大炭角菌化学成分的研究

王兴娜 ，刘吉开，谭仁祥

1江苏省农科院农产品加工所，南京210014;2 中国科学院昆明植物研究所，昆明650204;3南京大学生命科学院，南京210093

Introduction

Xylaria euglossa Fr.is a rare rot-wood inhabiting ascomycete，mainly occurring on stumps and fallen branches of forested areas in the Southwest of China. It is used as medicine in some aereas［1］.There is no report on the chemical components of Xylaria euglossa. We carried out a detailed chemical investigation on the fungus and isolated 10 compounds.

Materials and methods

General

Column chromatography (CC):silica gel (200-300 mesh)and Sephadex LH-20. Melting points:Boetius melting point apparatus.Optical rotation:Horiba SEPA-300 digital polarimeter. CD spectrum:CDJ-801 instrument，λ (△ε)in nm. UV spectrum:Shimadzu UV-2401PC spectrometer，λmaxin nm. IR spectrum:Bruker Tensor-27 spectrumeter，KBr pellets，υ in cm-1.1H NMR and13C NMR spectra:Bruker AV-400 and Bruker AM-500 spectrometer，J in Hz. MS:VG Autospec-3000 spectrometer and API QSTAR Pulsar 1 spectrometer.

Plant Material

Fresh X. euglossa was collected at the Ailao mountain of Yunnan province，P. R. China，and identified by Prof. Mu Zang，Kunming Institute of Botany，Chinese Academy of Sciences. The fruiting bodies of the fungus were preserved after dry. The voucher specimen(No.912)was deposited in the Herbarium of Kunming Institute of Botany，Chinese Academy of Sciences.

Extraction and isolation

The air-dried fruiting bodies of X. euglossa (0.5 kg)were crushed and extracted with chloroform/methanol(1/1，V/V)at room temperature. The combined extracts (25 g)were partitioned between chloroform and water.The chloroform soluble part (17 g)was subjected to silica gel CC. Gradient elution with chloroform/methanol from 100/0-0/100 (V/V)yielded nine fractions.Compound 6 was got from fraction Ⅱ(chloroform /methanol，100/1)after deposition. Then compounds 3 and 7 were isolated on silica gel (petroleum ether/acetone，85/15)and Sephadex LH-20 (chloroform/methanol，1/1)CC. Fraction Ⅲ (chloroform/methanol，100/2)was passed through silica gel CC with petroleum ether/acetone，and compound 9 was obtained at 95/5，compound 2 and 5 were obtained at 85/15. Compound 10 was got from fraction Ⅴ(chloroform/methanol，100/8)after deposition and compound 8 was obtained by using Sephadex LH-20 (chloroform/methanol，1/1). Fraction Ⅵ (chloroform/methanol，100/15)was passed through Sephadex LH-20 (chloroform/methanol，1/1)and the part with strong yellow and blue-white fluorescence under UV light at 254 nm was collected and passed through silica gel CC with petroleum ether/acetone.Compound 1 (15 mg)was obtained at 85/15 and following compound 4 (8 mg)was obtained at 70/30.

Results

Structure elucidation of compound 1

The1H NMR of 1 contained signals for two aliphatic Cmethyl groups，four methylene groups，four methoxy groups，four isolated aromatic protons，and two strongly chelated phenolic hydroxy groups. The proton at C-5 appeared as singlet (δ = 6.95)rather than a doublet. In the HMBC spectrum of 1 the protons at C-4'correlated with three aromatic quaternary carbons (C-10’，C-4'a，C-9'a). From these evidences the 7-10'connectivity was established.

Fig.1 Structure and selected HMBC correlations of compound 1

The CD spectrum of 1 exhibited a negative Cotton effect at longer wavelength and a positive one at shorter wavelength (a so called A-type curve). CD:200(0.03)，203 (0)，213 (-6.07)，223 (0)，230(5.73)，240 (2.36)，250 (1.82)，257 (2.36)，268(6.07)，275 (0)，283 (-6.07)，290 (-1.68)，320(0)，333 (0.88)，350 (0). Since the sign of the Cotton effect of dimeric preanthraquinons is only determined by the chiral axis，the absolute axial configuration could be safely assigned to compound 1. According to the rules of Prelog and Helmchen，conmpound 1 possessed P-configuration.

According to Oertel rules［2］，the occurrence of the 4'-methylene protons as well-separated AB-quartet (△δ= 0.32 ≥0.08)indicated an anti-relationship between the OH-group at C-3' and the bulk of the second anthraquinone moiety，accordingly，the 3'S-configuration. And in the1H NMR spectrum the 4-protons appeared near (△δ ＜ 0.08)，so 3R configuration. The structure of compound 1 was determined as xylarinone［3］.

Structure elucidation of compound 4

Fig.2 Structure of compound 4

Compound 4 was obtained as a white powder. Its molecular formula was determined as C23H31NO6from the quasi-molecular ion peak at m/z 418.2211 (［M +H］+，calcd.418.2229)in the positive-ion HR-ESIMS. Twenty-three signals in the13C NMR (DEPT)spectrum of compound 4 were recognized (9C，2CH，11CH2，1CH3)，including three carbonyl C-atoms(δ204.0，δ172.4，and 173.5)and six aromatic quartery C-atoms(δ 102.5，120.8，125.9，128.2，155.0，and 164.8). To fulfill the molecular formula of compound 4，the presence of one benzene ring，three carbonyl carbons (-COOH，-CO-，-CONH-)and two phenolic hydroxyl groups (δ13.7and δ10.0)were suggested. In the mass spectrum of 4，a significant fragment ion at m/z=181 (C12H21O)was recognized. The signals in1H NMR spectrum at δ2.97(CH2)and 3.12(CH2) were assigned to one isolated -CH2-CH2-group. According to the molecular formula and DEPT spectrum，eight -CH2-and one -CH3exist，also the H-14(δ 2.20)，C-12 (δ 129.0)correlated with C-13 (δ 151.4).Signals at δ6.13 (J =16，H-12)and 7.02 (H-13)showed a trans double bond exist. The correlations from H-9(δ 2.97)、H-10(δ 3.12)、H-12(δ 6.13)、H-13(δ 7.02)to C-11(δ 204.0)were observed in the HMBC spectrum. All these data suggested that a long chain –CH2-CH2-COCH=CH-(CH2)8CH3exist.(6.81 in the1H NMR spectrum showed a characteristic amide NH.13C NMR (DEPT)spectrum showed the presence of an amide at δ173.5 (C-1)and δ44.6 (C-3). In addition，the correlation between NH (δH6.81)and C-3a (δC125.9)、C-7a (δC128.2)in HMBC spectrum showed that NH、C-1、C-3 connected to benzene ring through C-3a、C-7a. Correlations from 6-OH(δ13.7)to C-5，6，7，from 4-OH (δ10.0)to C-4，5，3a in HMBC spectrum showed the positions of the two hydroxyl groups. Correlation from H-9 to C-4，6 showed the position of the long chain CH3(CH2)8CH=CHCOCH2CH2-to the benzene ring. Correlation from H-3 (δ 4.48)to C-1，C-7a，C-4 showed the position of NH and C-1，C-3. Compound 4 was determined as xylactam［4］.

Table 1 the1H NMR，13 C NMR，HMBC spectra of compound 4

3，9-dihydroxy-3-methyl-6，8-dimethoxy-dihydroanthracenone［5］(2)1H NMR (400 MHz，CDCl3)δ:2.59 (1H，d，J = 17.3 Hz，H-2α)，2.63 (1H，d，J= 17.3 Hz，H-2β)，2.82 (1H，d，J = 15.8 Hz，H-4α)，2.86 (1H，d，J = 15.8 Hz，H-4β)，6.39 (1H，d，J = 2.0 Hz，H-5)，6.24 (1H，d，J = 2.0 Hz，H-7)，6.70 (1H，s，H-10)，1.17 (3H，s，3-CH3)，3.71(3H，s，6-OCH3)，3.76 (3H，s，8-OCH3);13C NMR(100 MHz，CDCl3)δ:202.6 (C-1)，48.9 (C-2)，69.7 (C-3)，43.1 (C-4)，98.8 (C-5)，161.9 (C-6)，97.7 (C-7)，160.8 (C-8)，164.7 (C-9)，117.0(C-10)，136.9 (C-4a)，109.4 (C-8a)，109.8 (C-9a)，141.8 (C-10a)，27.6 (3-CH3)，55.0 (6-OCH3)，55.6 (8-OCH3).

1-hydroxy-6，8-dimethoxy-2-methylanthraquinone［6］(3)1H NMR (500 MHz，CDCl3)δ:7.05(1H，s，H-2)，7.54 (1H，s，H-4)，7.44 (1H，d，J =2.4 Hz，H-5)，6.77 (1H，d，J = 2.4 Hz，H-7)，2.41(3H，s，3-CH3)，4.01 (3H，s，6-OCH3)，3.97 (3H，s，8-OCH3);13C NMR (100 MHz，CDCl3)δ:162.9(C-1)，124.8 (C-2)，146.9 (C-3)，120.0 (C-4)，103.9 (C-5)，162.6 (C-6)，104.7 (C-7)，165.2 (C-8)，187.4 (C-9)，183.0 (C-10)，146.9 (C-4a)，115.2 (C-8a)，114.7 (C-9a)，137.7 (C-10a)，21.9(C3-CH3)，56.6 (6-OCH3)，56.0 (8-OCH3).

Neoechinulin A［7］(5)1H NMR (500 MHz，CDCl3)δ:8.35 (1H，s，H-N1)，6.05 (1H，m，H-2b)，5.18(2H，dd，J = 15.6，7.4 Hz，H-2c)，1.51 (3H，s，H-2d)，7.18 (1H，s，H-3a)，7.46 (s，H-3d)，4.28 (q，H-3e)，6.72 (s，H-3g)，1.58 (1H，d，J = 2.0 Hz，H-3h)，7.25 (1H，d，J = 7.1 Hz，H-4);13C NMR (100 MHz，CDCl3):δ 143.7 (C-2)，39.2 (C-2a)，144.3(C-2b)，113.3 (C-2c)，27.3 (C-2d)，102.9 (C-3)，111.9 (C-3a)，124.5 (C-3b)，159.8 (C-3c)，51.6(C-3e)，165.7 (C-3f)，20.8 (C-3h)，118.8 (C-4)，121.0 (C-5)，122.3 (C-6)，111.2 (C-7)，126.0 (C-8)，134.3 (C-9).

5α，8α-epidioxy-ergosta-6，22-dien-3β-ol［8］(6)1H NMR (400 MHz，CDCl3):δ 3.92 (1H，m，H-3)，6.24(1H，d，J = 8.4 Hz，H-6)，6.46 (1H，d，J = 8.4 Hz，H-7)，5.25 (1H，dd，J = 15.3，7.4 Hz，H-22)，5.13 (1H，dd，J = 15.3，7.4 Hz，H-23);13C NMR(100 MHz，CDCl3):δ 34.8 (C-1)，30.2 (C-2)，66.4(C-3)，37.0 (C-4)，79.4 (C-5)，135.4 (C-6)，130.7 (C-7)，82.1 (C-8)，51.2 (C-9)，36.9 (C-10)，20.6 (C-11)，39.4 (C-12)，44.6 (C-13)，51.7(C-14)，28.6 (C-15)，23.4 (C-16)，56.3 (C-17)，12.97 (C-18)，18.1 (C-19)，39.6 (C-20)，20.9 (C-21)，135.2 (C-22)，132.4 (C-23)，42.8 (C-24)，33.1 (C-25)，19.6 (C-26)，19.9 (C-27)，17.54 (C-28).

Ergosta-4，6，8(14)，22-tetraen-3-one［9］(7)1H NMR (400 MHz，CDCl3)δ:6.57 (1H，d，J = 9.4 Hz，H-7)，5.99 (1H，d，J = 9.4 Hz，H-6)，5.71(1H，s，H-4)，5.25 (2H，m，H-22，23);13C NMR(100 MHz，CDCl3)δ:199.2 (C-3)，164.2 (C-5)，155.9 (C-14)，135.0 (C-22)，133.9 (C-7)，132.7(C-23)，124.6 (C-6)，124.5 (C-8)，123.1 (C-4)，55.9 (C-17)，44.5 (C-9)，44.1 (C-13)，42.9 (C-24)，39.2 (C-20)，36.8 (C-10)，35.7 (C-12)，34.2(C-1)，34.1 (C-2)，33.1 (C-25)，27.6 (C-16)，25.4 (C-15)，21.2 (C-21)，19.9 (C-27)，19.6 (C-26)，19.0 (C-11)，18.9 (C-18)，17.6 (C-28)，16.7(C-19).

(22E，24R)-ergosta-7，22-dien-3β，5α，6β-triol［10］(8)1H NMR (400 MHz，DMSO-d6)δ:5.03 (1H，m，H-6)，3.72 (1H，m，H-3)，5.07 (1H，brd，J =2.4 Hz，H-7)，0.53 (3H，s，H-18)，0.89 (3H，s，H-19)，0.97 (3H，d，J = 6.6 Hz，H-21)，5.15 (1H，dd，J = 15.3，7.9 Hz，H-22)，5.22 (1H，dd，J =15.3，7.0 Hz，H-23)，0.79 (3H，d，J = 6.6 Hz，H-26)，0.78 (3H，d，J = 6.6 Hz，H-27)，0.87 (3H，d，J = 6.6 Hz，H-28);13C NMR (100 MHz，DMSO-d6)δ:31.2 (C-1)，32.5 (C-2)，66.0 (C-3)，40.0 (C-4)，74.5 (C-5)，72.2 (C-6)，119.5 (C-7)，139.8(C-8)，42.3 (C-9)，36.7 (C-10)，21.4 (C-11)，39.0 (C-12)，43.0 (C-13)，54.2 (C-14)，22.6 (C-15)，37.8 (C-16)，55.4 (C-17)，12.1 (C-18)，17.6(C-19)，40.1 (C-20)，21.0 (C-21)，135.4 (C-22)，131.4 (C-23)，42.1 (C-24)，32.5 (C-25)，19.6 (C-26)，19.9 (C-27)，17.3 (C-28).

Palmitic acid［11］(9)1H NMR (400 MHz，CDCl3)δ:2.32 (2H，q，J = 7.4 Hz，H-2)，1.61 (2H，m，H-3)，1.24 (26H，m，H-4～15)，0.85 (3H，q，J = 6.3 Hz，H-16);13C NMR (100 MHz，CDCl3)δ:179.8 (C-1)，34.0 (C-2)，24.7 (C-3)，29.0-29.6 (C-4～13)，31.9 (C-14)，22.7 (C-15)，14.0 (C-16).

(2S，2'R，3S，4R)-2-(2'-hydroxy-stearamide)docosane-1，3，4-triol［10］(10)1H NMR (400 MHz，C5D5N)δ:4.60 (1H，dd，J = 10.5，4.5 Hz，H-1a)，4.38 (1H，dd，J = 10.5，4.5 Hz，H-1b)，5.01 (1H，m，H-2)，4.28 (1H，dd，J = 6.5，4.5 Hz，H-3)，4.24(1H，m，H-4)，1.92 (2H，m，H-5)，1.72 (2H，m，H-6)，1.30-1.50 (18H，m，H-7～15)，0.90 (3H，t，J =6.8 Hz，H-16)，4.57 (1H，m，H-2')，2.21 (2H，m，H-3')，1.76 (2H，m，H-4')，1.30～1.50 (36 H，m，H-5'-22')，0.90 (3H，t，J = 6.8 Hz，H-23')，8.56(s，-NH);13C NMR (100 MHz，C5D5N)δ:62.2 (C-1)，53.2 (C-2)，77.0 (C-3)，73.1 (C-4)，35.8 (C-5)，29.6 (C-6)，29.7-30.4 (C-7～21)，14.2 (C-22)，175.3 (C-1')，72.6 (C-2')，35.7 (C-3')，26.6(C-4')，30.0-30.4 (C-5'-17')，14.2 (C-18').

1 Mao XL.Macrofungi in China.Henan:Henan Sciences and Technology Press，2000.571.

2 Elsworth C，Gill M，Gimenez A，et al.Pigments of fungi，Part 50.Structure，biosynthesis and stereochemistry of new dimeric dihydroanthracenones of the phlegmacin type from Cortinarius sinapicolor Cleland.J Chem Soc Perkin Trans 1，1999，2:119-126.

3 Wang XN，Tan RX，Wang F，et al.The first isolation of a phlegmacin type pigment from the ascomycete Xylaria euglossa.Z Naturforsch，2005，60b:333-336.

4 Wang XN，Tan RX，Liu JK.Xylactam，a new nitrogen-containing compound from the fruiting bodies of ascomcete Xylaria euglossa.J Antibiot，2005，58:268-270.

5 Steglich W，Oertel B.Untersuchungen zur constitution und verbeitung der farbstoffe von Cortinurius，untergattung phlegmacium.Sydowia Annales Mycologici，1984，37:284-295.

6 Kesava RB，Hanumaiah T，Rao CP，et al.Anthraquinones in Ventilago species. Phytochemistry，1983，22:2583-2585.

7 Nagasawa H，Isogai A，Suzuki A，et al.13C NMR spectra and stereochemistry of isoechinulins A，B and C.Agric Biol Chem，1979，43:1759-1763.

8 Du JC，Wang XN，Tan RX，et al.Chemical constituents of basidiomycete Coltricium nitidum.Chin Tradit Herb Drugs，2006，37:1297-1300.

9 Kobayashi M，Krishna MM，Ishida K，et al.Marine sterols XXI，occurrence of 3-oxo-4，6，8(14)-triunsaturated steroids in the sponge Dysidea herbacea.Chem Pharm Bull，1992，40:72-74.

10 Wang XN，Du JC，Tan RX，et al.Chemical constituents of basidiomycete Hydum repandum.Chin Tradit Herb Drugs，2005，36:1126-1130.

11 Inagaki M，Isobe R，Kawano Y，et al.Isolation and structures of three new ceramides from the Starfish acanthasterplanci.Europ J Org Chem，1998，1:129-131.