Triterpene glycosides from the Vietnamese sea cucumber Holothuria edulis

Abstract From the MeOH extract of the Vietnamese sea cucumber Holothuria edulis, eight triterpene glycosides (1–8), including one new compound namely holothurin A5 (1), were isolated by using various chromatographic separations. Their structures were established by spectroscopic experiments including 1D, 2D NMR and HR-ESI-MS. Holothurin A5 (1) has a hydroperoxy group at C-25. To the best of our knowledge, this is the first report of this group in triterpene saponins obtained from sea cucumbers to date. In addition, the in vitro cytotoxicity against five human cancer cell lines (HepG2, KB, LNCaP, MCF7 and SK-Mel2) of all isolated compounds was also evaluated using SRB assays. GRAPHICAL ABSTRACT


Introduction
Sea cucumbers (or holothurians), marine invertebrates from the class Holothuroidea containing approximately 1700 species and commonly known as trepang, bechede-mer or gamat, have long been utilized in the food and folk medicine systems of Asian and Middle Eastern communities because of its effectiveness against hypertension, asthma, rheumatism, cuts and burns, impotence and constipation. These invertebrates have a leathery skin and an elongated body containing a single branched gonad (Bordbar et al. 2011;Guo et al. 2015;Pangestuti and Arifin 2018). They are an important component of the marine ecosystem and distributed in all oceans the world over, generally living near corals, rocks or sea weeds in warm shallow waters. Most of the harvestable species of sea cucumbers, mainly targeted as beche-de-mer, belong to genera Bohadschia, Holothuria, Actinopyga, Isostichopus, Stichopus, Parastichopus, Thelenota and Cucumaria (Bordbar et al. 2011). Recently, some sea cucumbers were suggested to have a balanced nutritional quality suitable for human consumption (Roggatz et al. 2016;. Previous investigations on the Holothuria species have reported triterpene glycosides and glycosphingolipids as main constituents (Kornprobst 2014). Among Holothuria species, H. edulis is a little investigated species with a few triterpene glycosides reported to date (Kalinin and Stonik 1982;Kobayashi et al. 1991). This type of compounds is wellknown to have hemolytic, cytotoxic, antifungal and other biological activities caused by membranotropic action (Aminin et al. 2015). As part of our ongoing investigations on chemical constituents and cytotoxic activity of the Vietnamese sea cucumbers Vien et al. 2018), the current paper deals with the isolation, structure elucidation, and cytotoxic activity of eight triterpene glycosides (1-8, Figure 1) from the sea cucumber Holothuria edulis.
Holothurin A 5 (1) was isolated as a white amorphous powder. Its molecular formula was determined as C 54 H 85 NaO 28 S from the [M À Na]ion peak at m/z 1213.4952 in the negative HR-ESI-MS as well as the [M þ Na] þ sodium adduct ion peak at m/z 1259 in the positive ESI-MS. The fragment ion peak at m/z 97 [HSO 4 ]in the negative ESI-MS/ MS spectrum of the ion at m/z 1213 [M À Na]showed the presence of a sulfate group in 1. The NMR features indicated a holostane-type saponin, one of the main constituents of Holothuria sea cucumbers (Kornprobst 2014;Honey-Escandon et al. 2015). The 1 H NMR spectrum exhibited typical signals of four anomeric protons (each 1H, d, J ¼ 7.5 or 8.0 Hz) at d H 4.62 (H-1 0 ), 5.01 (H-1 00 ), 4.88 (H-1 000 ) and 5.25 (H-1 0000 ), which had HSQC correlations with the corresponding anomeric carbons at d C 105.0 (C-1 0 and C-1 00 ), 104.4 (C-1 000 ) and 105.1 (C-1 0000 ), confirming presence of four sugar moieties in 1. The completed 1 H and 13 C NMR data for all four sugar moieties (see Table S1, Supplementary material) were assigned by detailed analysis of the HSQC, HMBC, 1 H-1 H COSY, 1D and 2D TOCSY experiments. These data were similar to those of 2, 3 and 6 (Oleinikova et al. 1982;Thanh et al. 2006;Yuan et al. 2008), suggesting the tetrasaccharide chain as 3  Figure S4, Supplementary material) of H-1 0000 with C-3 000 , H-1 000 with C-4 00 and H-1 00 with C-2 0 confirmed the sequence of sugar moieties in the tetrasaccharide chain of 1.  Figure S11). The D-configuration of all the four sugar moieties was assigned on the basis of biosynthetic reasons, analogy with 2, 3 and 6, coexistence in the sea cucumber H. edulis, and reported holostane saponins from sea cucumbers (Kornprobst 2014;Honey-Escandon et al. 2015;Kalinin et al. 2015).
In addition, the 1 H and 13 C NMR data for the aglycone of 1 were similar to those of holothurin A 2 (2) (  H 3 -27), each 3H, s] in 1. The 13 C NMR peak at C-25 of 1 was strongly shifted downfield at d C 81.1 relative to that of leucospilotaside A (5) at d C 69.0 (Han et al. 2007) and holothurin B 4 at d C 69.5 (Silchenko et al. 2005), indicating presence of a hydroperoxy group at this carbon (Nakamura et al. 2007). The COSY correlations of H-22/H-23/H-24 as well as HMBC cross-peaks of H 3 -21 with C-17, C-20 and C-22; H 3 -26 and H 3 -27 with C-24 and C-25 clearly elucidated the side chain of 1 as shown in the Figure 1. The relative configurations of the triterpene skeleton of 1 were assigned to be identical to those of 2-8 from biosynthesis view with coexistence of them in the sea cucumber H. edulis. This assignment was also supported by the agreement of their 1 H and 13 C NMR data as well as by ROESY experiment (see Figure S9, Supplementary material). Finally, attachment of the tetrasaccharide chain at C-3 of the aglycon was demonstrated by HMBC cross-peak of the anomeric proton H-1 0 with C-3. Thus, the structure of 1 was established to be 3b sulfate-b-D-xylopyranosyl]-25-hydroperoxyholost-9(11),23E-diene-12a,17a-diol. The presence of hydroperoxy groups in triterpene saponins is relatively rare. To the best of our knowledge, this is the first report of this group in triterpene saponins obtained from sea cucumbers to date.
The cytotoxic activity against five human cancer cell lines as HepG2 (hepatoma cancer), KB (epidermoid carcinoma), LNCaP (prostate cancer), MCF7 (breast cancer), and SK-Mel2 (melanoma) of isolated compounds 1 -8 was evaluated by SRB assay (Monks et al. 1991) as previously described (Cuong et al. 2015). As the obtained results (see Table S2, Supplementary material), compounds 2, 6 and 7 showed strong cytotoxicity (IC 50 values from 0.75 ± 0.09 to 3.66 ± 0.41 mM) against all five cell lines, comparable to that of the positive control (ellipticine: IC 50 values from 1.42 ± 0.12 to 1.71 ± 0.24 mM). Compounds 1, 3 and 4 showed moderate cytotoxic effect against these cell lines with IC 50 values from 46.65 ± 2.28 to 82.75 ± 3.91 mM, whereas 5 and 8 revealed weak (IC 50 $ 90 mM) or less effect (IC 50 > 100 mM). Consideration the structures of 1 -5 suggested that presence of hydroperoxy, acetoxy or hydroxy group at C-25 might reduce cytotoxicity of these compounds against the tested cancer cells. In addition, comparison the cytotoxicity of compounds 6 -8 suggested that presence of the quinovose moiety in the sugar chain might play an important role for their cytotoxic effect on the tested cancer cells.

Biological material
The samples of the sea cucumber Holothuria (Halodeima) edulis Lesson, 1830 were collected at Cu Lao Cham Island, Quangnam, Vietnam, in August 2016, and identified by Prof. Do Cong Thung. The voucher specimens (CLC-QN-HS01) were deposited at the Institute of Marine Biochemistry and Institute of Marine Environment and Resources, VAST, Vietnam.

Supplementary material
General experimental procedures; 1D, 2D NMR and HR ESI mass spectra; key COSY, HMBC and ROESY correlations; plausible (-) ESI MS/MS fragmentation; 1 H and 13 C NMR data of the new compound 1 as well as IC 50 values of compounds 1-8.