Anti-inflammatory Asterosaponins from the Starfish Astropecten

Bioactive compounds derived from echinoderms. Ana R. Gomes , Ana C. Freitas , Teresa A. P. Rocha-Santos , Armando C. Duarte. RSC Adv. 2014 4 (56), ...
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Anti-inflammatory Asterosaponins from the Starfish Astropecten monacanthus Nguyen Phuong Thao,†,‡ Nguyen Xuan Cuong,† Bui Thi Thuy Luyen,†,‡ Nguyen Van Thanh,† Nguyen Xuan Nhiem,† Young-Sang Koh,§ Bui Minh Ly,⊥ Nguyen Hoai Nam,† Phan Van Kiem,† Chau Van Minh,*,† and Young Ho Kim*,‡ †

Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Caugiay, Hanoi, Vietnam College of Pharmacy, Chungnam National University, Daejeon 305-764, South Korea § School of Medicine, Brain Korea 21 Program, and Institute of Medical Science, Jeju National University, Jeju 690-756, South Korea ⊥ Nhatrang Institute of Technology Research and Application, 02 Hung Vuong, Nhatrang, Vietnam ‡

S Supporting Information *

ABSTRACT: Four new asterosaponins, astrosteriosides A−D (1−3 and 5), and two known compounds, psilasteroside (4) and marthasteroside B (6), were isolated from the MeOH extract of the edible Vietnamese starfish Astropecten monacanthus. Their structures were elucidated by chemical and spectroscopic methods including FTICRMS and 1D and 2D NMR experiments. The effects of the extracts and isolated compounds on pro-inflammatory cytokines were evaluated by measuring the production of IL-12 p40, IL-6, and TNF-α in LPS-stimulated bone marrow-derived dendritic cells. Compounds 1, 5, and 6 exhibited potent anti-inflammatory activity comparable to that of the positive control. Further studies are required to confirm efficacy in vivo and the mechanism of effects. Such potent anti-inflammatory activities render compounds 1, 5, and 6 important materials for further applications including complementary inflammation remedies and/or functional foods and nutraceuticals.

S

exhibit significant anti-inflammatory effects. The present study addresses the isolation, structure elucidation, and evaluation of the anti-inflammatory effects of six asterosaponins, including four new compounds, 1−3 and 5, from A. monacanthus.

tarfish (called also sea stars) are invertebrates belonging to the class Asteroidea, phylum Echinodermata. Research on starfish is a mature, largely explored area (with more than 30 years of activity) and has continuously expanded as prompted by the discovery of chemical constituents with unique structures and interesting pharmacological properties.1−3 The secondary metabolites from starfish are characterized by a diversity of polar steroids, including polyhydroxylated steroids and steroid glycosides. Starfish contain two main structural groups of steroid glycosides, namely, asterosaponins and glycosylated polyhydroxysteroids.1−5 Asterosaponins boast 3,6-dihydroxylation and Δ9(11) unsaturation of the aglycone with sulfonation at C-3 and a saccharide (usually five or six sugars) attachment at C-6. These compounds have exhibited a variety of biological activities, such as cytotoxic, hemolytic, antimicrobial, and antifouling effects.1−5 Starfish constituents exhibit pharmacological effects similar to those of sea cucumbers and have been historically used as tonic agents in Vietnamese folk medicines and medicinal foods.6 The edible starfish Astropecten monacanthus is abundant in the Vietnamese sea. However, no investigations of its chemical constituents and biological activities have been reported. In continuation of our recent investigations on Vietnamese starfish,7 the MeOH extract of A. monacanthus was found to © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION A MeOH extract of the starfish A. monacanthus (12.4 g) was suspended in H2O and successively partitioned with EtOAc and n-BuOH. The n-BuOH fraction (2.1 g) was separated by various chromatographic experiments to afford six asterosaponins. The known compounds, psilasteroside (4) 8 and marthasteroside B (6),9 were rapidly elucidated by comparing their observed and reported physical and spectroscopic data. Compound 1 was isolated as a white, amorphous powder with the molecular formula, C61H97NaO31S, determined by Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS). Its NMR features indicated an asterosaponin, one of the main constituents of starfish. The 1H and 13C NMR data of the aglycone of 1 were similar to those of psilasteroside (4),8 except for differences in the side chains, with the presence of a trisubstituted double bond [δC 157.4 (C, C-20) and δC 124.4 Received: June 20, 2013

A

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Chart 1

Figure 1. Key COSY (bold lines) and HMBC (H → C) correlations of compounds 1 and 5.

B

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Figure 2. Key ROESY correlations for the aglycone of 1.

structure of astrosterioside A (1) was elucidated as sodium 6αO-{α-L-arabinofuranosyl-(1→3)-β-D-fucopyranosyl-(1→2)-β-Dgalactopyranosyl-(1→4)-[β-D-quinovopyranosyl-(1→2)]-β-Dxylopyranosyl-(1→3)-β-D-quinovopyranosyl}-3β,6α-dihydroxy5α-cholesta-9(11),20(22)E-dien-23-on-3β-yl sulfate. The molecular formula of astrosterioside B (2) was identified as C61H101NaO32S by FTICRMS. Its 1H and 13C NMR data were close to those of psilasteroside (4),8 except for the presence of an oxymethine group [δC 66.7 (CH)/δH 4.43 (1H, m, H-23)] in 2 instead of the ketone group in 4. Consideration of the structure of 4 indicated that the additional oxymethine group was located at C-23 in 2. This was further supported by COSY and HMBC experiments. The 23R configuration was assigned based on the 13C NMR chemical shift observed at C23 (δC 66.7) of 2 and feroxoside B,11 which significantly differed from that of lethasterioside B at δC 69.1.10 The oligosaccharide chain of 2 also contained six sugar moieties with six anomeric carbons at δC 105.0 (C-1′), 104.2 (C-1″), 105.3 (C-1‴), 102.3 (C-1⁗), 107.0 (C-1⁗′), and 110.9 (C1⁗″), which correlated in the HSQC experiment with the relevant protons at δH 4.80 (1H, d, J = 7.0 Hz, H-1′), 5.05 (1H, d, J = 7.5 Hz, H-1″), 5.24 (1H, d, J = 7.0 Hz, H-1‴), 4.96 (1H, d, J = 7.0 Hz, H-1⁗), 4.85 (1H, d, J = 7.0 Hz, H-1⁗′), and 6.07 (1H, br s, H-1⁗″). The essentially identical 1H and 13C NMR data for the oligosaccharide part of 2 compared with those of 1 and psilasteroside (4),8 together with analysis of 2D NMR experiments (COSY, HMBC, HSQC, and ROESY) and acid hydrolysis (Experimental Section), confirmed that the same oligosaccharide chain was present in 1, 2, and 4. Based on these data, the structure of astrosterioside B (2) was characterized as sodium (20S,23R)-6α-O-{α-L-arabinofuranosyl-(1→3)-β-D-fucopyranosyl-(1→2)-β-D-galactopyranosyl-(1→4)-[β-D-quinovopyranosyl-(1→2)]-β-D-xylopyranosyl-(1→3)-β-D-quinovopyranosyl}-3β,6α,20,23-tetrahydroxy-5α-cholesta-9(11)-en-3βyl sulfate. The molecular formula of 3, C55H87NaO31S, was defined by FTICRMS. The NMR features were also typical of an asterosaponin. The oligosaccharide chain was identified as αL-arabinofuranosyl-(1→3)-β-D-fucopyranosyl-(1→2)-β-D-galactopyranosyl-(1→4)-[β-D-quinovopyranosyl-(1→2)]-β-D-xylopyranosyl-(1→3)-β-D-quinovopyranoside in the same manner as done for compounds 1 and 2. Comparison of the 1H and 13C NMR data of 3 with those of 1, 2, and 4 together with extensive 2D NMR analysis indicated that compounds 1−4 have the same steroidal nucleus but different side chains. The presence of a ketone at δC 208.5 (C-20) and a methyl group at δC 30.9 (C-21)/δH 2.05 (3H, s, H-21) indicated that the aglycone of 3 was asterone.12 Consequently, the structure of astrosterioside C

(CH, C-22)/δH 6.23 (1H, s, H-22)] in 1 instead of an oxygenated quaternary carbon and a methylene group in 4. In the HMBC spectrum, cross-peaks between H-21 (δH 2.28) and C-17 (δC 59.9), C-20 (δC 157.4), and C-22 (δC 124.4) and between H-22 (δH 6.23) and C-17 (δC 59.9), C-20 (δC 157.4), C-21 (δC 20.9), and C-23 (δC 200.6) clearly indicated the position of the double bond at C-20/C-22. The 13C NMR data in pyridine-d5 for the two oxymethine groups [δC 77.7 (C-3) and 80.1 (C-6)] and one trisubstituted double bond [δC 146.2 (C, C-9)/116.3 (CH, C-11)] of 1 were similar to those of psilasteroside (4) at δC 79.7 (C-3), 81.0 (C-6), 146.6 (C-9), and 117.8 (C-11) in CD3OD8 and lethasterioside A at δC 77.3 (C-3), 79.7 (C-6), 145.5 (C-9), and 116.3 (C-11) in pyridined5,10 respectively, supporting oxymethine groups at C-3 and C6 and a double bond at C-9/C-11. Detailed analysis of the other HMBC and COSY peaks (Figure 1) unambiguously identified the planar structure of the aglycone. In the ROESY spectrum, the correlation of H-3 (δH 4.85) with H-5 (δH 1.45) and that of H-14 (δH 1.33) with H-17 (δH 2.19) suggested an α-orientation for both H-3 and H-17. Spatial proximities were observed between H-6 (δH 3.79) and H-8 (δH 2.10)/H-19 (δH 0.91) and H-8 (δH 2.10) and H-18 (δH 0.57), indicating the β-orientation of H-6. Moreover, H-22 (δH 6.23) exhibited a strong correlation with H-17 (δH 2.19) but no correlations with H21 (δH 2.28), confirming an E configuration of the double bond at C-20/C-22 (Figure 2). In addition, the 13C NMR spectrum of 1 contained six anomeric carbon signals at δC 105.0 (C-1′), 104.2 (C-1″), 105.3 (C-1‴), 102.3 (C-1⁗), 107.0 (C-1⁗′), and 110.9 (C-1⁗″), which correlated with the corresponding anomeric protons at δH 4.80 (1H, d, J = 7.0 Hz, H-1′), 5.07 (1H, d, J = 7.5 Hz, H1″), 5.22 (1H, d, J = 7.0 Hz, H-1‴), 4.97 (1H, d, J = 7.5 Hz, H1⁗), 4.85 (1H, d, J = 7.0 Hz, H-1⁗′), and 6.07 (1H, br s, H1⁗″) in the HSQC spectrum, confirming the presence of six sugar moieties. The 1H and 13C NMR data of a pentose at δH 6.07 (1H, br s, H-1⁗″)/δC 110.9 (C-1⁗″), 82.4 (C-2⁗″), 78.8 (C-3⁗″), 87.5 (C-4⁗″), and 63.0 (C-5⁗″) were almost identical to those of psilasteroside (4)8 at δH 5.21 (1H, br s, H-1⁗″)/δC 111.1 (C-1⁗″), 82.9 (C-2⁗″), 78.9 (C-3⁗″), 86.7 (C-4⁗″), and 63.3 (C-5⁗″), indicating the presence of an arabinofuranosyl unit. A detailed comparison of the 1H and 13C NMR data for the oligosaccharide chain of 1 with those of psilasteroside (4)8 and a combination of the COSY, HMBC, HSQC, and ROESY data (Figure 1) indicated the same kinds and attachment positions for the six sugars as those observed for compound 4. The configurations of the sugar moieties were confirmed by acid hydrolysis of 1 followed by derivatization and HPLC analysis (Experimental Section). Consequently, the C

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Table 1. NMR Spectroscopic Data for the Aglycones of 1−3 and 5 1 pos.

δC,a,b type

1

35.9, CH2

2

29.4, CH2

3 4

77.7, CH 30.7, CH2

5 6 7

49.3, CH 80.1, CH 41.6, CH2

8 9 10 11 12

36.1, CH 146.2, C 38.4, C 116.3, CH 40.6, CH2

13 14 15

43.4, C 53.6, CH 25.4, CH2

16

25.4, CH2

17 18 19 20 21 22

59.9, CH 13.1, CH3 19.3, CH3 157.4, C 20.9, CH3 124.4, CH

23 24 25 26 27

2 δHa,c 1.35, 1.68, 1.83, 2.75, 4.85, 1.68, 3.42, 1.45, 3.79, 1.27, 2.68, 2.10,

m m m m m m m m m m m m

5.22, d (4.5) 1.98, m

1.33, 1.30, 1.80, 1.65, 1.93, 2.19, 0.57, 0.91,

m m m m m m s s

δC,a,b type 36.0, CH2 29.4, CH2 77.8, CH 30.7, CH2 49.4, CH 80.3, CH 41.6, CH2 35.5, CH 145.6, C 38.3, C 116.8, CH 42.5, CH2 41.6, C 54.1, CH 25.3, CH2 23.7, CH2

2.28, s 6.23, s

59.8, 13.7, 19.3, 74.6, 28.1, 51.6,

CH CH3 CH3 C CH3 CH2

200.6, C 53.8, CH2

2.37, m

66.7, CH 49.4, CH2

25.4, CH 22.7, CH3 22.7, CH3

2.25, m 0.92, d (6.0) 0.92, d (6.0)

24.8, CH 23.9, CH3 22.4, CH3

3 δHa,c 1.45, 1.65, 1.85, 2.35, 4.85, 1.68, 3.42, 1.45, 3.80, 1.30, 2.70, 2.13,

δC,b,d type

m m m m m m m m m m m m

35.3, CH2 28.4, CH2 75.3, CH 29.5, CH2 48.4, CH 78.0, CH 40.6, CH2

5.25, d (4.5) 2.10, m 2.30, m

34.9, CH 145.7, C 37.8, C 115.5, CH 39.5, CH2 41.9, C 52.9, CH 24.9, CH2

1.31, 1.29, 1.85, 2.05, 2.26, 1.98, 1.01, 0.97,

m m m m m m s s

1.66, 1.90, 1.96, 4.43, 1.40, 1.70, 2.10, 0.99, 1.00,

s m m m m m m d (6.0) d (6.0)

22.3, CH2 62.4, CH 12.8, CH3 19.0, CH3 208.5, C 30.9, CH3

5 δHc,d 1.30, 1.62, 1.40, 2.12, 3.85, 1.05, 2.37, 1.10, 3.45, 0.87, 2.26, 2.03,

m m m m m m m m m m m m

5.31, d (4.5) 2.17, m 2.26, m 1.36, 1.20, 1.70, 1.56, 2.04, 2.64, 0.43, 0.88,

m m m m m t (9.0) s s

2.05, s

δC,a,b type 36.0, CH2 29.5, CH2 77.7, CH 30.9, CH2 49.4, CH 80.9, CH 41.7, CH2 35.6, CH 145.7, C 38.3, C 116.5, CH 41.7, CH2 41.2, C 53.7, CH 25.3, CH2 28.6, CH2 56.2, 11.7, 19.3, 69.4, 30.1, 55.9,

CH CH3 CH3 C CH3 CH2

211.2, C 50.6, CH2 208.3, C 30.5, CH3

δHa,c 1.38, m 1.65, m 1.88, m 2.79e 4.88, m 1.70, m 3.50, m 1.50, m 3.81, m 1.25, m 2.72, m 2.01, m

5.17, d (4.5) 1.86, m 2.03, m 1.12, 1.00, 1.55, 1.07, 1.65, 1.08, 0.56, 0.94,

m m m m m m s s

1.51, s 2.80e

2.10, d (14.5) 2.45, d (14.5) 2.08, s

a

Measured in pyridine-d5. b125 MHz. c500 MHz. dMeasured in DMSO-d6. eOverlapped signals. Coupling constants (in parentheses) are in Hz. Assignments were done by HSQC, HMBC, COSY, and ROESY experiments.

HMBC spectrum, correlations of H-21 (δH 1.51) with C-17 (δC 56.2), C-20 (δC 69.4), and C-22 (δC 55.9) confirmed the position of the quaternary oxygenated carbon at C-20. Moreover, cross-peaks between H-22 (δH 2.80) and C-23 (δC 211.2), H-24 (δH 2.10 and 2.45) and C-23 (δC 211.2)/C-25 (δC 208.3), and H-26 (δH 2.08) and C-24 (δC 50.6)/C-25 (δC 208.3) confirmed the presence of two ketone groups at C-23 and C-25 (Figure 1). The 20R configuration was suggested by the marked difference between the 13C NMR chemical shifts at C-20 (δC 69.4) and C-21 (δC 30.1) of 5 relative to those of 2 at δC 74.6 (C-20) and 28.1 (C-21) and related compounds having a 20S configuration such as sodium (20S)-6α-O-{β-Dfucopyranosyl-(1→2)-α-L-arabinofuranosyl-(1→4)-[β-D-quinovopyranosyl-(1→2)]-β-D-xylopyranosyl-(1→3)-β-D-quinovopyranosyl}-20-hydroxy-23-oxo-5α-cholest-9(11)-en-3β-yl sulfate12 at δC 73.7 (C-20) and 27.0 (C-21) and ludiaquinoside8 at δC 74.6 (C-20) and 26.6 (C-21). Thus, the structure of astrosterioside D (5) was elucidated as sodium (20R)-6α-O-{βD-fucopyranosyl-(1→2)-β-D-fucopyranosyl-(1→4)-[β-D-quinovopyranosyl-(1→2)]-β-D-quinovopyranosyl-(1→3)-β-D-gluco-

(3) was characterized as sodium 6α-O-{α-L-arabinofuranosyl(1→3)-β-D-fucopyranosyl-(1→2)-β-D-galactopyranosyl-(1→4)[β-D-quinovopyranosyl-(1→2)]-β-D-xylopyranosyl-(1→3)-β-Dquinovopyranosyl}-5α-pregn-9(11)-en-20-on-3β-yl sulfate. Compound 5 was also isolated as a white, amorphous powder with spectroscopic features suggesting an asterosaponin. Its molecular formula, C56H89NaO29S, was determined by FTICRMS. The 1H and 13C NMR data of 5 were similar to those of marthasteroside B (6),9 except for differences in the signals of the side chains. Compounds 5 and 6 contained the same structures of the pentasaccharide chain and steroidal nucleus as determined by comparison of their 1H and 13C NMR data together with extensive analysis of 2D NMR spectra and acid hydrolysis. The side chain of 5 contained signals typical of one quaternary oxygenated [δC 69.4 (C-20)], two methyl [δC 30.1 (C-21)/δH 1.51 (3H, s, H-21) and δC 30.5 (C26)/δH 2.08 (3H, s, H-26)], two ketone [δC 211.2 (C-23) and 208.3 (C-25)], and two methylene groups [δC 55.9 (C-22)/δH 2.80 (2H, H-22) and δC 50.6 (C-24)/δH 2.10 (1H, d, J = 14.5 Hz, Ha-24) and 2.45 (1H, d, J = 14.5 Hz, Hb-24)]. In the D

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pyranosyl}-3β,6α,20-trihydroxy-27-nor-5α-cholesta-9(11)-en23,25-dion-3β-yl sulfate. The effects of the MeOH extract and n-BuOH fraction on pro-inflammatory cytokines were evaluated by measuring the production of IL-12 p40, IL-6, and TNF-α in LPS-stimulated bone marrow-derived dendritic cells (BMDCs). The MeOH extract showed significant inhibitory effects and the n-BuOH fraction exhibited potent inhibitory effects on the production of all three pro-inflammatory cytokines (Table 2). Among the

Table 3. Anti-inflammatory Effects of Compounds 1−6 on LPS-Stimulated BMDCs IC50 values (μM)a

Table 2. Anti-inflammatory Effects of the Extracts on LPSStimulated BMDCs a

IC50 values (μg/mL)a

a

extract

TNF-α

IL-6

IL-12 p40

MeOH extract n-BuOH fraction SB203580b

4.69 ± 0.17 2.25 ± 0.07 3.60 ± 0.12

2.10 ± 0.13 0.001 ± 0.000 1.70 ± 0.12

1.16 ± 0.02 0.25 ± 0.01 2.50 ± 0.10

compound

TNF-α

1 2 3 4 5 6 SB203580b

>50 >50 >50 >50 1.21 ± 0.06 >50 7.50 ± 0.21

IL-6 3.17 >50 30.49 >50 3.51 4.37 3.50

± 0.10 ± 1.25 ± 0.25 ± 0.26 ± 0.25

IL-12 p40 >50 >50 >50 >50 0.60 ± 0.02 >50 5.00 ± 0.15

IC50 values