Two new aloceramides from alocasia macrorrhiza

Journal of Chemistry, Vol. 43 (4), P. 513 - 516, 2005<br /> <br /> <br /> TWO NEW ALOCERAMIDES FROM ALOCASIA MACRORRHIZA<br /> Received 26th, July 2004<br /> NGUYEN QUYET TIEN1, PHAM HOANG NGOC1, PHAM HONG MINH1,<br /> PHAN VAN KIEM2 AND YOUNG HO KIM3<br /> 1<br /> Institute of Chemistry, Vietnamese Academy of Science and Technology<br /> 2<br /> Institute of Natural Products Chemistry, Vietnamese Academy of Science and Technology<br /> 3<br /> College of Pharmacy, Chungnam National University, Daejeon 305-764, Korea<br /> <br /> <br /> summary<br /> Two new aloceramides alomacrorrhiza A and alomacrorrhiza B were isolated from the<br /> ethanolic extract of the plant Alocasia macrorrhiza (L.) Schott. Their chemical structures were<br /> elucidated as (2S,3S,4R)-2N-[(2'R)-2'-hydroxy-hexacosanoyl]-tetradecane-1,3,4-triol (1) and<br /> (2S,3S,4R)-2N-[(2'R)-2'-hydroxy-hexacosanoyl]-hexadecane-1,3,4-triol (2) based on extensive<br /> 1D, 2D NMR, EI-MS, FAB-MS, HR-FAB-MS spectroscopic data and chemical degradation<br /> studies.<br /> Keywords: Araceae, Alocasia macrorrhiza, ceramide, alomacrorrhiza A, alomacrorrhiza B.<br /> <br /> I - INTRODUCTION polarimeter. EI-MS was obtained using a<br /> Hewlett Packard 5989 B MS spectrometer. FAB-<br /> Alocasia macrorrhiza (L.) Schott (Araceae) MS and HR-FAB-MS were obtained using a<br /> is widely distributed in Vietnam, and used as a JEOL JMS-DX 300 spectrometer. 1H-NMR<br /> folk medicine to treat inflammation, eczema and (500 MHz) and 13C-NMR (125 MHz) were<br /> abscess [2, 5]. Alocasin, an anti-fungal protein recorded on a Bruker AM500 FT-NMR<br /> and trypsin inhibitor has been isolated from the spectrometer and TMS was used as an internal<br /> giant taro A. macrorrhiza [1, 3, 7]. We report standard. Column chromatography (CC) was<br /> herein the isolation and structural elucidation of performed on silica gel (Kieselgel 60, 70 - 230<br /> two new ceramides, (2S,3S,4R)-2N-[(2'R)-2'- mesh and 230 - 400 mesh, Merck).<br /> hydroxy-hexacosanoyl]-tetradecane-1,3,4-triol Plant material<br /> (1) and (2S,3S,4R)-2N-[(2'R)-2'-hydroxy-hexa-<br /> co-sanoyl]-hexadecane-1,3,4-triol, (2) from the The roots of A. macrorrhiza were collected<br /> ethanolic extract of A. macrorrhiza. in Hoabinh province, Vietnam in December<br /> 1999 and identified by Prof. Nguyen Tien Ban,<br /> II - MATERIALS AND METHODS Institute of Ecology, Biological Resources,<br /> Vietnamese Academy of Science and<br /> General experimental procedures Technology (VAST). Voucher specimens are<br /> Melting points were determined using an deposited at the Institute Chemistry, VAST.<br /> Electrothermal IA-9200. IR spectrum was Extraction and isolation<br /> obtained on a Hitachi 270-30 type spectrometer<br /> with KBr discs. Optical rotations were The dried and powdered roots of A.<br /> determined on a JASCO DIP-1000 KUY macrorrhiza (2.0 kg) were extracted three times<br /> <br /> 513<br /> with hot EtOH repeatedly to give ethanolic NMR (500 MHz, CDCl3) : 4.18 (H, br s, H-2),<br /> extract (210.0 g), which was suspended in water 3.79 (3H, s, CH3-O), 0.89 (3H, t, 8.7); 13C-NMR<br /> and extracted using hexane, chloroform, ethyl (125 MHz, CDCl3) : 175.8 (C-1'), 70.4 (C-2'),<br /> acetate and n-butanol, respectively. The ethyl 52.4 (CH3-O), 34.4 (C-3'), 21.4 - 30.9 (C-4' to<br /> acetate extract (11.5 g) was subjected to C-13') and 14.1 (C-14').<br /> chromatography on a silica gel column, using<br /> chloroform-methanol (9 : 1) as eluent to yield Acetylation of 1<br /> six fractions (Fr. A-F). Fraction C (1.2 g) was Compound 1 (4 mg) was added to dry<br /> followed by CC on a YMC RP-8 column using a pyridine (0.25 ml) and Ac2O (0.5 ml) and left<br /> MeOH-H2O (10 : 1) as eluent to yield 1 (34.5 overnight. After usual workup, the reaction<br /> mg) and 2 (57 mg). mixture was chromatographed over silica gel<br /> (2S,3S,4R)-2N-[(2'R)-2'-hydroxy- [hexane-EtOAc (5 : 1)] to yield derivative 1a<br /> hexacosanoyl]-tetradecane-1,3,4-triol (1) (1.4 mg) as white crystals; mp 105 - 108oC;<br /> <br /> White amorphous powders; mp 112 - 114oC;<br /> [ ]25D +26.5o (c 0.1, MeOH); 1H-NMR (500<br /> [ ]25D +32.5o (c 1.00, MeOH); positive FAB-MS MHz, CDCl3) : 6.57 (d, J = 9.1 Hz, NH), 4.33 -<br /> 4.95 (m, 5H, carbinol protons), 2.18 (3H, s,<br /> m/z: 678.6 [M+Na]+; HR-FAB-MS m/z: OAc), 2.08 (3H, s, OAc), 2.05 (3H, s, OAc),<br /> 678.6013 [M+Na]+ (Calcd for C40H81NO5Na: 2.02 (3H, s, OAc) and 0.88 (6H, t, J = 8.7 Hz).<br /> 678.6012); 1H- and 13C-NMR (see table 1).<br /> Acetylation of 2<br /> (2S,3S,4R)-2N-[(2'R)-2'-hydroxy-<br /> hexacosanoyl]-hexadecane-1,3,4-triol (2) Compound 2 (4 mg) was acetylated as 1 to<br /> yield derivative 2a as white crystals; mp 108 -<br /> White amorphous powders; mp 107 - 109oC;<br /> 111 oC; [ ]D +31.0o (c 0.1, MeOH); 1H-NMR<br /> 25<br /> <br /> [ ]25D +42.5o (c 1.00, MeOH); positive FAB-MS (500 MHz, CDCl3) : 6.58 (d, J = 9.1 Hz, NH),<br /> m/z: 706.63 [M+Na]+; HR-FAB-MS m/z:<br /> 4.33-4.96 (m, 5H, carbinol protons), 2.19 (3H, s,<br /> 706.6321 [M+Na]+ (Calcd for C42H85NO5Na:<br /> OAc), 2.08 (3H, s, OAc), 2.06 (3H, s, OAc),<br /> 706.6325); 1H- and 13C-NMR (see table 1).<br /> 2.03 (3H, s, OAc) and 0.88 (6H, t, J = 8.7 Hz).<br /> Acid hydrolysis 1 and 2<br /> III - RESULTS AND DISCUSSION<br /> Each ceramide 1 (20 mg) and 2 (20 mg)<br /> were refluxed with 0.9 N HCl in 82% aqueous Repeated column chromatography on silica<br /> MeOH (15 ml) for 18 h. The resulting solutions gel and YMC RP-8 of the EtOAc extract of the<br /> were extracted with hexane, and combined dried and powdered roots of A. macrorrhiza<br /> organic phases were dried over Na2SO4. yielded two new ceramides 1 and 2. Compound<br /> Evaporation of the hexane yielded the same 1 and 2 formed as white amorphous powders.<br /> fatty acid methyl ester 3 as a white amorphous The IR spectrum of 1 and 2 exhibited hydroxyl<br /> powder. The H2O layers were neutralized with absorptions at 3434 cm-1 and amide functions at<br /> conc-NH4OH and extract with ether. The ether 1645 cm-1. The HR-FAB-MS of 1 provided the<br /> layers were dried over Na2SO4, filtered and then<br /> molecular formula C40H81NO5 (observed m/z:<br /> concentrated to yield the long chain bases.<br /> 678.6013 [M+Na]+; Calcd for C40H81NO5Na:<br /> 2-hydroxy-hexacosanoic acid methyl ester (3) 678.6012). The 1H- and 13C-NMR spectra of 1<br /> were typical of a ceramide processing a long<br /> A white amorphous powder; mp 60 - 62oC;<br /> chain base and 2-hydroxy fatty acid (table 1).<br /> [ ] -1.5o (c 0.5, CHCl3); EI-MS (70 eV) m/z<br /> 25<br /> D Assignments of all protons and carbons of 1<br /> (%): 426 [M]+ (9.3), 412 (22.7), 398 (61.0), 367 were made by 1H-1H COSY, HMQC and HMBC<br /> (8.0), 159 (15.4), 145 (23.9), 126 (13), 111 (13), spectra. The 1H-NMR of 1 showed a doublet at<br /> 97 (65.7), 83 (45.2), 57 (100) and 55 (82.3); 1H- 7.50 (d, J = 9.1 Hz) due to an NH proton, a<br /> <br /> 514<br /> broad singlet at 1.21 (methylene protons) and carbonyl group at C-1', three methine carbinol<br /> carbinol protons appearing between 3.40 and groups at C-2', C-3 and C-4, and methylene<br /> 3.86 suggesting it to be a ceramide. The 13C- carbinol at C-1. The stereochemistry of<br /> NMR spectrum of 1 showed carbonyl carbon ceramide 1 was determined as 2S, 3S, 4R, 2'R<br /> signals at 173.2 (s), carbinol carbons at 60.4 by comparison of the 1H, 13C-NMR data of 1<br /> (t), 71.0 (d), 71.0 (d) and 74.5 (d), methine with that of the other (2S,3S,4R,2'R)-<br /> carbon at 51.3 (d), methylene carbons at cerebrosides [6] and (2S,3S,4R,2'R)-phytos-<br /> phingosine moieties [4]. Compound 1 on<br /> 34.0-21.5 and two methyl carbons at 13.2 (q).<br /> In the 1H-1H COSY spectrum (table 1), the NH acetylation with Py-Ac2O (see experimental<br /> part) gave peracetyl derivative 1a that showed<br /> doublet at 7.50 showed a cross peak at 4.00<br /> attributed to the H-2 proton. The latter proton four acetyl groups at 2.18, 2.08, 2.05 and 2.02<br /> in the 1H-NMR. Furthermore, 1 was methan-<br /> showed coupling with two doublet doublet at<br /> olyzed with methanolic hydrochloric acid, 2-<br /> 3.72, 3.61 and a doublet doublet at 3.42,<br /> hydroxy-hexacosanoic acid methyl ester (3) was<br /> assigned to protons H-1 and H-3, respectively.<br /> obtained together with long-chain base (see<br /> The H-3 proton also showed coupling with the<br /> experimental part). Based on the above data, 1<br /> multiplet at 3.40 assigned to H-4.<br /> was elucidated as (2S,3S,4R)-2N-[(2'R)-2'-<br /> O hydroxy-hexacosanoyl]-tetradecane-1,3,4-triol,<br /> 1'<br /> 2'<br /> 3' 26' which we named alomacrorrhiza A.<br /> HN 20<br /> The molecular formula of 2 was deduced to<br /> OR be C42H85NO5 from the HR-FAB-MS (observed<br /> OR m/z: 706.6321 [M+Na]+; Calcd for<br /> 2 4<br /> C40H81NO5Na: 706.6325). The 1H- and 13C-<br /> RO 1 14<br /> n NMR spectra of 2 were very similar to those of<br /> 3<br /> 1 (table 1).<br /> Hb Ha OR<br /> Compound 1 was methanolyzed with<br /> 1 R = H, n = 7; 1a R = Ac, n = 7 methanolic hydrochloric acid, 2-hydroxy-<br /> 2 R = H, n = 9; 2a R = Ac, n = 9 hexacosanoic acid methyl ester (3) was obtained<br /> O together with the long-chain base. This<br /> confirmed that compounds 1 and 2 had the same<br /> 1' 3'<br /> H3C 2' N-acyl moiety. In addition, compound 2 on<br /> O (CH2)21 26' acetylation with Py-Ac2O gave peracetyl<br /> OH derivative 2a that showed four acetyl groups at<br /> 2.19, 2.08, 2.06 and 2.03 in the 1H-NMR. The<br /> 3<br /> stereochemistry of ceramide 2 was determined<br /> Figure 1. Structures of Compounds 1, 1a, 2, 2a<br /> as 2S, 3S, 4R, 2'R by comparison of the 1H-,<br /> and 3 13<br /> C-NMR data of 2 with that of 1 and the other<br /> The other carbinol proton appearing at (2S,3S,4R,2'R)-cerebrosides [6] and<br /> 3.86 showed only one cross peak to 2.00 m. (2S,3S,4R,2'R)-phytosphingosine moieties [4].<br /> The above 1H-1H correlation studies suggested These data led to the structure of 2 as<br /> the placement of three hydroxyl groups in long (2S,3S,4R)-2N-[(2'R)-2'-hydroxy-<br /> chain base and one hydroxyl group in N-acetyl hexacosanoyl]-hexadecane-1,3,4-triol, which we<br /> moiety. Moreover, the H-C long-range named alomacrorrhiza B.<br /> correlations between NH proton and carbon C-1'<br /> ( 173.2)/C-2' ( 71.0)/C-1 (60.4)/C-2 (51.3)/C- Acknowledgements: We are grateful to the<br /> 3 (74.5), and between proton H-2 ( 4.00) and KBSI for measuring the MS spectra and NMR<br /> Lab., Institute of Chemistry, VAST for NMR<br /> carbon C-3 ( 74.5)/C-4 (71.0) were observed in<br /> the HMBC. This confirmed the location of experiments. Thanks are due to Prof. Nguyen<br /> <br /> 515<br />  Table 1: 1H- and 13C-NMR spectral data for 1 and 2<br /> 1 2<br /> C 1 HMBC<br /> C<br /> a,b<br /> H<br /> a,c<br /> (J, Hz) H-1H COSY C<br /> a,b<br /> H<br /> a,c<br /> (J, Hz)<br /> (H to C)<br /> Long chain base<br /> - 7.50 d (9.1) H-2 C-1, 2, 3 - 7.50 d (9.1)<br /> NH<br /> 1', 2'<br /> 60.4 t 3.61 dd H-1b, 2 C-2, 3 60.6 t 3.61 dd<br /> 1a<br /> (6.1, 12.5) (6.1, 12.5)<br /> 3.72 dd H-1a, 2 C-2, 3 3.72 dd<br /> 1b<br /> (6.0, 12.8) (6.0, 12.8)<br /> 51.3 d 4.00 m H-1a, 1b, 3, C-1’, 3, 4 51.4 d 4.00 m<br /> 2<br /> NH<br /> 74.5 d 3.42 dd H-2, H-4 C-1, 2, 4 74.7 d 3.42 dd<br /> 3<br /> (3.2, 4.5) (3.2, 4.5)<br /> 4 71.0 d 3.40 m H-3, 5a, 5b C-2, 3 71.2 d 3.40 m<br /> 5a 31.8 t 1.55 m H-4, 6 31.9 t 1.55 m<br /> 5b 1.28 m H-4, 6 1.28 m<br /> 6 - 13 21.5 -30.8 t 1.26 br s 21.5-30.8 t 1.26 br s<br /> 14 13.2 q 0.89 t (8.7) H-13 13.2 q 0.89 t (8.7)<br /> N-acyl moiety<br /> 1' 173.2 s - 173.3 s -<br /> 71.0 d 3.86 dd H-3'a, 3'b C-1', 3' 71.0 d 3.86 dd<br /> 2'<br /> (8.0, 3.7) (8.0, 3.7)<br /> 3'a 34.0 t 2.00 m, H-2', 4' C-1', 2' 34.0 t 2.00 m<br /> 3'b 2.20 m H-2', 4' C-1', 2' 2.20 m<br /> 4'-25' 21.5 - 30.8 t 1.26 br s 21.5-30.8 t 1.26 br s<br /> 26' 13.2 q 0.89 t (8.7) H-25' 21.5-30.8 t 1.26 br s<br /> 27' 21.5-30.8 t 1.26 br s<br /> 28' 13.2 q 0.89 t (8.7)<br /> a<br /> In DMSO, b125 MHz, c500 MHz, Chemical shift ( ) in ppm. Assignments were assigned on the basis of<br /> DEPT, 1H-1H COSY, HMQC and HMBC spectra.<br /> <br /> Tien Ban, Institute of Ecology, Biological 4. S. S. Kang, J. S. Kim, K. H. Son, H. P. Kim<br /> Resources, VAST for the plant identi-fication. and H. W. Chang. Chem. Pharm. Bull., Vol.<br /> 49, P. 321 - 323 (2001).<br /> REFERENCES<br /> 5. D. T. Loi (ed.). Glossary of Vietnamese<br /> 1. J. H. Bradbury, B. C. Hammer. J. Agric. Medical Plants, Hanoi S&T Pub. (2001).<br /> Food Chem., Vol. 38, P. 1448 - 1453 6. J. Ryu, J. S. Kim and S. S. Kang.<br /> (1990). Cerebrosides from Longan Arillus. Ach.<br /> 2. V. V. Chi (ed.). Vietnamese Medical Plant Pharm. Res., Vol. 26, P. 138 - 142 (2003).<br /> Dictionary, Hanoi Medicine Pub. (1997). 7. H. X. Wang, T. B. Ng. Alocasin, an anti-<br /> 3. B. C. Hammer, D. C. Shaw and J. H. fungal protein from rhizomes of the giant<br /> Bradbury. Phytochemistry, Vol. 28, P. taro Alocasia macrorrhiza. Pro. Exp. & Pur.,<br /> 3019-3026 (1989). Vol. 28, P. 9 - 14 (2003).<br /> <br /> <br /> 516<br />