A NEW TRITERPENE GLYCOSIDE FROM THE SEA CUCUMBER HOLOTHURIA SCABRA COLLECTED IN VIETNAM

Bioassay guided fractionation led to the isolation of a new triterpene glycoside, holothurinogenin B (1) along with three known compounds, holothurin B (2), holothurin A (3), and holothurin A2 (4), from the methanol extract of the Vietnamese sea cucumber Holothuria scabra. Their structures were deduced from the spectral analysis (1D-NMR, 2D-NMR, MS) and chemical evidences.


INTRODUCTION
Sea cucumbers belong to the class Holothurioidea which widely distributed in Atlantic and Pacific Oceans.They have been used in Vietnamese traditional medicine for long time as tonics and delicacies [1].Pacific islanders used the holothurian body tissues as a toxin to kill fishes.To date, dozens of triterpene glycosides of holostane type have been identified from the holothurians [2,3].They expressed a broad spectrum of antifungal, antibacterial and cytotoxic activity [4,5].As a part of our on going study on bioactive substances from marine invertebrates, we have isolated several triterpene glycosides from the polar fractions of methanol extracts of the sea cucumber Holothuria scabra collected in Vietnam whose structures were elucidated by spectral data ( 1 H-NMR, 13 C-NMR, DEPT, 2D-NMR and ESI MS).

RESULTS AND DISCUSSION
Compound 1 was obtained as amorphous powders.The molecular formula was established as C 41 H 62 O 13 from the [M+H] + ion at m/z 762.7, [M+Na] + ion at m/z 785.3 in the positive ion mode ESI MS and from the [M-H] -ion at m/z 761.3 in the negative ion mode.The 13 C-NMR spectrum of 1 revealed that the aglycon part was similar to 22,25-oxidoholothurinogenin, an artificial genin from Holothuria leucospilota which possesses two olefinic double bonds and a lactone carbonyl group in the holostane nucleus [6,7].The 1 H-NMR, 13 C-NMR and DEPT spectra displayed resonances due to the presence of seven tertiary methyl groups, two olefinic bonds at C-7 (δ H 5.55, δ C 121.0/C-8(δ C 142.7) and C-9(δ C 148.8) /C-11(δ H 5.30, δ C 113.2) and C-11 (δ H 5.30, δ C 113.2, 148.8), and one lactone carbonyl group (δ C 178.3).The correlations of H-7 to C-6, H-11 to C-8, C-10, C12 and C-13 was obtained in the HMBC, these confirmed the position of two double bonds in the holostane skeleton at C-7/C-8 and C-9/C-11.The 13 C-NMR spectrum had a signal characteristic for the presence of a hydroxyl group at C-17 (δ C 86.9).The side chain in aglycon moiety of 1 was shown to be identical to that of holothurin B by comparison of the NMR spectra of their corresponding side part [8].The remaining part of the aglycon was confirmed by 1 H-NMR, 13 C-NMR, HSQC, and HMBC spectra (Table 1) and was identical to the artificial compound 22,25-oxidoholothurinogenin.The spectroscopic data of the aglycon and sugar moiety of 4 was coincident to that of 3 except for the data of the side chain.This suggested that 4 has the same holostane skeleton and tetrasaccharide chain composed of four sugar units with 3 but difference of the side chain subtitute.By inspecting the

R=
The isolation and determination of a new compound holothurinogenin B together with holothurin A, B, A 2 are precious for investigating the chemical diversity of marine organisms.Holothurin A and B are the major components of Holothuria scabra and many other holothurins [11].This class of compound shows typical cytotoxic activity which suggested for the development of anticancer agents in the years to come.

Animal materials
The

Extraction and isolation
Dried specimens of the sea cucumber were extracted three times with MeOH (7 days each time) and then concentrated under low pressure to obtain 150 g MeOH extract.The MeOH extract were suspended in water and partition with hexane, chloroform and n -butanol.All fractions were tested with cytotoxic activity with two cancer cell lines KB (Human epidermoid carcinoma) and Hep-2 (Human hepatocellular carcinoma) in an in vitro assay system.The CHCl 3 and BuOH fractions showed considerable activity and were selected for futher isolation of bioactive components.The BuOH fraction was chromatographed on silicagel column eluting with CHCl 3 -MeOH gradient (from 10:1 to 1:1) to give fraction B1, B2, and B3.Fraction B2 yeilded 20 mg of pure holothurinogenin B and 100 mg of holothurin B by using reversed phase YMC column with Acetone -H 2 O (3:1).Fration B3 was chromatographed using CHCl 3 -MeOH -H 2 O (20:10:1) to afford pure holothurin A (70 mg) and holothurin A 2 (6 mg).

Fig. 2 : 4 Table 2 :
Fig. 2: Structures of 2, 3, and 4 specimens of Holothuria scabra were collected at a deep of 3 -30 m in Catba, Haiphong province, North of Vietnam in Feb, 2006 and deep frozened until used.The sea cucumber Holothuria scabra was identified by Dr. Do Cong Thung, Institute of Marine Resources and Environment, Vietnamese Academy of Science and Technology, Vietnam.A voucher of specimen was deposited at Institute of Natural Products Chemistry, Vietnamese Academy of Science and Technology, Hanoi, Vietnam.

Table 1 :
[2,8]rs from the artificial genin by the presence of an oligosaccharide chain composed of two sugar units.The 1 H-NMR and 13 C-NMR spectra of 1 were similar to those of holothurin B which has carbonhydrate chain (a D-xylose attached to a D-quinovose) at C-3 (δ C 90.6) of the aglycon.The 1 H-NMR spectrum of 1 exhibited two anomeric proton signals at δ H 4.42 (d, J = 7.0 Hz, xylose) and δ H 4.55 (d, J = 7.5 Hz, quinovose) and a doublet at δ H 1.28 (J = 6.0 Hz) confirmed the position of a methyl group of the quinovose residue.The upfield shift at C-4 signal (δ C 71.1) demonstrated the absence of sulfate at C-4 of the xylose unit.The structure of sugar moiety was deduced by using 1 H-NMR, 13 C-NMR, HSQC and HMBC spectra (Table2).Based on the spectroscopic evidence and in comparison with published literature[2,8], compound 1 was elucidated to be 3β-O-[β-D-quinovopyranosyl-(1→2)-β-D-xylopyranosyl]-22,25-epoxyholosta-7,9(11)-diene-17-ol, which we named as holothurinogenin B. To our best knowledge, this compound was isolated for the first time from the nature.