Triterpene Tetraglycosides From Stichopus Herrmanni Semper,1868

Abstract

Using various column chromatographic methods, 5 triterpene tetraglycosides (1−5), including one new compound, namely holothurin A₆ (1), were obtained from the water soluble part of the methanol extract of the sea cucumber Stichopus herrmanni. Their structures were confirmed by careful analysis of the 1D and 2D NMR, and HR ESI QTOF mass spectra. Noteworthily, 24-dehydroechinoside A (3) showed potent cytotoxicity to 5 human cancer cell lines {HepG2 (hepatoma cancer), KB (epidermoid carcinoma), LNCaP (prostate cancer), MCF7 (breast cancer), and SK-Mel2 (melanoma)} with IC₅₀ values ranging from 0.19 ± 0.03 to 1.17 ± 0.18 µM.

Introduction

Within marine organisms, echinoderms are a source of a broad range of secondary metabolites having various biological activities. Of these metabolites, saponins represent the most abundant and diverse products in the phylum Echinodermata. Among these marine invertebrates, saponins are the most known and structurally diverse within sea cucumbers. The majority of them are holostane derivatives possessing an 18(20)-lanostane lactone as aglycon and a carbohydrate chain consisting of from 1 to 6 monosaccharide units. These compounds demonstrate interesting biological effects such as cytotoxic, ichthyotoxic, antifungal, and hemolytic activities, as well as a series of additional effects at subtoxic doses, including immunomodulatory and cancer preventive.^1,2

As a part of our ongoing investigations of saponins from Vietnamese sea cucumbers,^3–6 this paper deals with the isolation and structural elucidation of 5 triterpene tetraglycosides (1−5), including one new compound, holothurin A₆ (1), from the sea cucumber S herrmanni Semper, 1868. In addition, their cytotoxic activity was also evaluated against the human cancer cell lines HepG2 (hepatoma cancer), KB (epidermoid carcinoma), LNCaP (prostate cancer), MCF7 (breast cancer), and SK-Mel2 (melanoma).

Results and Discussion

Using various column chromatographic separations, 5 triterpene tetraglycosides (1−5) were obtained from the water soluble part of the sea cucumber Stichopus herrmanni (Figure 1). By careful analysis of the 1D and 2D NMR and ESI-MS data, as well as by comparison with literature data, the known saponins were elucidated as 24ξ-hydroxy-25-dehydroechinoside A (2), ⁷ 24-dehydroechinoside A (3), ⁸ holothurin A₂ (4),^6,9,10 and holothurin A₅ (5). ⁶

Figure 1.

The structures of 1‒5 and key COSY and HMBC interactions of 1.

Holothurin A₆ (1) was obtained as a white powder. Its HR ESI QTOF MS exhibited a quasi-molecular ion peak at m/z 1243.4767 [M + Na]⁺ (see Figure S9, Supplemental material), consistent with the molecular formula of C₅₄H₈₅NaO₂₇S. Similarly as in the case of 2−5, the ¹H and ¹³C NMR spectra of 1 were indicative for a triterpene tetraglycoside with characteristic signals of 4 anomeric protons/carbons at δ_H 4.63 (1H, d, J = 7.2 Hz, H-1′)/δ_C 105.1 (C-1′), δ_H 5.03 (1H, d, J = 7.8 Hz, H-1′′)/δ_C 105.1 (C-1′′), δ_H 4.89 (1H, d, J = 7.8 Hz, H-1′¢¢)/δ_C 104.6 (C-1′¢¢), and δ_H 5.25 (1H, d, J = 7.8 Hz, H-1′¢¢¢)/δ_C 105.3 (C-1′¢¢¢). Extensive analysis of the HSQC, ¹H–¹H COSY, HMBC, 1D and 2D TOCSY spectra allowed the assignment of the complete ¹H and ¹³C NMR spectroscopic data for all 4 sugar units (see Table 1). These data were extremely similar to those of holothurin A₅ (5), ⁶ suggesting that 1 and 5 possess the same tetrasaccharide chain: 3-O-methyl-β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→4)-β-D-quinovopyranosyl-(1→2)-4-O-sodium sulfate-β-D-xylopyranoside. The D-configuration of all the 4 sugar units was assigned due to biosynthetic reasons, and analogy with 2−5 coexistence in S herrmanni. The sequence of sugar units in the tetrasaccharide chain was assigned by HMBC interactions of H-1′¢¢¢ (δ_H 5.25) with C-3′¢¢ (δ_C 87.6), H-1′¢¢ (δ_H 4.89) with C-4′′ (δ_C 86.9) and H-1′′ (δ_H 5.03) with C-2′ (δ_C 82.8) (see Figure 1). This was also supported by ROESY correlations of H-1′¢¢¢ (δ_H 5.25) with H-3′¢¢ (δ_H 4.21), H-1′¢¢ (δ_H 4.89) with H-4′′ (δ_H 3.60), and H-1′′ (δ_H 5.03) with H-2′ (δ_H 4.00). Moreover, the ¹H and ¹³C NMR spectra for the triterpene part of 1 exhibited typical signals of 2 oxymethines [δ_C 88.4 (C-3) and 71.1 (C-12)/δ_H 3.07 (1H, dd, J = 3.6, 12.0 Hz, H-3) and 4.92 (1H, brd, J = 5.4 Hz, H-12)], 3 oxygenated quaternary carbons [δ_C 89.1 (C-17), 86.7 (C-20), and 69.6 (C-25)], one trisubstituted double bond [δ_C 153.9 (C, C-9) and 115.2 (CH, C-11)/δ_H 5.57 (1H, d, J = 4.2 Hz, H-11)], one trans disubstituted double bond [δ_C 120.6 (CH, C-23) and 143.5 (CH, C-24)/δ_H 6.60 (1H, m, H-23) and 5.96 (1H, d, J = 15.0 Hz, H-24)], one lactone carbonyl [δ_C 174.6 (C-18)], and 7 singlet methyls [δ_C 22.4 (C-19), 23.1 (C-21), 30.2 (C-26 and C-27), 16.6 (C-30), 27.9 (C-31), and 19.9 (C-32)/δ_H 1.30 (H-19), 1.70 (H-21), 1.50 (H-26), 1.49 (H-27), 1.01 (H-30), 1.19 (H-31), and 1.59 (H-32), each 3H, s]. The ¹H and ¹³C NMR data for the triterpene part of 1 were also similar to those of holothurin A₅ (5), ⁶ except for the remarkable difference for the side chains. The ¹³C NMR signal for C-25 of 1 was shifted upfield to δ_C 69.6 versus that of holothurin A₅ (5) ⁶ at δ_C 81.1, indicating the presence of a hydroxy substituent at C-25.^11,12 The relative configuration of the aglycon of 1 was assigned to be the same as those of 2–5 from a biosynthetic viewpoint with their coexistence in S herrmanni. This was also suggested by the similarity of their ¹H and ¹³C NMR spectroscopic data and by the ROESY spectrum, with key spatial proximities of H-3 (δ_H 3.07) with H-5 (δ_H 0.92) and H-31 (δ_H 1.19), H-19 (δ_H 1.30) with H-8 (δ_H 3.29) and H-30 (δ_H 1.01), and H-12 (δ_H 4.92) with H-21 (δ_H 1.70) (see Figure 2). Finally, HMBC interaction of the anomeric proton H-1′ (δ_H 4.63) with C-3 (δ_C 88.4) and the ROE-correlation of H-1′ (δ_H 4.63) with H-3 (δ_H 3.07) confirmed the location of the tetrasaccharide chain at C-3 of the aglycon. Thus, the structure (23E)-3β-O-[3-O-methyl-β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→4)-β-D-quinovopyranosyl-(1→2)-4-O-sodium sulfate-β-D-xylopyranosyl]-holost-9(11),23E-diene-12α,17α,25-triol was elucidated for 1 (Supplemental Figures S1-S9).

Figure 2.

Key ROESY correlations of 1.

Table 1.

¹H and ¹³C NMR Spectroscopic Data of 1.

C	δ C	δH mult. (J in Hz)	C	δ C	δH mult. (J in Hz)
Aglycon			4-O-Sulfo-Xyl
1	36.2	1.28 m/1.73 m	1′	105.1	4.63 d (7.2)
2	26.8	1.83 m/2.02 m	2′	82.8	4.00 dd (7.2, 9.0)
3	88.4	3.07 dd (3.6, 12.0)	3′	75.6	4.30 t (9.0)
4	39.8	-	4′	75.9	5.14 m
5	52.5	0.92 br d (10.2)	5′	64.3	3.71 dd (9.0, 11.4) 4.75 dd (5.4, 11.4)
6	21.0	1.49 m/1.70 m	Qui
7	28.2	1.45 m/1.71 m	1′′	105.1	5.03 d (7.8)
8	40.7	3.29 m	2′′	76.1	3.96 dd (7.8, 9.0)
9	153.9	-	3′′	75.4	4.04 t (9.0)
10	39.5	-	4′′	86.9	3.60 t (9.0)
11	115.2	5.57 br d (4.2)	5′′	71.6	3.68 dd (9.0, 5.4)
12	71.1	4.92 br d (5.4)	6′′	18.0	1.66 d (6.0)
13	58.5	-	Glc
14	46.2	-	1‴	104.6	4.89 d (7.8)
15	36.5	1.35 m/1.78 m	2‴	73.7	3.98 dd (7.8, 9.0)
16	35.6	2.30 m/2.70 m	3‴	87.6	4.21 t (9.0)
17	89.1	-	4‴	69.6	3.94a
18	174.6	-	5‴	77.7	3.95a
19	22.4	1.30 s	6‴	62.0	4.10 dd (4.2, 11.4) 4.42 br d (11.4)
20	86.7	-	3-OMe-Glc
21	23.1	1.70 s	1″″	105.3	5.25 d (7.8)
22	41.3	2.68 m	2″″	74.9	3.93 dd (7.8, 9.0)
23	120.6	6.60 m	3″″	87.7	3.68 t (9.0)
24	143.5	5.96 d (15.0)	4″″	70.5	4.01 t (9.0)
25	69.6	-	5″″	78.2	3.95 m
26	30.2	1.50 s	6″″	62.0	4.18 dd (4.8, 11.4) 4.43 br d (10.8)
27	30.2	1.49 s	OMe	60.7	3.82 s
30	16.6	1.01 s
31	27.9	1.19 s
32	19.9	1.59 s

^a

Overlapped signals. All data were confirmed by HSQC, COSY, HMBC, ROESY, and 1D and 2D TOCSY spectra.

The cytotoxic activity of compounds 1−3 against the human cancer cell lines HepG2 (hepatoma cancer), KB (epidermoid carcinoma), LNCaP (prostate cancer), MCF7 (breast cancer), and SK-Mel2 (melanoma) was evaluated by SRB assay, ¹³ following previously described steps. ³ Potent cytotoxicity was observed for 24-dehydroechinoside A (3) (IC₅₀ values from 0.19 ± 0.03 to 1.17 ± 0.18 µM) against all the 5 cell lines, comparable to that of the positive control (ellipticine: IC₅₀ values from 1.26 ± 0.16 to 1.83 ± 0.20 µM) (Table 2). Similar cytotoxic effect was also reported for holothurin A₂ (4) (IC₅₀ values from 0.75 ± 0.09 to 0.96 ± 0.09 µM). ⁶ Strong cytotoxic activity was observed for 24ξ-hydroxy-25-dehydroechinoside A (2) on all 5 cell lines (IC₅₀ values from 3.99 ± 0.58 to 11.67 ± 1.19 µM) and for holothurin A₆ (1) on MCF7 (IC₅₀ = 14.31 ± 1.17 µM) and SK-Mel2 (IC₅₀ = 13.87 ± 1.39 µM) cell lines. Compound 1 exhibited moderate activity against LNCaP (IC₅₀ = 48.57 ± 2.49 µM) and HepG2 (IC₅₀ = 52.62 ± 3.45 µM) cell lines, and weak effect on the KB (IC₅₀ = 97.86 ± 1.50 µM) cell line. Previously, holothurin A₅ (5) was found to exhibit either moderate or weak activity against the tested cell lines (IC₅₀ values from 46.65 ± 2.28 to 66.22 ± 6.32 µM). ⁶ In addition, 1 showed no cytotoxic effect (IC₅₀ > 100 µM) on the normal human embryonic kidney cell line (HEK-293A), whereas 2 and 3 revealed cytotoxicity with IC₅₀ values of 7.70 ± 0.24 and 0.27 ± 0.03 µM, respectively.

Table 2.

The Cytotoxicity of 1−3.

Compounds	IC50 (µM)
	LNCaP	HepG2	KB	MCF7	SK-Mel2	HEK-293A
1	48.57 ± 2.49	52.62 ± 3.45	97.86 ± 1.50	14.31 ± 1.17	13.87 ± 1.39	>100
2	3.99 ± 0.58	7.42 ± 0.38	11.67 ± 1.19	6.30 ± 0.69	5.59 ± 0.52	7.70 ± 0.24
3	0.19 ± 0.04	0.30 ± 0.05	1.17 ± 0.18	0.19 ± 0.03	0.32 ± 0.03	0.27 ± 0.03
Ellipticinea	1.50 ± 0.16	1.26 ± 0.16	1.83 ± 0.20	1.30 ± 0.16	1.58 ± 0.20	1.30 ± 0.16

^a

Positive control. Data are the means ± SD of triplicate experiments.

Experimental

General

Refer to Supplemental Material.

Marine Organism Material

S herrmanni Semper, 1868 samples were collected at Bai Giau island (coordinates: 12°40′44′′−109°20′33′′) and Khai Luong island (coordinates: 12°35′06′′−109°24′44′′) in Van Phong, Khanhhoa, Vietnam, during July 2020. Its scientific name was identified by one of our authors, Prof Do Cong Thung. Voucher specimens (No. DLB-2020-DG06) were deposited at the IMBC and the IMER, VAST.

Extraction and Isolation

Refer to Supplemental Material.

Holothurin A₆ (1)

White amorphous powder.

[α]_D²⁵ −21 (c 0.1, MeOH);

¹H (pyridine-d₅, 600 MHz) and ¹³C NMR (pyridine-d₅, 150 MHz) data given in Table 1;

HR ESI QTOF MS m/z 1243.4767 [M + Na]⁺ (calcd. for C₅₄H₈₅Na₂O₂₇S⁺, 1243.4783).

Cytotoxic Assays

Refer to Supplemental Material.

Conclusions

Five triterpene tetraglycosides, with one new compound holothurin A₆ (1), were isolated and structurally elucidated from the Vietnamese sea cucumber S herrmanni. Among the isolated compounds, 24-dehydroechinoside A (3) and holothurin A₂ (4) showed potent cytotoxicity on the human cancer cell lines HepG2, KB, LNCaP, MCF7, and SK-Mel2. Consideration of the cytotoxicity and structures of 1−5 suggested that the side chain might play an important role in the cytotoxicity of these saponins against the tested cancer cell lines and the presence of an oxygenated substituent in the side chains might reduce the cytotoxicity of these compounds.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X221105369 - Supplemental material for Triterpene Tetraglycosides From Stichopus Herrmanni Semper, 1868

Supplemental material, sj-docx-1-npx-10.1177_1934578X221105369 for Triterpene Tetraglycosides From Stichopus Herrmanni Semper, 1868 by Le Thi Vien, Tran Thi Hong Hanh, Tran Hong Quang and Nguyen Van Thanh, Do Thi Thao, Nguyen Xuan Cuong, Nguyen Hoai Nam, Do Cong Thung, Phan Van Kiem in Natural Product Communications