Armillaramide, a new sphingolipid from the fungus Armillaria mellea

Article abstract of DOI:10.1016/S0367-326X(01)00319-7

A new C₁₈-phytosphingosine ceramide containing non-hydroxy fatty acid, armillaramide (1), has been isolated together with ergosterol peroxide from the fruiting bodies of the basidiomycete Armillaria mellea. Its structure was established as (2S,3S,4R)-2-hexadecanoylamino-octadecane-1,3,4-triol by spectroscopic and chemical methods.

Full text of DOI:10.1016/S0367-326X(01)00319-7

Ž
.
Fitoterapia 72 2001 858᎐864
Armillaramide, a new sphingolipid from the
fungus Armillaria mellea
Jin-Ming Gaoa,b, Xue Yanga, Chen-Ying Wanga,
U
Ji-Kai Liua,
aLaboratory of Phytochemisty, Kunming Institute of Botany, Chinese Academy of Sciences,
Kunming 650204, PR China
bCollege of Life Sciences, Northwest Science & Technology Uni¨ersity of Agriculture and Forestry,
Yangling, Shaanxi 712100, PR China
Received 19 April 2001; accepted in revised form 23 May 2001
Abstract
A new C18-phytosphingosine ceramide containing non-hydroxy fatty acid, armillaramide
Ž .
1 , has been isolated together with ergosterol peroxide from the fruiting bodies of the
Ž
.
basidiomycete Armillaria mellea. Its structure was established as 2S,3S,4R -2-hexadecanoyl-
amino-octadecane-1,3,4-triol by spectroscopic and chemical methods. ᮊ 2001 Elsevier Sci-
ence B.V. All rights reserved.
Ž
.
Keywords: Armillaria mellea; Armillaramide; 2S,3S,4R -2-hexadecanoylamino-octadecane-1,3,4-triol;
Ceramides; Sphingolipids; Sterols
1. Introduction
Ž
.
Ž
.
Armillaria mellea Vahl. Ex Fr Quel. Tricholomataceae is a fungus symbiotic
´
Ž
.
with the Chinese medicinal herb Gastrodia elata Blume Orchidaceae . A range of
biologically active sesquiterpenoid aryl esters with the protoilludane skeleton have
been isolated from the artificially-cultured mycelium of different strains of this
basidiomycete 1 . The crude drug containing extract of artificially cultured
mycelium of A. mellea in Chinese market is used for the treatment of geriatric
w x
patients with palsy, dizziness, headache, neurasthenia, insomnia, numbness in
U
Corresponding author. Tel.: q86-871-5216327; fax: q86-871-5150227.
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.
E-mail address: jkliu@mail.kib.ac.cn J.K. Liu .
0367-326Xr01r$ - see front matter ᮊ 2001 Elsevier Science B.V. All rights reserved.
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.
PII: S 0 3 6 7 - 3 2 6 X 0 1 0 0 3 1 9 - 7

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J.-M. Gao et al. rFitoterapia 72 2001 858᎐864
859
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limbs, and infantile convulsion 2 . This arouses our interest to know what kind of
secondary metabolites occurs in the fruiting bodies of this fungus.
Sphingolipids are found to be important building blocks of the plasma mem-
brane of eukaryotic cells. Their function is to anchor lipid-bound carbohydrates to
cell surfaces and to create an epidermal water permeability barrier, as well as to
participate in antigen᎐antibody reactions and transmission of biological informa-
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tion 3 . Recently, some sphingolipids have been reported to exhibit antihepatotoxic
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4 , antitumor and immunostimulatory activities 5 . Because of their promising
biological activities, they have aroused much increasing research interest.
In the course of our investigation on the sphingolipid composition of higher
fungi collected from Yunnan Province of China, two antifungal cerebrosides from
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w x
Russula ochroleuca 6 , a cerebroside from Polyporus ellisii 7 , and a ceramide from
R. cyanoxantha 8 have recently been reported. In continuation of our preceding
w x
studies on the basidiomycete-derived bioactive secondary metabolites, the chemical
constituents of A. mellea were investigated. A new ceramide 1 was obtained by
fractionation of the ethyl acetate extract of the fruiting bodies of this fungus. In
this paper, the isolation and structure elucidation of the new compound are
described.

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J.-M. Gao et al. rFitoterapia 72 2001 858᎐864
Table 1
13
NMR data for 1 in pyridine-d5 500 MHz for 1H-NMR, 125 MHz for C-NMR
Ž
.
13
1
Ž
.
Ž
.
J in Hz
Position
1
C DEPT
H
1H-1H COSY
H-2
Ž
.
Ž
Ž
.
.
62.28 CH2
4.57 dd, J 12.4, 3.8
4.40 dd, J 12.4, 4.0
Ž .
4.98 m
Ž
Ž
Ž
Ž
.
.
.
2
3
4
5
53.79 CH
76.86 CH
73.18 CH
34.09 CH2
26.68 CH2
22.98᎐32.19 11 CH2
14.29 CH3
H-1, H-3, NH
H-2, H-4
H-3, H-5
H-4
Ž
Ž
.
4.31 dd, J 4.8, 3.9
.
.
.
4.23 m
.
.
Ž
2.20 m
Ž
Ž
1.93 m
6
Ž
.
.
Ž
Ž
.
7᎐17
18
NHCO
1Ј
2Ј
3Ј
1.30 br s
0.90 t, J 5.6
8.13 d, J 6.7
Ž
.
.
Ž
.
H-2
Ž .
173.44 C
36.91 CH2
26.44 CH2
22.98᎐32.19 11 CH2
Ž
Ž
.
.
Ž
.
2.43 t, J 6.0
Ž .
1.83 m
1.30 br s
0.90 t, J 5.6
H-3Ј
H-2Ј
Ž
Ž
Ž
.
4Ј᎐15Ј
16Ј
Ž
.
.
14.29 CH3
2. Results and discussion
The molecular formula of compound 1 was determined as C34 H69 NO4 by high
resolution EI-MS. The IR spectrum exhibited the absorption bands of hydroxyls,
amide NH, and long aliphatic chains. The triacetyl derivative 1a, obtained from 1
by reaction with pyridine-Ac2O, still showed a sharp IR absorption band at 3331
1
cmy1, further indicating the presence of amide NH. The H-NMR spectrum of 1
Ž
.
Table 1 showed the presence of two terminal methyls at ␦ 0.90, methylenes at ␦
Ž
.
Ž
.
1.30 approx. 36H, br s , an amide proton signal at ␦ 8.13 d, J s 6.7 Hz . The
13
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.
C-NMR spectrum of 1 Table 1 showed one quaternary carbon at ␦ 173.44
Ž
.
Ž
.
Ž
.
Ž
.
CONH , three methines at ␦ 53.79 CHNH , 76.86 CHOH and 73.18 CHOH ,
Ž
.
and one methylene at ␦ 62.28 CH2OH . All of the above spectral data revealed
that 1 was a phytosphingosine-type sphingolipid 5,8 .
w
x
Ž
.
The presence of a triplet signal at ␦ 2.43 2H, J s 6.0 Hz due to the methylene
protons connected to amide carbonyl indicated that the N-acyl moiety in 1 was a
w x
Ž
.
non-hydroxy fatty acid. Methanolysis 8 of 1 yielded methyl palmitate 1b detected
by GCrMS. The existence of the palmitoyl moiety was also confirmed
Ž
.
by high resolution EI-MS at mrz 239.2372 calcd. 239.2374 for C16 H31O
as well as significant fragment ions at mrz 239 CH3 CH2 14CO q, 256
w
Ž
.
x
w
Ž
.
xq
w
Ž
.
xq
CH3 CH2 14CONH2 q H , and 298 M-H2O-CH3 CH2 14CO in the EI-MS.
Moreover, the positive mode FAB-MS of the peracetate 1a showed the molecular
ion at mrz 681 M in addition to the ion at mrz 682 M q H q. The positive ion
q
Ž
.
Ž
.
q
Ž
.
FAB-MS also displayed the intense peak at mrz 622 M q H-AcOH in addition
q
q
Ž
.
Ž
.
to the fragment ions at mrz 562 M q H-2 = AcOH , 502 M q H-3 = AcOH .
The prominent ion peak at mrz 264 M-CO CH2 14CH3-3 = AcOH q H q formed
Ž
Ž
.
.
Ž
.
by elimination of a fatty acyl group from the long-chain base moiety 681᎐417 ,
established that the molecular weight of the fatty acid is 256 and that the base is a

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J.-M. Gao et al. rFitoterapia 72 2001 858᎐864
861
C18-phytosphingosine containing three hydroxyls and an amino group. This conclu-
sion was in turn supported by the presence of the prominent ion peak at mrz 318
in the positive FAB-MS of 1 and confirmed by peracetylation of the methanolysis
product of 1 to afford a tetra-acetylphytosphingosine, i.e. 2-acetoamino-1,3,4-tri-
acetoxyoctadecane 1c , the 1H-NMR and EI-MS spectra of 1c was found to be
Ž
.
w
x
identical to that of the compound reported in literature 8,9 .
The proton assignments were further supported by analysis of 1H-1H COSY.
The relative stereochemistry of 1 at C-2, C-3 and C-4 was proposed as 2S, 3S and
1
w
Ž
.
4R, since H-NMR spectral data of 1 1-Ha at ␦ 4.57 dd, J s 12.4, 3.8 Hz , 1-Hb
Ž
.
Ž
.
Ž
at ␦ 4.40 dd, J s 12.4, 4.0 Hz , 2-H at ␦ 4.98 m , 3-H at ␦ 4.31 dd, J s 4.8, 3.9
.
Ž
.x
Hz , and 4-H at ␦ 4.23 m were in good agreement with those of natural and
Ž
.
Ž
.
synthetic ceramides, 2S,3S,4R -2- 2Ј-hydroxytetracosanoylamino hexadecane-
w
Ž
1,3,4-triol isolated from starfish Acanthaster planci 1-Ha at ␦ 4.52 dd, J s 10.7,
.
Ž
.
Ž
.
Ž
4.5 Hz , 1-Hb at ␦ 4.43 dd, J s 10.6, 5.0 Hz , 2-H at ␦ 5.12 m , 3-H at ␦ 4.36 dd,
.
Ž
.x w
x
J s 4.6, 6.6 Hz , and 4-H at ␦ 4.29 m 10,11 . Furthermore, the chemical shifts
w
Ž
and coupling constants of 1-H, 2-H, 3-H and 4-H for 1a 1-Ha at ␦ 4.29 dd,
.
Ž
.
Ž .
.x
J s 11.6, 4.6 Hz , 1-Hb at ␦ 4.01 dd, J s 11.6, 3.1 Hz , 2-H at ␦ 4.47 m , 3-H at ␦
Ž
.
Ž
5.12 dd, J s 8.7, 3.1 Hz and 4-H at ␦ 4.93 dt, J s 9.8, 3.1 Hz were in full
Ž
.
agreement with those of 2S, 3S, 4R -2-acetoamino-1, 3,4-triacetoxyheptadecane
w
Ž
.
Ž
.
1-Ha at ␦ 4.29 dd, J s 11.6, 4.3 Hz , 1-Hb at ␦ 4.00 dd, J s 11.6, 3.1 Hz , 2-H at
Ž
.
Ž
.
Ž
␦ 4.47 m , 3-H at ␦ 5.10 dd, J s 8.5, 3.1 Hz and 4-H at ␦ 4.93 dt, J s 9.8, 3.1
.x w x Žw x Žw x
.
.
D q 10.9
Hz 5 . In addition, the optical rotations of 1
␣
D q 14.4 and 1c
␣
Ž
.
were also in accordance with those reported for the known ceramide 2S,3S,4R -2-
Ž
.
Ž
w x
2Ј-hydroxytetracosanoylamino hexadecane-1,3,4-triol natural
D q 9.1 10,11 . These data suggested that 1 has the same absolute
configuration at asymmetric centers 2, 3 and 4. Accordingly, the above evidence
resulted in the establishment of the structure of 1 as 2S,3S,4R -2-hexadecanoyl-
amino-octadecane-1,3,4-triol.
␣
D q 11.5; syn-
w x
. w
x
thetic
␣
Ž
.
Comparison of the physicochemical properties with the reported data allowed to
w
identify the other new-isolated compound as ergosterol peroxide 5␣,8␣-epidioxy-
w x
Ž
.
x
22 E,24R -ergosta-6,22-dien-3␤-ol , previously isolated from R. ochroleuca 6 and
w x
w x
w x
R. cyanoxantha 8 and reported to possess anticomplementary 6 , antitumor 12 ,
w
x
and antimycobacterial 13 activities.
To the best of our knowledge, this is the first isolation of this type of ceramide
from higher fungi and a new natural product with the configuration of 2S,3S,4R.
Ž
.
The compound with the same molecular formula C34 H69 NO4 has been previously
w
x
obtained by synthesis, but no data of its stereochemistry have been reported 14 .
3. Experimental
3.1. General experimental procedures
Melting points were obtained on an XRC-1 apparatus and uncorrected. The
optical rotations were measured with a Horiba SEPA-300 polarimeter. NMR
13
1
1
spectra 1H-, C-NMR and H- H COSY were recorded on Bruker AM-400 and
Ž
.

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J.-M. Gao et al. rFitoterapia 72 2001 858᎐864
862
DRX-500 NMR instrument with TMS as an internal standard. MS spectra were
carried out with a VG Autospec-3000 mass spectrometer. IR spectra were obtained
in KBr pellets on a Bio-Rad FTS-135 infrared spectrophotometer. GC-MS was
performed on a Finnigan 4510 GC-MS spectrometer employing the electron impact
Ž
.
Ž
.
Ž
.
EI mode ionizing potential 70 eV and a capillary column 30 m = 0.25 mm
packed with 5% phenyl and 95% methylsilicone on HP-5. Helium was used as
Ž
.
carrier gas, column temperature 160᎐240ЊC rate of temperature: 5ЊCrmin .
Ž
TLC was carried out on plates precoated with silica gel F254 Qingdao Marine
.
Chemical Ltd., PR China and detection was achieved by spraying with 10% copper
sulfate in orthophosphoric acid solution followed by heating for 10᎐15 min.
3.2. Fungal material
The fresh fruiting bodies of A. mellea were collected from ZhongDian of
Yunnan Province, in August 1998 and identified by Profs P.G. Liu and X.H. Wang,
Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, PR China.
A voucher specimen has been deposited at the Herbarium of Kunming Institute of
Botany.
3.3. Extraction and isolation
Ž
.
The fresh fruiting bodies dry wt. 712 g of A. mellea were extracted four times
Ž
.
with EtOAc 4 = 1.5 l at room temperature. The combined EtOAc layer was
concentrated to dryness in vacuo to give a residue 30 g which was subjected to
Ž
.
Si-gel CC with a gradient elution of n-hexane-Et2O.
Ž
.
The fraction 4.61 g eluted with n-hexane᎐Et2O 8:2, was passed through
Ž
.
vacuum liquid chromatography VLC using petroleum ether᎐acetone 20:1᎐9:1,
and the fraction eluted with 15:1 gave ergosterol peroxide 125 mg as colorless
Ž
.
23
D
Ž
.
w x
Ž
.
needles n-hexane᎐AcOEt , mp 182᎐184ЊC, ␣
y34 c 0.6, CHCl3 ; IR, EI-MS,
1H and 13C-NMR spectra were in full agreement with those of an authentic sample
w
x
6,7 .
The eluate 1.27 g obtained from n-hexane᎐Et2O 6:4 was passed through VLC
with CHCl3᎐MeOH 15:1᎐2:1 as eluents to provide eight fractions. Fraction 3 was
Ž
.
Ž
.
repeatedly crystallized from n-hexane᎐Et2O to afford compound 1 26.8 mg .
26
D
Ž .
w x
Ž
Armillaramide 1 . White amorphous powder, mp 113᎐117ЊC; ␣
q14.4 c
.
Ž
.
0.68, pyridine ; IR bands KBr : 3376, 3220, 2919, 2851, 2487, 1615, 1557, 1469,
1380, 1078, 1050, 720, 639, 530 cmy1; H- and C-NMR: see Table 1; EI-MS 70
1
13
Ž
.
w
xq Ž .
w
xq Ž .
1 537 4.5 , 328 74 , 311 35 , 299 19 , 298
Ž
.
Ž
.
Ž
.
Ž .
eV mrz: 555 M
3 , 553 M-2H
Ž
Ž
.
Ž
.
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž .
60 , 281 65 , 280 26 , 256 80 , 257 29 , 239 13 , 211 2.5 , 111 12 , 97 31 , 83
.
Ž
Ž
.
w
xq
Ž
.
w
42 , 69 49 , 60 100 ; positive FAB-MS mrz: 556 M q 1
100 , 538 M q 1-
xq Ž .
Ž .
Ž
.
Ž .
w
xq
Ž
calcd. for
H2O
5 , 318 2 , 282 2.5 , 256 4 ; HREIMS mrz; 555.5187 M
.
C34 H69 NO4, 555.5226 .
3.4. Acetylation of 1
Ž
.
Ž
.
A solution of 1 3.9 mg in Ac2O᎐pyridine 1:1 1 ml was left at room

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J.-M. Gao et al. rFitoterapia 72 2001 858᎐864
863
temperature overnight, then diluted with 2 ml of water and extracted with EtOAc
Ž
.
3 = 5 ml . The extract was washed with brine, dried over Na2 SO4, and evaporated
Ž
.
to dryness. The residue obtained was Si-gel CC petroleum ether᎐EtOAc 8:2 to
give 4.5 mg of 1a.
(
)
Ž
.
1,3,4-Triacetoxy- 2S,3S,4R -2-hexadecanoylamino-octadecane 1a . White solid; IR
Ž
.
bands KBr : 3331, 2922, 2846, 1737, 1679, 1529, 1468, 1440, 1372, 1272, 1246, 1224,
1103, 1048, 720, 660 cmy1; H-NMR 400 MHz, CDCl3 : ␦ 5.96 1H, d, J 9.2 Hz,
1
Ž
.
.
Ž
.
.
Ž
Ž
.
Ž
NH , 5.10 1H, dd, J 8.7, 3.1 Hz, 3-H , 4.93 1H, dt, J 9.8, 3.1 Hz, 4-H , 4.47 m,
Ž
.
Ž
.
2-H , 4.29 1H, dd, J 11.6, 4.6 Hz, 1-Ha , 4.01 1H, dd, J 11.6, 3.1 Hz, 1-Hb , 2.22
Ž
.
Ž
.
Ž
.
Ž
2H, t, J 7.4 Hz, 2Ј-H2 , 2.10 3H, s, OAc , 2.06 6H, s, 2 = OAc , 1.66 4H, m,
.
Ž
.
Ž
.
5-H2 and 6-H2 , 1.12᎐1.27 n = CH2 , br s , 0.90 6H, t, 6.6 Hz, 2 = CH3 ; positive
FAB-MS mrz: 682 M q 1 100 , 622 47 , 562 3 , 502 0.2 , 264 18 .
w
xq
Ž
.
Ž
.
Ž .
Ž
.
Ž .
3.5. Methanolysis of 1 and acetylation of the long chain base
Ž
.
Compound 1 10 mg was refluxed with 1.5 ml of 0.9 N HCl in 82% aq. MeOH
for 16 h. The reaction mixture was extracted with n-hexane. The n-hexane layer
Ž
.
was concentrated and Si-gel CC n-hexanerEtOAc 9:1᎐7:3 to give a fatty acid
methyl ester 1b, which was subjected to GC-MS. The analysis showed only a single
peak assigned to methyl hexadecanoate, major ion peaks at mrz 270 M and 211
M-COOMe . The aqueous layer was neutralized with sat. Na2CO3, concentrated
w
xq
w
xq
to dryness, and then heated with Ac2O᎐pyridine 1:1 for 1.5 h at 70ЊC. The reaction
mixture was diluted with water and extracted with EtOAc. The residue of the
Ž
.
extract was Si-gel CC n-hexanerEtOAc 8:2 to furnish 1c.
27
Ž
.
w x
Ž
q10.9 c 0.67,
2-Acetoamino-1,3,4-triacetoxyoctadecane 1c . White solid, ␣
.
Ž
.
w
xq Ž .
Ž .
ŽD.
Ž
.
CHCl3 ; EI-MS 70 eV mrz: 486 M q 1
1 , 426 2 , 366 9 , 305 24.5 , 245
1
Ž
.
Ž
.
.
Ž
.
Ž
0.5 ; H-NMR 400 MHz, CDCl3 : ␦ 5.97 1H, d, J 9.2 Hz, NH , 5.10 1H, dd, J
Ž
.
Ž
.
Ž
8.5, 3.1 Hz, 3-H , 4.93 1H, dt, J 9.8, 3.1 Hz, 4-H , 4.47 1H, m, 2-H , 4.29 1H, dd,
.
Ž
.
Ž
.
J 11.6, 4.3 Hz, 1-Ha , 4.00 1H, dd, J 11.6, 3.1 Hz, 1-Hb , 2.08 3H, s, 3-OAc , 2.05
Ž
.
Ž
.
Ž
.
Ž
6H, s, 1-OAc, 4-OAc , 2.03 3H, s, HNAc , 1.12᎐1.70 26H, m , 0.88 3H, t, J 6.1
.
Hz, CH3 .
Acknowledgements
We wish to acknowledge financial support from the Natural Science Foundation
Ž
.
of Yunnan Province 2000B0066M . We are grateful to Ms X.H. Wang, Profs P.G.
Liu and D.G. Ji for their help in this project.
References
w x
1
Donnellly DMX, Konishi T, Dunne O, Cremin P. Phytochemistry 1997;44:1473.
Yang JS, Chen YW, Feng XZ et al. Planta Med 1989;55:564.
Kolter T, Sandhoff K. Angew Chem Int Ed Engl 1999;38:1532.
w x
2
w x
3
w x
4
Kim SY, Choi YH, Huh H, Kim JW, Kim YC, Lee HS. J Nat Prod 1997;60:274.

(
)
864
w x
J.-M. Gao et al. rFitoterapia 72 2001 858᎐864
5
Natori T, Morita M, Akimoto K, Koezuka Y. Tetrahedron 1994;50:2771.
Gao JM, Dong ZJ, Yang X, Liu JK. Acta Bot Yunnan 2001;23:86.
Gao JM, Hu L, Dong ZJ, Liu JK. Lipids 2001;36: in press .
Gao JM, Dong ZJ, Liu JK. Lipids 2001;36:175.
w x
6
w x
Ž
.
7
w x
8
w x
9
Lourenco A, Lobo AM, Rodriguez B, Jimeno ML. Phytochemistry 1996;43:617.
w
w
w
w
w
x
10 Inagaki M, Isobe R, Kawano Y, Miyamoto T, Komori T, Higuchi R. Eur J Org Chem 1998:129.
x
11 Sugiyama S, Honda M, Higuchi R, Komori T. Liebigs Ann Chem 1991: 349.
x
12 Kahlos K, Kangas L, Hiltunen R. Planta Med 1989;55:389.
x
13 Cantrell C, Rajab MS, Franzblau SG, Fronczek FR, Fischer NH. Planta Med 1999;65:732.
x
Ž
.
14 Michel P, Bernadette L, Didier S. Eur Pat Appl EP 736 522 Chem Abstr 1996;125:308691.