全 文 ：红汁乳菇中一个新的?类色素?
房立真1 , 2 , 杨婉秋1 , 2 , 董泽军1 , 邵红军1 , 2 , 刘吉开1
??
(1 中国科学院昆明植物研究所植物化学与西部植物资源持续利用国家重点实验室 , 云南 昆明 650204;
2 中国科学院研究生院 , 北京 100049 )
摘要 : 从担子菌红汁乳菇的子实体中分离得到一个新的绿色?类色素 , 其化学结构通过波谱学方法包括二
维核磁共振鉴定为 : 1 - [ (15 E ) -丁烯-17-酮 ]-4-甲基-7-异丙基?。同时还从该菌中分离得到其它三个红紫色
的?类色素。
关键词 : 红汁乳菇 ; ? ; 担子菌 ; 色素
中图分类号 : Q 946 文献标识码 : A 文章编号 : 0253 - 2700 (2007) 01 - 122 - 03
A New Azulene Pigment from the Fruiting Bodies of the
Basidiomycete Lactarius hatsudake (Russulaceae)
FANG Li-Zhen1 , 2 , YANG Wan-Qiu1 , 2 , DONG Ze-Jun1 , SHAO Hong-Jun1 , 2 , LIU Ji-Kai1 *
(1 State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Instituteof Botany, Chinese Academy
of Sciences, Kunming 650204 , China; 2 GraduateSchool of the ChineseAcademy of Sciences, Beijing 100049 , China)
Abstract: A new green azulene-type pigment, was isolated from the fruiting bodies of the basidiomycete Lactarius hat-
sudake together with three red-purple azulene pigments . The structure of the new compound 1 was determined as 1 -
[ (15 E )-buten-17-one ]-4-methyl-7-isopropylazulene (1 ) by spectral means including 2D-NMR .
Key words: Lactarius hatsudake; Azulene; Basidiomycete; Pigment .
Lactarius hatsudake belonging to the family Rus-
sulaceae of Basidiomycete is a common mushroom .
Similar to L . deliciosus, its latex of the fruiting bodies
is orange-colored, but then slowly ( within minutes)
darkens, and eventually turns blue-green if the fruiting
bodies are cut or broken ( Yuan and Sun, 1995 ) .
These colors in L . deliciosus have previously been
shown to bedue to guaianesesquiterpenes . Lactarovio-
lin, lactarazulene and lactarofulvene have been isolated
fromspecimens of L. deliciosus in different areas of the
world ( Heilbronner and Schmid, 1954; Vokac et al .
1970; Bertelli and Crabtree, 1968 ) , however there are
few reportsonpigments from L . hatsudake . Inour previ-
ous investigation of bioactive metabolites of the fruiting
bodies of L . hatsudake in Yunnan province of China,
three red-purple azulene pigments, 1-formyl-4-methyl-7-
isopropylazulene (2) , 1-formyl-4-methyl-7 - (1-hydroxy-1-
methylethyl ) azulene( 3 ) , 4-methyl-7-isopropyl-azulene-
1-carboxylic acid (4) were isolated (Fang et al . 2006) .
Recently in our re-investigation of pigments from L . h-
atsudake, another azulene-typegreenpigment has been
isolated . This paper deals with the isolation and struc-
tural elucidation of this pigment, 1 -[ ( 15 E )-buten-
17 -one]-4-methyl-7-isopropylazulene ( 1) .
云 南 植 物 研 究 2007 , 29 (1) : 122～124
Acta Botanica Yunnanica

?
?? ?Corresponding author ( phone: + 86 - 871 - 5216327; E-mail : jkliu@ mail . kib. ac. cn)
Received date: 2006 - 06 - 19 , Accepted date: 2006 - 09 - 18
作者简介 : 房立真 (1973 - ) 男 , 博士研究生 , 主要从事高等真菌化学研究和有机合成。 ?
Foundation item: This project was supported by theNational Natural Science Foundation of China (30225048 and 30470027 ) and the Natural Sci-
ence Foundation of Yunnan Province (2005C0052M and 2005C0011R)
Results and Discussion
Compound 1 was obtained as a green solid with a
molecular formula C18 H20 O determined from pseudo-
molecular ion at m?z 253 .1593 [ M + H ] + in HR-
TOF-MS . Its 13 C-NMR spectrum included signals four
methyls, eight methynes, and six quaternary carbons .
The 1 H-NMR spectrumof 1 exhibited amethyl group at
δ2 .87 (3H , s, Me - 14 ) , aisopropyl groupatδ3.27
(1H, m, J = 6 .9, H - 11) andδ1.39 (6H, d, J =
6.9, Me- 12, Me- 13) , five signalsof aromatic protons
atδ8.26 (1H, d, J = 4.2 , H - 2) , 7. 46 (1H, d, J
= 4.2, H - 3) , 7.38 (1H, d, J = 10 .8, H - 5) , 7.73
(1H, dd, J = 10 .8, 1.7 , H - 6) , and 8 .74 (1H, d,
J = 1 .7, H - 8) , respectively . It was characteristic for
thepresence of an azulene skeleton as well as displayed
anα, β-unsaturated keto moiety with a singlet for one
methyl atδ 2.34 (1H, s, H - 1) linked to a carbonyl
group and two doublets for two couplingprotons atδ8.30
(1H, d, J = 15.7, H - 15) andδ6 .80 (1H, d, J =
15.7, H - 16) . The E configuration for side chain dou-
ble bondof 1 was suggestedby its coupling constant ( J
= 15 .7 ) . The 13 C-NMR spectrum of 1 showed total
seventeen carbon signals, thirteen of which were in
good accordance with those of 2 , the known guaine
sesquiterpene except the signal differences atδ 135 .4
( d, C - 15) , δ123 .7 ( d, C - 16) , δ197 .4 (s, C
- 17) , δ27 .5 (q, C - 18) , which indicated the pr-
esence of anα, β-unsaturated keto instead of the alde-
hyde group of 1 . The all key correlations of HMBC
spectrumof 1 between H - 16 and C - 1 , H - 15 and C
- 9 , H - 15 and C - 2 , not only confirmed the assign-
ments, but also clearly suggested that theα, β-unsat-
urated group was attached to C - 1 position of azulene
moiety ( Fig . 2 ) . Combined with the evidences of
HSQC and COSY , compound 1 was elucidated as 1 -
[ ( 15 E )-buten-17-one] -4-methyl-7-isopropylazulene .
Comparison of the physicochemical properties with
reported data (Fang et al . 2006) allowed us to identify
the compounds2 , 3 and 4 , isolated fromthesamefun-
gus, as 1-formyl-4-methyl-7-isopropylazulene ( 2 ) , 1-
formyl-4-methyl-7 - (1-hydroxy-1-methylethyl ) azulene
(3 ) and 4-methyl-7-isopropylazulene-1-carboxylic acid
(4) , respectively .
It was worthwhile to note that the13 C-NMR signals
of C - 1 and C - 9 in guaine sespuiterpenes ( i . e . com-
pounds 2 , 3 , 4 , Fig . 1) werenot able to be assigned
directly because of no other correlations but the undis-
tinguished ones which all related to H - 2 , H - 3, H - 8
and H - 15 in their HMBC spectra . Signal existences of
1 H- and 13 C-NMR ofα, β-unsaturated moiety in 1 al-
lowedus to assign all the
1
H- and
13
C-NMR signals of
the azulene skeleton easily by the key correlations of
HMBC (see Fig . 1) , and the assignment errors in pre-
vious literature could be corrected ( Koul , 1985 ) .
Compound 1 was responsible for the blue-green color of
the fruiting bodies of L . hatsudake . Although it might
be a work-up product, but according to literature (Ber-
gendoff and Sterner, 1988) the formation and transfor-
mation of such compounds in Lactarius species were as-
sumed to be enzymatic as they never havebeenobserved
in vitro, and the compound 2 was notobserved to react
with acetone under our isolation conditions either .
Experimental Part
General CC: Column chromatography . Melting point
was obtained on an XRC-1 micro-meltingpoint apparatus and un-
Fig . 1 Structures of compounds 1 - 4 Fig . 2 The key HMBC correlations of compound 1
931 期 FANG Li-Zhen et al .: A New Azulene Pigment fromthe Fruiting Bodies of the Basidiomycete . . .
Table 1 1 H- and 13C-NMR Data (400 and 100 MHz, resp .) for 1 in CD3COCD3 , and 2 in CDCl3 (δ in ppm, J in Hz)
1 ?
No . H C
2
No . H C
1 ?124 .7 (s) 1 125 V. 4 (s)
2 ?8 .26 ( d, 4 . 4) 134 .4 ( d) 2 8 . 11 ( d, 4 . 2 ) 141 Q. 4 ( d)
3 ?7 .46 ( d, 4 . 4) 117 .4 ( d) 3 7 . 19 ( d, 4 . 2 ) 115 Q. 4 ( d)
4 ?147 .4 (s) 4 147 V. 7 (s)
5 ?7 .38 ( d, 10 L. 8 ) 130 .1 ( d) 5 7 . 43 ( d, 10 ?. 6 ) 131 Q. 2 ( d)
6 ?7 .73 ( dd, 10 q. 8 , 1 2. 7) 137 .6 ( d) 6 7 . 66 ( dd, 10 6. 6 , 1 . 8 ) 137 Q. 3 ( d)
7 ?145 .4 (s) 7 149 V. 5 (s)
8 ?8 .74 ( d, 1 . 7) 134 .2 ( d) 8 9 . 72 ( d, 1 . 8 ) 137 Q. 5 ( d)
9 ?139 .3 (s) 9 139 O. 5 (s)
10 ?142 .7 (s) 10 144 V. 1 (s)
11 ?3 .27 ( m, 6 7. 9 ) 39 z. 0 ( d) 11 3 . 19 ( m, 6 . 9) 38 . 4 ( d)
12 ?1 .39 ( d, 6 . 9) 24 z. 6 ( q) 12 1 . 38 ( d, 6 . 9 ) 24 . 4 ( q)
13 ?1 .39 ( d, 6 . 9) 24 z. 6 ( q) 13 1 . 38 ( d, 6 . 9 ) 24 . 4 ( q)
14 ?2 .87 ( s) 24 z. 3 ( q) 14 2 . 86 (s) 24 . 6 ( q)
15 ?8 .30 ( d, 15 L. 7 ) 135 .4 ( d) 15 10 .30 (s) 186 Q. 3 ( d)
16 ?6 .80 ( d, 15 L. 7 ) 123 .7 ( d)
17 ?197 .4 (s)
18 ?2 .34 ( s) 27 z. 5 ( q)
Assignments were based on HMBC , HSQC and 1 H-1 H COSY spectra .
corrected . TLC: visualization byheating silica-gel plates spayed
with 10% H2 SO4 in EtOH . IR Spectra: Perkin-Elmer 577
spectrometer; KBr pellets; in cm- 1 . 1 H- and 13 C-NMR Spec-
tra: DRX-500 spectrometers; δ in ppm, J in Hz . MS: VG
Autospec-3000 spectrometer; m?z ( rel . int .) . UV spectra:
Shimdzu UV-2411 PC spectrophotometer; λMeOHmax nm ( logε) .
Mushroom Material The fresh fruiting bodies of L . h-
atsudake were purchased at market in Wuding county of Yunnan
province, China, in August, 2005 and identified by Prof . ZANG
MU . Thevoucher specimen was deposited at the herbarium of the
Kunming Instituteof Botany, ChineseAcademy of Sciences .
Extraction and Isolation The fresh fruiting bodies of
L . hatsudake ( 5 kg) were extracted six timeswith acetone . The
combined extracts were evaporated to give deep brown syrup,
which was partitioned between H2 O and EtOAc . The organic
layer was concentrated in vacuo and the residue (43 g) was sub-
jected to repeated CC ( silica gel ) employing a gradient elution
with petroleumether andacetone . Thefraction (8 g) eluted with
petroleumether?acetone 100∶20 ( v?v) was further separated by
repeated CC (silicagel ; chloroform?petroleumether?acetone30∶
10∶1 (v?v?v) ) and purified through reversed phasechromatogra-
phy ( RP-8 , H2 O?MeOH 1∶4 v?v) to afford 1 ( 6 mg) , 2 ( 47
mg) , 3 (13 mg) and 4 (7 mg) .
Compound 1 , C18 H20 O, green solid; mp: 67 - 69℃ (ace-
tone) ; Rf = 0.72 ( petroleum ether?CHCl3?MeOH = 5∶15∶1 V?V?
V) ; UV?VISλMeOHmax nm ( logε) : 208 .5 (4 . 16) , 237 (4 .34) ,
308 (4 .20) , 321 (4 .21) , 418 (4.42) , 574 (2 .92) ; HR-TOF-
MS ( pos .) : m?z 253 .1593 ( [M + H ] + ; C18 H21 O, calcd .
253 .1592 ) . IR ( KBr ) : 2960 , 1728 , 1653 , 1629 , 1596 ,
1528 , 1498 , 1463 , 1259 , 1210 , 963 , 921 , 789 , 650 cm- 1 .
EI-MS m?z: 252 [M ] + ( 73 ) , 237 [M-Me] + ( 100 ) , 222
[M-C2 H6 ] + ( 10 ) , 209 ( 71 ) , 195 ( 48 ) , 179 ( 44 ) , 165
(53) , 152 (34) , 139 (9) , 119 (8) , 105 (12) , 91 (12 ) , 83
(13) , 71 (14) , 57 (31) ; 1 H- and 13 C-NMR: seeTable .
Compound 2 , C15 H16 O, red-purple solid; mp: 59 - 60℃
(acetone) ; Rf = 0 .70 ( petroleumether?CHCl3?MeOH = 5∶15∶1
V?V?V ) ; EI-MS m?z: 212 [ M ] + ( 90 ) , 197 [ M-Me] +
(92 ) , 183 [ M-C2 H5 ] + ( 7 ) , 169 ( 29 ) , 153 ( 31 ) , 141
(20 ) , 115 (16) , 97 ( 19 ) , 85 (59) , 71 ( 83) , 57 ( 100 ) ;
1
H- and
13
C-NMR: seeTable .
References:
Berg ?endorff O, Sterner O, 1988 . The sespuiterpenesof Lactarius delici-
osus and Lactarius deterrimus [ J ] . Phytochemistry, 27 : 97—100
Bert ?elli DJ , Crabtree JH , 1968 . Naturally occurring fluven hydrocar-
bons [ J ] . Tetrahedron, 24 : 2079—2089
Fang ?LZ, Shao HJ , Yang WQ et al . 2006 . Two new azulene pigments
from the fruiting bodies of Basidiomycete Lactarious hatsudake [ J ] .
Helv Chim Acta, 89 : 1463—1466
Heil ?bronner E , Schmid RW, 1954 . Zur kenntnis der sesquiterpeneund
azulene [ J ] . Helv ChimActa, 37 : 2018—2039
Koul ?SK , 1985 . A C-formylated azulene from Lactarious deterrimus
[ J ] . Phytochemistry, 24 : 181—182
Voka ?c K , Samek Z , Herout V et al . 1970 . The structureof two native
orange substances from Lactarius hatsudake [ J ] . Collect Czech
ChemCommun, 35 : 1296—1301
Yuan ?MS , Sun PQ, 1995 . Lactarius hatsudake [ B] . Sichuan Mush-
rooms ( 四川蕈菌 ) , 236
04 云 南 植 物 研 究 29 卷