Variegatusides: New Non-Sulphated Triterpene Glycosides from the Sea Cucumber Stichopus variegates Semper

Four new triterpene glycosides, variegatusides C–F (1–4), together with three structurally known triterpene glycosides, variegatusides A and B (5, 6), and holothurin B (7), were isolated from the sea cucumber Stichopus variegates Semper (Holothuriidae), collected from the South China Sea. Their structures were elucidated on the basis of extensive spectral analysis (nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESIMS)) and chemical evidence. Variegatusides C–F exhibit the same structural feature consisting of the presence of a 23-hydroxyl group at the holostane-type triterpene aglycone side chain. Variegatuside C (1) has a double bond (24, 25) in this same chain, while variegatuside D (2) exhibits a 8(9)-ene bond in the holostane-type triterpene aglycone, which has not been extracted from other sea cucumber species. Compound 4 is a native compound from the sea cucumber S. variegates Semper, which has been reported to be desacetylstichloroside B1. Except for holothurin B, these glycosides have no sulfate group in their sugar chain and show potent antifungal activities in vitro biotests.


Introduction
Triterpene glycosides are the predominant secondary metabolites of holothurians and are responsible for their general toxicity. These glycosides have been reported to have a wide spectrum of biological effects, including antifungal, cytotoxic, hemolytic, and immunomodulatory activities [1,2]. In the search for new pharmacologically active substances from marine organisms, attention has been paid to echinoderms, and among them, to sea cucumbers (class Holothuridea). More than 100 of these glycosides have been described, and the majority are usually lanosterol type triterpenes with an 18(20) lactone and a sugar chain of up to six monosaccharide units (princinally D-xylose, D-glucose, D-quinovose, D-3-O-methylglucose and D-3-O-methylxylose) linked to the C-3 of the aglycone [3]. As a continuation of our studies on the structure and biological role of triterpene oligoglycosides from holothurians [4][5][6][7][8][9][10], we have firstly investigated the ethanol (EtOH) extracts of the Stichopodidae-type sea cucumber Stichopus variegates Semper collected from the Hainan province in the South China sea which is used as a tonic in China [11]. Herein, we report the isolation and structure elucidation of the four new, unprecedented, non-sulfated triterpene glycosides, variegatusides C (1), D (2), E (3) and F (4). All isolated compounds revealed different antifungal activities.

Results and Discussion
The 70% ethanolic extract of S. variegates (15 kg, wet weight) was suspended in H 2 O and partitioned successively with petroleum ether and n-BuOH. The n-BuOH extract was subjected to silica gel and reversed-phase silica (Lichroprep RP-18, 40-63 μm). Final purification of individual compounds was achieved by reversed-phase HPLC on Zobax SB C-18 to give variegatusides C (1), D (2), E (3), F (4) and three known compounds 5-7 ( Figure 1). Structures of the glycosides have been elucidated by extensive analysis (NMR and ESIMS) and chemical method.
Variegatuside C (1), obtained as colorless amorphous powder, was positive to Liebermann-Burchard and Molish test. Its molecular formula was determined as C 53 H 84 O 22 from the pseudomolecular ion peak at m/z 1071.5419 [M − H] + in negative ion mode HRESIMS and at m/z 1095 [M + Na] + in positive ion mode ESIMS and 13 C NMR. The IR spectrum showed the presence of hydroxyl (3417 cm −1 ), carbonyl (1761 cm −1 ), and olefinic (1652 cm −1 ) groups.
The 1 H NMR, 13 C NMR and DEPT spectra of glycoside 1 showed the close aglycone with desacetylstichloroside B 1 [12] and variegatuside A [13], differing only by the presence of an olefinic bond at 24(25). The 1 H and 13 C NMR spectral data of 1 (Table 1) suggested the presence of a triterpene aglycone with two olefinic bonds, one ester, and one hydroxyl group bonded to an oligosaccharide chain composed of four sugar units. Resonances for a 7(8)-double bond at δ C 143.6 (C-8), 120.3 (C-7); δ H 5.62 (m, H-7) and one 24(25)-double bond at δ C 131.2 (C-25), 123.2 (C-24); δ H 5.01 (d, J = 7.8 Hz, H-24) were present. The positions of two C=C bonds in 7,8 and 24,25 were corroborated by the HMBC correlations H-32/C-8, H-9/C-8, H-5/C-7, H-9/C-7, and H-27/C-25, H-24/C-25, H-27/C-24, H-23/C-24, H-22/C-24, respectively. The 13 C NMR chemical shift inventory of 1 closely parallel that of frondoside A [14] except for signals assigned to C-22 (δ C 48.9) and C-23 (δ C 66.0) which are shifted downfield by δ 9.77 and 43.24, respectively, in agreement with the presence of an OH group at C-23. The side chain of aglycone is thus comparable to that of stichlorogenol [10], a C-23-hydroxy aglycone from the sea cucumber Stichopus chloronotus for which stereochemistry has been confirmed by X-ray crystallography [12]. Comparable signals in the 13 C NMR spectrum (pyridine-d 5 ) of the latter are observed at δ C 47.62 and 65.70 [15], suggesting the 23S configuration in variegatuside C (1).   The presence of four monosaccharide units in the sugar chain of the glycoside 1 was deduced from its 13 C NMR and DEPT spectra, which showed four anomeric carbons at 105.8, 105.9, 105.0, and 105.6 ppm, correlated by HMQC to their corresponding anomeric protons at 4.83 (d, J = 7.2 Hz), 5.28 (d, J = 7.8 Hz), 5.11 (d, J = 7.8 Hz), and 5.25 (d, J = 7.2 Hz) ppm ( Table 1). The coupling constants of the anomeric protons were indicative in all cases of a β-configuration for the glycosidic bonds [16]. The monosaccharide units in 1 were identified as xylose, glucose, and 3-O-methylglucose in a 2:1:1 ratio by acidic hydrolysis with aqueous 2 mol/L trifluoroacetic acid and preparation of the corresponding standard aldonitrile peracetates (Sigma), which were analyzed by GC-MS. The NMR spectral data of the carbohydrate part of glycoside 1 were coincident with those of thelenotoside B from Thelenota ananas [17], indicating that these two glycosides contain the same carbohydrate chain.
The DQFCOSY experiment allowed the sequential assignment of most of the resonances of each sugar ring, starting from the easily distinguished signals due to anomeric protons. Complete assignment was achieved by a combination of DQFCOSY and TOCSY results. The HMQC experiment correlated all proton resonances with those of their corresponding carbons. These data (Table 2) indicated that the four sugar residues are in their pyranose forms. The locations of the interglycosidic linkages were deduced from the chemical shifts of Xyl 1 C-2 (δ C 83.7), Glu C-3 (δ C 80.4) and Xyl 2 C-3 (δ C 87.6), which were downfield relative to shifts expected for the corresponding methyl glycopyranosides [16]. The sequence of the sugar residues in 1 was determined by analysis of HMBC correlations: Xyl 1 H-1/C-3 of the aglycone, Glu H-1/Xyl 1 C-2, Xyl 2 H-1/ Glu C-3 and MeGlu H-1/Xyl 2 C-3. This conclusion was also confirmed by the NOESY correlations as shown in Figure 2.   The IR spectrum showed the presence of hydroxyl (3355 cm −1 ), carbonyl (1760 cm −1 ), and olefinic (1633 cm −1 ) groups.
The 13 C NMR spectral data of the aglycone parts of the glycoside 2 (Table 1) were found to be identical to those of the aglycone of variegatuside B [13], which had been previously identified as 23(S)-hydroxylholosta-8(9)-ene-3β-ol. And in the downfield region of the 13 C NMR spectrum of 2, signals at δ C 131.1 and 135.6 are present. Such signals were not characteristic for a 7(8)-or 9(11)-double bond. The DEPT and HMQC spectra of variegatuside D (2) indicated that these signals are signals of quaternary carbons belong to a 8(9)-double bond. This structure for the glycoside 2 was confirmed by the NMR spectra and 1 H-1 H COSY, HMBC, HMQC, TOCSY and NOESY.
The five β-monosaccharide units were identified as xylose, glucose, and 3-O-methyl glucose in a 2:2:1 ratio based on the 1 H and 13 C NMR spectra, which showed five anomeric carbons The NMR data of compound 3 (Table 3) suggested that the aglycone of 3 was quite comparable to those of variegatuside A [13], differing only from the position of an olefinic bond at 9(11). Resonances for a 9(11)-double bond [δ C 151.2 (C-9) and 111.0 (C-11); δ H 5.60 (1H, brd, H-11)] were present. The position of C=C bond in 9, 11 was corroborated by the HMBC correlations Me-19/C-9, H-5/C-9, and H-2/C-11. This conclusion was confirmed from the TOCSY and 1 H-1 H COSY spectrum.
The presence of six monosaccharide units in the sugar chain of the glycoside 3 was deduced from its 13 C NMR and DEPT spectra, which showed six anomeric carbons at δ C 104.  Table 4). The oligosaccharide chain of 3 was quite identical to that of desacetystichloroside B 1 [12]. The monosaccharide units in 3 were identified as xylose, glucose, and 3-O-methylglucose in a 1:1:1 ratio by acidic hydrolysis with aqueous 2 mol/L trifluoroacetic acid followed by GC-MS analysis of the corresponding aldonitrile peracetates.    −1 ), carbonyl (1750 cm −1 ), and olefinic (1635 cm −1 ) groups.
The analysis of 13 C NMR spectral data of the aglycone moiety of variegatuside F (4) showed the presence of the signal at δ C 180.7 (C-18) characteristic for the 18(20)-lactone. The signals of carbons C-22~C-27 were coincident with the corresponding signals in the 13 C NMR spectrum of variegatuside A [13]. This indicated its side chains have a hydroxyl group at δ C 65.5 (C-23) ( Table 3). Glycoside 4 is a native compound from the sea cucumber S. variegates Semper which has been reported as desacetylstichloroside B 1 [12]. The 1 H and 13 C NMR spectral data of 4 (Table 3) suggested the presence of a triterpene aglycone with an olefinic bond, one ester, and one hydroxyl group bonded to an oligosacchaide chain. Resonances for a 7 (8) Table 3).
Comparison of the NMR spectra of carbohydrate moieties of variegatuside E (3) and variegatuside F (4) ( Table 4)   Some triterpene glycosides hitherto isolated from sea cucumber exhibited significant antifungal activity [15]. Glycosides 1-7 exhibited selective antifungal activities against six strains while 2, 3 had significant growth inhibitory activities against six strains (Table 5). These facts suggest that the Δ 25 terminal double bond may increase the activity. The component of the carbohydrate chain seems play an important role whereas the position of trisubstituted double bond in aglycone moiety (Δ 7 , Δ 8 or Δ 9 (11) ) contributes little to the bioactivity. Therefore, more extensive studies are needed before a clear structure-activity relationship can be reached.

General Experimental Procedures
Melting points were determined on an XT5-XMT apparatus. Optical rotations were measured on a Perkin-Elmer-341 polarimeter (PerkinElmer). IR spectra were recorded on a Bruker Vector-22 infrared spectrometer (Bruker). NMR spectra were obtained from Varian Inova-600 spectrometer with standard pulse sequence. ESI-and HR-ESI-MS were acquired on Micromass Quattro mass spectrometer (Varian). GC-MS were performed on a Finnigan Voyager apparatus (Finnigan Voyager, San Jose, CA, USA) using a DB-5 column (30 m × 0.25 mm i.d., 0.25 μm). HPLC was carried out on an Agilent 1100 liquid chromatograph (Agilent Technologies, Palo Alto, CA, America) equipped with a refractive index detector using a Zorbax 300 SB-C 18

Extraction and Isolation
The sea cucumber (15 kg, wet weight) were crumbled and extracted at room temperature three times with 70% ethanol (20 L, 7 days for each extraction). The combined extracts were concentrated to leave a rufous residue, which was suspended in H 2 O and then partitioned successively with petroleum ether and n-BuOH. The n-BuOH fraction (17.6 g) was chromatographed on silica gel eluting with CHCl 3 -MeOH-H 2 O (8.2:1.8:1 to 6.5:3.5:1, lower phase) gradient to give three fractions (A-C) based on thin-layer chromatography (TLC) analysis. Fractions B and C mainly contained triterpene glycosides. Fr. B (2.1g) was subjected to reversed-phase silica MPLC (Lichroprep RP-C 18 , 40-63 μm,) eluting with an aq. MeOH (30%-70%) gradient to give 5 sub-fractions, and the Fr. B3 (165 mg) was