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A new triterpene glycoside from sea cucumber Holothuria leucospilota
Chinese Chemical Letters 18 (2007) 161–164 www.elsevier.com/locate/cclet
A new triterpene glycoside from sea cucumber Holothuria leucospilota Hua Han, Yang Hua Yi *, Ling Li, Xiao Hua Wang, Bao Shu Liu, Peng Sun, Min Xiang Pan Research Center for Marine Drugs, School of Pharmacy, Second Military Medical University, Shanghai 200433, China Received 30 August 2006
Abstract A new triterpene glycoside, leucospilotaside A, along with a known saponin, isolated from sea cucumber Holothuria leucospilota, and its structure was elucidated as 3b-O-[4-O-sodiumsulfate-b-D-quinovopyranosyl-(1 ! 2)-b-D-xylopyranosyl]holosta-22-ketone-9-en-17a,25a-diol (1) by extensive spectroscopic analysis and chemical methods. Leucospilotaside A (1) has a ketone carbonyl group (22) in the aglycon side chain. # 2007 Yang Hua Yi. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Sea cucumber; Holothuria leucospilota; Triterpene glycoside; Leucospilotaside A
Sea cucumber saponins known as holothurins are a rich source of triterpene glycosides. The triterpene is usually of the lanosterol-type with a 18(20)-lactone, and a sugar chain with up to six monosaccharide linked to the C-3 of the aglycon. These compounds exhibit wide spectra of biological activity, such as antifungal, cytotoxic, hemolytic, cytostatic and immunomodulatory effects [1–3]. We have focused our attention on biologically active triterpene glycosides from sea cucumbers [1,2]. Here we report the isolation and structure elucidation of a new monosulfated glycoside, leucospilotaside A (1), along with the known holothurin B (2). The sea cucumber, identified as Holothuria leucospilota [4] by Prof. Yu Lin LIAO (Institute of Oceanology, Chinese Academy of Science, Qingdao), were collected along the coast of Hainan Province in the South China Sea in spring 2005. A voucher specimen (reg. No. HYSC-2005-03) is preserved at Research Center for Marine Drugs, School of Pharmacy, Second Military Medical University, Shanghai. The 80% ethanolic extract of H. leucospilota was sequentially submitted to column chromatography on DA-101 resin (Nankai University, Tianjin), silic gel, and reversed-phase silica (lichroprep RP-18, 40–63 mm). Finally, purification of these compounds was achieved by reversed-phase HPLC on zobax SB C-18 to give the pure leucospilotaside A (1), along with the main component of the glycosidic fraction. Leucospilotaside A (1), a white amorphous powder, ½a20 D þ 6:2 (c 0.7, pyridine), mp 247.5–249.5 8C, was positive to Liebemann-Burchard and molish test. The molecular formula was established as C41H63O18NaS from the [M+Na]+ * Corresponding author. E-mail address: [email protected] (Y.H. Yi). 1001-8417/$ – see front matter # 2007 Yang Hua Yi. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2006.12.027
162
H. Han et al. / Chinese Chemical Letters 18 (2007) 161–164
Table 1 13 C and 1H NMR chemical shifts for the aglycon moieties of leucospilotaside A (1) and holothurin B (2) (d ppm) Position
dC 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
HMBC
1 a
36.5 27.0 88.7 40.1 52.8 21.2 28.4 40.9 154.3 39.8 115.3 71.1 58.7 46.3 36.8
dH (J in Hz) t t d s d t t d s s d d s s t
16
37.6 t
17 18 19 20 21 22 23 24 25 26 27 30 31 32
92.4 s 173.7 s 22. 6 q 87.6 s 21.5 q 209.2 s 34.8 t 39.51 t 69.0 s 30.0 q 29.7 q 16.8 q 28.2 q 18.6 q
a b
b
0.98 m 1.51 m 1.73 m 3.27 m
C-8, -10, -12 C-9, -11, -14,-18
1.82 m 2.40 dd (9.2, 14.6) 2.20 m
36.4 27.4 88.7 40.0 52.7 21.2 27.3 40.9 153.8 39.7 115.5 71.5 58.8 45.9 36.8
t t d s d t t d s s d d s s t
35.6 t
1.36 s
C-1, -5, -9, -10
1.91 s
C-17, -20, -22
3.19 m 1.19 m
C-20, -22,-24, -25 C-22, -23, -26, -27
1.40 1.39 1.12 1.26 1.60
C-24, -25, -27 C-24, -25, -26 C-3, -4, -5, -31 C-3, -4, -5, -30 C-8, -13, -14, -15
s s s s s
dH (J in Hz) b
dCa
1.33 m; 1.82 m 1.86 m 3.16 dd (4.0, 11.6)
5.60 d (4.1) 4.94 d (4.0)
2
89.7 174.4 22.5 86.6 18.8 80.7 27.4 38.4 81.3 28.6 27.0 16.7 28.1 20.3
s s q s q d t t s q q q q q
1.43 m;1.84 m 2.08 m 3.16 (4.0,11.6) 1.03 1.50 1.34 3.37
m m m m
5.62 d (4.0) 4.97 d
1.82 m 2.96 m 2.40 m
1.39 s 1.77 4.33 1.99 1.60
s m m m
1.20 1.22 1.14 1.28 1.67
s s s s s
Recorded at 100 MHz in C5D5N–D2O (4:1); multiplicity by DEPT. Recorded at 400 MHz in C5D5N–D2O (4:1).
ion at m/z 921.3534 (calcd. 921.3531) in the positive ion mode HRESIMS and [M-Na] ion at m/z 876 in the negative ion mode ESIMS. The fragment ion peak at m/z 801 [M+Na-NaHSO4]+ in the positive ion mode ESIMS/MS indicated the presence of a sulfate group in the glycoside. This was supported by extensive analysis of 1D NMR and 2D NMR (1H–1H COSY, NOESY, HMQC, HMBC) and MS. IR spectrum showed the presence of hydroxyl (3419 cm1), carbonyl (1749 cm1, 1730 cm1), olefinic (1653 cm1), and sulfate (1065 cm1) groups. The data of the 1H, 13C NMR and DEPT suggested the presence of a triterpenoid aglycon with a 9(11)-double bond [dC 154.3 (s, C-9) and 115.3 (d, C-11), dH 5.60 (d, H-11)], one oxomethine [dC 88.7 (d, C-3), dH 3.16 (dd, H-3)] and one ketone carbonyl group (dC 209.2, s, C-22). The assignments of the NMR signals associated with the aglycon moiety (Table 1) were derived and confirmed by 1H–1H COSY, TOCSY, HMBC, and NOESY experiments, and comparison with those of the aglycons of the known related compounds (from H. leucospilota) [5,6], which had been identified as holothurigenol. Correlation from H-21 to C-22, H-23 to C-22 in the HMBC spectrum indicated that ketone carbonyl group located at C-22 of the aglycon. The presence of D-xylose and D-quinovose for the glycoside 1 in a ratio of 1:1 was established by acid hydrolysis followed by GC–MS analysis of the corresponding aldononitrile peracetate [7]. The two anomeric carbons and protons in glycoside 1 was confirmed by its 13C and 1H NMR spectra at dC 105.2, 105.8 and dH 4.72 (d, J = 7.6 Hz), 5.08 (d, J = 7.6 Hz) (Table 2). Assignment of the sugar moieties was performed by 1H–1H COSY, TOCSY, HMBC, and NOESY experiments, which showed cross-peaks between H-1 of xylose and C-3 of the aglycon, and H-1 of quinovose
H. Han et al. / Chinese Chemical Letters 18 (2007) 161–164
163
Table 2 1 H and 13C NMR data for the sugar moieties of leucospilotaside A (1) (d ppm) Position
dCa
dH (J in Hz) b
HMBC
Xyl (1 ! C-3) 1 2 3 4 5
105.2 83.1 75.5 76.9 64.2
4.72 4.05 4.32 4.03 3.74
d (7.6) m m m m, 4.76 m
C-3 C-1 C-4 C-2 C-1
Qui (1 ! 2Xyl) 1 2 3 4 5 6
105.8 77.7 76.7 76.1 73.4 19.9
5.08 4.08 5.10 3.66 3.77 1.62
d (7.6) m m m m s
C-2 Xyl C-3 Qui C-4 Qui C-3, 5 Qui
a b
Qui, C-3Xyl Xyl, C-2 Xyl Xyl Xyl
C-4, 5 Qui
Recorded at 100 MHz in C5D5N–D2O (4:1); multiplicity by DEPT. Recorded at 400 MHz in C5D5N–D2O (4:1).
Fig. 1. The structure of leucospilotaside A (1).
and C-2 of xylose. The site of the sulfate group in the sugar unit of 1 was determined from chemical shift of the signal of C-40 (xyl) which was downfield relative to shift, expected for corresponding desulfated derivative of 1. The a configuration of H-1 at the anomeric carbons was deduced from the coupling constant values. These data were also confirmed by the NOESY spectrum. All these data confirmed the structure of leucospilotaside A (1) as 3b-O-[4-O-sodiumsulfate-b-D-quinovopyranosyl-(1!2)-b-D-xylopyranosyl]holosta-22-ketone-9-en-17a,25a-diol (1) (Fig. 1). Acknowledgments This research was supported by the State Foundation for High-tech Project ‘‘863’’ from the Ministory of Science and Technology, China (No. 2001AA624100). We thank Professor Yu Lin Liao for taxonomic identification of the sea cucumber. References [1] Z.R. Zou, Y.H. Yi, H.M. Wu, et al. J. Nat. Prod. 66 (2003) 1055. [2] Z.R. Zou, Y.H. Yi, H.M. Wu, et al. J. Nat. Prod. 68 (2005) 540.
164 [3] [4] [5] [6] [7]
H. Han et al. / Chinese Chemical Letters 18 (2007) 161–164 A.P. Murray, C. Muniain, A.M. Seldes, et al. Tetrahedron 57 (2001) 9563. Y.L. Liao, Chinese Fauna Echinodermata Holothuroidea, Beijing, Science Press, 1997, p. 174. I. Kitagawa, T. Nishino, M. Kobayashi, et al. Chem. Pharm. Bull. 29 (1981) 1942. I. Kitagawa, T. Nishino, M. Kobayashi, et al. Chem. Pharm. Bull. 29 (1981) 1951. S.A. Avilov, A.S. Antonov, A.S. Silchenko, et al. J. Nat. Prod. 66 (2003) 910.
A new triterpene glycoside from sea cucumber Holothuria leucospilota Hua Han, Yang Hua Yi *, Ling Li, Xiao Hua Wang, Bao Shu Liu, Peng Sun, Min Xiang Pan Research Center for Marine Drugs, School of Pharmacy, Second Military Medical University, Shanghai 200433, China Received 30 August 2006
Abstract A new triterpene glycoside, leucospilotaside A, along with a known saponin, isolated from sea cucumber Holothuria leucospilota, and its structure was elucidated as 3b-O-[4-O-sodiumsulfate-b-D-quinovopyranosyl-(1 ! 2)-b-D-xylopyranosyl]holosta-22-ketone-9-en-17a,25a-diol (1) by extensive spectroscopic analysis and chemical methods. Leucospilotaside A (1) has a ketone carbonyl group (22) in the aglycon side chain. # 2007 Yang Hua Yi. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Sea cucumber; Holothuria leucospilota; Triterpene glycoside; Leucospilotaside A
Sea cucumber saponins known as holothurins are a rich source of triterpene glycosides. The triterpene is usually of the lanosterol-type with a 18(20)-lactone, and a sugar chain with up to six monosaccharide linked to the C-3 of the aglycon. These compounds exhibit wide spectra of biological activity, such as antifungal, cytotoxic, hemolytic, cytostatic and immunomodulatory effects [1–3]. We have focused our attention on biologically active triterpene glycosides from sea cucumbers [1,2]. Here we report the isolation and structure elucidation of a new monosulfated glycoside, leucospilotaside A (1), along with the known holothurin B (2). The sea cucumber, identified as Holothuria leucospilota [4] by Prof. Yu Lin LIAO (Institute of Oceanology, Chinese Academy of Science, Qingdao), were collected along the coast of Hainan Province in the South China Sea in spring 2005. A voucher specimen (reg. No. HYSC-2005-03) is preserved at Research Center for Marine Drugs, School of Pharmacy, Second Military Medical University, Shanghai. The 80% ethanolic extract of H. leucospilota was sequentially submitted to column chromatography on DA-101 resin (Nankai University, Tianjin), silic gel, and reversed-phase silica (lichroprep RP-18, 40–63 mm). Finally, purification of these compounds was achieved by reversed-phase HPLC on zobax SB C-18 to give the pure leucospilotaside A (1), along with the main component of the glycosidic fraction. Leucospilotaside A (1), a white amorphous powder, ½a20 D þ 6:2 (c 0.7, pyridine), mp 247.5–249.5 8C, was positive to Liebemann-Burchard and molish test. The molecular formula was established as C41H63O18NaS from the [M+Na]+ * Corresponding author. E-mail address: [email protected] (Y.H. Yi). 1001-8417/$ – see front matter # 2007 Yang Hua Yi. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2006.12.027
162
H. Han et al. / Chinese Chemical Letters 18 (2007) 161–164
Table 1 13 C and 1H NMR chemical shifts for the aglycon moieties of leucospilotaside A (1) and holothurin B (2) (d ppm) Position
dC 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
HMBC
1 a
36.5 27.0 88.7 40.1 52.8 21.2 28.4 40.9 154.3 39.8 115.3 71.1 58.7 46.3 36.8
dH (J in Hz) t t d s d t t d s s d d s s t
16
37.6 t
17 18 19 20 21 22 23 24 25 26 27 30 31 32
92.4 s 173.7 s 22. 6 q 87.6 s 21.5 q 209.2 s 34.8 t 39.51 t 69.0 s 30.0 q 29.7 q 16.8 q 28.2 q 18.6 q
a b
b
0.98 m 1.51 m 1.73 m 3.27 m
C-8, -10, -12 C-9, -11, -14,-18
1.82 m 2.40 dd (9.2, 14.6) 2.20 m
36.4 27.4 88.7 40.0 52.7 21.2 27.3 40.9 153.8 39.7 115.5 71.5 58.8 45.9 36.8
t t d s d t t d s s d d s s t
35.6 t
1.36 s
C-1, -5, -9, -10
1.91 s
C-17, -20, -22
3.19 m 1.19 m
C-20, -22,-24, -25 C-22, -23, -26, -27
1.40 1.39 1.12 1.26 1.60
C-24, -25, -27 C-24, -25, -26 C-3, -4, -5, -31 C-3, -4, -5, -30 C-8, -13, -14, -15
s s s s s
dH (J in Hz) b
dCa
1.33 m; 1.82 m 1.86 m 3.16 dd (4.0, 11.6)
5.60 d (4.1) 4.94 d (4.0)
2
89.7 174.4 22.5 86.6 18.8 80.7 27.4 38.4 81.3 28.6 27.0 16.7 28.1 20.3
s s q s q d t t s q q q q q
1.43 m;1.84 m 2.08 m 3.16 (4.0,11.6) 1.03 1.50 1.34 3.37
m m m m
5.62 d (4.0) 4.97 d
1.82 m 2.96 m 2.40 m
1.39 s 1.77 4.33 1.99 1.60
s m m m
1.20 1.22 1.14 1.28 1.67
s s s s s
Recorded at 100 MHz in C5D5N–D2O (4:1); multiplicity by DEPT. Recorded at 400 MHz in C5D5N–D2O (4:1).
ion at m/z 921.3534 (calcd. 921.3531) in the positive ion mode HRESIMS and [M-Na] ion at m/z 876 in the negative ion mode ESIMS. The fragment ion peak at m/z 801 [M+Na-NaHSO4]+ in the positive ion mode ESIMS/MS indicated the presence of a sulfate group in the glycoside. This was supported by extensive analysis of 1D NMR and 2D NMR (1H–1H COSY, NOESY, HMQC, HMBC) and MS. IR spectrum showed the presence of hydroxyl (3419 cm1), carbonyl (1749 cm1, 1730 cm1), olefinic (1653 cm1), and sulfate (1065 cm1) groups. The data of the 1H, 13C NMR and DEPT suggested the presence of a triterpenoid aglycon with a 9(11)-double bond [dC 154.3 (s, C-9) and 115.3 (d, C-11), dH 5.60 (d, H-11)], one oxomethine [dC 88.7 (d, C-3), dH 3.16 (dd, H-3)] and one ketone carbonyl group (dC 209.2, s, C-22). The assignments of the NMR signals associated with the aglycon moiety (Table 1) were derived and confirmed by 1H–1H COSY, TOCSY, HMBC, and NOESY experiments, and comparison with those of the aglycons of the known related compounds (from H. leucospilota) [5,6], which had been identified as holothurigenol. Correlation from H-21 to C-22, H-23 to C-22 in the HMBC spectrum indicated that ketone carbonyl group located at C-22 of the aglycon. The presence of D-xylose and D-quinovose for the glycoside 1 in a ratio of 1:1 was established by acid hydrolysis followed by GC–MS analysis of the corresponding aldononitrile peracetate [7]. The two anomeric carbons and protons in glycoside 1 was confirmed by its 13C and 1H NMR spectra at dC 105.2, 105.8 and dH 4.72 (d, J = 7.6 Hz), 5.08 (d, J = 7.6 Hz) (Table 2). Assignment of the sugar moieties was performed by 1H–1H COSY, TOCSY, HMBC, and NOESY experiments, which showed cross-peaks between H-1 of xylose and C-3 of the aglycon, and H-1 of quinovose
H. Han et al. / Chinese Chemical Letters 18 (2007) 161–164
163
Table 2 1 H and 13C NMR data for the sugar moieties of leucospilotaside A (1) (d ppm) Position
dCa
dH (J in Hz) b
HMBC
Xyl (1 ! C-3) 1 2 3 4 5
105.2 83.1 75.5 76.9 64.2
4.72 4.05 4.32 4.03 3.74
d (7.6) m m m m, 4.76 m
C-3 C-1 C-4 C-2 C-1
Qui (1 ! 2Xyl) 1 2 3 4 5 6
105.8 77.7 76.7 76.1 73.4 19.9
5.08 4.08 5.10 3.66 3.77 1.62
d (7.6) m m m m s
C-2 Xyl C-3 Qui C-4 Qui C-3, 5 Qui
a b
Qui, C-3Xyl Xyl, C-2 Xyl Xyl Xyl
C-4, 5 Qui
Recorded at 100 MHz in C5D5N–D2O (4:1); multiplicity by DEPT. Recorded at 400 MHz in C5D5N–D2O (4:1).
Fig. 1. The structure of leucospilotaside A (1).
and C-2 of xylose. The site of the sulfate group in the sugar unit of 1 was determined from chemical shift of the signal of C-40 (xyl) which was downfield relative to shift, expected for corresponding desulfated derivative of 1. The a configuration of H-1 at the anomeric carbons was deduced from the coupling constant values. These data were also confirmed by the NOESY spectrum. All these data confirmed the structure of leucospilotaside A (1) as 3b-O-[4-O-sodiumsulfate-b-D-quinovopyranosyl-(1!2)-b-D-xylopyranosyl]holosta-22-ketone-9-en-17a,25a-diol (1) (Fig. 1). Acknowledgments This research was supported by the State Foundation for High-tech Project ‘‘863’’ from the Ministory of Science and Technology, China (No. 2001AA624100). We thank Professor Yu Lin Liao for taxonomic identification of the sea cucumber. References [1] Z.R. Zou, Y.H. Yi, H.M. Wu, et al. J. Nat. Prod. 66 (2003) 1055. [2] Z.R. Zou, Y.H. Yi, H.M. Wu, et al. J. Nat. Prod. 68 (2005) 540.
164 [3] [4] [5] [6] [7]
H. Han et al. / Chinese Chemical Letters 18 (2007) 161–164 A.P. Murray, C. Muniain, A.M. Seldes, et al. Tetrahedron 57 (2001) 9563. Y.L. Liao, Chinese Fauna Echinodermata Holothuroidea, Beijing, Science Press, 1997, p. 174. I. Kitagawa, T. Nishino, M. Kobayashi, et al. Chem. Pharm. Bull. 29 (1981) 1942. I. Kitagawa, T. Nishino, M. Kobayashi, et al. Chem. Pharm. Bull. 29 (1981) 1951. S.A. Avilov, A.S. Antonov, A.S. Silchenko, et al. J. Nat. Prod. 66 (2003) 910.