Analysis of saponins as bioactive zoochemicals from the marine functional food sea cucumber...

1 23

European Food Research andTechnologyZeitschrift für Lebensmittel-Untersuchung und -Forschung A ISSN 1438-2377Volume 238Number 6 Eur Food Res Technol (2014)238:937-955DOI 10.1007/s00217-014-2171-6

Analysis of saponins as bioactivezoochemicals from the marine functionalfood sea cucumber Bohadschia cousteaui

Mohamed Elbandy, Jung Rae Rho &Raffat Afifi

1 23

Your article is protected by copyright and

all rights are held exclusively by Springer-

Verlag Berlin Heidelberg. This e-offprint is

for personal use only and shall not be self-

archived in electronic repositories. If you wish

to self-archive your article, please use the

accepted manuscript version for posting on

your own website. You may further deposit

the accepted manuscript version in any

repository, provided it is only made publicly

available 12 months after official publication

or later and provided acknowledgement is

given to the original source of publication

and a link is inserted to the published article

on Springer's website. The link must be

accompanied by the following text: "The final

publication is available at link.springer.com”.

1 3

Eur Food Res Technol (2014) 238:937–955DOI 10.1007/s00217-014-2171-6

ORIgInal PaPER

Analysis of saponins as bioactive zoochemicals from the marine functional food sea cucumber Bohadschia cousteaui

Mohamed Elbandy · Jung Rae Rho · Raffat Afifi

Received: 13 november 2013 / Revised: 15 January 2014 / accepted: 16 January 2014 / Published online: 12 February 2014 © Springer-Verlag Berlin Heidelberg 2014

their therapeutic values as a functional food than for their seafood taste.

Keywords Bohadschia cousteaui · Sea cucumber · Saponins · Holostane-type triterpene · Coustesides · Bioactive compounds · Marine functional food

Introduction

In recent decades, the concept of functional foods has offered a new and practical approach to achieving optimal health by promoting the use of natural products with physi-ological benefits, thus reducing the risk of various chronic diseases [1, 2]. Most of the currently available functional foods and therapeutic agents are derived either directly or indirectly from naturally occurring sources, especially the terrestrial food plants and marine species [2–4]. Sea cucumber, Bohadschia cousteaui, belongs to the genus Bohadschia of the Holothuriidae family. There are about 22 species of sea cucumber in Egyptian waters. B. coust-eaui is widely distributed in the Red Sea [5]. Sea cucumber has been a traditional healthy food for thousands of years. It has been used in asian traditional medicine for a long time as tonics and delicacies. Sea cucumber as a type of sea foods, known as Beche-de-mer or trepang, suffers from over catch because of its commercial exploitation as marine functional food [6]. Sea cucumbers have high-value com-ponents and bioactives as well as biological and medicinal properties of these multipurpose marine invertebrates, as one of the potential sources for functional foods and nutra-ceuticals. nutritionally, sea cucumbers have an impressive profile of valuable nutrients such as vitamin a, vitamin B1 (thiamine), vitamin B2 (riboflavin) and vitamin B3 (nia-cin), essential amino acids such as leucine and lysine and

Abstract The edible portion of sea cucumber, body walls, is a source of natural bioactive compounds. Triter-pene saponins are the main chemical constituents in sea cucumber that have potential interest for the body health and food industry. Twenty-one lanostane-type non-sul-phated triterpene glycosides were isolated from the metha-nol/methylene chloride extract of the body walls of Bohad-schia cousteaui. Ten new saponins called coustesides a (1), B (3), C (9), D (10), E (11), F (12), g (15), H (16), I (17) and J (18), including two pentasaccharide and eight hexa-saccharide saponins, together with eleven known triterpene glycosides, were isolated by reversed-phase semi-pre-parative HPlC. Their structures were mainly determined by 1D- and 2D-nMR (1H, 13C, COSY, TOCSY, HSQC, HMBC and ROESY) as well as MS experiments and acid hydrolysis. Most of the isolated compounds showed good antifungal activity against Candida albicans. Moreover, sea cucumber B. cousteaui is a rich source of biologically active saponins. Therefore, sea cucumbers are eaten for

M. Elbandy (*) Department of Food Sciences and Technology, Faculty of Environmental agricultural Sciences, Suez Canal University, Elarish, north Sinai, Egypte-mail: [email protected]

M. Elbandy Department of Clinical nutrition, Faculty of applied Medical Sciences, Jizan University, Jizan, KSa

J. R. Rho Department of Oceanography, Kunsan national University, Kunsan 673-701, Jeonbuk, Korea

R. afifi Department of Marine Sciences, Faculty of Science, Suez Canal University, Ismailia, Egypt

Author's personal copy

938 Eur Food Res Technol (2014) 238:937–955

1 3

minerals, especially calcium, magnesium, iron and zinc. For modern applications, dried sea cucumber used as a nutritional supplement is prepared in capsules or tablets [7, 8]. Therapeutic properties and medicinal benefits of sea cucumbers can be linked to the presence of a wide array of bioactives, especially triterpene glycosides (saponins), chondroitin sulphates, glycosaminoglycan (gags), sul-phated polysaccharides, sterols (glycosides and sulphates), phenolics, cereberosides, lectins, peptides, glycoprotein, glycosphingolipids, and essential fatty acids [8, 9]. Triter-pene glycosides are the most important secondary metab-olites of sea cucumbers because of their biological activi-ties, including antifungal, cytotoxic, haemolytic, cytostatic and immunomodulatory effects. However, food and non-food sources of saponins have come into renewed focus in recent years due to increasing evidence of their benefits such as cholesterol-lowering and anticancer properties [10, 11]. Due to the presence of a lipid-soluble aglycone and water-soluble sugar chain (s) in their structure (amphiphilic nature), saponins are surface-active compounds with deter-gent, wetting, emulsifying and foaming properties. There-fore, saponins may be used as natural food additives. The commercial potential of saponins has resulted in the devel-opment of new process/processing strategies and re-evalu-ation of existing technologies for their extraction, concen-tration, fractionation, purification and structure elucidation [12].The present study is a part of our continuing investi-gation on bioactive compounds of functional foods [13, 14]. Sea cucumber and its extracts are frequently claimed to be part of nutritional supplements or functional foods or even as a complement for the treatment of certain diseases [9]. Thus, the general purpose of this work is to deepen our knowledge on the chemical composition of saponins as the main bioactive constituents of the sea cucumber B. cousteaui. This paper completes the previous report [15] of the first chemical study of this specie. The results would be a first step to assess the suitability of this sea food, as a source of natural bioactive compounds potentially use-ful for the food industry, to prepare functional foods or as a source of natural food additives and pharmaceutical products.

Materials and methods

general experimental procedures

Optical rotations were measured on a P-1010 digital polarimeter (Jasco, Easton, MD, USa). IR spectra were recorded on a FT/IR-4100 spectrometer (Jasco, Tokyo, Japan). UV spectra were recorded on a Cary 50 Bio spec-trometer (Varian, Palo alto, USa). The 1D- and 2D-nMR spectra were obtained on a VnMRS 500 spectrometer

(Varian, Palo alto, Ca, USa) working at 500 MHz for 1H and 125 MHz for 13C. all nMR chemical shifts were referenced to CD3OD at δH 3.30 and δC 49.0 as an inter-nal standard. HRFaB mass spectra were acquired with a JMS-700 model (MStation, JEOl, Tokyo, Japan). TlC was performed on silica gel F245 plates (Merck, Darm-stadt, germany), detection by spraying with 10 % H2SO4 followed by heating. Following are the solvent systems: for saponins, CHCl3/MeOH/acOH/H2O 15:8:3:2 (a); for sapogenins, CHCl3/MeOH 9:1 (b); for monosaccharides, CHCl3/MeOH/H2O 8:5:1 (c); Following are the spray rea-gents: for saponins, Komarowsky reagent, 2 % 4-hydroxy-benzaldehyde in MeOH/50 % H2SO4 soln. 5:1 [16]; for the sugars, diphenylamine/phosphoric acid reagent. HPlC was performed using a Rheodyne syringe-loading sample injector with a 100-μl sample loop, manual HPlC injec-tion syringe 100 μl (Hamilton, Bonaduz, Switzerland), a 355 refractive index detector (Varian, Palo alto, Ca, USa) using the YMC ODS-a column (10 × 250 mm, S-5 μm) (YMC Company ltd., Kyoto, Japan) and the Varian polaris nH2 column (4.6 × 250 mm, S-5 μm) (Varian, Palo alto, Ca, USa), isocratic solvent system 70 % aq. MeOH; flow rate 2 ml/min; methanol, HPlC grade (Merck, nJ, USa). Water was purified by a Milli-Q system (Millipore, Bed-ford, Ma, USa). all other solvents were distilled prior to use. authentic sugars (quinovose, glucose, xylose and Me-glucose) were purchased from (Fluka, Buchs, Switzerland) and (Sigma Chemical Co, St. louis, MO, USa). all other chemicals and reagents were purchased locally and were of analytical grade.

Sample materials

The specimens of B. cousteaui were collected from the Red Sea, gulf of aqaba in December 2007 and deep-fro-zen until used. The sea cucumber B. cousteaui was identi-fied by Mr Mohamed ahmed (Marine Science Dept., Suez Canal University, Ismailia, Egypt.). a voucher of specimen was deposited at the Museum of Marine Science Depart-ment, Suez Canal University.

Extraction and isolation

The body walls of B. cousteaui were cut into small pieces and lyophilized prior to extraction. The freeze-dried tis-sues were macerated overnight 4 times into sufficient volumes of methanol/methylene chloride (1:1). Extracts were combined and allowed to evaporate in a rotavapour at 40 °C until dry. The dry extracts (30 g) were suspended in water and successively partitioned with n-hexane and n-butanol. all the fractions were evaporated under reduced pressure at 40 °C until dryness. Dried hexane and butanol fractions (14 and 10 g, respectively) were kept in vials


939Eur Food Res Technol (2014) 238:937–955

1 3

for further analysis. The n-butanol fraction was redis-solved in 85 % aq MeOH and partitioned with n-hexane (dried hexane fraction 6.1 g). The 85 % aq MeOH fraction (crude glycoside containing mixture, 2.2 g) was chroma-tographed under vacuum over reversed silica gel column (10 × 15 cm) with 15–40 μm particle size, eluted with gra-dient of MeOH/H2O (50, 60, 70, 80, 90 and 100 %, 500 ml each) to yield six fractions [a (350 mg); B (70 mg); C (80 mg); D (200 mg); E (400 mg) and F (600 mg)]. Frac-tions D, E and F were selected for further separation on the basis of 1H-nMR monitoring. These fractions were subjected to ODS HPlC (Fig. 1) using isocratic solvent system (70 % aq MeOH; 2 ml/min) to afford twenty-one compounds, including ten new saponins named coust-esides a (1), B (3), C (9), D (10), E (11), F (12), g (15), H (16), I (17) and J (18) in addition to eleven known com-pounds 2, 4, 5, 6, 7, 8, 13, 14, 19, 20 and 21. Compounds 12 (10.0 mg), (Rt 75.70 min); 14 (9.0 mg), (Rt 40.80 min); and 21 (8.0 mg), (Rt 42.70 min) were isolated as pure com-pounds, while the other eighteen saponins were isolated in pairs as nine mixtures. Purities or compound ratios were carefully checked and determined in each mixture by using quantitative nuclear magnetic resonance (qnMR) and quantitative mass spectrometry (qMS) as simple relative quantification methods. qnMR is based on the fact that the signal intensities of a given nMR resonance are directly proportional to the molar amount of that nucleus in the sample [17]. Furthermore, qMS- or MS-based quantitative assay is also founded on the fact that the peak areas and heights are really relative to the concentration of various molecules present in the sample [18]. Thus, the calcula-tion of relative peak intensities of nMR plots and relative peak areas and heights of MS spectra of compound pairs in each mixture enables us to determine compound ratios or purity. These nine compound pairs were (1, 11), (12 mg, ratio 2:1), (Rt 59.10 min); (2, 13), (16 mg, ratio 1:1), (Rt 83.51 min); (3, 15), (10 mg, ratio 3:2), (Rt 64.72 min); (4, 16), (9 mg, ratio 1:1), (Rt 70.80 min); (5, 17), (15 mg,

ratio 3:2), (Rt 87.80 min); (6, 18), (70 mg, ratio 1:1), (Rt 124.70 min); (7, 19), (12 mg, ratio 1:1), (Rt 32.00 min); and (8, 20), (10 mg, ratio 1:1), (Rt 35.40 min); and (9, 10), (10 mg, ratio 3:2), (Rt 49.20 min), respectively.

Cousteside a (1): white amorphous powder; IR (film) 3,396, 1,749, 1,650, 1,074 cm−1; 1H- and 13C-nMR data (CD3OD) (see Tables 1, 2, 3, 4, 5 and 6); FaB-MS (posi-tive ion mode) m/z 1,464 [M + na]+; HR-FaB-MS (posi-tive ion mode) m/z 1,463.6773 [M + na]+ (calcd. for C67H108O33na, 1,463.6770).

Cousteside B (3): white amorphous powder; IR (film) 3,392, 1,743, 1,652, 1,073 cm−1; 1H- and 13C-nMR data (CD3OD) (see Tables 1, 2, 3, 4, 5 and 6); FaB-MS (posi-tive ion mode) m/z 1,446 [M + na]+; HR-FaB-MS (posi-tive ion mode) m/z 1,445.6667 [M + na]+ (calcd. for C67H106O32na, 1,445.6659).

Cousteside C (9): white amorphous powder; IR (film) 3,396, 1,733, 1,650, 1,074 cm−1; 1H- and 13C-nMR data (CD3OD) (see Tables 1, 2, 3, 4, 5 and 6); FaB-MS (posi-tive ion mode) m/z 1,304[M + na]+; HR-FaB-MS (posi-tive ion mode) m/z 1,303.6037 [M + na]+ (calcd. for C60H96O29na, 1,303.6031).

Cousteside D (10): white amorphous powder; IR (film) 3,396, 1,733, 1,652, 1,074 cm−1; 1H- and 13C-nMR data (CD3OD) (see Tables 1, 2, 3, 4, 5 and 6); FaB-MS (posi-tive ion mode) m/z 1,346 [M + na]+; HR-FaB-MS (posi-tive ion mode) m/z 1,345.6143 [M + na]+ (calcd. for C62H98O30na, 1,345.6135).

Cousteside E (11) white amorphous powder; IR (film) 3,395, 1,731, 1,651, 1,072 cm−1; 1H- and 13C-nMR data (CD3OD) (see Tables 1, 2, 3, 4, 5 and 6); FaB-MS (posi-tive ion mode) m/z 1,288 [M + na]+; HR-FaB-MS (posi-tive ion mode) m/z 1,287.6088 [M + na]+ (calcd. for C60H96O28na, 1,287.6082).

Cousteside F (12) white amorphous powder; IR (film) 3,396, 1,732, 1,650, 1,070 cm−1; 1H- and 13C-nMR data (CD3OD) (see Tables 1, 2, 3, 4, 5 and 6); FaB-MS (posi-tive ion mode) m/z 1,288 [M + na]+; HR-FaB-MS

Fig. 1 Semi-preparative HPlC chromatogram of the fraction F using YMC ODS-a column (10 × 250 mm, S-5 μm), isocratic solvent system (70 % aq MeOH). Flow rate: 2 ml/min. RI detector



1 3

Table 1 1H- and 13C-nMR data of the aglycon portion of compounds 1, 3, 9 and 10 (CD3OD, δ in ppm)

δ H (mult, J in Hz) δ13C δ H (mult, J in Hz) δ13C δ H (mult, J in Hz) δ13C δ H (mult, J in Hz) δ13C

Position 1 3 9 10

1a 1.52 (1 H, m) 37.4 (CH2) 1.51 (1 H, m) 37.5 (CH2) 1.48 (1 H, m) 37.2 (CH2) 1.48 (1 H, m) 37.2 (CH2)

1b 1.84 (1 H, m) 1.82 (1 H, m) 1.81 (1 H, m) 1.81 (1 H, m)


2b 1.97 (1 H, m) 1.94 (1 H, m) 1.95 (1 H, m) 1.95 (1 H, m)

3 3.16 (1 H, m) 90.7 (CH) 3.11 (1 H, dd, J = 5.9, 12.2)

90.4 (CH) 3.12 (1 H, m) 90.4 (CH) 3.12 (1 H, m) 90.3 (CH)

4 40.9 (C) 40.9 (C) 40.9 (C) 40.9 (C)

5 0.95 (1 H, m) 53.9 (CH) 0.94 (1 H, m) 53.9 (CH) 0.95 (1 H, d, J = 11.0)

53.9 (CH) 0.95 (1 H, d, J = 11.0)

53.8 (CH)


6b 1.73 (1 H, m) 1.73 (1 H, m) 1.73 (1 H, m) 1.73 (1 H, m)


7b 1.75 (1 H, m) 1.74 (1 H, m) 1.74 (1 H, m) 1.74 (1 H, m)

8 3.02 (1 H, dd, J = 5.4, 13.7)

41.3 (CH) 3.02 (1 H, dd, J = 5.9, 13.2)

41.3 (CH) 3.10 (1 H, m) 41.8 (CH) 3.10 (1 H, m) 41.6 (CH)

9 155.0 (C) 155.3 (C) 154.9 (C) 154.9 (C)

10 40.5 (C) 40.8 (C) 40.6 (C) 40.5 (C)

11 5.44 (1 H, br d, J = 4.1)

115.9 (CH) 5.43 (1 H, br d, J = 4.0)

115.8 (CH) 5.34 (1 H, br d, J = 4.7)

115.2 (CH) 5.34 (1 H, br d, J = 4.7)

115.2 (CH)

12 4.13 (1 H, br d, J = 4.1)

69.4 (CH) 4.13 (1 H, br d, J = 4.0)

69.5 (CH) 4.52 (1 H, d, J = 4.7)

72.9 (CH) 4.57 (1 H, d, J = 4.7)

72.6 (CH)

13 65.3 (C) 65.5 (C) 60.0 (C) 59.9 (C)

14 47.4 (C) 47.6 (C) 42.6 (C) 43.4 (C)


15b 1.62 (1 H, m) 1.62 (1 H, m) 2.16 (1 H, m) 2.30 (1 H, m)

16 1.97 (1 H, m) 24.9 (CH2) 1.98 (1 H, m) 25.0 (CH2) 4.69 (1 H, q-like, J = 7.6)

86.3 (CH) 5.71 (1 H, q-like, J = 7.6)

86.5 (CH)

2.01 (1 H, m) 2.00 (1 H, m)

17 2.94 (1 H, q-like, J = 5.4)

47.9 (CH) 2.93 (1 H, dd, J = 2.9, 10.8)

47.9 (CH) 89.8 (C) 89.4 (C)

18 179.5 (C) 179.6 (C) 176.8 (C) 175.8 (C)

19 1.15 (3H, s) 22.7 (CH3) 1.15 (3H, s) 22.8 (CH3) 1.14 (3H, s) 22.9 (CH3) 1.14 (3H, s) 22.8 (CH3)

20 86.7 (C) 86.6 (C) 90.0 (C) 90.1 (C)


22a 1.72 (1 H, m) 40.1 (CH2) 2.58 (1 H, d, J = 7.3)

43.5 (CH2) 1.89 (1 H, m) 37.9 (CH2) 1.82 (1 H, m) 38.5 (CH2)

22b 2.01 (1 H, m) 2.58 (1 H, d, J = 7.3)

2.54 (1 H, ddd, J = 4.1)

2.42 (1 H, m)

23a 2.00 (1 H, m) 24.0 (CH2) 5.60 (1 H, dt, J = 15.7, 7.3)

125.4 (CH) 2.07 (1 H, m) 24.7 (CH2) 2.06 (1 H, m) 24.5 (CH2)

23b 2.03 (1 H, m) 2.11 (1 H, m) 2.10 (1 H, m)

24 5.15 (1 H, t-like, J = 7.3)

124.6 (CH) 6.30 (1 H, d, J = 15.7)

138.8 (CH) 5.12 (1 H, t-like, J = 6.6)

126.0 (CH) 5.10 (1 H, t-like, J = 6.2)

125.4 (CH)

25 133.0 (C) 143.3 (C) 132.9 (C) 132.0 (C)

26 1.69 (3H, s) 25.9 (CH3) 4.94 (2H, br s) 116.4 (CH2) 1.67 (3H, s) 25.9 (CH3) 1.68 (3H, s) 25.8 (CH3)





Co 171.3



1 3

(positive ion mode) m/z 1,287.6088 [M + na]+ (calcd. for C60H96O28na, 1,287.6082).

Cousteside g (15) white amorphous powder; IR (film) 3,392, 1,733, 1,652, 1,073 cm−1; 1H- and 13C-nMR data (CD3OD) (see Tables 1, 2, 3, 4, 5 and 6); FaB-MS (posi-tive ion mode) m/z 1,270 [M + na]+; HR-FaB-MS (posi-tive ion mode) m/z 1,269.5983 [M + na]+ (calcd. for C60H94O27na, 1,269.5975).

Cousteside H (16) white amorphous powder; IR (film) 3,385, 1,730, 1,649, 1,073 cm−1; 1H- and 13C-nMR data (CD3OD) (see Tables 1, 2, 3, 4, 5 and 6); FaB-MS (posi-tive ion mode) m/z 1,272 [M + na]+; HR-FaB-MS (posi-tive ion mode) m/z 1,271.6139 [M + na]+ (calcd. for C60H96O27na, 1,271.6135).

Cousteside I (17) white amorphous powder; IR (film) 3,394, 1,733, 1,652, 1,074 cm−1; 1H- and 13C-nMR data (CD3OD) (see Tables 1, 2, 3, 4, 5 and 6); FaB-MS (posi-tive ion mode) m/z 1,290[M + na]+; HR-FaB-MS (posi-tive ion mode) m/z 1,289.6254 [M + na]+ (calcd. for C60H98O28na, 1,289.6248).

Cousteside J (18) white amorphous powder; IR (film) 3,395, 1,740, 1,652, 1,072 cm−1; 1H- and 13C-nMR data (CD3OD) (see Tables 1, 2, 3, 4, 5 and 6); FaB-MS (posi-tive ion mode) m/z 1,274[M + na]+; HR-FaB-MS (posi-tive ion mode) m/z 1,273.6296 [M + na]+ (calcd. for C60H98O27na, 1,273.6298).

acid hydrolysis and sugar analysis

Solutions of isolated saponins (each 5.0 mg) in H2O (2 ml) and 2 n CF3COOH (5 ml) were refluxed on a water bath at 75 °C for 6 h. Then, the reaction mixture was diluted with H2O (10 ml) and extracted with EtOac (3 × 20 ml). The combined EtOac extracts were washed with H2O and evaporated to dryness to afford the aglycon. The aqueous layer was repeatedly evaporated to dryness with MeOH, and the residue was dissolved in CH3Cn-H2O (1:1) and then analysed by TlC (c) and HPlC (Varian Polaris nH2 column (4.6 × 250 mm, S-5 μm; CH3Cn-H2O (90:10)). HPlC analysis was conducted by using an isocratic elution of CH3Cn-H2O (90:10) with RI detection, and the flow rate was set at 0.9 ml/min. Coelution experiments with stand-ard sugar samples allowed the identification of quinovose (tR = 9.97 min), methyl glucose (tR = 10.54 min), xylose (tR = 11.17 min) and glucose (tR = 19.74 min). Coinjection of each hydrolysate with standard d-quinovose, d-methyl

glucose, d-xylose and d-glucose gave consistent peaks. and then each of these elutes was individually collected, evaporated to dryness and dissolved in H2O, and then, opti-cal rotation was recorded at room temperature.

antifungal activity

The screening of isolated saponins for antifungal activity was done by the disc diffusion method, which is usually used as a preliminary check [19]. The appropriate solidi-fied medium (PDa for fungal strains) was inoculated with Candida albicans and spread over the plates using a ster-ile swab in order to get a uniform fungal growth on both control and test plates. after agar inoculation, sterile fil-ter discs (autoclaved at 121 °C for 15 min) (Whatman no 1, 6 mm diameter) were impregnated with 10 μl of stock solutions of pure compounds and different saponin mix-tures (1 mg/ml) and placed on the agar surface using ster-ile forceps. Filter disc moistened with DMSO solution was placed on the seeded Petri dish as a negative control. Filter discs loaded with ampicillin solution 10 μl (1 mg/ml) were used as a reference control. The dishes were then incubated at 25 °C, for 48 h. after the incubation period, the mean diameter of inhibition halo where C. albicans did not grow (clearly visible inhibition zone) was measured in millime-tres, for each disc, and evaluated for susceptibility or resist-ance using the comparative standard method. Blanks were prepared by adding 10 μl of DMSO solution to the filter discs. The effectiveness of each saponin or saponin mixture was calculated by measuring the diameter (in mm) of the zone of microorganism growth inhibition around the disc. Each assay in these experiments was repeated three times, and the results (mm of zone of inhibition) were expressed as average values (±standard deviation).

Results and discussion

Structure elucidation of biologically active saponins is the first step to study the relationship between chemical struc-ture and biological activity. Identification of the chemical structure may help to obtain information about which moi-eties in the saponin molecule are important to obtain potent and specific drugs. Moreover, they may give an evidence to the mechanism of the action of sea cucumber-derived drugs or sea cucumber as functional food [20]. Furthermore, the

Table 1 continued


Position 1 3 9 10

Me 2.20 (3H, s) 21.4



1 3

Table 2 1H- and 13C-nMR data of the aglycon portion of compounds 11–12, 15 and 16 (CD3OD, δ in ppm)


Position 11 12 15 16


1b 1.84 (1 H, m) 1.80 (1 H, m) 1.82 (1 H, m) 1.82 (1 H, m)


2b 1.97 (1 H, m) 1.95 (1 H, m) 1.94 (1 H, m) 1.95 (1 H, m)

3 3.16 (1 H, m) 90.7 (CH) 3.11 (1 H, m) 90.4 (CH) 3.11 (1 H, dd, J = 5.9, 12.2)

90.4 (CH) 3.12 (1 H, m) 90.4 (CH)

4 40.9 (C) 40.9 (C) 40.9 (C) 40.8 (C)

5 0.95 (1 H, m) 53.9 (CH) 0.95 (1 H, m) 53.9 (CH) 0.94 (1 H, m) 53.9 (CH) 0.94 (1 H, d, J = 12.0)

53.9 (CH)


6b 1.73 (1 H, m) 1.75 (1 H, m) 1.73 (1 H, m) 1.72 (1 H, m)


7b 1.75 (1 H, m) 1.75 (1 H, m) 1.74 (1 H, m) 1.74 (1 H, m)

8 3.02 (1 H, dd, J = 5.4, 13.7)

41.3 (CH) 3.00 (1 H, dd, J = 5.6, 13.0)

41.9 (CH) 3.02 (1 H, dd, J = 5.9, 13.2)

41.3 (CH) 3.02 (1 H, dd, J = 4.7, 13.0)

41.3 (CH)

9 155.0 (C) 155.5 (C) 155.3 (C) 155.1 (C)

10 40.5 (C) 40.5 (C) 40.8 (C) 40.5 (C)

11 5.44 (1 H, br d, J = 4.1)

115.9 (CH) 5.36 (1 H, br d, J = 4.7)

115.7 (CH) 5.43 (1 H, br d, J = 4.0)

115.8 (CH) 5.44 (1 H, br d, J = 4.7)

115.8 (CH)

12 4.13 (1 H, br d, J = 4.1)

69.4 (CH) 4.53 (1 H, br d, J = 4.7)

72.6 (CH) 4.13 (1 H, br d, J = 4.0)

69.5 (CH) 4.13 (1 H, br d, J = 4.7)

69.5 (CH)

13 65.3 (C) 59.8 (C) 65.5 (C) 65.3 (C)

14 47.4 (C) 47.0 (C) 47.6 (C) 47.4 (C)


15b 1.62 (1 H, m) 1.60 (1 H, m) 1.62 (1 H, m) 1.61 (1 H, m)

16 1.97 (1 H, m) 24.9 (CH2) 2.07 (1 H, m) 36.4 (CH2) 1.98 (1 H, m) 25.0 (CH2) 1.93 (1 H, m) 24.8 (CH2)

2.01 (1 H, m) 2.53 (1 H, m) 2.00 (1 H, m) 1.99 (1 H, m)

17 2.94 (1 H, q-like, J = 5.4)

47.9 (CH) 88.5 (C) 2.93 (1 H, dd, J = 2.9, 10.8)

47.9 (CH) 2.93 (1 H, q-like, J = 5.4)

47.8 (CH)

18 179.5 (C) 176.7 (C) 179.6 (C) 179.4 (C)


20 86.7 (C) 90.1 (C) 86.6 (C) 86.8 (C)


22a 1.72 (1 H, m) 40.1 (CH2) 1.74 (1 H, m) 39.5 (CH2) 2.58 (1 H, d, J = 7.3)

43.5 (CH2) 1.68 (1 H, m) 39.5 (CH2)

22b 2.01 (1 H, m) 2.38 (1 H, m) 2.58 (1 H, d, J = 7.3)

1.72 (1 H, m)

23a 2.00 (1 H, m) 24.0 (CH2) 2.06 (1 H, m) 23.9 (CH2) 5.60 (1 H, dt, J = 15.7, 7.3)

125.4 (CH) 1.47 (1 H, m) 23.2 (CH2)

23b 2.03 (1 H, m) 2.16 (1 H, m) 1.50 (1 H, m)

24 5.15 (1 H, t-like, J = 7.3)

124.6 (CH) 5.14 (1 H, t-like, J = 7.1)

124.8 (CH) 6.30 (1 H, d, J = 15.7)

138.8 (CH) 2.06 (1 H, m)2.09 (1 H, m)

39.0 (CH2)

25 133.0 (C) 133.0 (C) 143.3 (C) 146.3 (C)

26 1.69 (3H, s) 25.9 (CH3) 1.69 (3H, s) 26.0 (CH3) 4.94 (2H, br s) 116.4 (CH2) 4.72 (3H, s) 111.1 (CH2)







1 3

saponin analysis may indicate the suitability of this sea cucumber as a source of natural functional compounds or food additives for the food industry.

Structure elucidation of the isolated saponins

The methanol/methylene chloride extract of the body walls of sea cucumber B. cousteaui was successively partitioned

between H2O and n-hexane and n-butanol. The BuOH frac-tion was redissolved in 85 % aq MeOH and partitioned with n-hexane. The methanolic fraction was submitted to flash column chromatography on reversed silica gel eluting with a MeOH:H2O (50:50; 60:40; 70:30; 80:20; 90:10; and 100:00, 500 ml each) gradient to give six fractions (a–F). Fractions D, E and F were further purified by reversed-phase semi-preparative HPlC (YMC ODS-a column

Table 3 1H- and 13C-nMR data of the aglycon portion of compounds 17 and 18 (CD3OD, δ in ppm)

δH (mult, J in Hz) δ13C δ H (mult, J in Hz) δ13C

Position 17 18

1a 1.51 (1 H, m) 37.4 (CH2) 1.50 (1 H, m) 37.4 (CH2)

1b 1.83 (1 H, m) 1.82 (1 H, m)

2a 1.78 (1 H, m) 27.7 (CH2) 1.76 (1 H, m) 27.7 (CH2)

2b 1.96 (1 H, m) 1.95 (1 H, m)

3 3.15 (1 H, dd, J = 4.4, 12.7) 90.8 (CH) 3.11(1 H, m) 90.3 (CH)

4 40.9 (C) 40.8 (C)

5 0.95 (1 H, m) 53.9 (CH) 0.94 (1 H, m) 53.9 (CH)

6a 1.55 (1 H, m) 22.1 (CH2) 1.54 (1 H, m) 22.1 (CH2)

6b 1.72 (1 H, m) 1.72 (1 H, m)

7a 1.35 (1 H, m) 29.6 (CH2) 1.34 (1 H, m) 29.6 (CH2)

7b 1.75 (1 H, m) 1.75 (1 H, m)

8 3.02 (1 H, dd, J = 6.1, 13.2) 41.3 (CH) 3.02 (1 H, dd, J = 5.1, 13.1) 41.3 (CH)

9 155.0 (C) 155.0 (C)

10 40.5 (C) 40.5 (C)

11 5.44 (1 H, br d, J = 4.7) 115.9 (CH) 5.44 (1 H, br d, J = 4.4) 115.8 (CH)

12 4.13 (1 H, br d, J = 4.7) 69.5 (CH) 4.13 (1 H, br d, J = 4.4) 69.4 (CH)

13 65.3 (C) 65.2 (C)

14 47.4 (C) 47.4 (C)

15a 1.18 (1 H, m) 38.0 (CH2) 1.18 (1 H, m) 37.9 (CH2)

15b 1.61 (1 H, m) 1.60 (1 H, m)

16 1.95 (1 H, m) 24.9 (CH2) 1.95 (1 H, m) 24.9 (CH2)

1.97 (1 H, m) 1.97 (1 H, m)

17 2.92 (1 H, q-like, J = 4.7) 47.9 (CH) 2.92 (1 H, q-like, J = 4.7) 47.8 (CH)

18 179.5 (C) 179.5 (C)

19 1.15 (3H, s) 22.8 (CH3) 1.15 (3H, s) 22.8 (CH3)

20 86.9 (C) 86.8 (C)

21 1.44 (3H, s) 26.8 (CH3) 1.44 (3H, s) 26.8 (CH3)

22a 1.69 (1 H, m) 40.3 (CH2) 1.70 (1 H, m) 40.3 (CH2)

22b 1.72 (1 H, m) 1.72 (1 H, m)

23a 1.24 (1 H, m) 23.1 (CH2) 1.24 (1 H, m) 23.0 (CH2)

23b 1.35 (1 H, m) 1.33 (1 H, m)

24 1.22 (1 H, m) 40.5 (CH2) 1.21 (1 H, m) 40.5 (CH2)

1.25 (1 H, m) 1.25 (1 H, m)

25 1.56 (1 H, m) 29.0 (CH) 1.56 (1 H, m) 28.9 (CH)

26 0.91 (3H, s) 23.0 (CH3) 0.91 (3H, s) 22.9 (CH3)

27 0.91 (3H, s) 23.0 (CH3) 0.92 (3H, s) 23.0 (CH3)

28 1.09 (3H, s) 28.6 (CH3) 1.08 (3H, s) 28.6 (CH3)

29 0.90 (3H, s) 17.1 (CH3) 0.90 (3H, s) 17.1 (CH3)

30 0.97 (3H, s) 22.2 (CH3) 0.98 (3H, s) 22.1 (CH3)



1 3

Table 4 1H- and 13C-nMR data of the sugar portion of compounds 1, 3, 9 and 10 (CD3OD, δ in ppm)

δH (mult, J in Hz) δ13C δH (mult, J in Hz) δ13C δH (mult, J in Hz) δ13C δH (mult, J in Hz) δ13C

Position 1 3 9 10

Xyl

1 4.45 (1H, d, J = 7.0)

105.9 (CH) 4.42 (1H, d, J = 7.0)

105.8 (CH) 4.42 (1H, d, J = 7.0)

105.8 (CH) 4.42 (1H, d, J = 7.0)

105.8 (CH)

2 3.61(1H, m) 81.2 (CH) 3.52(1H, m) 82.7 (CH) 3.51(1H, m) 82.7 (CH) 3.51(1H, m) 82.7 (CH)

3 3.70(1H, m) 76.3 (CH) 3.68(1H, m) 76.2 (CH) 3.69 (1H, m) 76.3 (CH) 3.69(1H, m) 76.3 (CH)

4 3.71(1H, m) 78.5 (CH) 3.70(1H, m) 78.4 (CH) 3.70(1H, m) 78.5 (CH) 3.70(1H, m) 78.5 (CH)

5a 3.30(1H, m) 64.2 (CH2) 3.31 (1H, m) 64.1 (CH2) 3.31 (1H, m) 64.1 (CH2) 3.31 (1H, m) 64.1 (CH2)

5b 4.02 (1H, dd, J = 4.4, 11.2)

4.01 (1H, dd, J = 5.4, 12.2)

4.00 (1H, dd, J = 4.7, 12.0)

4.00 (1H, dd, J = 4.7, 12.0)

Qui or glc glc Qui Qui Qui

1 4.72 (1H, d, J = 7.8)

104.7 (CH) 4.61 (1H, d, J = 7.8)

105.2(CH) 4.61 (1H, d, J = 7.8)

105.2 (CH) 4.61 (1H, d, J = 7.8)

105.2 (CH)

2 3.27 (1H, m) 76.2 (CH) 3.28 (1H, m) 76.4 (CH) 3.28 (1H, m) 76.5 (CH) 3.28 (1H, m) 76.5 (CH)




6 3.78 (1H, dd, J = 4.4, 11.7)

62.2 (CH2) 1.35 (3H, d, J = 6.1)

18.1 (CH3) 1.35 (3H, d, J = 6.1)

18.1 (CH3) 1.35 (3H, d, J = 6.1)

18.1 (CH3)

3.91 (1H, dd, J = 2.5, 12.5)

glc 1

1 4.44 (1H, d, J = 7.8)

104.7 (CH) 4.41 (1H, d, J = 7.8)

104.7 (CH) 4.40 (1H, d, J = 8.1)

104.7 (CH) 4.40 (1H, d, J = 8.1)

104.7 (CH)





6a 3.62 (1H, m) 62.5 (CH2) 3.63 (1H, m) 62.4 (CH2) 3.63 (1H, m) 62.4 (CH2) 3.63 (1H, m) 62.4 (CH2)

6b 3.85 (1H, m) 3.85 (1H, m) 3.85 (1H, m) 3.85 (1H, m)

Me-glc 1

1 4.57 (1H, d, J = 8.0)

105.2 (CH) 4.57 (1H, d, J = 7.8)

105.2 (CH) 4.57 (1H, d, J = 7.6)

105.2 (CH) 4.57 (1H, d, J = 7.6)

105.2 (CH)



4 3.33 (1H, m) 71.1 (CH) 3.31 (1H, m) 71.1 (CH) 3.32 (1H, m)) 71.1 (CH) 3.32 (1H, m) 71.1 (CH)


6a 3.61 (1H, m) 62.3 (CH2) 3.61 (1H, m) 62.5 (CH2) 3.61(1H, m) 62.5 (CH2) 3.61 (1H, m) 62.5 (CH2)

6b 3.88 (1H, m) 3.86 (1H, m) 3.86 (1H, m) 3.86 (1H, m)

3-MeO 3.62 (3H, s) 61.2 3.62 (3H, s) 61.1 3.63 (3H, s) 3.63 (3H, s) 61.1

glc 2

1 4.42 (1H, d, J = 7.6)

102.9 (CH) 4.43 (1H, d, J = 7.8)

102.9 (CH) 4.36 (1H, d, J = 7.8)

103.4 (CH) 4.36 (1H, d, J = 7.8)

103.4 (CH)

2 3.40 (1H, m) 74.2 (CH) 3.41(1H, m) 74.5 (CH) 3.19 (1H, m) 74.7 (CH) 3.19 (1H, m) 74.7 (CH)





6b 4.42 (1H, d, J = 7.6)

3.85 (1H, m) 3.87 (1H, m) 3.87 (1H, m)



1 3

(10 × 250 mm, S-5 μm), 70 % aq. MeOH, 2 ml/min). as a result, ten new triterpene glycosides, namely coustesides a (1), B (3), C (9), D (10), E (11), F (12), g (15), H (16), I (17) and J (18) were isolated together with eleven known compounds 2, 4–8, 13–14, and 19–21(Fig. 2). Structural elucidation of the saponins was mainly determined by 1D- and 2D-nMR experiments (1H, 13C, DEPT, COSY, TOCSY, ROESY, HSQC and HMBC) and FaB-MS.

Cousteside a (1) was isolated as a white amorphous powder. It was a major saponin in a mixture with com-pound 11 (ratio 2: 1). Its molecular formula was established as C67H108O33 on the basis of a combination of a peak at m/z 1,463.6773 [M +na] + (calcd. for C67H108O33na, 1,463.6770) of HRFaBMS and the 13C-nMR spectrum. The IR absorptions of 1 implied the presence of hydroxyl (3,396 cm−1), lactone carbonyl (1,749 cm−1), olefinic (1,650 cm−1) and ether (1,074 cm−1) groups. The 1H-nMR, 13C-nMR and DEPT spectra displayed resonance due to seven tertiary methyl groups [δ 0.91 (3H, s), 0.98 (3H, s), 1.09 (3H, s), 1.15 (3H, s), 1.46 (3H, s), 1.64 (3H, s) and 1.69 (3H, s)], two olefinic bonds [δ 5.44 (1 H, br d, 4.1 Hz), δ115.9 (C-11)], and [δ 5.15 (1 H, t-like, 7.3 Hz), δ124.6 (C-24)], one lactone carbonyl group [δ179.5(C-18)], and one doublet [δ 4.13 (1 H, br d, 4.1 Hz), δ 69.4 (C-12)] that was ascribed to a methine proton linked to a carbon-bearing α hydroxyl group and suggested that the aglycone of 1 had a holostane triterpenoid skeleton with a 9 (11), 24 (25)-dien-12-ol moiety (Fig. 2). The relative stereochemis-try of all chiral centres of the aglycone was established with the aid of a ROESY experiment (Fig. 3). ROESY spectrum showed correlations between H-3 and H-1 α, [δ 1.52 (1 H, m)], H-5 α and Η-28, confirming the β-configuration at C-3. The 12 α-configuration of the hydroxyl group was confirmed by cross-peak between [δ 4.13 (1 H, br d, 4.1 Hz) and 1.46 (3 H, s)] in the ROESY spectrum and from the coupling constant for H-12 with H-11 (4.1 Hz)

[21]. The correlation between H-12 and H-11 in the ROESY spectrum also evidenced the α-configuration of the hydrogen at C-17. Further confirmation was obtained by the coupling constant between H-12 and H-11 (d = 4.1) [22]. The full assignment of all the 1H- and 13C-nMR sig-nals by 2D-nMR experiments associated with the aglycon moiety of 1 resulted in the establishment of its structure as a holosta-3β-12α-dihydroxy-9 (11), 24 (25)-dien (Tables 1, 2, 3), (Fig. 2). Most of the signals were in good agreement with literature data of the aglycon of pervicoside B and impatienside a previously reported from the sea cucumbers Holothuria pervicax and Holothuria impatiens, respec-tively [21, 23].

The 1H-nMR spectrum of cousteside a (1) displayed sig-nals for six anomeric protons at [δ 4.72 (1 H, d, 7.8 Hz), 4.57 (1 H, d, 8.0 Hz), 4.57 (1 H, d, 8.0 Hz), 4.45 (1 H, d, 7.0 Hz), 4.44 (1 H, d, 7.8 Hz) and 4.42 (1 H, d, 7.6 Hz)], which cor-related with the HSQC spectrum with 13C-nMR signals at [δ 104.7, 105.2, 105.2, 105.9, 104.7 and 102.9], respec-tively. The ring protons of the monosaccharide residues were assigned starting from the easily distinguishable signals due to anomeric protons by means of the COSY, TOCSY, HSQC and HMBC nMR spectra (Tables 4, 5, 6), and the sequence of the oligosaccharide chain was obtained from the ROESY (Fig. 3) and HMBC spectra (Fig. 4). The cou-pling constants of the anomeric protons were indicative of β-configuration of the glycosidic bonds in all cases. Evalu-ation of spin–spin couplings and chemical shifts allowed the identification of three β-glucopyranosyl (glc), two β-3-O-methyl-glucopyranosyl (Me-glc) and one β-xylopyranosyl (Xyl) units. all sugars of glycosides from B. cousteaui belong to the D-series as established by both HPlC analy-ses with authentic sugars after acid hydrolysis and optical rotation measurement for each isolated residue. The sugar chain was composed of glucose (d-glc), 3-O-methyl-glu-cose (d-Me-glc) and xylose (d-Xyl) in the ratio of 3:2:1.

m Overlapped with other signals

Table 4 continued

δH (mult, J in Hz) δ13C δH (mult, J in Hz) δ13C δH (mult, J in Hz) δ13C δH (mult, J in Hz) δ13C

Position 1 3 9 10

Me-glc 2

1 4.57 (1H, d, J = 8.0)

105.2 (CH) 4.56 (1H, d, J = 7.8)

105.2 (CH)

2 3.30 (1H, m) 75.4 (CH) 3.31(1H, m) 75.4 (CH)

3 3.10 (1H, m) 87.6 (CH) 3.10 (1H, m) 87.5 (CH)

4 3.34 (1H, m) 71.1 (CH) 3.32 (1H, m) 71.1 (CH)

5 3.30 (1H, m) 78.0 (CH) 3.30 (1H, m) 78.0 (CH)

6a 3.61 (1H, m) 62.3 (CH2) 3.32 (1H, m) 62.5 (CH2)

3.88 (1H, m) 3.86 (1H, m)

3-MeO 3.62 (3H, s) 61.2 3.62 (3H, s) 61.1



1 3

Table 5 1H- and 13C-nMR data of the sugar portion of compounds 11–12, 15 and 16 (CD3OD, δ in ppm)


δH (mult, J in Hz) δ 13C δH (mult, J in Hz) δ13C δH (mult, J in Hz) δ13C δH (mult, J in Hz) δ13C

Position 11 12 15 16

Xyl

1 4.45 (1H, d, J = 7.0)

105.9 (CH) 4.42 (1H, d, J = 7.1)

105.8 (CH) 4.42 (1H, d, J = 7.0)

105.8 (CH) 4.42 (1H, d, 5.4) 105.8 (CH)

2 3.61(1H, m) 81.2 (CH) 3.52 (1H, m) 82.8 (CH) 3.52 (1H, m) 82.7 (CH) 3.52 (1H, m) 82.7 (CH)




5b 4.02 (1H, dd, J = 4.4, 11.2)

4.01 (1H, dd, J = 4.7, 12.0)

4.01 (1H, dd, J = 5.4, 12.2)

4.01 (1H, dd, J = 4.7, 12.0)

Qui or glc glc Qui Qui Qui

1 4.72 (1H, d, J = 7.8)

104.7 (CH) 4.61 (1H, d, J = 7.8)

105.2(CH) 4.61 (1H, d, J = 7.8)

105.2(CH) 4.61 (1H, d, J = 7.8)

105.2(CH)





6 3.78 (1H, dd, J = 4.4, 11.7)

62.2 (CH2) 1.35 (3H, d, J = 6.1)

18.2 (CH3) 1.35 (3H, d, J = 6.1)

18.1 (CH3) 1.35 (3H, d, J = 6.1)

18.1 (CH3)

3.91 (1H, dd, J = 2.5, 12.5)

glc 1

1 4.44 (1H, d, J = 7.8)

104.7 (CH) 4.41 (1H, d, J = 7.8)

104.7 (CH) 4.41 (1H, d, J = 7.8)

104.7 (CH) 4.41 (1H, d, J = 7.8)

104.7 (CH)






6b 3.85 (1H, m) 3.85 (1H, m) 3.85 (1H, m) 3.85 (1H, m)

Me-glc 1

1 4.57 (1H, d, J = 8.0)

105.2 (CH) 4.57 (1H, d, J = 7.8)

105.2 (CH) 4.57 (1H, d, J = 7.8)

105.2 (CH) 4.57 (1H, d, J = 7.8)

105.2 (CH)






6b 3.88 (1H, m) 3.86 (1H, m) 3.86 (1H, m) 3.86 (1H, m)

3-MeO 3.62 (3H, s) 61.2 3.62 (3H, s) 61.2 3.62 (3H, s) 61.1 3.62 (3H, s) 61.1

glc 2

1 4.36 (1H, d, J = 7.8)

103.5 (CH) 4.36 (1H, d, 7.8) 103.4 (CH) 4.36 (1H, d, J = 7.8)

103.4 (CH) 4.36 (1H, d, 7.8) 103.4 (CH)






6b 4.44 (1H, d, J = 7.8)

3.87 (1H, m) 3.85 (1H, m) 3.87 (1H, m)



1 3

The D-configuration of these three carbohydrates units was in accordance with that most often encountered among the sea cucumber glycosides [24, 25]. The interglycosidic link-ages were deduced by analysis of the chemical shifts of the aglycon at [δ 90.7 (C-3)], for Xyl at [δ 81.2 (C-2) and 78.5 (C-4)], for glc at [δ 81.4 (C-4)], for glc 1 at [δ 87.5 (C-3)] and for glc 2 at [δ 87.6 (C-3)] (Tables 4, 5, 6), which

were shifted downfield relative to resonance expected for the corresponding glucopyranosides [26]. The structure of the carbohydrate chain of 1 was corroborated by the HMBC spectrum (Fig. 4), which showed a cross-peak between the proton signal of Xyl at δ 4.45 (H-1), and the carbon of the aglycon at δ 90.7 (C-3) indicated that the Xyl was con-nected to C-3 of the aglycon. Similarly, the location of the

Table 6 1H- and 13C-nMR data of the sugar portion of compounds 17 and 18 (CD3OD, δ in ppm)


δH (mult, J in Hz) δ 13C δH (mult, J in Hz) δ13C

Position 17 18

Xyl

1 4.45 (1H, d, J = 7.0) 105.9 (CH) 4.42 (1H, d, J = 7.0) 105.8 (CH)

2 3.61 (1H, m) 81.3 (CH) 3.52 (1H, m) 82.9 (CH)

3 3.70 (1H, m) 76.5 (CH) 3.70 (1H, m) 76.3 (CH)

4 3.70 (1H, m) 78.5 (CH) 3.70 (1H, m) 78.6 (CH)

5a 3.30 (1H, m) 64.2 (CH2) 3.31 (1H, m) 64.1 (CH2)

5b 4.02 (1H, dd, J = 4.0, 11.5) 4.01 (1H, dd, J = 4.4, 11.5)

Qui or glc glc Qui

1 4.72 (1H, d, J = 7.8) 104.7(CH) 4.60 (1H, d, J = 7.8) 105.2(CH)

2 3.27 76.2 (CH) 3.29a 76.4 (CH)

3 3.51a 76.1 (CH) 3.48 75.9 (CH)

4 3.48 81.5 (CH) 3.16 (1H, bs) 86.8 (CH)

5 3.38 (1H, bs) 77.0 (CH) 3.45 (1H, d, 3.4) 72.5 (CH)

6a 3.78 (1H, dd, J = 4.7, 12.0) 62.6 (CH2) 1.35 (3H, d, J = 6.1) 18.2 (CH3)

6b 3.91 (1H, dd, J = 2.5, 12.2)

glc 1

1 4.43 (1H, d, J = 7.8) 104.4 (CH) 4.41 (1H, d, J = 8.0) 104.7 (CH)

2 3.40(1H, m) 74.4 (CH) 3.40(1H, m) 74.0 (CH)

3 3.55 (1H, m) 87.6 (CH) 3.55(1H, m) 87.5 (CH)

4 3.40 (1H, m) 69.8 (CH) 3.38 (1H, m) 69.9 (CH)

5 3.32 (1H, m) 78.1(CH) 3.38 (1H, m) 77.8 (CH)

6a 3.63 (1H, m) 62.4 (CH2) 3.63 (1H, m) 62.4 (CH2)

6b 3.85 (1H, m) 3.85 (1H, m)

Me-glc

1 4.57 (1H, d, J = 7.8) 105.2 (CH) 4.57 (1H, d, J = 7.8) 105.2 (CH)

2 3.30 (1H, m) 75.4 (CH) 3.32 (1H, m) 75.4 (CH)

3 3.10 (1H, m) 87.7 (CH) 3.11 (1H, m) 87.6 (CH)

4 3.32 (1H, m) 71.2 (CH) 3.31 (1H, m) 71.1 (CH)

5 3.29 (1H, m) 78.0 (CH) 3.32 (1H, m) 78.0 (CH)

6a 3.65 (1H, m) 62.4 (CH2) 3.61 (1H, m) 62.5 (CH2)

6b 3.97 (1H, m) 3.86 (1H, m)

3-MeO 3.62 (3H, s) 61.2 3.62 (3H, s) 61.1

glc 2

1 4.36 (1H, d, J = 7.8) 103.4 (CH) 4.36 (1H, d, J = 7.8) 103.4 (CH)

2 3.19 (1H, m) 74.7 (CH) 3.20 (1H, m) 74.6 (CH)

3 3.28 (1H, m) 78.1 (CH) 3.31 (1H, m) 78.0 (CH)

4 3.26 (1H, m) 71.5 (CH) 3.27 (1H, m) 71.5 (CH)

5 3.34 (1H, m) 77.9 (CH) 3.35 (1H, m) 77.8 (CH)

6a 3.63 (1H, m) 62.6 (CH2) 3.63 (1H, m) 62.6 (CH2)

6b 3.85 (1H, m) 3.85 (1H, m)



1 3

Fig. 2 Structure of saponin compounds 1–21



1 3

interglycosidic linkage in the oligosaccharide chain was assigned on the basis of cross-peaks between the proton signal of glc at δ 4.72 (H-1) and the carbon of the Xyl at δ 81.2 (C-2), the proton signal of glc 1 at δ 4.44 (H-1) and the carbon of the glc at δ 81.4 (C-4), the proton signal of Me-glc at δ 4.57 (H-1) and the carbon of the glc 1 at δ 87.5 (C-3), the proton signal of glc 2 at δ 4.42 (H-1) and the car-bon of the Xyl at δ 78.5 (C-4) and the proton signal of Me-glc 2 at δ 4.57 (H-1) and the carbon of the glc 2 at δ 87.6 (C-3). The exact sequence of the sugars and their points of attachment were also confirmed by the ROESY cross-peaks between the proton signals of Xyl at δ 4.45 (H-1)/aglycon δ 3.16 (H-3), glc at δ 4.72 (H-1)/Xyl δ 3.61 (H-2), glc 1 at δ 4.44 (H-1)/glc δ 3.48 (H-4), Me-glc 1 at δ 4.57 (H-1)/glc 1 δ 3.55 (H-3), glc 2 at δ 4.42 (H-1)/Xyl δ 3.71(H-4) and Me-glc 2 at δ 4.57 (H-1)/glc 2 δ 3.54 (H-3). Oligosaccha-ride chain structure was almost superimposable with that of arguside C isolated from Bohadschia argus Jaeger. The only difference between cousteside a (1) and arguside C is the presence of the double bond at Δ24(25) in 1 [27] (Fig. 2). On the basis of the above observations, the structure of cousteside a (1) was established as 3-O-{(3-O-methyl-β-d-glucopyranosyl)-(1 → 3)-β-d-glucopyranosyl)-(1 → 4)-β-d-glucopyranosyl)-(1 → 2)-[(3-O-methyl-β-d-glucopyranosyl)- (1 → 3)-β-d-glucopyranosyl)-(1 → 4)]-β-d-xylopyranosyl} holosta-3β,12α-dihydroxy-9 (11), 24 (25)-diene.

Cousteside B (3) was isolated as a white amorphous powder. It was a major saponin in a mixture with com-pound 15 (ratio 3: 2). Its molecular formula was established

as C67H106O32 on the basis of a combination of a peak at m/z 1,445.6667 [M +na] + (calcd. for C67H106O32na, 1,445.6659) of HRFaBMS and the 13C-nMR spectrum. The IR absorptions of 3 showed the presence of hydroxyl (3,392 cm−1), lactone carbonyl (1,743 cm−1), olefinic (1,652 cm−1) and ether (1,073 cm−1) groups. The 1H-nMR, 13C-nMR and DEPT spectra of the aglycon part dis-played resonance due to six tertiary methyl groups [δ 0.91 (3H, s), 0.98 (3H, s), 1.07 (3H, s), 1.15 (3H, s), 1.42 (3H, s) and 1.84 (3H, s)], three olefinic bonds [δ 5.43 (1 H, br d, 4.0 Hz), δ115.8 (C-11)], [δ 5.60 (1 H, dt,15.7, 7.3 Hz), δ125.4 (C-23)]; 6.30 (1 H, d, 15.7 Hz), δ138.8 (C-24)] and [δ 4.94 (2H, br s), δ116.4 (C-26)]. One lactone carbonyl group [δ179.6 (C-18)] and one doublet [δ 4.13 (1 H, br d, 4.0 Hz), δ 69.5 (C-12)] that was attributed to a methine pro-ton are linked to a carbon-bearing α hydroxyl group, sug-gesting that the aglycon of 3 belongs to the holostane type. In the side chain, the position of double bonds at Δ23 (24) and Δ25 (26) was deduced from the analysis of the COSY and HMBC experiments. In the COSY spectrum, the pro-ton at δ 5.60 (1 H, dt, 15.7, 7.3) (H-23) showed correlations with two protons at δ 2.58 (1 H, d, 7.3 Hz) (H-22) and 6.30 (1 H, d, 15.7 Hz) (H-24), while the methylene proton at δ 4.94 (2H, br s) (H-26) showed that only one COSY correla-tion with methyl proton at δ 1.84 (3H, s) (H-27) indicated that the location of the first double bond was adjacent to proton 22 and the second one was terminal, respectively. Furthermore, this was also confirmed by the HMBC plot that showed correlations between H-22 and C-17, C-20,

Fig. 3 The key ROESY correlations and relative configuration of compound 1



1 3

C-21, C-23, C-24; H-23 and C-20, C-22, C-24, C-25; H-24 and C-22, C-25, C-26, C-27; and H-27 and C-24, C-25, C-26. The E stereochemistry of the Δ23 (24) double bond was deduced from the large coupling constant for H-23 with H-24 (d, 15.7 Hz). The ROESY spectrum allowed us to establish the relative configuration of all stereogenic cen-tres of the aglycon, which was similar to cousteside a (1). after extensive 2D-nMR studies, the aglycon was identi-fied as a holosta-3β-12α-dihydroxy-9 (11), 23 (24) E, 25 (26)-triene (Tables 1, 2, 3), (Fig. 2). The presence of two double bonds in these positions in the side chain of the aglycon is a new structural feature among sea cucumber glycosides [28, 29].

The 1H-nMR spectrum of cousteside B (3) displayed signals for six anomeric protons at [δ 4.61 (1 H, d, 7.8 Hz), 4.57 (1 H, d, 7.8 Hz), 4.56 (1 H, d, 7.8 Hz), 4.43 (1 H, d, 7.8 Hz), 4.42 (1 H, d, 7.0 Hz), and 4.41(1 H, d, 7.8 Hz)], which correlated with the HSQC spectrum with 13C-nMR signals at [δ 105.2, 105.2, 105.2, 102.9, 105.8, and 104.7], respectively. The COSY experiment (Tables 4, 5, 6) allowed the sequential assignment of most of the resonance

for each sugar ring, starting from the easily distinguish-able signals due to anomeric H-atoms. Complete assign-ment was achieved by combination of COSY and TOCSY results. The HSQC experiment correlated all H-atoms reso-nance with those of their corresponding C-atoms. Data of the above experiments (Tables 4, 5, 6) indicated that the sugar residues are in their pyranose forms, the location of the interglycosidic linkage was deduced from the chemical shifts, and the sequence of the oligosaccharide chain was obtained from the HMBC and ROESY spectra. Evaluation of spin–spin couplings and chemical shifts allowed the iden-tification of two β-glucopyranosyl (glc), two β-3-O-methyl-glucopyranosyl (Me-glc), one β-quinopyranosyl (Qui) and one β-xylopyranosyl (Xyl) units. This was confirmed by both HPlC analyses with authentic sugars after acid hydrolysis of 3 and optical rotation measurement for each isolated residue, indicating that the sugar chain was com-posed of glucose (d-glc), 3-O-methyl-glucose (d-Me-glc), quinovose (d-Qui) and xylose (d-Xyl) in the ratio of 2:2:1:1. The D-configuration of these four carbohydrates units was in accordance with that most often encountered among the sea

Fig. 4 Selected HMBC and COSY correlations of compound 1



1 3

cucumber glycosides. Oligosaccharide chain structure was almost superimposable with that of arguside C isolated from B. argus Jaeger. The only difference between cousteside B (3) and arguside C is the presence of the double bond at Δ24

(25) in 3 [27]. So the difference between cousteside B (3) and cousteside a (1) in the sugar chain structure is the existence of quinovose moiety in position 2 of xylose in cousteside B (3) instead of glucose unit at the same position in cousteside a (1). On the basis of the above observations, the structure of cousteside B (3) was established as 3-O-{(3-O-methyl-β-d-glucopyranosyl)-(1 → 3)-β-d-glucopyranosyl)-(1 → 4)-β-d-quinopyranosyl)-(1 → 2)-[(3-O-methyl-β-d-glucopyra-nosyl)-(1 → 3)-β-d-glucopyranosyl)-(1 → 4)]-β-d-xylopyranosyl}holosta-3β,12α-dihydroxy-9 (11), 23 (24), 25 (26)-triene.

Cousteside C (9) was isolated as a white amorphous powder. It was a major saponin in a mixture with com-pound 10 (ratio 3: 2). Its molecular formula was estab-lished as C60H96O29 on the basis of a combination of a peak at m/z 1,303.6037 [M +na] + (calcd. for C60H96O29na, 1,303.6031) of HRFaBMS and the 13C-nMR spectrum. The IR absorptions of 9 showed the presence of hydroxyl (3,396 cm−1), lactone carbonyl (1,733 cm−1), olefinic (1,650 cm−1) and ether (1,074 cm−1) groups. an exami-nation of the 1H-nMR and 13C-nMR spectra of 9 sug-gested the presence of a triterpene aglycon with seven tertiary methyls, two olefinic bonds, one lactone carbonyl group and one doublet that was ascribed to methine pro-ton linked to an oxygenated carbon and suggested that the aglycon of cousteside C (9) has a holostane triterpenoid skeleton which is very similar to that of cousteside a (1) with only two differences at positions 16 and 17. The nMR spectrum of 9 showed resonances due to two additional hydroxyl groups. There is a hydroxyl at C-17, the sig-nal of C-17 was shifted downfield from 47.9 to 89.8 ppm, and the carbon C-17 (δ = 89.8) showed long-range corre-lations with the protons at δ 1.58 (H-21,3H, s) and δ 4.52 (H-12, 1 H, d = 4.7) in the HMBC spectrum. a correla-tion between H-12 and H-21 in the ROESY spectrum evi-denced the α-configuration of the hydroxyl group at C-17 [30]. The nMR spectra of 9 also showed resonances due to an extra hydroxyl group linked to C-16, the signal of C-16 was shifted downfield from δ 24.9 to 86.3 ppm, and also the signals of the methylene group, H-16α and H-16β, were shifted downfield from δ 1.97 (1 H, m) and 2.01(1 H, m) in compound 1 to a methine proton, δ 4.69 (1 H, q-like = 7.6) ppm in compound 9. The location of the hydroxyl group at C-16 was deduced from the chemical shift of the H-16 signal δ 4.69 (1 H, q-like = 7.6), which showed coupling to signals at δ 2.16 (H-15α, m), 1.28 (H-15β, m) in the TOCSY spectrum. The 16 β configuration of the hydroxyl group was confirmed by ROESY experiment and from the coupling constants for H-16α with H-15α (7.6 Hz). The

relative stereochemistry of all other chiral centres of the aglycon was established with the aid of a ROESY experi-ments and was identical with that of the aglycon part of compound 1 as previously mentioned. all the nMR signals associated with the aglycon moiety were unambiguously assigned by COSY, TOCSY, HSQC and HMBC experi-ments (Tables 1, 2, 3). The aglycon of 9 is similar to that reported from Bohadschia marmorata despite the pres-ence of a hydroxyl group in 9 instead of methylene group in 17α-hydroxy impatienside a at C-16 [31]. Thus, the structure of the aglycon part of 9 was identified as holosta-3β,12α,16β,17α-tetrahydroxy-9 (11), 24 (25)-diene. This aglycon may be reported for the first time from sea cucum-ber saponins (Fig. 2).

The carbohydrate chain of 9 consisted of five monosac-charide residues as deduced from the 1D- and 2D-nMR spectra. The 1H-nMR spectrum of cousteside C (9) dis-played signals for five anomeric protons at [δ 4.61 (1 H, d, 7.8 Hz), 4.57 (1 H, d, 7.6 Hz), 4.42 (1 H, d, 7.0 Hz), 4.40 (1 H, d, 8.1 Hz) and 4.36 (1 H, d, 7.8 Hz)], which corre-lated with the HSQC spectrum with 13C-nMR signals at [δ 105.2, 105.2, 105.8, 104.7 and 103.4], respectively. HPlC analysis with authentic sugars was conducted after acid hydrolysis of 9 and optical rotation measurement for each isolated residues, indicating that the sugar chain was composed of glucose (d-glc), 3-O-methyl-glucose (d-Me-glc), quinovose (d-Qui) and xylose (d-Xyl) in the ratio of 2:1:1:1. The positions of interglycosidic linkages were deduced from ROESY and HMBC. From the exten-sive 1D- and 2D-nMR experiments, it was concluded that the carbohydrate chain of 9 (Tables 4, 5, 6) was identi-cal with that reported of 17-dehydroxyholothurinoside a, griseaside a [32], arguside F, impatienside B and pervi-coside D [33] isolated from Holothuria grisea and Holo-thuria axiloga, respectively. The assignments of all the 1H- and 13C-nMR signals of 9 were successfully carried out with 2D-nMR experiments (Tables 1, 2, 3, 4, 5, 6). Thus, the structure of cousteside C (9) was established as 3-O-{(3-O-methyl-β-d-glucopyranosyl)-(1 → 3)-β-d-glucopyranosyl)-(1 → 4)-β-d-quinopyranosyl)-(1 → 2)- [β-d-glucopyranosyl)-(1 → 4)]-β-d-xylopyranosyl}holosta-3β,12α,16β,17α-tetrahydroxy-9 (11), 24 (25)-diene.

Cousteside D (10) was isolated as a white amorphous powder. It was a minor saponin in a mixture with com-pound 9 (ratio 3: 2). Its molecular formula was established as C62H98O30 on the basis of a combination of a peak at m/z 1,345.6143[M +na] + (calcd. for C62H98O30na, 1,345.6135) of FaB-MS and the 13C-nMR spectrum, indicating a molecular weight of 1,346. The IR absorp-tions of 10 showed the presence of hydroxyl (3,396 cm−1), lactone carbonyl (1,733 cm−1), olefinic (1,652 cm−1) and ether (1,074 cm−1) groups. The 1H- and 13C-nMR data of 10 (Tables 1, 2, 3, 4, 5, 6) assigned from COSY,



1 3

TOCSY, HSQC, ROESY and HMBC experiments were superimposable with those of 9, except for the appear-ance of one difference corresponding to the replacement of a hydroxyl group by an acetoxy group in 10 (Fig. 2). The nMR spectra of 10 showed resonances due to an ace-toxy group [δC 171.3 and 21.4; δH 2.20 (3H, s)]. The sig-nal of C-16 was slightly shifted downfield from δ 86.3 to 86.5 ppm, but the signal of H-16 was shifted downfield from δ 4.69 (1 H, q-like = 7.6) in compound 9 to 5.71 (1 H, q-like = 7.6) ppm in compound 10. The location of the acetyl group at C-16 was deduced from the chemical shift of the H-16 signal δ 5.71 (1 H, q-like = 7.6), which showed coupling to signals at δ 2.30 (H-15α, m), 1.20 (H-15β, m) in the TOCSY spectrum, and correlation with the carbonyl signal at δ = 171.3 in the HMBC spectrum. The 16β configuration of the acetoxy group was con-firmed by correlation between H-15α and H-16α (δ = 2.30 and 5.71) in the ROESY spectrum and from the coupling constants for H-16α with H-15α (7.6 Hz). all the nMR signals associated with the aglycon moiety were unam-biguously assigned by COSY, TOCSY, HSQC and HMBC experiments (Tables 1, 2, 3). These data suggested that the aglycon of 10 is a 16β-acetoxy holosta-3β,12α,17α-trihydroxy-9 (11), 24 (25)-diene. The presence of hydroxyl and acetoxy groups at positions 16 and 17, together with this side chain of the aglycon, is a new structural feature among sea cucumber glycosides (Fig. 2). Comparison of 1D with 2D-nMR data of 10 with those of 9 suggested that 10 bore identical carbohydrate chain linked to C-3 of the aglycon as those of 9. On the basis of the above observa-tions, the structures of cousteside D (10) were established as 3-O-{(3-O-methyl-β-d-glucopyranosyl)-(1 → 3)-β-d-glucopyranosyl)-(1 → 4)-β-d-quinopyranosyl)-(1 → 2)- [β-D-glucopyranosyl)-(1 → 4)]-β-d-xylopyranosyl} holosta-3β,12α,17α-trihydroxy-16β-acetoxy,9 (11), 24 (25)-diene.

Cousteside E (11) was isolated as a white amorphous powder. It was a minor saponin in a mixture with com-pound 1 (ratio 2:1). Its molecular formula was established as C60H96O28 on the basis of a combination of a peak at m/z 1,287.6088[M +na] + (calcd. for C60H96O28na, 1,287.6082) of HRFaBMS and the 13C-nMR spectrum, 176 mass units lower than those of 1. The 1H- and 13C-nMR data of 11, assigned by extensive 2D-nMR analyses, allowed the identification of the same aglycon as in 1, a holosta-3β-12α-dihydroxy-9 (11), 24 (25)-dien (Tables 1, 2, 3, 4, 5, 6). The presence of five monosaccharide units was suggested by the five anomeric resonances [δ 4.72 (1 H, d, 7.8 Hz), 4.57 (1 H, d, 8.0 Hz), 4.45 (1 H, d, 7.0 Hz), 4.44 (1 H, d, 7.8 Hz), and 4.36 (1 H, d, 7.8 Hz)], which were HSQC-correlated with [δ 104.7, 105.2, 105.9, 104.7 and 103.5], respectively (Tables 4, 5, 6). Comparison of the 2D-nMR spectra (COSY, TOCSY, HSQC, HMBC

and ROESY) of 11 with those of 1 revealed the presence of terminal glucose (glc 2) in 11 instead of 1,3-substitu-tion glucose (glc 2) as seen in 1 (Tables 4, 5, 6), (Fig. 2). The sugar chain was composed of glucose (d-glc), 3-O-methyl-glucose (d-Me-glc) and xylose (d-Xyl) in the ratio of 3:1:1, respectively. The absence of 3-O-methyl glucose unit was also confirmed by the 176 mass units in 11 lower than those of 1. The presence of three glucose, one 3-O-methyl glucose and one xylose units in these positions of the carbohydrate chain that consists only of five sugars is a new structural feature among sea cucum-ber glycosides. Extensive study of the 2D-nMR spectra of cousteside E (11) led to the establishment of its structure as 3-O-{(3-O-methyl-β-d-glucopyranosyl)-(1 → 3)-β-d-glucopyranosyl)-(1 → 4)-β-d-glucopyranosyl)-(1 → 2)- [β-d-glucopyranosyl)-(1 → 4)]-β-d-xylopyranosyl}holosta-3β,12α-dihydroxy,9 (11), 24 (25)-diene.

Cousteside F (12) was isolated pure as a white amor-phous powder. Its molecular formula was established as C60H96O28 on the basis of a combination of a peak at m/z 1,287.6088 [M +na] + (calcd. for C60H96O28na, 1,287.6082) of HRFaBMS and the 13C-nMR spec-trum. The full assignment of all the 1H- and 13C-nMR signals by 2D-nMR experiments associated with the aglycon moiety of 12 suggested that the aglycon struc-ture is a holosta-3β-12-17α-trihydroxy-9 (11), 24 (25)-dien (Tables 1, 2, 3), (Fig. 2). Most of the signals were in good agreement with literature data of the aglycon of 17α-hydroxy impatienside a, previously reported from the sea cucumbers B. marmorata [31]. The 1H- and 13C-nMR spectra of 12 due to the sugar moieties (Tables 4, 5, 6) were superimposable with those of 9. Further investigation of 1D and 2D spectral data resulted in the determination of the structure of cousteside F (12) as 3-O-{(3-O-methyl-β-d-glucopyranosyl)-(1 → 3)-β-d-glucopyranosyl)-(1 → 4)-β-d-quinopyranosyl)-(1 → 2)- [β-d-glucopyranosyl)-(1 → 4)]-β-d-xylopyranosyl}holosta-3β,12α,17α-trihydroxy,9 (11), 24 (25)-diene.

Cousteside g (15) was isolated as a white amorphous powder. It was a minor saponin in a mixture with com-pound 3 (ratio 3: 2). Its molecular formula was established as C60H94O27 on the basis of a combination of a peak at m/z 1,269.5983 [M +na] + (calcd. for C60H94O27na, 1,269.5975) of HRFaBMS and the 13C-nMR spec-trum, 176 mass units lower than those of 3. The 1H- and 13C-nMR signals corresponding to the aglycon part of 15 were superimposable with those previously described for 3. The extensive investigation of 1D- and 2D-nMR data of 15 resulted in the determination of its aglycon struc-ture as a holosta-3β-12α-dihydroxy-9 (11), 23 (24) E, 25 (26)-triene (Tables 1, 2, 3), (Fig. 2). The 1H- and 13C-nMR spectra of 15 due to the sugar moieties (Tables 4, 5, 6) were superimposable with those of 9. The full assignment of all



1 3

the 1H- and 13C-nMR signals of 15 was successfully car-ried out with 2D-nMR experiments. Thus, the structure of cousteside g (15) was established as 3-O-{(3-O-methyl-β-d-glucopyranosyl)-(1 → 3)-β-d-glucopyranosyl)-(1 → 4)-β-d-quinopyranosyl)-(1 → 2)-[β-d-glucopyranosyl)-(1 → 4)]- β-d-xylopyranosyl}holosta-3β,12α-dihydroxy-9 (11), 23 (24), 25 (26)-triene.

Cousteside H (16) was isolated as a white amorphous powder. It was in a mixture with compound 4 (ratio 1: 1). Its molecular formula was established as C60H96O27 on the basis of a combination of a peak at m/z 1,271.6139 [M +na] + (calcd. for C60H96O27na, 1,271.6135) of HRFaBMS and the 13C-nMR spectrum, indicating a molecular weight of 1,249, 176 mass units less than those of 4. The full assignments of the C- and H-atoms of the aglycon part by 2D-nMR investigations were in good agreement with those previously reported for a holosta-3β-12α-dihydroxy-9 (11), 25 (26)-dien (Tables 1, 2, 3), (Fig. 2) in a triterpene glyco-side, marmoratoside isolated from the sea cucumber B. mar-morata [31]. The 1H- and 13C-nMR signals corresponding to the sugar chain part of 16 were superimposable with those previously mentioned of 9 (Tables 4, 5, 6). On the basis of the 2D-nMR spectral data, and MS and acid hydrolyses, the structure of cousteside H (16) was established as 3-O-{(3-O-methyl-β-d-glucopyranosyl)-(1 → 3)-β-d-glucopyranosyl)- (1 → 4)-β-d-quinopyranosyl)-(1 → 2)-[β-d-gluco-pyranosyl)-(1 → 4)]-β-d-xylopyranosyl}holosta-3β,12α-dihydroxy,9 (11), 25 (26)-diene.

Cousteside I (17) was isolated as a white amorphous powder. It was a minor saponin in a mixture with com-pound 5 (ratio 3: 2). Its molecular formula was established as C60H98O28 on the basis of a combination of a peak at m/z 1289.6254 [M +na] + (calcd. for C60H98O28na, 1,289.6248) of HRFaBMS and the 13C-nMR spectrum, indicating a molecular weight of 1,267, 176 mass units

less than those of 5. Extensive study of the 2D-nMR spec-tra of 17 led to the establishment of its aglycon structure as a holosta-3β,12α-dihydroxy,9-ene (Fig. 2). The 1H- and 13C-nMR data were in good agreement with those reported to the aglycon part of bivittoside a, B and D isolated from the sea cucumber Bohadschia bivittata Mitsukuri [34] and arguside C isolated from the sea cucumber B. argus Jaeger [27]. Comparison of 2D-nMR (COSY, TOCSY, HSQC, HMBC and ROESY) of 17 with those of 11 revealed that the oligosaccharide chains were identical. On the basis of detailed 1D- and 2D-nMR analyses (Tables 1, 2, 3, 4, 5, 6), the structure of cousteside I (17) was, thus, elucidated as 3-O-{(3-O-methyl-β-d-glucopyranosyl)-(1 → 3)-β-d-glucopyranosyl)-(1 → 4)-β-d-glucopyranosyl)-(1 → 2)- [β-d-glucopyranosyl)-(1 → 4)]-β-d-xylopyranosyl} holosta-3β,12α-dihydroxy,9-ene.

Cousteside J (18) was isolated as a white amorphous powder. It was in a mixture with compound 6 (ratio 1: 1). Its molecular formula was established as C60H98O27 on the basis of a combination of a peak at m/z 1,273.6296 [M +na] + (calcd. for C60H98O27na, 1,273.6298) of HRFaBMS and the 13C-nMR spectrum, indicating a molecular weight of 1,251, 16 mass units less than those of 17. Comparison of the nMR spectra of 17 and 18 showed that 18 had the same aglycon part structure, but there was only one difference at the sugar chain. Compound 18 had a quinovose moiety instead of the glucose unit which was attached to xylose in position 2 in 17 (Fig. 2). Therefore, further investigation of 1H- and 13C-nMR signals by 2D-nMR experiments of 18 revealed that the carbohy-drate chain was identical with those of 9. The full assign-ment of 1D and 2D spectral data and MS resulted in the determination of the structure of cousteside J (18) as 3-O-{(3-O-methyl-β-d-glucopyranosyl)-(1 → 3)-β-d-glucopyranosyl)-(1 → 4)-β-d-quinopyranosyl)-(1 → 2)- [β-d-glucopyranosyl)-(1 → 4)]-β-d-xylopyranosyl}holosta-3β,12α-dihydroxy,9-ene.

Compounds 2, 4–8, 13–14 and 19–21 (Fig. 2) were identified by comparison of their 1D- and 2D-nMR and MS data with those reported for these known compounds. Triterpene glycoside, impatienside a (2), was previously reported from the sea cucumber H. impatiens [23], whereas marmoratoside a (4) and bivittoside D (6) have previously been isolated from the sea cucumber B. marmorata [31]. arguside C (5) was isolated earlier from the sea cucum-ber B. argus [27]. arguside F (13) and impatienside B (14) were previously found in the sea cucumber Holothuria (Microthele) axiloga [33]. Holothurinoside I (7), holothuri-noside H (8), holothurinoside a (19) and desholothurin a (21) were characterized in the sea cucumber Holothuria forskali [35]. also, 17-dehydroxyholothurinoside a (20) has previously been reported from the sea cucumber H. gri-sea [32].

Table 7 antifungal activity of the tested saponin mixtures by disc diffusion method

a The diameter of the filter paper disc (6 mm) is included

Compounds 1 mg/ml Mean inhibition zone diameter (mm) aafter 48 h of C. albicans incubation

ampicillin 13.8 ± 0.1

1, 11 15.7 ± 0.01

2, 13 15.4 ± 0.15

3, 15 14.9 ± 0.05

4, 16 14.7 ± 0.05

5, 17 11.5 ± 0.12

6, 18 10.7 ± 0.05

9, 10 16.3 ± 0.05

12 11.0 ± 0.10

14 18.0 ± 0.01



1 3

antifungal activity

Some triterpene glycosides hitherto isolated from sea cucumber exhibited antifungal activity. Herein, we report the antifungal activities of two triterpene glycosides 12 and 14 and seven saponins pairs (1, 11), (ratio 2:1); (2, 13), (ratio 1:1); (3, 15), (ratio 3:2); (4, 16), (ratio 1:1); (5, 17), (ratio 3:2); (6, 18), (ratio 1:1) and (9, 10), (ratio 3:2) isolated from the sea cucumber B. cousteaui against C. albicans. ampicillin was used as positive control. The results of the screening test are summarized in (Table 7). The antifungal activity was qualitatively assessed by the presence or absence of the inhibition zone. Our results (Table 7) indicated that all tested glycosides showed anti-fungal activity at concentration (10 μg/disc) and the diam-eter of the inhibition zones ranged from 10.7 to 18.0 mm. as shown in Table 7, the pure compound 14 exhibited the highest antifungal activity, while the smallest inhibition zone was obtained by saponin pair (6, 18). It also was noted that the saponin pairs were sufficiently potent to inhibit the growth of C. albicans. On the basis of the data available, the antifungal activity of sea cucumber glycosides is very sensitive to their precise structures. Our study revealed that the highest effective compounds contain aglycon with dou-ble bond at Δ24 (25).Therefore, more extensive studies are needed before a clear structure–activity relationship can be reached. Meanwhile, sea cucumber glycosides continue to be an interesting source of antifungal compounds.

Conclusions

Twenty-one lanostane-type non-sulphated triterpene gly-cosides were isolated from the methanol/methylene chlo-ride extract of the body walls of B. cousteaui. Ten new saponins called cousteside A (1), 3-O-{(3-O-methyl-β-d-glucopyranosyl)-(1 → 3)-β-d-glucopyranosyl)-(1 → 4)-β-d-glucopyranosyl)-(1 → 2)-[(3-O-methyl-β-d-glucopyranosyl)- (1 → 3)-β-d-glucopyranosyl)-(1 → 4)]-β-d-xylopyranosyl} holosta-3β,12α-dihydroxy-9 (11), 24 (25)-diene; cousteside B (3), 3-O-{(3-O-methyl-β-d-glucopyranosyl)-(1 → 3)-β-d-glucopyranosyl)-(1 → 4)-β-d-quinopyranosyl)-(1 → 2)- [(3-O-methyl-β-d-glucopyranosyl)-(1 → 3)-β-d-gluco-pyranosyl)-(1 → 4)]-β-d-xylopyranosyl}holosta-3β,12α-dihydroxy-9 (11), 23 (24), 25 (26)-triene; cousteside C (9), 3-O-{(3-O-methyl-β-d-glucopyranosyl)-(1 → 3)-β-d-glucopyranosyl)-(1 → 4)- β-d-quinopyranosyl)-(1 → 2)-[β-d-glucopyranosyl)-(1 → 4)]-β-d-xylopyranosyl}holosta-3β,12α,16β,17α-tetrahydroxy-9 (11), 24 (25)-diene; cousteside D (10), 3-O-{(3-O-methyl-β-d-glucopyranosyl)- (1 → 3)-β-d-glucopyranosyl)-(1 → 4)-β-d-quinopyranosyl)- (1 → 2)-[β-d-glucopyranosyl)-(1 → 4)]-β-d-xylo-pyranosyl}holosta-3β,12α,17α-trihydroxy-16β-acetoxy,9 (11),

24 (25)-diene; cousteside E (11), 3-O-{(3-O-methyl-β-d-glucopyranosyl)-(1 → 3)-β-d-glucopyranosyl)-(1 → 4)-β-d-glucopyranosyl)-(1 → 2)-[β-d-glucopyranosyl)-(1 → 4)]- β-d-xylopyranosyl}holosta-3β,12α-dihydroxy,9 (11), 24 (25)- diene; cousteside F (12), 3-O-{(3-O-methyl-β-d-gluco-pyranosyl)-(1 → 3)-β-d-glucopyranosyl)-(1 → 4)-β-d-quinopyranosyl)-(1 → 2)-[β-d-glucopyranosyl)-(1 → 4)]-β-d-xylopyranosyl}holosta-3β,12α,17α-trihydroxy,9 (11), 24 (25)-diene; cousteside G (15), 3-O-{(3-O-methyl-β-d-glucopyranosyl)-(1 → 3)-β-d-glucopyranosyl)-(1 → 4)-β-d-quinopyranosyl)-(1 → 2)-[β-d-glucopyranosyl)-(1 → 4)]-β-d-xylopyranosyl}holosta-3β,12α-dihydroxy-9 (11), 23 (24), 25 (26)-triene; cousteside H (16), 3-O-{(3-O-methyl-β-d-glucopyranosyl)-(1 → 3)-β-d-glucopyranosyl)-(1 → 4)-β-d-quinopyranosyl)-(1 → 2)-[β-d-glucopyranosyl)-(1 → 4)]- β-D-xylopyranosyl}holosta-3β,12α-dihydroxy,9 (11), 25 (26)- diene; cousteside I (17), 3-O-{(3-O-methyl-β-d-gluco-pyranosyl)-(1 → 3)-β-d-glucopyranosyl)-(1 → 4)-β-d-glucopyranosyl)-(1 → 2)-[β-d-glucopyranosyl)-(1 → 4)]-β-d-xylopyranosyl}holosta-3β ,12α -dihydroxy,9-ene; and cousteside J (18), 3-O-{(3-O-methyl-β-d-gluco-pyranosyl)-(1 → 3)-β-d-glucopyranosyl)-(1 → 4)-β-d-quinopyranosyl)-(1 → 2)-[β-d-d-glucopyranosyl)-(1 → 4)]- β-d-xylopyranosyl}holosta-3β,12α-dihydroxy,9-ene, together with eleven known triterpene glycosides (2, 4–8, 13–14 and 19–21), were isolated by reversed-phase semi-preparative HPlC (Fig. 2).

Most of the isolated compounds showed good antifun-gal activity against c. albicans. These saponins are of great interest for potential functional food ingredient, dietary supplement, food additives, food preservative and drug development as well as ingredients of new leads and com-mercially successful products for various industrial appli-cations, especially pharmaceuticals, agrochemicals, func-tional foods and nutraceuticals. Sea cucumber B. cousteaui is one of the potential marine animals with high food and medicinal value. The potential medicinal properties of this animal are ascribed to the presence of functional compo-nents with promising multiple biological activities.

Conflict of interest none.

Compliance with Ethics Requirements This article does not con-tain any studies with human or animal subjects.

References

1. Webb gP (2006) an overview of dietary supplements and func-tional food. In dietary supplements and functional foods, 1st edn. Blackwell Publishing, Oxford, UK, pp 1–35

2. Shahidi F (2009) nutraceuticals and functional foods: whole ver-sus processed foods. Trends Food Sci Tech 20:376–387

3. Hu SY (2005) Food plants of China. Chinese University Press, Hong Kong, China, p 3–11, 275–278



1 3

4. Venugopal V (2009) Marine habitat and resources. In: Venugopal V (ed) Marine products for healthcare: functional and bioactive nutraceutical compounds from the ocean. CRC Press Taylor & Francis group, Boca Raton, Fl, pp 23–50

5. lawrence aJ (2006) Darwin initiative for the sustainable use of sea cucumber in Egypt. Darwin Final Report Part 4 Oct 04

6. Chen J (2003) Overview of sea cucumber farming and sea ranch-ing practices in China. SPC Beche-de-mer Inform Bull 18:18–23

7. Jiaxin C (2003) Overview of sea cucumber farming and sea ranching practices in China. SPC Beche-de-mer Inform Bull 18:1–6

8. Zhong Y, Khan Ma, Shahidi F (2007) Compositional characteris-tics and antioxidant properties of fresh and processed sea cucum-ber (Cucumaria frondosa). J agri Food Chem 55:1188–1192

9. Bordbar S, anwar F, Saari n (2001) High-value components and bioactives from sea cucumbers for functional foods–a Review. Mar Drugs 9:1761–1805

10. gurfinkel DM, Rao aV (2003) Soysaponins: the relationship between chemical structure and colon anticarcinogenic activity. nutr Cancer 47:24–33

11. Kim SW, Park SK, Kang SI, Kang HC, Oh HJ, Bae CY, Bae DH (2003) Hypocholesterolemic property of Yucca schidigera and Quillaja saponaria extracts in human body. arch Pharmacol Res 26:1042–1046

12. Özlem gÜ, giuseppe M (2007) Saponins: properties, applica-tions and processing. Crit Rev Food Sci nutr 47:231–258

13. Elbandy M, Kang OH, Kwon DY, Rho JR (2009) Two new anti-inflammatory triterpene saponins from the Egyptian medicinal food black cumin (seeds of Nigella sativa). Bull Korean Chem Soc 30:1811–1816

14. Elbandy M, ashoush I (2012) Phytochemicals in pomegranate seeds and their effect as hypolipidemic agent in hypercholester-olemic rats. World J Dairy Food Sci 7(1):85–92

15. Elbandy M, Rho JR, afifi R. (2011) analysis of triterpene sapo-nins of the nutraceutical foods sea cucumber, (Bohadschia cous-teaui) by using nuclear magnetic resonance and mass spectrom-etry. In Proceeding of ICFST‒ China 2011, 9th International Conference Food Science Technology, China: Hangzhou

16. Elbandy M, Miyamoto T, Delaude C, lacaille-Dubois Ma (2002) Five new medicagenic acid saponins from Muraltia ononidifolia. Helv Chim acta 85:2721–2728

17. naoki Sa, atsuko TB, Takako SC, Kazunori aD (2010) Devel-opment of an absolute quantification method for organic com-pounds using quantitative nMR (qnMR) and improvement of the reliability of food analysis. Tetrahedron 57:9563–9568

18. Xue Rn, Fan l, Rieser MJ, El-Shourbagy Ta (2007) Recent advances in high throughput quantitative bioanalysis by lC-MS/MS. J Pharmaceut Biomed anal 44:342–355

19. nCClS (2004) Method for antifungal disk diffusion suscepti-bility testing of yeasts; approved guideline. nCClS document M44-a (ISBn 1-56238-532-1). nCClS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USa

20. Van Rossum JMV (1963) The relation between chemical struc-ture and biological activity–a Review. J Pharml Pharmacol 15(1):285–316

21. Kitagawa I, Kobayashi M, Inamoto T, Fuchida M, Kyogoku Y (1989) Marine natural products. XIV. Structures of echinosides a and B, antifungal lanostane oligosides from the sea cucumber Actinopyga echinities (Jaeger). Chem Pharma Bull 33:5214–5224

22. Kitagawa I, Kobayashi M, Hori M, Kyogoku Y (1985) Marine natural products. VIII. Four lanostane-type triterpene oligoglyco-sides, bivittosides a, B, C, and D, from the okinawan sea cucum-ber Bohadschia bivittata Mitsukuri. Chem Pharma Bull 37:61–67

23. Sun P, liu BS, Yi YH, li l, gui M, Tang HF, Zhang DZ, Zhang Sl (2007) a new cytotoxic lanostane-type triterpene glycoside from the sea cucumber Holothuria impatiens. Chem Biodivers 4:450–457

24. Stonik Va, Kalinin VI, avilov Sa (1999) Toxins from sea cucum-bers (Holothuroids): chemical structures, properties, taxonomic distribution, biosynthesis and evolution. J nat Toxins 8:235–248

25. avilov Sa, Kalinin VI, Smirnov aV (2004) Use of triterpene gly-cosides for resolving taxonomic problems in the sea cucumber genus Cucumaria (Holothurioidea, Echinodermata). Biochem Syst Ecol 32:715–733

26. Breitmaier E, Voelter W (1987) Carbon-13 nMR Spectroscopy. VCH, Weinheim

27. liu BS, Yi YH, li l, Sun P, Yuan WH, Sun gQ, Han H, Xue M (2008) argusides B and C, two new cytotoxic triterpene glyco-sides from the sea cucumber Bohadschia argus Jaeger. Chem Biodivers 5:1228–1297

28. Kalinin VI, Silchenko aS, avilov Sa, Stonic Va, Smirnov aV (2005) Sea cucumber triterpene glycosides, the recent progress in structural elucidation and chemotaxonomy. Phytochem Rev 4:221–236

29. Wu J, Yi YH, Tang HF, Wu HM, Zhou ZR (2007) Hillasides a and B, two new cytotoxic triterpene glycosides from the sea cucumber Holothuria hilla lesson. J asian nat Prod Res 9:609–615

30. Murray aP, Muniain C, Seldes aM, Maier MS (2001) Patagonic-oside a: a novel antifungal disulfated triterpene glycoside from the sea cucumber Psolus patagonicus. Tetrahedron 57:9563–9568

31. Yuan WH, Yi YH, Tang HF, liu BS, Wang Zl, Sun gQ, Zhang W, li l, Sun P (2009) antifungal triterpene glycosides from the sea cucumber Bohadschia marmorata. Planta Med 75:168–173

32. Sun gQ, li l, Yi YH, Yuan WH, liu BS, Weng YY, Zhang Sl, Sun P, Wang Zl (2008) Two new cytotoxic nonsulfated pentasac-charide holostane (= 20-hydroxylanostan-18-oic acid γ-lactone) glycosides from the sea cucumber Holothuria grisea. Helv Chim acta 91:145–1460

33. Yuan WH, Yi YH, Tan RX, Wang Zl, Sun gQ, Xue M, Zhang HW, Tang HF (2009) antifungal triterpene glycosides from the sea cucumber Holothuria (Microthele) axiloga. Planta Med 75:647–653

34. Ohta T, Hikino H (1981) Structures of four new triterpenoidal oli-goglycosides, bivittoside a, B, C and D from the sea cucumber Bohadschia bivittata Mitsukuri. Chem Pharma Bull 29:282–285

35. Dyck SV, gerbaux P, Flammang P (2009) Elucidation of molecu-lar diversity and body distribution of saponins in the sea cucum-ber Holothuria forskali (Echinodermata) by mass spectrometry. Comparative Biochem Physiol Part B 152:124–134


Analysis of saponins as bioactive zoochemicals from the marine functional food sea cucumber...

Documents

Transcript of Analysis of saponins as bioactive zoochemicals from the marine functional food sea cucumber...