12-620-0678doc - 中国科学技术协会.pdf

620 药学学报 Acta Pharmaceutica Sinica 2009, 44 (6): 620624 Antifungal active triterpene glycosides from sea cucumber Holothuria scabra HAN Hua1, 2, YI Yang- hua1*, LI Ling1, LIU Bao- shu1, LA Ming- ping1, ZHANG Hong- wei1 (1. Research Center for Marine Drugs, School of Pharmacy, Second Military Medical University, Shanghai 200433, China; 2. College of Animal Sciences, Zhejiang University, Hangzhou 310029, China) Abstract: To study the new antifungal active triterpene glycosides of sea cucumber Holothuria scabra. Triterpene glycosides from Holothuria scabra were separated and purified by silica gel chromatography, reversed- phase silica ge1 chromatography and RP- HPLC. Their structures were elucidated on the basis of spectral data and chemical evidence. Three triterpene glycosides were identified as scabraside A (1), echinoside A (2) and holothurin A1 (3). Scabraside A (1) is a new triterpene glycoside, and compounds 2 and 3 were isolated from Holothuria scabra for the first time. They showed antifungal activities (1MIC8016 gmL1). Key words: Holothuria scabra; triterpene glycoside; antifungal activity CLC number: R284.1 Document code: A Article ID: 0513- 4870 (2009) 06- 0620- 05 糙海参中具有抗真菌活性的三萜皂苷 韩 华1, 2, 易杨华1*, 李 玲1, 刘宝姝1, 喇明平1, 张宏伟1 (1. 第二军医大学药学院海洋药物研究中心, 上海 200433; 2. 浙江大学动物科学学院, 浙江 杭州 310029) 摘要: 为了研究糙海参体内的化学成分，寻找结构新颖具有抗真菌活性的三萜皂苷类成分。应用多种色谱分离技术对糙海参体内的化学成分进行分离纯化，根据化合物的理化性质、波谱数据及化学方法鉴定其结构。分离得到 3 个三萜皂苷化合物，分别为 scabraside A (1)、echinoside A (2) 和 holothurin A1 (3)，并对其抗真菌活性进行了研究 (1MIC8016 gmL1)。化合物 1 为新的三萜皂苷化合物，化合物 2 和 3 为首次从该海参中分离得到，它们均显示显著的抗真菌活性。 关键词: 糙海参; 三萜皂苷; 抗真菌活性 Sea cucumber Holothuria scabra Jaeger (Holo-thuriidae) is distributed abundantly in South China sea. In order to find new biologically active substances from marine organisms, we have been studying kinds of sea cucumbers of echinoderm17. Triterpene glycosides are the predominant secondary metabolites of the sea cucumber, and exhibit wide spectra of biological Received 2008- 11- 19. Project supported by the “863” Hi- tech Research and Development Program of China (No.2006AA09Z417) and National Natural Science Foundation of China (No.20772155). *Corresponding author Tel: 86- 21- 65384988, Fax: 86- 21- 65483662, E- mail: activities, such as antifungal, cytotoxic, hemolytic, cytostatic and immunomodulatory functions8, 9. They are usually the triterpenoid of the lanosterol- type with an 18(20)- lactone and the sugar chain of up to six mono-saccharide units is generally linked to the C- 3 of the aglycone, which is composed of D- xylose, D- quinovose, D- glucose, and 3- O- methyl- D- glucose10. We here report the isolation and the antifungal activity of three glycosides from the sea cucumber H. scabra and the structural elucidation of the new triterpene glycosides. These compounds showed antifungal activity against seven fungi trains tested. HAN Hua, et al: Antifungal active triterpene glycosides from sea cucumber Holothuria scabra 621 Results and discussion Structure elucidation Scabraside A (1) was positive in the Liebermann- Burchard and Molish tests. Its molecular formula was determined as C54H85O26SNa from pseudomolecular ion peak at m/z 1 227.484 9 M + Na+ (calcd. for C54H85Na2O26S: 1 227.484 5) in positive- ion mode HR- ESI- MS and at m/z 1181 M Na in negative- ion mode ESI- MS. A fragment ion peak at m/z 1 107 M OSO3Na + Na H+ indicated the presence of a sulfate group in 1, which was confirmed by the IR spectrum with absorption bands at 1 255 and 1 071 cm1. An examination of the 1H and 13C NMR spectra of 1 (Table 1) indicated the presence of a triterpene aglycone with six methyls, two olefinic bonds and one lactone carbonyl group, which had a close similarity to the aglycone of echinoside A11, but 1 differed from echinoside A at C- 25 and C- 26. The positions of a disubstituted terminal double bond at 25(26) was deduced from the NMR signals at C 145.5 (C- 25), 110.8 (C- 26); H 4.66 (s, H- 26) together with the analysis of the TOCSY and HMBC experiments. The HMBC spectrum showed cross- peaks H- 23/C- 25, H- 24/C- 25, H- 27/C- 25 and H- 27/C- 26. The 1H NMR spectrum also showed an olefinic methyl signal (H 1.67 (s, H- 27) ) and five methyl groups (H 1.23 (s, H- 31), 1.03 (s, H- 30), 1.65 (s, H- 32), 1.36 (s, H- 19) and 1.66 (s, H- 21) ). The double bond at 9(11) was deduced from the NMR signals at C 154.0 (C- 9), 115.6 (C- 11); H 5.57 (d, J = 4.2 Hz, H- 11) together with the analysis of the TOCSY and HMBC experiments. The HMBC spectrum showed cross- peaks H- 7/C- 9, H- 19/C- 9, H- 8/C- 11, H- 12/C- 11, and in the TOCSY spectrum, two protons (H 5.57 (H- 11) and 4.95 (H- 12) ) comprised a two- spin system. A signal characteristic for an oxygenated methine (C 71.3 (C- 12); H 4.95 (dd, J = 5.6, 12.0 Hz, H- 12) ) in the holostane nucleus indicated - configuration of the allylic OH group at C- 1212. Therefore, a 12- hydroxylated 9(11) terpenoid aglycone was identified. The presence of four - sugars unit in 1 was deduced from the 13C and 1H NMR spectra, which showed four anomeric carbon and four anomeric proton resonances with coupling constant doublets (J values) 7.27.8 Hz. The sugar moiety was confirmed to be D- xylose (Xyl), D- quinovose (Qui), D- glucose (Glc) and 3- O- methylglucose (MeGlc) in a ratio of 1111 by acidic hydrolysis (aq. 2 molL1 CF3COOH) followed by GC/MS analysis of the corresponding aldononitrile peracetates and by comparing the GC retention time of the corresponding aldononitrile peracetates with those of the authentic samples prepared in the same manner12. The 1H and 13C NMR signals attributable to the sugar unit were assigned by the 2D NMR experiments and the data indicated that sugar residues were all in pyranose form. The sequence of the sugar residues in 1 was determined by analysis of HMBC correlations: Xyl H- 1/C- 3 of the aglycone, Qui H- 1/Xyl C- 2, Glc H- 1/Qui C- 4 and MeGlc H- 1/Glc C- 3. The position of the sulfate group was determined by comparing 13C NMR data of 1 with those of known glycosides13. A downfield esterification shift was observed for the signal of Xyl C- 4 (from 68.2 to 75.5). On the basis of the above data, the structure of 1 was deduced as 3- O- 3- O- methyl- - D- glucopyranosyl- (1 3)- - D- glucopyranosyl- (1 4)- - D- quinovopyranosyl- (1 2)- 4- O- sulfate- - D- xylopyransyl- holosta- 9 (11), 25- diene- 3, 12, 17- triol and named scabraside A, see Figure 1 and 2. Table 1 1H (600 MHz) and 13C NMR(150 MHz) data (, J in Hz) for glycoside 1 in C5D5Na Aglycon C H (J) Sugar C H (J) 1 36.3 1.39 (m), 1.81 (m) Xyl 2 27.0 1.86 (m), 2.05 (m) 1 105.2 4.65 (d, J = 7.2) 3 88.7 3.13 (dd, J = 4.8, 12.0) 2 83.4 4.05 (m) 4 40.0 3 75.3 4.26 (m) 5 52.7 0.97 (d, J = 10.2) 4 75.5 5.03 (m) 6 28.3 1.69 (m), 1.52 (m) 5 64.3 3.69 (m), 4.72 (m) 7 21.2 1.45 (m), 1.74 (m) Qui 8 40.9 3.33 (br d, J = 10.4) 1 105.4 5.01 d, J = 7.8) 9 154.0 2 76.3 3.95 (m) 10 39.7 3 75.8 4.03 (m) 11 115.6 5.57 (d, J = 4.2) 4 86.8 3.59 (m) 12 71.3 4.95 (dd, J = 5.6, 12.0) 5 71.9 3.67 (m) 13 58.6 6 18.1 1.69 d, J = 5.4) 14 46.4 Glc 15 36.7 1.79 (m), 1.36 (m) 1 104.9 4.94 d, J = 7.8) 16 36.1 2.28 (m), 2.61 (m) 2 73.9 4.01 (m) 17 89.3 3 87.9 4.21 (m) 18 174.7 4 69.5 3.99 (m) 19 22.6 1.36 (s) 5 77.8 3.91 (m) 20 87.1 6 61.8 4.46 (m), 4.27 (m) 21 23.0 1.66 (s) MeGlc 22 38.2 1.79 (m), 1.85 (m) 1 105.8 5.30 d, J = 7.8) 23 22.4 0.83 (m), 0.89 (m) 2 75.0 4.02 (m) 24 38.4 1.92 (m), 1.99 (m) 3 88.0 3.68 (m) 25 145.5 4 70.6 4.02 (m) 26 110.8 4.66 (s) 5 78.3 3.94 (m) 27 22.2 1.67 (s) 6 62.1 4.19 (m), 4.26 (m) 30 16.7 1.03 (s) OMe 60.8 3.84 (s) 31 28.1 1.23 (s) 32 20.1 1.65 (s) aAssignments aided by DQFCOSY, TOCSY, HMQC, HMBC and NOESY experiments 622 药学学报 Acta Pharmaceutica Sinica 2009, 44 (6): 620624 Figure 1 The structures of compounds 13 Figure 2 Key HMBC correlations of compound 1 Table 2 Antifungal effects of triterpene glycosides (MIC80: gmL1) C. albicans C. pseudotropicalis C. neoformans T. rubrum M. gypseum F. compacta A. fumigatus 1 8 4 8 8 4 8 2 2 4 2 8 4 4 8 1 3 16 8 8 8 8 8 8 FCZ 0.25 1 2 2 64 8 64 ITCZ 0.125 0.125 2 0.125 0.125 1 0.5 TBNF 32 0.125 0.125 0.125 0.125 0.125 0.125 Antifungal activity Some triterpene glycosides hitherto isolated from sea cucumber exhibited antifungal activity. Herein, we report the antifungal activities of three triterpene glycosides isolated from the sea cucumber H. scabra Jeager against seven strains: Candida albicans, Cryptococcus neoformans, Candida pseudotropicalis, Trichophyton rubrum, Fonsecaea compacta, Aspergillus fumigatus, and Microsporum gypseum. Fluconazole (FCZ), Terbinafine (TBNF) and Itraconazole (ITCZ) were used as positive controls. Our results (Table 2) indicated that three glycosides showed antifungal activity against seven fungi strains with MIC80 in the range of HAN Hua, et al: Antifungal active triterpene glycosides from sea cucumber Holothuria scabra 623 116 gmL1. On the basis of the data available, the antifungal activity of sea cucumber glycosides is very sensitive to their precise functionalization, and perhaps they show different sensitivities against different fungi strains. 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. Experimental General experimental procedures Melting points were determined on an XT5- XMT apparatus. Optical rotations were measured with a Perkin- Elmer 341 polarimeter. IR spectra were recorded on a Bruker Vector 22 infrared spectrometer. NMR spectra were recorded in C5D5N on a Varian Inova- 600 spectrometer, and the 2D NMR spectra were obtained using standard pulse sequences. ESI- MS and HR- ESI- MS were recorded on a Micromass Quattro mass spectrometer. GC/MS were performed on a Finnigan Voyager apparatus using a DB- 5 column (30 m 0.25 mm ID, 0.25 m) with an initial temperature of 150 for 2 min and then temperature programming to 300 at a rate of 15 min1. Semipreparative HPLC was carried out on an Agilent 1100 liquid chromatograph equipped with a refractive index detector using a Zorbax 300 SB- C18 column (25 cm 9.4 mm ID). Column chromatographies were performed on silica gel (200300 mesh, 1040 m; Yantai, P. R. China) and ODS (4063 m; Merck) and Sephadex LH- 20 (Pharmacia). Fractions were monitored by TLC (precoated silica gel GF254 plates (1040 m; Yantai), and spots were visualized by heating silica gel plates sprayed with 15% H2SO4 in EtOH. Seven strains Candida albicans, Cryptococcus neoformans, Candida pseudotropicalis, Trichophyton rubrum, Fonsecaea compacta, Aspergillus fumigatus, and Microsporum gypseum. Positive controls: Fluconazole (FCZ), Terbinafine (TBNF) and Itraconazole (ITCZ). Animal material Specimens of H. scabra Jeaber were collected from offshore waters of Hainan Island in the South China Sea in May 2006, and authenticated by Professor LIAO Yu- lin (Institute of Oceanology, Chinese Academy of Science, P.R.China). A voucher specimen (No. HY200605) was deposited at the Research Center for Marine Drugs, School of Pharmacy, Second Military Medical University. Extraction and isolation The sea cucumbers of 3 kg (dry weight) were powdered and refluxed four times each of 1 h with 60% ethanol (6 L 4). The extract was concentrated, and the residue (420 g) was suspended in H2O, passed through a DA101 resin column (2 kg, 15 cm 105 cm, Nankai University, Tianjin, China) and then eluted with H2O (5 L), 70% EtOH (10 L), and 95% EtOH (5 L), separately. The glycoside fraction was eluted with 70% ethanol. The combined extracts were concentrated. The glycoside fraction (crude glycoside- containing mixture, 70 g) was chromatographed over silica gel CC (200300 mesh, 2 100 g), stepwise eluted with CHCl3- MeOH- H2O (8 21 to 6.53.51) gradient to give fraction A (2.43 g), B (3 g), C (1.13 g), D (3.8 g) and E (2.23 g). Fraction D and E were further purified by MPLC with a column of reversed- phase silica (Lichroprep RP- C18, 4063 m). Final purification of the glycosides in fraction D and E was achieved by HPLC (Zorbax 300 SB- C18). Fraction D afforded 10.7 mg of pure glycoside 1 (tR = 21 min) and 204.5 mg of 2 (tR = 26.5 min) using MeOH- H2O (6238) as the mobile phase and a flow rate of 1.5 mLmin1. Fraction E gave 22.5 mg of pure glycoside 3 (tR = 19.7 min) using MeOH- H2O (5743) as the mobile phase and a flow rate of 1.5 mLmin1. Acid hydrolysis of the compounds 13 Each of the glycosides (1 mg) was heated with 2 molL1 trifluoroacetic acid (1 mL) at 120 for 2 h. The reaction mixture was evaporated to dryness, and the residue was partitioned between CH2Cl2 and H2O. The aqueous phase was concentrated under reduced pressure. Then pyridine (1 mL) and NH2OHHCl (2 mg) were added to the dried residue, and the mixture was heated at 90 for 30 min. Then, Ac2O (0.8 mL) was added, and heating was continued at 90 for 1 h. The solution was concentrated, and the resulting aldononitrile peracetates were analyzed by GC- MS using standard aldononitrile peracetates as reference samples. D- xylose, D- quinovose, D- glucose and D- 3- O- methylglucose were identified in a 1111 ratio for all three glycosides. Identification Compound 1 Colorless amorphous powder, mp 234236 , 20 D: 11.3 (c 0.4, MeOH); IR (KBr) max: 3 418, 1 760, 1 652, 1 228, 1 069 cm1; ESI (+)- MS (m/z): 1 227 M+Na+, ESI ()- MS (m/z): 1 181 MNa; HR- ESI (+)- MS (m/z): 1 227.484 9 M+Na+ 624 药学学报 Acta Pharmaceutica Sinica 2009, 44 (6): 620624 (calcd. for C54H85Na2O26S: 1 227.484 5). For 1H and 13C NMR data, see Table 1. Compound 2 Obtained as a colourless crystal. And it gave a positive reaction to Liebermann- Burchard and Molish tests. Mp 250252 , 20 D: 10.2 (c 0.34, MeOH); IR (KBr) max: 3 420, 1 763, 1 653, 1 260, 1 071 cm1; ESI (+)- MS (m/z): 1 229 M+Na+, ESI ()- MS (m/z): 1 183 MNa; HR- ESI (+)- MS (m/z): 1 229.500 4 M+Na+ (calcd. for C54H87Na2O26S: 1 229.500 2). It was deduced as echinoside A by comparison of the NMR spectra with those of echinoside A11. Compound 3 Obtained as a colourless crystal. And it gave a positive reaction to Liebermann- Burchard and Molish tests. Mp 236 238 , 20 D: 15.3 (c 0.31, MeOH); IR (KBr) max: 3 421, 1 770, 1 635, 1 266, 1 074 cm1; ESI (+)- MS (m/z): 1 245 M+Na+ (C54H87Na2O27S), ESI ()- MS (m/z): 1 199 MNa. It was deduced as holothurin A1 by comparison of the NMR spectra with