22/C-17, C-21 and the key ROESY

correlations H₃-21/H-16, H-17, H-22; and H₂-22/H-16 supported the total structure of the

side chain (Figure 2A,B). The NMR spectroscopic data of aglycon part of 2 were coincident

with those for known cucumariosides A₁₀and I₄from the sea cucumber E. fraudatrix with

23,24,25,26,27-pentanorlanosta-7,20(22)-diene-18(16)-lactone-3β-ol aglycon [23,24], but the

carbohydrate chain was different.

On the basis of extensive 2D NMR and MS analysis of glycosides

gested that oligosaccharide moiety of are identical to those of glycosides

the basis of all the above-mentioned data, the structure of paciﬁcusoside B (

1

–

3

, we have sug-

and . On

) was eluci-

-D-glucopyranosyl-(1 4)-[ -D-

-D-xylopyranosyl}-23,24,25,26,27-

1

2

3

dated to be 3

xylopyranosyl-(1

pentanorlanosta-7,20(22)-diene-18(16)-lactone.

β

-O-{3-O-methyl-

β

-D-xylopyranosyl-(1

→

3)-

β

→

β

→

2)]- -D-quinovopyranosyl-(1

β

→

2)-

β

The molecular formula of 3

was determined to be C₆₀H₉₄O₂₆from the [M+Na]⁺sodiated-

molecular ion peak at m/z 1253.5920 (calcd for [C₆₀H₉₄O₂₆Na]⁺, 1253.5926) in the (+)HRESIMS

and from the [M–H]⁻ion peak at m/z 1229.5952 (calcd for [C₆₀H₉₃O₂₆]⁻, 1229.5961) in the

(–)HRESIMS (Figure S18). The IR spectrum of compound

3 showed the presence of hy-

droxy (3427 cm⁻¹), lactone carbonyl (1745 cm⁻¹), and olefinic (1635 cm⁻¹) groups (Figure S19).

1

The thorough comparison of the H and ¹³C NMR data of compound

3 with those of 1

(Figures S4, S5, S20, and S21) showed that they differed from each other only in the sig-

nals of their side chains (Table 1 ). The proton and carbon signals of aglycon side chain

of 3 demonstrated the presence of three methyl groups (δ_H1.53 s, 1.59 s, 1.65 s; δ_C28.5,

17.9, 25.7) and one double bond (δ_H5.12 m; δ_C124.4, 132.1). The protons sequence from

H-22 to H-27, correlated with the corresponding carbon atoms of the side chain of 3, was

assigned using the COSY and HSQC experiments (Table 1 , Figures S22 and S23). The key

HMBC correlations H₃-21/C-17, C-20, C-22; H-22/C-17, C-20; H-23/C-20, C-21; H-24/C-26,

C-27; H₃-26/C-24, C-25, C-27, and H₃-27/C-24, C-25, C-26 and key ROESY correlations H₃-

21/H-17, H-22 and H-24/H₃-27, H₃-26 supported the total structure of the ∆

²⁴-lanostane

side chain (Figure 1 ). Thus, aglycon of glycoside 3 was identical to aglycon of known cu-

cumarioside A₁from E. fraudatrix [25 ]. Therefore, the structure of pacificusoside C (3) was

established as 3

xylopyranosyl-(1

7,24-diene.

β

-O-{3-O-methyl-

β

-D-xylopyranosyl-(1

→

3)-

β

-D-glucopyranosyl-(1

→

4)-[β-D-

→

2)]- -D-quinovopyranosyl-(1

β

→

2)- -D-xylopyranosyl}-16β

β

-acetoxyholosta-

3.2. In Vitro Anticancer Activity of Compounds 1–6

The cytotoxicity of investigated compounds 1–6 was tested against human embry-

onic kidney HEK 293 cells, colorectal carcinoma HT-29 cells, melanoma RPMI-7951 cells,

and breast cancer MDA-MB-231 cells using MTS assay. The results of the experiment

are expressed in IC₅₀values (Table 3). The compounds

ity against the tested cell lines (Table 3, column a). Compound

cytotoxic activity against HEK 293 (IC₅₀= 6.7 0.2), HT-29 (IC₅₀= 6.2

231 (IC₅₀= 6.4 ± 0.4) cells and moderate effect against RPMI-7951 cells (IC₅₀= 23.7 ± 0.4).

Compound inhibits cell viability of all tested cell lines with IC₅₀value more than 20

(Table 3, column a). In contrast, compound was nontoxic against all types of cells at

concentrations range up to 40 M (Table 3, column a). Cisplatin, a well-known chemother-

apeutic drug, was used as the reference cytotoxic drug (positive control). IC₅₀of cisplatin

4–

6

reveal high cytotoxic activ-

has comparable high

0.3), and MDA-MB-

3

±

2

µM

1

µ

against HEK 293, HT-29, RPMI-7951, and MDA-MB-231 cells was 64.6 µM, 95.4 µM,

48.4 µM, and 7.8 µM, respectively, after 48 h of treatment (data not shown).

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Table 3. Cytotoxic activities of compounds

mean ± standard deviation.

1–6 and their mixtures with cholesterol. Values are indicated as

Inhibiting Concentration, (IC₅₀), µM

HT-29 RPMI-7951

Compound

HEK 293

MDA-MB-231

a

b

a

b

a

b

a

b

1

2

3

4

5

6

>40

n.d.

>40

24.2 ± 4.0

17.3 ± 0.8

19.5 ± 1.2

13.2 ± 0.5

>40

n.d.

>40

19.4 ± 3.3

14.3 ± 3.0

15.9 ± 0.26

10.9 ± 0.05

>40

n.d.

>40

n.d.

>40

20.0 ± 4.7

12.8 ± 0.7

12.9 ± 2.5

10.5 ± 1.8

28.6 ± 2.1

6.7 ± 0.2

5.5 ± 0.3

7.9 ± 0.9

6.3 ± 0.6

26.2 ± 2.5

6.2 ± 0.3

5.3 ± 0.2

5.5 ± 0.6

7.8 ± 0.7

20.4 ± 1.6

23.7 ± 0.4

5.8 ± 1.2

5.9 ± 0.9

5.8 ± 2.6

21.5 ± 0.5

6.4 ± 0.4

3.6 ± 0.2

4.4 ± 0.09

5.5 ± 0.1

16.4 ± 3.1

17.0 ± 2.8

11.4 ± 0.8

IC₅₀, the concentration of compounds that caused a 50% reduction in cell viability of tested normal and cancer cells. a IC₅₀of individual

compounds 1–6 for 24 h. b IC₅₀of 1–6 combined with cholesterol for 24 h. n.d.—not determined.

Such potent cytotoxicity of compounds 3–6 can be explained by the fact that the

studied triterpene glycosides are capable of interacting with cholesterol of cellular mem-

branes, which leads to cell death as a result of disruption or loss of the integrity of the cell

membrane. Since cholesterol is known for its function of stabilizing the cell membrane, the

loss of cholesterol itself or the loss of its ability to perform this function is undoubtedly

a reason of the observed instability of the cellular membranes and membranolysis of the

target cells [26

analyzed the inﬂuence of triterpene glycosides

viability. It was found that the cytotoxic activity of

greatly reduced compared with activity of the investigated individual triterpene glycosides

(Table 3, column b). The IC₅₀value of compounds was increased by approximately

3 times, and compound —by 1.5 times (Table 3, column b). Obtained data suggested

that mechanism of cytotoxic effect of triterpene glycosides may be related to bind-

–

28]. Since the triterpene saponins are able to complex cholesterol [29], we

combined with cholesterol on the cell

combined with cholesterol was

2–6

3–5

6

2–6

ing of membrane cholesterol resulting in membranolysis and cell death. Several works

conﬁrmed that triterpene glycosides from sea cucumbers possess potent membranolytic

activity [30,31].

It was demonstrated that the investigated compounds

10, 20, and 40 M) did not show selective cytotoxicity on cancer cells, because the

viability of normal cells was also suppressed. That is why we investigated the effect

of compounds on colony formation at the nontoxic concentrations of 0.1, 0.5, and

1–6 (at concentrations 5,

µ

1–6

1 µM, at which they did not influence the viability of normal HEK 293 cells (data

not shown).

Compounds 1, 2, and 6 at a concentration of 1 µM possess moderate inhibitory

activity on the model of the colony formation of the cancer cells, namely, 1, 2, and

6 decreased number of colonies of HT-29 by 21, 22, and 28%, respectively; RPMI-7951—

by 27%, 26%, and 45%, respectively; MDA-MB-231—by 28, 25, and 40%, respectively

(Figure 3). Compounds

HT-29, RPMI-7951, and MDA-MB-231 cells at concentrations of 0.5

Figure 3). To the best of our knowledge this is the first evidence that triterpene

3

,

4

, and

5

almost completely suppressed colony growth of

µ

M and 1

µM

(

glycosides at nontoxic concentrations significantly inhibited the colony formation of

human cancer cells.

Biomolecules 2021, 11, 427

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Figure 3. Effect of compounds 1–6 on colony formation of human colorectal carcinoma HT-29, melanoma RPMI-7951, and

breast cancer MDA-MB-231 cell lines. HT-29, RPMI-7951, and MDA-MB-231 cells (2.4

×

10⁴/mL) treate_◦d with/without

investigated (0.1, 0.5, and 1 µM) and subjected into a soft agar. The culture was maintained at 37 C in a 5% CO₂

1–

6

atmosphere for 2 weeks. The colonies were counted under a microscope with the aid of the ImageJ software program

n = 6 for control and each compound, n—quantity of photos). The asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001) indicate a

(

signiﬁcant decrease in colony formation in cells treated with compounds compared with the nontreated cells (control).

4. Conclusions

The isolation of a series of new triterpene glycosides from starﬁsh is a rare case.

Previously, triterpene glycosides, characteristic metabolites of sea cucumbers, were isolated

only from two species of starﬁsh: A. rollestoni Bell and C. granulatus [20

A–C ( ), new glycosides from the starﬁsh S. paciﬁcus have a great structural similarity

with the triterpene glycosides earlier obtained from the sea cucumber E. fraudatrix. In

particular, the isolated glycosides contain the holostane type of aglycons with 16β-

,21]. Paciﬁcusosides

1–3

1,

3

acetoxy group and 7(8)-double bond and glycoside 2 contains the rare nonholostane type

aglycon, 23,24,25,26,27-pentanorlanosta-7,20(22)-diene-18(16)-lactone-3β-ol.

The oligoglycoside chains of 1–3 have 3-O-methyl-D-xylose as a terminal monosaccha-

ride unit. This monosaccharide residue was previously found only in the sea cucumber E.

fraudatrix. It was previously suggested that this structural peculiarity is a chemotaxonomic

marker of E. fraudatrix [32]. The ﬁnding of 3-O-methyl-D-xylose unit in paciﬁcusosides A–C

(1–3) from the starﬁsh shows that these glycosides are food markers. Probably, these com-

pounds are partly metabolized in the starﬁsh glycosides from the sea cucumber, belonging

to the genus Eupentacta and obtained with diet. The studied starﬁsh has the synonymized

name: Solaster endeca paciﬁcus (Djakonov, 1938). It was reported that in the Atlantic Ocean

S. endeca paciﬁcus feeds on other starﬁsh and bivalve mollusks, but in the Paciﬁc, its diet is

mainly sea cucumbers and other invertebrates [33]. Without doubt, the starﬁsh S. paciﬁcus

feeds on sea cucumbers, as was reported earlier.

At the same time, glycoside 1 has a novel unique type of side chain, containing the

aldehyde group in the triterpene aglycon. This structural fragment was not previously

Biomolecules 2021, 11, 427

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found in the sea cucumber triterpene glycosides or sea star steroidal glycosides. Obviously,

paciﬁcusoside A is a metabolite, formed by the transformation of a toxic glycoside from

a diet in the starﬁsh. It is of particular interest that modiﬁed glycoside (1) is signiﬁcantly

less cytotoxic when compared with intact cucumariosides from E. fraudatrix. We assumed

that the formation of an unusual glycoside, paciﬁcusoside A (1), from related glycosides

of the starﬁsh is carried out in two stages. At the ﬁrst stage, cucumarioside C₂(5) from

the sea cucumber E. fraudatrix, most probably the biosynthetic precursor of paciﬁcusoside

A (1), is oxidized by oxygenases similar to P₄₅₀cytochromes. At the second stage, the

shortening of the

with the formation of the

Figure 4). Presumably, the cleavage of part of the side chain occurs under the action of

∆

^22,24-lanostane side chain of compound

²²-24-aldo-25,26,27-trinorlanostane side chain in glycoside 1

5 occurs in the starﬁsh S. paciﬁcus

∆

(

speciﬁc oxygenases, similarly to the formation of pregnenolone from cholesterol under

the action of cytochrome P450_sccin mammals during the synthesis of progesterone. The

existence of such oxygenases in sea stars can be conﬁrmed by the detection of (24R)-5

α-

cholestane-3 ,4 ,6 ,8,15 ,24,25-heptaol, which has hydroxy groups at C-24 and C-25 in

β

cholestane side chain [34] as in the intermediate on Figure 4.

Figure 4. Hypothetic scheme of the biosynthesis of aglycon side chain of compound 1.

We believe that the formation of similar compounds with an aldehyde group in the side

chain of glycosides 3 and 4 is also possible, but requires more time after feeding. Moreover,

the slow metabolism of the toxic triterpene glycosides in starﬁsh allows them to use the

accumulated dietary ichthyotoxic compounds to defend themselves against predators.

Thus, the oxidation with a subsequent decrease in the number of carbon atoms in the

side chain of the triterpene glycosides partly led to the decrease in the cytotoxicity of the

modiﬁed compounds. Indeed, based on the obtained data, one can see a pattern between

the structure and biological activity of the isolated compounds. The highest cytotoxicity

against all types of cancer and normal cells, as well as the highest inhibitory effect on colony

formation of cancer cells, were demonstrated by glycosides 3–5, which have

and cis- and trans-

^22,24-lanostane side chains in the triterpene aglycon, respectively. At

the same time, the IC₅₀of paciﬁcusoside A (1) on all cell types was more than 40 M, and

∆

²⁴-lanostane

∆

µ

this compound also slightly inhibited the colony formation of all types of cancer cells in a

soft agar assay. According to the data of the colony formation assay, the glycosides 2 and

6, which have a triterpene aglycon with a rearranged 18(16)-lactone, are less active than

glycosides 3–5, which have a holostane triterpene aglycon with an 18(20)-lactone.

Finally, we once more conﬁrmed the hypothesis that triterpene glycosides are membra-

nolytic and exert their cytotoxic effect by binding the components of the plasma membrane

of target cells. In fact, when cholesterol was combined with the compounds 1–6, the cyto-

toxicity of all glycosides decreased markedly. This is likely due to the fact that some of the

molecules of triterpene glycosides 1–6 bind the cholesterol added to these compounds.

On the other hand, glycosides 3–5, which showed signiﬁcant or complete inhibition

the colony formation of cancer cells at nontoxic concentrations, can be further studied as

potential anticancer agents.

Supplementary Materials: The following are available online at https://ww w .mdpi.com/2218-273

X/11/3/427/s1, Figure S1: (–)HRESIMS spectrum of paciﬁcusoside A (1); Figure S2: IR spectrum of

1

paciﬁcusoside A (1) in KBr; Figure S3: UV spectrum of paciﬁcusoside A (1) in MeOH; Figure S4: H

NMR spectrum of paciﬁcusoside A (1) in C₅D₅N; Figure S5: ¹³C NMR spectrum of paciﬁcusoside A

Biomolecules 2021, 11, 427

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1

(1) in C₅D₅N; Figure S6: H-¹H COSY spectrum of paciﬁcusoside A (1) in C₅D₅N; Figure S7: HSQC

spectrum of paciﬁcusoside A (1) in C₅D₅N; Figure S8: HMBC spectrum of paciﬁcusoside A (1) in

C₅D₅N; Figure S9: ROESY spectrum of paciﬁcusoside A (1) in C₅D₅N; Figure S10: (–)HRESIMS

spectrum of paciﬁcusoside B (2); Figure S11: IR spectrum of paciﬁcusoside B (2) in KBr; Figure S12:

¹H NMR spectrum of paciﬁcusoside B (2) in C₅D₅N; Figure S13: ¹³C NMR spectrum of paciﬁcusoside

B (2) in C₅D₅N; Figure S14: ¹H-¹H-COSY spectrum of paciﬁcusoside B (2) in C₅D₅N; Figure S15:

HSQC spectrum of paciﬁcusoside B (2) in C₅D₅N; Figure S16: HMBC spectrum of paciﬁcusoside B (2)

in C₅D₅N; Figure S17: ROESY spectrum of paciﬁcusoside B (2) in C₅D₅N; Figure S18: (–)HRESIMS

spectrum of paciﬁcusoside C (3); Figure S19: IR spectrum of paciﬁcusoside C (3) in KBr, Figure S20:

¹H NMR spectrum of paciﬁcusoside C (3) in C₅D₅N; Figure S21: ¹³C NMR spectrum of paciﬁcusoside

C (3) in C₅D₅N; Figure S22: ¹H-¹H COSY spectrum of paciﬁcusoside C (3) in C₅D₅N; Figure S23:

HSQC spectrum of paciﬁcusoside C; (3) in C₅D₅N; Figure S24: HMBC spectrum of paciﬁcusoside C;

(3) in C₅D₅N; Figure S25: ROESY spectrum of paciﬁcusoside C; (3) in C₅D₅N.

Author Contributions: T.V.M. isolated the metabolites, elucidated their structure, and prepared the

manuscript; A.A.K. and N.V.I. analyzed the compounds and edited the manuscript; A.I.K. performed

the acquisition and interpretation of NMR spectra; P.S.D. did the acquisition and interpretation of

mass spectra; A.S.K. investigated the cytotoxic and anticancer activity of the compounds; O.S.M.

described the results of the biological activity of the compounds and prepared the manuscript.; V.A.S.

edited the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding: The isolation and establishment of the chemical structures were partially supported by

Grant No. 20-03-00014 from the RFBR (Russian Foundation for Basic Research). The study on the

cytotoxic and the anticancer activities of starﬁsh metabolites was supported by Grant No. 18-74-10028

from the RSF (Russian Science Foundation).

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

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

corresponding authors.

Acknowledgments: The study was carried out on the equipment of the Collective Facilities Center

“The Far Eastern Center for Structural Molecular Research (NMR/MS) of PIBOC FEB RAS”. We

are grateful to B.B. Grebnev (G.B. Elyakov Paciﬁc Institute of Bioorganic Chemistry FEB RAS,

Vladivostok, Russia) for species identiﬁcation of the starﬁsh, V.I. Kalinin for reading and discussion

of the manuscript, E.V. Evtushenko for providing sample of 3-O-Me-Xyl, V.P. Glazunov for providing

IR spectra of compounds and N.Y. Kim for providing UV spectra of compounds.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

1.

2.

3.

4.

Gomes, A.R.; Freitas, A.C.; Rocha-Santos, T.A.P.; Duarte, A.C. Bioactive compounds derived from echinoderms. RSC Adv. 2014, 4,

29365–29382. [CrossRef]

Mondol, M.A.M.; Shin, H.J.; Rahman, M.A.; Islam, M.T. Sea cucumber glycosides: Chemical structures, producing species and

important biological properties. Mar. Drugs 2017, 15, 317. [CrossRef] [PubMed]

Kalinin, V.I.; Silchenko, A.S.; Avilov, S.A.; Stonik, V.A. Non-holostane aglycones of sea cucumber triterpene glycosides. Structure,

biosynthesis, evolution. Steroids 2019, 147, 42–51. [CrossRef]

Kalinin, V.I.; Aminin, D.L.; Avilov, S.A.; Silchenko, A.S.; Stonik, V.A. Triterpene glycosides from sea cucumbers (Holothurioidea,

Echinodermata), biological activities and functions. In Studies in Natural Product Chemistry (Bioactive Natural Products); Atta-ur-

Rahman, Ed.; Elsevier Science Publisher: Amsterdam, The Netherlands, 2008; Volume 35, pp. 135–196.

Careaga, V.P.; Maier, M.S. Cytotoxic triterpene glycosides from sea cucumbers. In Handbook of Anticancer Drugs from Marine Origin;

Kim, S.-K., Ed.; Springer International Publishing: Cham, Switzerland, 2015; pp. 515–528.

Chludil, H.D.; Murray, A.P.; Seldes, A.M.; Maier, M.S. Biologically active triterpene glycosides from sea cucumbers (Holothuroidea,

Echinodermata). In Studies in Natural Products Chemistry; Atta-ur-Rahman, Ed.; Elsevier Science Publisher: Amsterdam, The

Netherlands, 2003; Volume 28, pp. 587–615.

5.

6.

7.

8.

Kim, S.K.; Himaya, S.W.A. Chapter 20-Triterpene glycosides from sea cucumbers and their biological activities. Adv. Food Nutr.

Res. 2012, 63, 297–319. [CrossRef]

Maier, M.S.; Roccatagliata, A.J.; Kuriss, A.; Chludil, H.; Seldes, A.M.; Pujol, C.A.; Damonte, E.B. Two new cytotoxic and virucidal

trisulfated triterpene glycosides from the Antarctic sea cucumber Staurocucumis liouvillei. J. Nat. Prod. 2001, 64, 732–736. [CrossRef]

Biomolecules 2021, 11, 427

15 of 15

9.

Aminin, D.L.; Menchinskaya, E.S.; Pisliagin, E.A.; Silchenko, A.S.; Avilov, S.A.; Kalinin, V.I. Anticancer activity of sea cucumber

triterpene glycosides. Mar. Drugs 2015, 13, 1202–1223. [CrossRef]

10. Aminin, D.L.; Pislyagin, E.A.; Menchinskaya, E.S.; Silchenko, A.S.; Avilov, S.A.; Kalinin, V.I. Immunomodulatory and anticancer

activity of sea cucumber triterpene glycosides. In Studies in Natural Products Chemistry (Bioactive Natural Products); Atta-ur-Rahman,

Ed.; Elsevier Science Publisher: Amsterdam, The Netherlands, 2014; Volume 41, pp. 75–94.

11. Minale, L.; Riccio, R.; Zollo, F. Steroidal oligoglycosides and polyhydroxysteroids from Echinoderms. Fortschr. Chem. Org. Nat.

1993, 62, 75–308. [CrossRef]

12. Stonik, V.A. Marine polar steroids. Russ. Chem. Rev. 2001, 70, 673–715. [CrossRef]

13. Iorizzi, M.; De Marino, S.; Zollo, F. Steroidal oligoglycosides from the Asteroidea. Curr. Org. Chem. 2001, 5, 951–973. [CrossRef]

14. Stonik, V.A.; Ivanchina, N.V.; Kicha, A.A. New polar steroids from starﬁsh. Nat. Prod. Commun. 2008, 3, 1587–1610. [CrossRef]

15. Dong, G.; Xu, T.H.; Yang, B.; Lin, X.P.; Zhou, X.F.; Yang, X.W.; Liu, Y.H. Chemical constituents and bioactivities of starﬁsh. Chem.

Biodivers. 2011, 8, 740–791. [CrossRef]

16. Ivanchina, N.V.; Kicha, A.A.; Stonik, V.A. Steroid glycosides from marine organisms. Steroids 2011, 76, 425–454. [CrossRef]

[PubMed]

17. Ivanchina, N.V.; Kicha, A.A.; Malyarenko, T.V.; Stonik, V.A. Advances in Natural Products Discovery; Gomes, A.R., Rocha-Santos, T.,

Duarte, A., Eds.; Nova Science Publishers: New York, NY, USA, 2017; Volume 6, pp. 191–224.

18. Stonik, V.A.; Kicha, A.A.; Malyarenko, T.V.; Ivanchina, N.V. Asterosaponins: Structures, taxonomic distribution, biogenesis and

biological activities. Mar. Drugs 2020, 18, 584. [CrossRef]

19. Xia, J.M.; Miao, Z.; Xie, C.L.; Zhang, J.W.; Yang, X.W. Chemical constituents and bioactivities of starﬁsh: An update. Chem.

Biodivers. 2020, 17, e1900638. [CrossRef]

20. Zhan, Y.C.; Sun, Y.; Li, W.; Lin, Y.; Sha, Y.; Pei, Y.H. A new triterpene glycoside from Asterias rollentoni. J. Asian Nat. Prod. Res.

2006, 8, 631–636. [CrossRef]

21. Ivanchina, N.V.; Kalinovsky, A.I.; Malyarenko, T.V.; Kicha, A.A.; Dmitrenok, P.S. A holothurian triterpene glycoside holothurin

A₂(= echinoside A) isolated from the starﬁsh Choriaster granulatus. Nat. Prod. Commun. 2019, 14, 1–3. [CrossRef]

22. Aﬁyatullov, S.S.; Kalinovsky, A.I.; Stonik, V.A. Structures of cucumariosides C₁and C₂–two new triterpene glycosides from the

holothurian Eupentacta fraudatrix. Chem. Nat. Comp. 1987, 23, 691–696. [CrossRef]

23. Silchenko, A.S.; Kalinovsky, A.I.; Avilov, S.A.; Andrijaschenko, P.V.; Dmitrenok, P.S.; Martyyas, E.A.; Kalinin, V.I. Triterpene

glycosides from the sea cucumber Eupentacta fraudatrix. Structure and cytotoxic action of cucumariosides A₂, A₇, A₉, A₁₀, A₁₁

,

A₁₃and A₁₄, seven new minor non-sulfated tetraosides and an aglycone with an uncommon 18-hydroxy group. Nat. Prod.

Commun. 2012, 7, 845–852. [CrossRef]

24. Silchenko, A.S.; Kalinovsky, A.I.; Avilov, S.A.; Andryjaschenko, P.V.; Dmitrenok, P.S.; Martyyas, E.A.; Kalinin, V.I. Triterpene

glycosides from the sea cucumber Eupentacta fraudatrix. Structure and biological action of cucumariosides I₁, I₃, I₄, three new

minor disulfated pentaosides. Nat. Prod. Commun. 2013, 8, 1053–1058. [CrossRef] [PubMed]

25. Silchenko, A.S.; Kalinovsky, A.I.; Avilov, S.A.; Andryjaschenko, P.V.; Dmitrenok, P.S.; Martyyas, E.A.; Kalinin, V.I. Triterpene

glycosides from the sea cucumber Eupentacta fraudatrix. Structure and biological action of cucumariosides A₁, A₃, A₄, A₅, A₆, A₁₂

and A₁₅, seven new minor non-sulfated tetraosides and unprecedented 25-keto, 27-norholostane aglycone. Nat. Prod. Commun.

2012, 7, 517–525. [CrossRef]

26. Schlösser, E. Interaction of saponins with cholesterol, lecithin, and albumin. Can. J. Physiol. Pharmacol. 1969, 47, 487–490.

[CrossRef] [PubMed]

27. De Geyter, E.; Swevers, L.; Soin, T.; Geelen, D.; Smagghe, G. Saponins do not affect the ecdysteroid receptor complex but cause

membrane permeation in insect culture cell lines. J. Insect. Physiol. 2012, 58, 18–23. [CrossRef]

28. Böttger, S.; Melzig, M.F. The inﬂuence of saponins on cell membrane cholesterol. Bioorg. Med. Chem. 2013, 21, 7118–7124.

[CrossRef] [PubMed]

29. Wojciechowski, K.; Orczyk, M.; Gutberlet, T.; Geue, T. Complexation of phospholipids and cholesterol by triterpenic saponins in

bulk and in monolayers. Biochim. Biophys. Acta Biomembr. 2016, 1858, 363–373. [CrossRef]

30. Wang, J.; Han, H.; Chen, X.; Yi, Y.; Sun, H. Cytotoxic and apoptosis-inducing activity of triterpene glycosides from Holothuria

scabra and Cucumaria frondosa against HepG2 cells. Mar. Drugs 2014, 12, 4274–4290. [CrossRef] [PubMed]

31. Popov, A.M.; Kalinovskaia, N.I.; Kuznetsova, T.A.; Agafonova, I.G.; Anisimov, M.M. Role of sterols in the membranotropic

activity of triterpene glycosides. Antibiotiki 1983, 28, 656–659.

32. Popov, R.S.; Ivanchina, N.V.; Silchenko, A.S.; Avilov, S.A.; Kalinin, V.I.; Dolmatov, I.Y.; Stonik, V.A.; Dmitrenok, P.S. Metabolite

proﬁling of triterpene glycosides of the Far Eastern sea cucumber Eupentacta fraudatrix and their distribution in various body

components using LC-ESI QTOF-MS. Mar. Drugs 2017, 15, 302. [CrossRef]

33. Sea Stars of the Paciﬁc Northwest. Available online: https://seastarsofthepaciﬁcnorthwest.info/northern-sun-star.html (accessed

on 16 February 2021).

34. Ivanchina, N.V.; Kicha, A.A.; Kalinovsky, A.I.; Dmitrenok, P.S.; Dmitrenok, A.S.; Chaikina, E.L.; Stonik, V.A.; Gavagin, M.;

Cimino, G. Polar steroids compounds from the Far Eastern starﬁsh Henricia leviuscula. J. Nat. Prod. 2006, 69, 224–228. [CrossRef]

Article Doi

New triterpene glycosides from the far eastern starfish solaster pacificus and their biological activity

DOI: 10.3390/biom11030427

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Full text of DOI:10.3390/biom11030427

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