Armillaramide, a new sphingolipid from the fungus Armillaria mellea

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

Source and publish data:

Fitoterapia p. 858 - 864 (2001)

Update date:2022-08-04

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Authors:

Gao, Jin-Ming

Yang, Xue

Wang, Chen-Ying

Liu, Ji-Kai

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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

Fitoterapia 72 2001 858᎐864

Armillaramide, a new sphingolipid from the

fungus Armillaria mellea

Jin-Ming Gao^a,b, Xue Yang^a, Chen-Ying Wang^a,

Ji-Kai Liu^a,

^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

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-

Keywords: Armillaria mellea; Armillaramide; 2S,3S,4R -2-hexadecanoylamino-octadecane-1,3,4-triol;

Ceramides; Sphingolipids; Sterols

1. Introduction

Armillaria mellea Vahl. Ex Fr Quel. Tricholomataceae is a fungus symbiotic

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

Corresponding author. Tel.: q86-871-5216327; fax: q86-871-5150227.

E-mail address: jkliu@mail.kib.ac.cn J.K. Liu .

PII: S 0 3 6 7 - 3 2 6 X 0 1 0 0 3 1 9 - 7

(

)

J.-M. Gao et al. rFitoterapia 72 2001 858᎐864

859

w x

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-

w x

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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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

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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.

(

)

860

J.-M. Gao et al. rFitoterapia 72 2001 858᎐864

Table 1

NMR data for 1 in pyridine-d₅500 MHz for ¹H-NMR, 125 MHz for C-NMR

J in Hz

Position

C DEPT

¹H-¹H COSY

H-2

62.28 CH₂

4.57 dd, J 12.4, 3.8

4.40 dd, J 12.4, 4.0

Ž .

4.98 m

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

4.31 dd, J 4.8, 3.9

4.23 m

2.20 m

1.93 m

7᎐17

NHCO

1Ј

2Ј

3Ј

1.30 br s

0.90 t, J 5.6

8.13 d, J 6.7

H-2

Ž .

173.44 C

36.91 CH₂

26.44 CH₂

22.98᎐32.19 11 CH₂

2.43 t, J 6.0

Ž .

1.83 m

1.30 br s

0.90 t, J 5.6

H-3Ј

H-2Ј

4Ј᎐15Ј

16Ј

14.29 CH₃

2. Results and discussion

The molecular formula of compound 1 was determined as C₃₄H₆₉NO₄by 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

cm^y1, further indicating the presence of amide NH. The H-NMR spectrum of 1

Table 1 showed the presence of two terminal methyls at ␦ 0.90, methylenes at ␦

1.30 approx. 36H, br s , an amide proton signal at ␦ 8.13 d, J s 6.7 Hz . The

C-NMR spectrum of 1 Table 1 showed one quaternary carbon at ␦ 173.44

CONH , three methines at ␦ 53.79 CHNH , 76.86 CHOH and 73.18 CHOH ,

and one methylene at ␦ 62.28 CH₂OH . All of the above spectral data revealed

that 1 was a phytosphingosine-type sphingolipid 5,8 .

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

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non-hydroxy fatty acid. Methanolysis 8 of 1 yielded methyl palmitate 1b detected

by GCrMS. The existence of the palmitoyl moiety was also confirmed

by high resolution EI-MS at mrz 239.2372 calcd. 239.2374 for C₁₆H₃₁O

as well as significant fragment ions at mrz 239 CH₃CH_{2 14}CO ^q, 256

x^q

CH₃CH_{2 14}CONH₂q H , and 298 M-H₂O-CH₃CH_{2 14}CO 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

FAB-MS also displayed the intense peak at mrz 622 M q H-AcOH in addition

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 CH_{2 14}CH₃-3 = AcOH q H ^qformed

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

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

4R, since H-NMR spectral data of 1 1-Ha at ␦ 4.57 dd, J s 12.4, 3.8 Hz , 1-Hb

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

Hz , and 4-H at ␦ 4.23 m were in good agreement with those of natural and

synthetic ceramides, 2S,3S,4R -2- 2Ј-hydroxytetracosanoylamino hexadecane-

1,3,4-triol isolated from starfish Acanthaster planci 1-Ha at ␦ 4.52 dd, J s 10.7,

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,

.x w

J s 4.6, 6.6 Hz , and 4-H at ␦ 4.29 m 10,11 . Furthermore, the chemical shifts

and coupling constants of 1-H, 2-H, 3-H and 4-H for 1a 1-Ha at ␦ 4.29 dd,

Ž .

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 ␦

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

agreement with those of 2S, 3S, 4R -2-acetoamino-1, 3,4-triacetoxyheptadecane

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

␦ 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

_Dq 10.9

Hz 5 . In addition, the optical rotations of 1

␣

_Dq 14.4 and 1c

␣

were also in accordance with those reported for the known ceramide 2S,3S,4R -2-

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2Ј-hydroxytetracosanoylamino hexadecane-1,3,4-triol natural

_Dq 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.

␣

_Dq 11.5; syn-

w x

. w

thetic

␣

Comparison of the physicochemical properties with the reported data allowed to

identify the other new-isolated compound as ergosterol peroxide 5␣,8␣-epidioxy-

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22 E,24R -ergosta-6,22-dien-3␤-ol , previously isolated from R. ochroleuca 6 and

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R. cyanoxantha 8 and reported to possess anticomplementary 6 , antitumor 12 ,

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.

The compound with the same molecular formula C₃₄H₆₉NO₄has been previously

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

spectra ¹H-, C-NMR and H- H COSY were recorded on Bruker AM-400 and

( )

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

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

carrier gas, column temperature 160᎐240ЊC rate of temperature: 5ЊCrmin .

TLC was carried out on plates precoated with silica gel F₂₅₄Qingdao 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

The fresh fruiting bodies dry wt. 712 g of A. mellea were extracted four times

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

Si-gel CC with a gradient elution of n-hexane-Et₂O.

The fraction 4.61 g eluted with n-hexane᎐Et₂O 8:2, was passed through

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

w x

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

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

repeatedly crystallized from n-hexane᎐Et₂O to afford compound 1 26.8 mg .

Ž .

w x

Armillaramide 1 . White amorphous powder, mp 113᎐117ЊC; ␣

q14.4 c

0.68, pyridine ; IR bands KBr : 3376, 3220, 2919, 2851, 2487, 1615, 1557, 1469,

1380, 1078, 1050, 720, 639, 530 cm^y1; H- and C-NMR: see Table 1; EI-MS 70

x^qŽ .

1 537 4.5 , 328 74 , 311 35 , 299 19 , 298

Ž .

eV mrz: 555 M

3 , 553 M-2H

Ž .

60 , 281 65 , 280 26 , 256 80 , 257 29 , 239 13 , 211 2.5 , 111 12 , 97 31 , 83

x^q

42 , 69 49 , 60 100 ; positive FAB-MS mrz: 556 M q 1

100 , 538 M q 1-

x^qŽ .

Ž .

x^q

calcd. for

H₂O

5 , 318 2 , 282 2.5 , 256 4 ; HREIMS mrz; 555.5187 M

C₃₄H₆₉NO₄, 555.5226 .

3.4. Acetylation of 1

A solution of 1 3.9 mg in Ac₂O᎐pyridine 1:1 1 ml was left at room

(

)

J.-M. Gao et al. rFitoterapia 72 2001 858᎐864

863

temperature overnight, then diluted with 2 ml of water and extracted with EtOAc

3 = 5 ml . The extract was washed with brine, dried over Na₂SO₄, and evaporated

to dryness. The residue obtained was Si-gel CC petroleum ether᎐EtOAc 8:2 to

give 4.5 mg of 1a.

(

)

1,3,4-Triacetoxy- 2S,3S,4R -2-hexadecanoylamino-octadecane 1a . White solid; IR

bands KBr : 3331, 2922, 2846, 1737, 1679, 1529, 1468, 1440, 1372, 1272, 1246, 1224,

1103, 1048, 720, 660 cm^y1; H-NMR 400 MHz, CDCl₃: ␦ 5.96 1H, d, J 9.2 Hz,

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,

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

2H, t, J 7.4 Hz, 2Ј-H₂, 2.10 3H, s, OAc , 2.06 6H, s, 2 = OAc , 1.66 4H, m,

5-H₂and 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 .

x^q

Ž .

3.5. Methanolysis of 1 and acetylation of the long chain base

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

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

x^q

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

extract was Si-gel CC n-hexanerEtOAc 8:2 to furnish 1c.

w x

q10.9 c 0.67,

2-Acetoamino-1,3,4-triacetoxyoctadecane 1c . White solid, ␣

x^qŽ .

Ž .

Ž^D.

CHCl₃; EI-MS 70 eV mrz: 486 M q 1

1 , 426 2 , 366 9 , 305 24.5 , 245

0.5 ; H-NMR 400 MHz, CDCl₃: ␦ 5.97 1H, d, J 9.2 Hz, NH , 5.10 1H, dd, J

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,

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

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

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

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

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

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

Lourenco A, Lobo AM, Rodriguez B, Jimeno ML. Phytochemistry 1996;43:617.

10 Inagaki M, Isobe R, Kawano Y, Miyamoto T, Komori T, Higuchi R. Eur J Org Chem 1998:129.

11 Sugiyama S, Honda M, Higuchi R, Komori T. Liebigs Ann Chem 1991: 349.

12 Kahlos K, Kangas L, Hiltunen R. Planta Med 1989;55:389.

13 Cantrell C, Rajab MS, Franzblau SG, Fronczek FR, Fischer NH. Planta Med 1999;65:732.

14 Michel P, Bernadette L, Didier S. Eur Pat Appl EP 736 522 Chem Abstr 1996;125:308691.

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Article Doi

Armillaramide, a new sphingolipid from the fungus Armillaria mellea

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

Source and publish data:

Authors:

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

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

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