three cerebrosides from the sea cucumber cucumaria frondosa

Chinese Journal of Natural Medicines 2012, 10(2): 0105−0109doi: 10.3724/SP.J.1009.2012.00105

ChineseJournal of Natural Medicines

Three cerebrosides from the sea cucumber Cucumaria frondosa

LA Ming-Ping1, SHAO Jun-Jie2, JIAO Jian2, YI Yang-Hua1* 1Research Center for Marine Drugs, Second Military Medical University, Shanghai 200433, China; 2Dalian Haiyantang Biology Co., Ltd., Dalian 116021, China

Available online 20 Mar. 2012

[ABSTRACT] AIM: To study the chemical constituents of the sea cucumber Cucumaria frondosa. METHOD: Three sphingos-ine-type glucocerebrosides, CF-3-1, CF-3-2 and CF-3-3 were isolated by means of high performance liquid chromatography (HPLC) from a cerebroside molecular species 3 which was obtained from the less polar fraction of the 65% EtOH extract of the sea cucumber Cucumaria frondosa Gunnerus. RESULT: The structures of these cerebrosides were determined on the basis of chemical and spectro-scopic evidences. CONCLUSION: Three compounds were isolated from the sea cucumber Cucumaria frondosa for the first time. CF-3-3 and CF-3-2 were obtained as pure compounds for the first time. [KEY WORDS] Glycosphingolipids; Cerebrosides; Sea Cucumber; Cucumaria frondosa

[CLC Number] R284.1 [Document code] A [Article ID] 1672-3651(2012)02-0105-05

1 Introduction

The sea cucumber Cucumaria frondosa Gunnerus (Cu- cumariidae, Dendrochirotida), is distributed abundantly in the Arctic, including the coast of Russia, Canada, Norway and Britain [1]. In our laboratory, isolation and characterization of homogeneous cerebrosides from C. frondosa were conducted in the hope of discovering the biologically active compounds. We report herein the isolation and structural elucidation of three cerebrosides from the body walls of C. frondosa.

2 Isolation and structural elucidation of cerebrosides from molecular species 3

Molecular species 3 is separated by reversed-phase HPLC into twelve peaks, and is recovered to give eight compounds, three of which (CF-3-1, CF-3-2 and CF-3-3) reveal a single molecular ion peak as [M + Na]+ in the posi-

[Received on] 3-May-2011 [Research Funding] This project was supported by International Science and Technology Cooperation Project of China (No. 2010DFB30810) [*Corresponding author] YI Yang-Hua: Prof., Tel: 86-21-65384988, Fax: 86-21-65483662, E-mail: [email protected] These authors have no any conflict of interest to declare. Copyright © 2012, China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

tive-ion ESI-MS spectra, and [M - H]− in the negative- ion ESI-MS spectra. The 13C NMR spectra of CF-3-1, CF-3-2 and CF-3-3 are essentially identical with those of molecular species 3. This proves that these three compounds are cere-broside components of molecular species 3.

Molecular species 3 undergoes methanolysis upon treatment with methanolic hydrochloric acid to yield a mix-ture of fatty acid methyl esters (FAM) and long-chain bases (LCB), together with methyl D-glucopyranoside. Analysis of the FAM mixture by GC-MS reveals the presence of five components, which are characterized as methyl hexa-decanoate, methyl octadecanoate, methyl 2- hydroxydoco-sanoate, methyl 2-hydroxytricosenoate and methyl 2-hydroxytetracosenoate. CF-3-1, CF-3-2 and CF-3-3 were methanolyzed independently with methanolic hydrochloric acid to yield fatty acid methyl esters (FAM) FAM-1, FAM-2, FAM-3 and long-chain bases-1 (LCB-1) together with methyl D-glucopyranoside-1, LCB-2 together with methyl D-glucopyranoside-2, LCB-3 together with methyl D-glucopyranoside-3 respectively. When FAM-1, FAM-2 and FAM-3 were analyzed independently by GC-MS, a sin-gle FAM was detected from FAM-1, FAM-2 and FAM-3, which are characterized as methyl 2-hydroxytetracosenoate. Accordingly, FAM-1, FAM-2 and FAM-3 are the same components, and CF-3-1, CF-3-2 and CF-3-3 are pure cere-brosides. The location and geometries of the double bonds of FAM-1, FAM-2 and FAM-3 were determined as follows: The positive-ion ESI-MS spectra of the dimethyl disulfide

LA Ming-Ping, et al. /Chinese Journal of Natural Medicines 2012, 10(2): 105−109

(DMDS) derivatives of FAM-1, FAM-2 and FAM-3 show [M + Na]+ at m/z 513, and the EI-MS spectra show remark-able fragment-ion peaks at m/z 173 due to cleavage of the bond between the carbons bearing a methylthio group (Scheme 2). These data indicate that the double bonds in the fatty acid residues of CF-3-1, CF-3-2 and CF-3-3 are located at C-15′ (Scheme 1). Furthermore, it has been reported [2] that the geometry of the double bond in a long-chain alkene can be determined on the basis of the 13C NMR chemical shift of the methylene carbon next to the olefinic carbon, namely the carbon signal observed at δC 26-27 in Z type and δC 31-32 in E type. The geometry is also inferred from the 1H NMR sig-nals due to the olefinic protons, namely the proton signals

appearing as a triplet-like in the Z type and as a multiplet in the E type. When the 13C- and 1H NMR spectra of CF-3-1 were assigned in detail, the signals of the two methylene carbons (C-14 and C-17) next to the olefinic carbons were observed at δC 27.3 and 26.5, and the signals of olefinic pro-tons (15′-H and 16′-H) appear at δH 5.31 as a triplet-like. Thus, the olefinic unit in the fatty acid residues of CF-3-1, CF-3-2 and CF-3-3 is characterized as Z type. In the 13C- NMR spectrum of CF-3-3, the carbon signals for the terminal methyl groups were observed at δC 14.8 (normal form). CF-3-3 has normal-type fatty acids and normal-type LCB, namely CF-3-1 and CF-3-2 also have normal-type fatty acids, as shown in Scheme 1.

Scheme 1

Scheme 2

Fig. 1 EI-MS fragmentation of DMDS derivatives of FAM-1, FAM-2 and FAM-3

In the 13C NMR spectrum of CF-3-1, the carbon signals for the terminal methyl groups were observed at δC 14.2 (normal form), δC 11.5 and 19.4 (ante-iso form). CF-3-1 had normal-type fatty acids and ante-iso-type LCB. Taking the molecular masses of CF-3-1 (829) and the fatty acid compo-nents from FAM-l into account, the long-chain base of com-pounds CF-3-1 is 2-amino-1, 3, 4-trihydroxy-14-methyl- hexadecane

The 1H- and 13C NMR spectra of CF-3-1 are in good agreement with those of the known glucocerebroside CE-3-1[3], which was obtained from the related sea cucumber Cucumaria echinata and is composed of (2S, 3S, 4R)-phyto- sphingosines with branching methyl groups, normal-(2R)- 2-hydroxy fatty acids, and β-D-glucopyranose (Scheme 1). Therefore, the structures of the homogeneous components of CF-3-1 is 1-O-(β-D-glucopyranosyl)-(2S, 3S, 4R)-2-[(2R, 15Z)-2-hydroxy-15-tetracosen oylamino]-14-methylhexade- cane-1, 3, 4-triol.

The 1H NMR signals of the basic structure of CF-3-2 and CF-3-3 are in good agreement with those of the known cerebroside molecular species SJC-2 [4], which is composed of (2S, 3R, 4E)-sphingosine, fatty acid, and β-D-glucopyranose subunits, and is a component of the related sea cu-cumber Stichopus japonicus. In addition, the absolute con-figuration of the glucose unit was verified as being of D-form by means of the Hara method [5]. These results suggest that CF-3-2 and CF-3-3 has the same absolute configuration as that of SJC-2 for the basic moiety at C-2, -3, -4 and -5, and for the glucopyranose (Scheme 1).

In the 13C NMR spectrum of CF-3-2 , the carbon signals for the terminal methyl groups were observed at δC 14.2 (normal form), and at δC 11.5 and 19.3 (ante-iso form). CF-3-2 had normal-type fatty acids and ante-iso-type LCB. LCB-2 by GC-MS showed what is characterized as 2-amino -1, 3-dihydroxy-4-heptadecene. Taking the molecular masses of CF-3-2 (811) and the fatty acid components from FAM into account, the long-chain base of compounds CF-3-2 is 2-amino-1, 3-dihydroxy-14-methyl-4-hexadecene.

Therefore, the structure of the homogeneous components


of CF-3-2 is 1-O-(β-D-glucopyranosyl)-(2S, 3R, 4E)-2-[(2R, 15Z)-2-hydroxy-15-tetracosenoylamino]-15-methyl-4-hexadecene-1, 3-diol. A ganglioside molecular species SJC-2 which include CF-3-2 have been obtained from the related sea cucumber Stichopus japonicus [4].

In the 13C NMR spectrum of CF-3-3, the carbon signals for the terminal methyl groups were observed at δC 14.8 (normal form). CF-3-3 had normal-type fatty acids and nor-mal-type LCBs. Furthermore, CF-3-3 was suggested to con-tain two olefinic groups in the fatty acid and LCB side chains, since four olefinic carbon signals (δC 130.2, 130.2, 129.8, and 131.0) were observed in addition to the carbon signals due to the double bond at C-4/C-5. Taking the molecular masses of CF-3-3 (823) and the fatty acid components from FAM into account, the long-chain base of compounds CF-3-2 is de-terimined 2-amino-1, 3-dihydroxy- octadecadiene. The loca-tion and geometries of the double bonds in the side chains of CF-3-3 were determined as follows.

The mass spectra of the dimethyl disulfide (DMDS) de-rivatives [5] of FAM from CF-3-3 [(A) in Scheme 2] and CF-3-3 [(C) in Scheme 3] show remarkable fragment-ion peaks at m/z 173 for A and m/z 173 and 187 for C respec-tively due to cleavage of the bond between the carbons bear-ing a methylthio group (Scheme 2). These data indicate that the double bonds in the fatty acid and LCB residues of CF-3-3 are located at C-15′ and C-8, respectively, as shown in Scheme 1.

Scheme 3

Fig. 2 EI-MS fragmentation of DMDS derivatives of CF-3-3

When the 1H NMR spectra of CF-3-3 were assigned in detail, the peak area of the proton signals appearing as a trip-

let-like (δH 5.32) was two times of the proton signals appear-ing as a multiplet (δH 5.47, 5.82). Furthermore, the proton signals at δH 5.32 were assigned to the olefinic groups at C-8 and C-15′. Thus, the olefinic groups at C-8 and C-15′ of CE-3-3 are characterized as being of cis (Z) geometries (Scheme 1).

On the basis of the above facts into account, the struc-tures of CF-3-3 is 1-O-(β-D-glucopyranosyl)-(2S, 3R, 4E, 8Z)-2-[(2R, 15Z)-2-hydroxy-15-tetracosenoylamino]-4, 8-octa-decadiene-1, 3-diol.

3 Experimental

Commercial silica gel (Yantai, China, 200–300 mesh; 400–500 mesh) was used for column chromatography. Pre-coated silica gel plates (Yantai, China, HSGF-254) were used for analytical thin-layer chromatography (TLC). Spots were detected on TLC under UV or by heating after spraying with anisaldehyde-sulphuric acid reagent. The NMR spectra were recorded at 300 K on a Bruker drx 400 spectrometer. Chemi-cal shifts were reported in parts per million (δ), with use of the residual C5D5N signal (δH 8.80) as an internal standard for 1H NMR and C5D5N (δC 150.00) for 13C NMR; coupling constants (J) in Hz. Infrared spectra were recorded in thin polymer films on a Nexus 470 FT-IR spectro- photometer (Nicolet, USA); peaks are reported in cm-1. The mass spectra and high resolution mass spectra were performed on a Q-TOF Micro mass spectrometer, resolution 5000 (Waters, USA). An isopropyl alcohol solution of sodium iodide (2 mg·mL−1) was used as a reference compound. Semi-preparative RP-HPLC was performed on an Agilent 1100 system equipped with a refractive index detector using a YMC Pack ODS-A column (particle size 5 μm, 250 mm × 10 mm).

Separation and structural elucidation of molecular spe-cies 3

Separation of molecular species 3: The body walls of the sea cucumber Cucumaria frondosa Gunnerus (1.8 Kg) were chopped and extracted with 65% EtOH (5 L), and the EtOH solution was concentrated in vacuo. The residue was parti-tioned between H2O and n-BuOH; the organic layer was separated and concentrated in vacuo to give the fraction (4 g). This fraction was chromatographed on silica gel (solvents CHCl3; CHCl3/MeOH, 20 : l and CHCl3/MeOH, 10 : l) to give three fractions (1-3). Fraction 3 was refined by re-versed-phase (C-18, solvent 95% MeOH) column chroma-tography to give molecular species 3 (174 mg).

Molecular species 3: Amorphous powder, mp 135-138

°C, IRυ KBrmax cm−1: 3420 (OH), 1645, 1540 (amide).

Methanolysis of molecular species 3: Molecular species 3 (5.0 mg) was heated with 10% HCl in MeOH (1 mL) at 70 °C for 18 h. The reaction mixture was then extracted with n-hexane, and the extract was concentrated in vacuo to yield a mixture of FAM. The MeOH layer was concentrated in vacuo to give a mixture of LCB and methyl glycoside.


GC-MS analysis of FAM mixture from molecular spe-cies 3: A FAM mixture from molecular species 3 was sub-jected to GC-MS [column temp. 150-300 °C (rate of temperature increase 5 °C·min−1)]. The results were as follows: methyl hexadecanoate, tR = 10.2 min; m/z 270 [M]+, 227 [M - 43]+; methyl octadecanoate, tR = 11.2 min; m/z 298 [M]+, 255 [M - 43]+; methyl 2-hydroxydocosanoate, tR =13.5 min; m/z 370 [M]+, 311 [M - 59]+; methyl 2-hydroxytricoseoate, tR = 13.9 min; m/z 382 [M]+, 323 [M - 59]+; methyl 2- hy-droxytetracosenoate, tR = 14.5 min, m/z 396 [M]+, 337 [M - 59]+.

CF-3-1 Amorphous powder, mp 135−137 °C, [ɑ] 25D

+5.8° (c 0.13, 1-PrOH). ESI-MS+ (positive-ion mode); m/z 852 [M + Na]+. ESI-MS− (negative-ion mode); m/z 828 [M - H]−. CF-3-1 was found to be identical to CE-3-1 {ref. [3]:

Amorphous powder, mp 138-140 °C, [α] 25D +5.8 (c 0.13,

1-PrOH)}. Methanolysis of CF-3-1: CF-3-1 was methanolyzed in

the same manner as described for molecular species 3, to yield a mixture of FAM-1. The MeOH layer was concentrated in vacuo to give a mixture of LCB-1 and methyl glyco-side.

GC-MS analysis of FAM-1: FAM-1 was subjected to GC-MS using the same conditions as described for the FAM mixture obtained from molecular species 3. The results were as follows: methyl 2-hydroxytetracosenoate, tR = 15.3 min; m/z 396 [M]+, 337 [M - 59]+.

DMDS derivatives of FAM-1: FAM-1 (methyl tetra- cosenoate, 1 mg) was dissolved in carbon disulfide (0.2 mL) and dimethyl disulfide (DMDS, 0.2 mL) and iodine (1 mg) were added to the solution. The resulting mixture was kept at 60 °C for 40 h in a small-volume sealed vial. The reaction was subsequently quenched with aqueous Na2S2O3 (5%), and the mixture was extracted with n-hexane (0.3 mL). The ex-tract was concentrated and the residue (DMDS derivative) was analyzed by ESI-MS and EI-MS. ESI-MS (positive-ion mode); m/z 513 [M + Na]+. EI-MS (positive-ion mode); m/z 490 [M]+, 317, 173.

GC-MS Analysis LCB-1 from CF-3-1: A mixture of LCB-1 and methyl glycoside from CF-3-1 was heated with Ac2–pyridine (1 : 1) for 4 h at 65 °C, diluted with H2O, ex-tracted with CHCl3, The CHCl3 layer was washed with H2O and concentrated in vacuo and analyzed using GC-MS [col-umn temperature 180-300 °C (rate of temp. increase 5 °C·min−1)]. LCB-1 (GC-MS): 2-amino-1, 3, 4-trihydroxy- 14-methyl-hexadecane, tR = 14.02 min; m/z 326 [M - 193]+, 285 [M - 234]+, 132. Methyl glycoside (GC-MS): methyl β-glucopyranosides was detected.

CF-3-2 Amorphous powder, mp 136-139 °C, [α] 25D

+6.1 (c 0.13, 1-PrOH). ESI-MS+ (positive-ion mode) m/z 834 [M + Na]+. ESI-MS− (negative-ion mode); m/z 810 [M - H]−.

Methanolysis of CF-3-2: CF-3-2 was methanolyzed in

the same manner as described for CF-3, to yield a mixture of FAM-2. The MeOH layer was concentrated in vacuo to give a mixture of LCB-2 and methyl glycoside.

GC-MS analysis of FAM-2: FAM-2 was subjected to GC-MS using the same conditions as described for the FAM mixture obtained from CF-3. The results were as follows: methyl 2-hydroxytetracosenoate, tR = 15.3 min; m/z 396 [M]+, 337 [M - 59]+.

DMDS derivatives of FAM-2: FAM-2 was converted to its DMDS derivative in the same manner as described for FAM-1. the DMDS derivative of FAM-2 was analyzed by ESI-MS and EI-MS. ESI-MS+ (positive-ion mode); m/z 513 [M + Na]+. EI-MS- (positive-ion mode); m/z 490 [M]+, 317, 173.

GC-MS analysis LCB-2 from CF-3-2: The residue (mixture of the LCB and methyl glycoside) from CF-3-2 was acetylateed, and the reaction mixture was analyzed using GC-MS in the same manner as described for CF-3-1. LCB-2 (GC-MS): 2-amino-1, 3-dihydroxy-14-methyl-4-hexadecene, tR = 14.85 min; m/z 326 [M - 103]+, 297 [M - 132]+, 236 [M - 193]+, 132. Methyl glycoside (GC-MS): methyl β-glucopyranosides was detected.

CF-3-3 Amorphous powder, mp 139-141 °C, [α] 25D

+5.8 (c 0.13, 1-PrOH). ESI-MS+ (positive-ion mode); m/z 846 [M + Na]+. ESI-MS- (negative-ion mode); m/z: 822 [M - H] −.

Methanolysis of CF-3-3: CF-3-3 was methanolyzed in the same manner as described for CF-3-1, to yield a mixture of FAM-3. The MeOH layer was concentrated in vacuo to give a mixture of LCB-3 and methyl glycoside.

GC-MS analysis of FAM-3: FAM-3 was subjected to GC-MS [column temp. 150-300 °C (rate of temp. increase 5 °C·min−1)]. The results were as follows: methyl octadecanoate, tR = 14.1 min, m/z 298 [M]+, 255 [M - 43]+; methyl icosanoate, tR = 15.3 min, m/z 396 [M]+, 337 [M - 59]+.

DMDS derivatives of FAM-3: FAM-3 was converted to its DMDS derivative in the same manner as described for FAM-1. the DMDS derivative of FAM-3 was analyzed by ESI-MS and EI-MS. ESI-MS+ (positive-ion mode); m/z 513 [M + Na]+. EI-MS+ (positive-ion mode); m/z 490 [M]+, 317, 173.

GC-MS analysis LCB-3 from CF-3-3: The residue (mixture of the LCB-3 and methyl glycoside) from CF-3-3 was acetylateed, and the reaction mixture was analyzed using GC-MS in the same manner as described for CF-3-1. LCB-3 (GC-MS): 2-amino-1, 3-dihydroxy-4-hexadecene, tR = 15.05 min, m/z 338 [M - 103]+, 309 [M - 132]+, 248 [M - 193]+. Methyl glycoside (GC-MS): methyl β-glucopyranosides was detected.

DMDS Derivatives of CF-3-3: FAM-3 was converted to its DMDS derivative in the same manner as described for FAM-1. The residue (CF-3-3 DMDS derivative) was ana-lyzed by EI-MS. EI-MS+ (positive-ion mode) m/z 187, 173.


References

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[3] Yamadaa K, Haraa E, Miyamotoa T, et al. Isolation and struc-ture of biologically active glycosphingolipids from the sea cu-

cumber Cucumaria echinata [J]. Eur J Org Chem, 1998, 1998(2): 371-378

[4] Kisa F, Yamada K, Kaneko M, etc. Constituents of Holothur-oidea, 14. Isolation and Structure of New Glucocerebroside Molecular Species from the Sea Cucumber Stichopus japonicus [J]. Chem Pharm Bull, 2005, 53(4): 382- 386

[5] Hara S, Okabe H, Mihashi K. Gas-liquid chromatographic separation of aldose enantiomers as trimethylsilyl ethers of methyl 2-(polyhydroxyalkyl)-thiazolidine-4(R)-carboxylates [J]. Chem Pharm Bull, 1987, 35(2): 501-506.

叶瓜参中的三个脑苷脂

喇明平 1, 邵俊杰 2, 焦健 2, 易杨华 1* 1第二军医大学药学院海洋药物研究中心, 上海 200433; 2大连海晏堂生物有限公司, 大连 116021

【摘要】目的：研究叶瓜参化学成分。方法：对叶瓜参的 65%乙醇提取物中小极性部分进行行分离纯化, 得到脑苷脂分

子种 3, 对脑苷脂分子种 3 应用高效液相色谱进一步分离纯化, 得到 3 个脑苷脂单体, CF-3-1 (1)、CF-3-2 (2) 和 CF-3-3 (3)。结

果：根据化合物的波谱数据和化学方法鉴定了其结构。结论：三个化合物为首次从叶瓜参中分离得到, 首次得到化合物 2 和 3的单体化合物。

【关键词】鞘糖脂; 脑苷脂; 海参; 叶瓜参

【基金项目】国家国际科学技术合作基金(No. 2010DFB30810)

three cerebrosides from the sea cucumber cucumaria frondosa

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