全 文 ：Diphaladine A , 扁枝石松中一个新的石松生物碱?
吴兴德1 , 何 隽2 , 许 刚2 , 彭丽艳2 , 宋流东1 ** , 赵勤实2
??
(1 昆明医学院药学院 , 云南 昆明 650031; 2 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)
摘要 : 从扁枝石松 ( Diphasiastrumcomplanatum) 的 95%乙醇提取物中分离得到 1 个新的石松生物碱 , 经波
谱技术鉴定其结构 , 命名为 diphaladine A (1)。同时还分离得到其它 9 个已知石松生物碱类化合物 , 其中
obscurumine A ( 2 ) , L20 , lycoposerramine-K , des-N-methyl-β-obscurine, des-N-methyl-α-obscurine, lycoflexion, 和
phlegmariurine B 等 7 个化合物均为首次从该植物中分离得到。
关键词 : 扁枝石松 ; 石松生物碱 ; diphaladine A
中图分类号 : Q 946 文献标识码 : A 文章编号 : 0253 - 2700 (2009) 01 - 093 - 04
Diphaladine A , a New Lycopodium Alkaloid from
Diphasiastrum complanatum (Lycopodiaceae )
WU Xing-De
1
, HE Juan
2
, XU Gang
2
, PENG Li-Yan
2
,
SONG Liu-Dong
1 **
, ZHAO Qin-Shi
2 **
(1 Collegeof Pharmacology, Kunming Medical University, Kunming 650031 , China; 2 State Key Laboratory of Phytochemistry and
Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204 , China)
Abstract: A new Lycopodium alkaloid, diphaladine A (1 ) , was isolated from the 95 % EtOH extract of Diphasiastrum
complanatum together with other nine known alkaloids . Their structures were elucidated on the basis of extensive spectro-
scopic analysis . To the best of our knowledge, obscurumineA (2 ) , L20 , lycoposerramine-K , des-N-methyl-β-obscurine,
des-N-methyl-α-obscurine, lycoflexion, and phlegmariurine B were all isolated fromthis plant for the first time .
Key words: Diphasiastrumcomplanatum; Lycopodium alkaloids; diphaladine A
Lycopodiumalkaloids are a groupof natural prod-
ucts with unique heterocyclic ring systems, which have
attracted great interest from biogenetic ( Ayer and Tri-
fonov, 1994) , synthetic (Cassayer et al. , 2002; Wil-
liams et al. , 1994 ) , and biological (Liu et al. , 1986)
points of view . Among them, huperzine A wasreported
to be a potent acetylcholinesterase ( ACHE ) inhibitor
and have the ability to increase the efficiency of learn-
ing and memory in animals ( Liu et al. , 1986 ) .
DiphasiastrumcomplanatumHolubbelongs to the fami-
ly Lycopodiaceae and is widely distributed in Yunnan,
Sichuan, and Guizhou Provinces, and used as a tradi-
tional Chinese herbal medicine for the treatment of ar-
thritic pain, quadriplegia, and contusion ( Jiangsu In-
stitute of Botany, 1990 ) . In previous phytochemical
investigations, several Lycopodium alkaloids have been
reported fromthis plant ( Ishiuchi et al. , 2006; Kubota
et al. , 2007 ) . As a part of our systematic research
云 南 植 物 研 究 2009 , 31 (1) : 93～96
Acta Botanica Yunnanica DOI : 10 .3724?SP. J . 1143 .2009.08197
?
?? ?Author for correspondences; E-mail : qinshizhao@mail . kib. ac. cn, ynsld@126 . com; Tel: + 86 - 871 - 5223058
Received date: 2008 - 11 - 03 , Accepted date: 2008 - 11 - 25
作者简介 : 吴兴德 (1984 - ) 男 , 在读硕士研究生 , 主要从事植物化学成分研究。 ?
Foun ?dation items: Theproject ( P-06-04 ) of StateKeyLaboratory of Phytochemistry andPlant Resources in West China, Kunming Instituteof Bota-
ny, Chinese Academy of Sciences, and the 973 Program (No . 2009CB522300 )
works on ferns ( Li et al. , 2006; 2007) , the chemical
constituents of D. complanatumwas studied and anew
Lycopodiumalkaloid, as well as nine known alkaloids
wereobtained . In this paper, we reported the isolation
and structural elucidation of diphaladine A ( Fig . 1) .
Fig . 1 Structures of compounds 1 and 2
Results and Discussion
Compound 1 wasobtained as colorlessoil . Its mo-
lecular formula, C16 H25 NO4 , was deduced on thebasis
of HR-ESI-MS ( [M + H] + at m?z296 .1862; calcd .
296 .1861 ) . The IR absorptions at 1705 and 3175 -
3485 cm- 1 indicated the presence of ketone and
hydroxyl groups, respectively . In theEI-MS, the frag-
ment peak ion at m?z 279 ( [M-16 ] + ) , due to the
loss of one O-atom, suggested the existenceof aN-ox-
ide function . The
1
H and
13
C NMR spectra (Table 1)
of 1 showed 16 carbon resonances due to a ketone car-
bonyl (δC 209 .3) , two quaternary carbons, four me-
thines, eight methylenes, and one methyl group (δC
22 .3 , δH 0 .94 ) . Among them, two methylenes (δC
63 .0 , δH 3 .03 and 3 .61; δC 55 .7 , δH 3 .03 and
4 .41) andonequaternary carbon (δC 73 .5) canbeas-
cribable to bearing a nitrogen oxide function (Kashiwa-
ba et al. , 1998) , whereas one quaternary carbon (δC
72 .0) and one methine (δC 72 .2 , δH 4 .24 ) were as-
cribed as oxygen-bearing atoms . Considering the char-
acteristic NMR data discussed above, along with the
knowledge about the structural types of Lycopodiumal-
kaloids isolated from D. complanatum and related pla-
nts previously (Kubota et al. , 2007; Takayama et al. ,
2003) , compound 1 can be ascribed to be a lycopod-
ine-typeLycopodiumalkaloid .
Table 1 1 H and 13 C NMR data for compounds 1 and 2 in CDCl3
No
1 e
δH δC
2 ?
δH δC
1 iα 3 m. 03 ( overlapped) 63 ?. 0 (t) 2 ?. 92 (1H , dd, 4 . 8 , 13 .3) 64 .2 ( t)
1 kβ 3 m. 61 ( overlapped) 3 ?. 62 (1H , dt, 5 . 0 , 13 . 5)
2 iα 1 m. 91 ( 1H , m) 21 ?. 3 (t) 1 ?. 91 (1H , m) 22 .0 ( t)
2 kβ 1 m. 95 ( 1H , m) 1 ?. 98 (1H , m)
3 iα 2 m. 12 ( 1H , m) 17 ?. 4 (t) 2 ?. 12 (1H , br d, 14 v. 3 ) 18 .7 ( t)
3 kβ 1 m. 65 ( 1H , m) 1 ?. 69 (1H , m)
4 ?3 m. 87 ( 1H , dd, 2 u. 2 , 10 M. 0 ) 48 .5 (d) 3 ?. 30 (1H , m) 49 .0 ( d)
5 ?209 ?. 3 (s) 209 .2 ( s)
6 iα 3 m. 61 ( overlapped) 44 ?. 9 (t) 3 ?. 66 (1H , m) 44 .8 ( t)
6 kβ 2 m. 31 ( 1H , br d 13 ?. 6 ) 2 ?. 36 (1H , dd, 1 . 7 , 17 .1)
7 ?2 m. 23 ( 1H , m) 40 .3 (d) 2 ?. 77 (1H , m) 42 .5 ( d)
8 Lexo 2 m. 07 ( 1H , dt, 2 e. 8 , 10 =. 4 ) 36 ?. 8 (t) 2 ?. 01 (1H , m) 36 .4 ( t)
8 7endo 1 m. 27 ( 1H , br d, 10 ?. 4) 1 ?. 37 (1H , br d, 12 v. 4 )
9 iα 4 m. 41 ( 1H , dt, 2 e. 2 , 10 =. 2 ) 55 ?. 7 (t) 4 ?. 31 (1H , dt, 3 . 7 , 13 . 0) 60 .7 ( t)
9 kβ 3 m. 03 ( overlapped) 2 ?. 98 (1H , dd, 4 . 7 , 12 .8)
10 |α 3 m. 20 ( 1H , m) 26 ?. 4 (t) 2 ?. 79 (1H , m) 17 .4 ( t)
10 }β 1 m. 79 ( 1H , br d, 11 ?. 8) 1 ?. 81 (1H , br d, 6 Q. 7 )
11 |α 4 m. 24 ( 1H , s) 72 ?. 2 (d) 1 ?. 53 (1H , br d, 13 v. 5 ) 30 .3 ( t)
11 }β 2 ?. 48 (1H , dt, 4 . 6 , 13 . 7)
12 ?72 .0 (s) 74 .6 (s)
13 ?73 .5 (s) 73 .1 (s)
14 ^exo 2 m. 51 ( 1H , t, 10 e. 4 ) 30 ?. 1 (t) 2 ?. 52 (1H , t, 12 . 2) 30 .6 ( t)
14 Jendo 1 m. 98 ( 1H , m) 2 ?. 08 (1H , dd, 4 . 5 , 13 .4)
15 ?1 m. 56 ( 1H , m) 24 .6 (d) 1 ?. 49 (1H , m) 25 .9 ( d)
16 ?0 m. 94 ( 3H , d , 6 P. 2 ) 22 .3 (q) 0 ?. 97 (3H , d, 6 . 2) 22 .8 ( q)
49 云 南 植 物 研 究 31 卷
A careful comparisonof the1 H and 13 C NMR data
of 1 with those of obscurumine A ( 2) (Table 1) indi-
cated that the two compounds werevery similar to each
other except for the presence of an O-bearing methine
(δC 72 .2) in 1 insteadof themethylenegroup at C - 11
(δC 30 .3 ) in 2 . In addition, the downfield chemical
shift of C - 10 fromδC 17 .4 in 2 toδC 26 .4 in 1 , and
the upfield chemical shift of C - 9 fromδC 60 .7 in 2 to
δC 55 .7 in 1 , also indicated that the C - 11 of 1 was
substituted by a hydroxyl group, which can be con-
firmed by the HMBC correlations between H - 11 and C
- 7 , C - 9 , and C - 12 . ( Fig . 2 ) . Furthermore, the
HMBC correlations fromH - 1 to C - 9 andC - 13 , from
H - 3 , H - 7 , and H - 14 to C - 13 , from H - 3 , H - 6
to C - 5 , and from H - 14 to C - 4 ( Fig . 2) also con-
firmed the structure .
Fig . 2 Key HMBC and ROESY correlations of compound 1
The relative stereochemistry of 1 was elucidated
by ROESY experiment ( Fig . 2 ) . The ROESY cross-
peaksof H - 9αwith H - 2α and H - 4 suggested the
quinolizine partial structure was cis-oriented (Tan and
Zhu, 2004; Tong et al. , 2003; Ortega et al. ,
2004) ) . In addition, the ROESY correlations of H - 4
with H - 6αand H - 11 suggested the hydroxyl group at
C - 11 was β-oriented . Thus, the structure of com-
pound 1 was elucidated and named as diphaladine A .
The known alkaloids wereobscurumine A (Morita
et al. , 2005 ) , lycopodine (Yuan et al. , 1995 ) , L20
(Takayama et al. , 2003) , lycoposerramine-K ( Takay-
ama et al. , 2003 ) , lycodine ( Nakashima et al. ,
1975) , des-N-methyl-β-obscurine (Ayer and Kasitu,
1989) , des-N-methyl-α-obscurine (Ayer and Kasitu,
1989) , lycoflexion ( Takayama et al. , 2002 ) , and
phlegmariurineB ( Tan et al. , 2002 ) . The structures
were determined bycomparisonof thespectroscopic da-
ta with those reported in the literature .
Experimental
General experimental procedures Optical rotations were
recorded on a SEPA-300 polarimeter . IR spectra were deter-
mined on aBio-Rad FTS-135 spectrophotometer with KBr pelets .
The MS spectra were performed on a VG Autospec-3000 spec-
trometer . 1D NMR and2D NMR were recorded on aBruker AV-
400 or DRX-500 Spectrometer with TMS as internal standard .
Column chromatography was performed over silica gel ( 200 - 300
mesh, 10 - 40μm, Qingdao Marine Chemical Inc ., China) and
Sephadex LH-20 (AmershamBiosciences, Sweden) .
Plant Material The whole plant of D. complanatumwas
collected fromLvchuncountry, Yunnan Province, P . R .China,
in June2007 , andwas identified byProf . Cheng Xiao . A vouch-
er specimen ( No . 20070603 ) was deposited at the State Key
Laboratoryof Phytochemistry and Plant ResourcesinWest China,
Kunming Institute of Botany, Chinese Academy of Sciences .
Extraction and Isolation The dried and powdered plant
materials of D. complanatum (10 kg) were extracted three times
with 95% EtOH . After removal of solvent in a vacuum, the
crude extract was dissolved in H2 O to form a suspension, which
was adjusted with 5‰ HCl to pH 2 , and then extracted with
EtOAc . The aqueous phase was adjusted to pH 10 with NH3·
H2 O and extracted with CHCl3 to give crude alkaloids ( 4. 8 g) .
Thecrude alkaloidswere subjected to a silica gel column, using
petroleumether-acetone (from 1∶0 to 0∶1) as eluent, to obtain
four fractions ( Fr. 1-Fr. 4 ) . Fr. 2 was further separated using
Sephadex LH-20 eluted with CHCl3 -MeOH (1∶1 ) and repeated
column chromatographywith petroleumether-EtOAc-Et2 NH (50∶
1∶0 .01 ) to yield lycopodine ( 20 mg) and lycodine (2 . 8 mg) ;
Fr. 3 was subjectedto column chromatographyover silica gel with
CHCl3 -MeOH (from 100∶1 to 10∶1) , followed by purification
over Sephadex LH-20 eluted with CHCl3 -MeOH (1∶1) to obtain
L20 ( 50 mg) , lycoposerramine-K ( 5 . 5 mg) , des-N-methyl-α-
obscurine (30 mg) , and lycoflexion (70 mg) ; Fr. 4 was subject-
edto repeatedcolumnchromatographyover silicagel with CHCl3 -
MeOH (from30∶1 to 5∶1) and petroleum ether-acetone-Et2 NH
(10∶1∶0 .01) , followed by Sephadex LH-20 column chromatog-
raphy with MeOH to afford compound 1 (5 mg) , des-N-methyl-
β-obscurine (35 mg) , obscurumine A ( 3 mg) , and phlegmariu-
rine B (9 mg) .
Diphaladine A : colorless oil , C16 H25 NO4 ; [α]23.3D = +
11 .9 ( c = 0 .12 , CHCl3 ) ; IR (KBr) : 3421 , 3215 , 2956 ,
591 期 WU Xing-De et al. : Diphaladine A , a NewLycopodiumAlkaloid from Diphasiastrumcomplanatum . . .
2872 , 1705 , 1631 , 1456 , 1317 , 1057 , 987 , 753 cm- 1 ; 1 H
(400 MHz, CDCl3 ) and 13 C-NMR (100 MHZ , CDCl3 ) data see
table1; EI-MS m?z ( % ) : 295 [M] + (5 ) , 279 (20 ) , 262
(100) , 259 (24) , 242 (69) , 149 (44) , 148 (33) , 55 (24) ;
HR-ESI-MS: 296 .1862 [ M + H ] + ( calcd . for C16 H26 NO4 ,
296 .1861) .
Acknowledgements : The authors aregrateful to the members of
the Analytical Group in State Key Laboratory of Phytochemistry
and Plant Resources in West China, Kunming Institute of Bota-
ny, for the spectral measurements .
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