全 文 ：Isolation and structure determ ination of cypritibetquinone A and B,
two new phenanthraquinones from Cypripedium tibeticum
LIU Dong, JU Jian-hua, ZOU Zhong-jie, LIN G eng, YANG Jun-shan*
(Institute of Medicina l P lan tDevelopm ent, Chinese Academ y ofM ed ical S ciences and
Pek ing Un ionM edica l College, B eijing 100094 , Ch ina)
Abstract:A mi 　 To study the chem ical constituents o f Cypriped ium tibeticum. M ethods
Compoundsw e re isola ted by repeated silica gel chromatography and purified on Sephadex LH-20 and
struc tures w ere de te rm ined by spectra l analysis. Results　Cypritibe tquinones A and B w ere iso la ted from
the ethy l ace tate residue and the ir struc tures we re determ ined as 7-hydroxy-2-me thoxy-1, 4-
phenan thraqu inone (1) and 7-hydroxy-2, 10-dimethoxy-1, 4-phenanthraquinone (2), respective ly, by
ex tensive spectral analy ses. Conclusion　Cypritibe tqu inones A and B are tw o new phenanthraquinones.
Key words:Cypripedium tibeticum;cypritibetquinone A;cypritibe tquinone B;phenanthraquinones
CLC number:R284. 2　　　Document code:A　　　Article ID:0513 - 4870(2005)03 -0255 - 03
Received date:2004-06-28.
* Correspond ing au thor　Tel:86 -10 - 62899707,
Fax:86 - 10 -62898425,
E-m ail:junshanyang@ hotm ai.l com
西藏杓兰中两个新菲醌类化合物的分离与结构鉴定
刘　东 , 鞠建华 , 邹忠杰 , 林　耕 , 杨峻山*
(中国医学科学院 、中国协和医科大学 药用植物研究所 , 北京 100094)
摘要:目的　研究西藏杓兰的化学成分。方法　用硅胶柱色谱反复进行分离和 Sephadex LH-20纯化 , 并通过波
谱分析方法鉴定化合物结构。结果　从乙酸乙酯部位分离得到两个菲醌类化合物:西藏杓兰醌 A 和西藏杓兰醌 B。
鉴定其结构为:7-羟基-2-甲氧基-1, 4-菲醌(1);7-羟基-2, 10-二甲氧基-1, 4-菲醌(2)。结论　西藏杓兰醌 A 和西藏杓
兰醌 B均为新的菲醌类化合物。
关键词:西藏杓兰;西藏杓兰醌 A;西藏杓兰醌 B;菲醌
　 　 Cypripedium tibeticum K ing e t Ro lfe
(O rch idaceae) is a robust terrestrial he rb 13-15 cm
tall, w ith a sho rt creeping rhizome, found in
northw este rn Yunnan, western S ichuan prov ince s, and
southeaste rn T ibet Autonomous Region of the Peop le′s
Repub lic o f China. A s a Chine se fo lk herb medicine,
it po ssesses actions of d iu resis, de tumescence,
sedation, analgesia and can “ invigorate ” blood
circula tion. Ex tract o f the rhizomes is used inmedicine
for the trea tment of lumbago, edema in low e r limb ,
trauma tic in jury, gono rrhea, and leukorrhea [ 1] . No
investiga tion on the constituents has been reported
prev iously. In the course o f our chem ica l study on th is
medicinal plant, tw o new phenanthraquinones,
cypritibetquinones A (1) and B (2), were iso lated
from the ethano l ex tract o f the rhizomes ofC. tibeticum ,
the ir struc tures a re described herein.
Compound 1　Red amo rphous powder, has the
molecula r fo rmu la o f C15H10O4 , as deduced from its
HREI-MS , and requ ired eleven sites o f unsaturation.
It show ed UV abso rptions at 394, 298, and 228 nm
( logε3.53, 4.22, and 4.53) and IR abso rptions a t
3 300 (hydroxy l), 1 662 , 1 634 (che lated quinone
carbony l), 1 625 , 1 590, and 1 495 cm -1 (aromatic
ring), indicated that 1 was a hydroxy-1, 4-quinone
de rivative
[ 2 - 4]
.
The
1
H NMR spectrum o f 1 exhibited signals
arising from a me thoxy l g roup a tδ3.83(3H , s), a
pheno lic hydroxy l group atδ10.40(1H , br. s), an
255 药学学报 Ac ta Pharm aceu tica S in ica 2005, 40(3):255 - 257
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ABX-sy stem a roma tic protons atδ9.31(1H , d, J =9.5
H z), 7.31(1H , dd, J =9.5, 2.3 Hz) and 7.21(1H ,
d, J =2.3H z), an iso lated aroma tic pro ton atδ6.22
(1H , s), and a pair o fortho-coupled p ro tons atδ8.02
(1H , d, J =8.6 H z) and 7.91(1H , d, J =8.6 H z)
which are typ ica l of H-9 and H-10 of
phenanthraqu inone or phenanth rene de rivatives. O f the
th ree ABX-sy stem pro tons, one appea ring at a
re lative ly downfie ld position as an ortho-coup led
doub let at δ9.31 is typ ica l of H-5 as in o ther 1, 4-
phenanthraqu inones. Th is signal w as assigned to H-5
which coup led w ith H-6 appearing as an ortho-meta-
coupled double t atδ7.31. The chem ica l shift and the
splitting patterns o fH-6 further indica ted tha tH-8 w as
also unsubstitu ted, which , acco rding ly resonated as a
m eta-coupled doub le t atδ7.21. ThusH-6 andH-8 of
1 must be flanked by an oxygen substituent a t C-7.
Because the hydroxy l g roup a t δ10.40 w as not
chelated, it w as assumed to be located at C-7,
resu lting in theme thoxyl g roup atC-2 orC-3. Thisw as
supported by the appearance of a [M - 71] + peak at
m /z 183 and a [M - 99] + peak a tm /z 155 in the
E IM S spec trum o f 1, a common feature in such 2-
methoxy-1, 4-qu inones or 3-me thoxy-1, 4-quinones[ 2 -5] .
Locations o f the me thoxy l g roup a t C-2 and
hydroxy l group at C-7 w e re de te rm ined by careful
analyses of theHMQC andHMBC spectra, as show n in
Figure 1. Hence, the struc ture o f compound 1 was
elucidated as 7-hyd roxy-2-methoxy-1, 4-phenanthra-
quinone.
F igure 1　K ey HMBC corre lations of compound 1
Compound 2　Red amorphous powde r, has the
mo lecu lar fo rmu la o f C16H12O5 as determ ined by
HRE IM S , 30 mass units greater than that of 1,
co rresponding to ame thoxy l deriva tive o f 1. It showed
charac te ristic UV and IR abso rp tions very sim ilar to
those o f 1 , suggesting that 2 m igh t be ana logous
compound to 1.
The
1
H NMR spec trum of 2were closely sim ilar to
that of 1, which, revealed an ABX-type o f protons due
to H-5 , H-6 and H-8 , an iso lated benzene pro ton
assignable to H-3, a long w ith an aroma tic me thoxy l
g roup a t C-2 [ δ3.81 (3H , s)] and a phenolic
hydroxy l group at C-7 [ δ 10.35 (1H , br. s)] .
How ever, it show ed an aroma tic proton atδ7.58(1H ,
s) and aromatic me thoxy l g roup atδ3.92(3H , s) in
p lace of the tw o ortho-coupled 9-and 10-protons in 1,
sugge sting tha t2 has amethoxy l group substituted a tC-
9 or C-10.
Loca tions of the m ethoxy l and hydroxy l g roups in
2 we re de term ined by NOESY experimen ts. In the
NOESY spectrum of 2, sign ificant corre lations w ere
observed be tw een H-3 /2-OM e, H-5 /H-6 /7-OH , 7-
OH /H-8 /H-9, and H-9 /10-OM e. M ore convincing
evidence in suppo rt o f the structure 2 for
cypritibetquinone B w as provided by ca refu l analyses o f
its
13
C NMRw ithHMQC andHMBC spectra, as shown
in Figure 2. H ence, the structure of 2 was e lucidated
as 7-hydroxy-2, 10-dime thoxy-1, 4-phenanth raquinone.
A s far as w e cou ld exhaust from literature,
cypritibetquinone B (2) is the first examp le o f a ring-B
oxygena ted phenanquinone de rivative from a natu ra l
sou rce.
F igure 2　Key HMBC corre lations and NOESY
of compound 2
Expermi ental
Melting po in ts w ere de term ined on a Fishe r-Johns
appa ra tus and w ere unco rrec ted. UV spec tra w ere
measu red on a Ph ilips PYE Unican PU8800
spectrometer. IR spectra we re recorded on a Perkin-
E lmer 983G spec tromete r. NMR spectra w ere
measu red in py ridine-d5 on a B ruke r AM-500
spectrometer (500MH z for 1H NMR and 125MHz for
13
C NMR), using TM S as in ternal standard. An
Autospec-U ltim aETOF spectrome te rw as used to reco rd
the E I-MS and HRE I-MS. S i ge l 60H (400 - 500
mesh) from Q ingdao Ha iyang Chem ical G roup Co. ,
Q ingdao, Shandong Prov ince, People′s Republic o f
Ch ina, were used fo r co lumn chrom atog raphy.
The rh izomes o f C. tibeticum we re co llected a t
L ijiang, Yunnan province, People′s Repub lic o f
Ch ina, in N ovember 1999, and identified by Professor
CHEN X in-qi, Institute o f Bo tany, ChineseA cademy
256 药学学报 Ac ta Pharm aceu tica S in ica 2005, 40(3):255 - 257
Tab le 1　NMR Data for Compounds 1 and 2 (500MHz, DMSO-d6 , J in H z)
No.
1
δ1H δ13C
2
δ1H δ13C N o.
1
δ1H δ13C
2
δ1H δ13C
1 180. 2 179. 1 8 7.21 d(2.3) 109. 7 7. 12 d(2. 0) 108.4
2 158. 3 159. 5 8a 138. 9 140.6
3 6. 22 s 111. 0 6. 20 s 109. 2 9 8.02 d(8.6) 132. 2 7. 58 s 112.6
4 188. 3 188. 6 10 7.91 d(8.6) 121. 8 155.7
4 a 126. 7 130. 2 10a 128. 2 119.6
4b 123. 3 119. 3 7-OH 10.40 s 10. 35 s
5 9. 31 d(9. 5) 129. 6 9. 07 d(9. 5) 130. 2 2-OCH3 3.83 s 56. 3 3. 81 s 56.7
6 7. 31 dd(9. 5, 2.3) 122. 4 7. 10 dd(9. 5, 2. 0) 120. 6 10-OCH3 3. 92 s 56.4
7 157. 5 158. 3
of Sciences. A vouche r specimen (HB-99-1124) is
deposited at the Institu te of M ed icina l P lant
Development, Ch inese Academy o f M ed ica l Sciences
and Pek ing UnionM edicalC ollege.
Extraction and isolation　Dried p lant material
(3.4 kg)was extrac ted w ith e thano l under re flux three
times to a ffo rd E tOH extrac t (445 g). Suspended in
w a ter, it w as ex tracted w ith petro leum e ther, e thy l
acetate and n-bu tano l, separa te ly. The e thy l aceta te
residue ( 31.0 g ) was sub jected to co lumn
chromatography on silica ge l 60H (400 - 500 mesh)
w ith a CHC l3-MeOH (100∶0 - 1∶1) grad ient system
repeated ly and purified on Sephadex LH-20 w ith
CHC l3-MeOH (1∶1) to affo rd compounds 1 (14 mg)
and 2 (54mg).
Cypr itibetquinoneA (7-hydroxy-2-m ethoxy-1,
4-phenanthraquinone) ( 1) 　 Red powde r;mp
260 - 262℃; IR (KB r) cm -1: 3 300, 2 960,
1 662, 1 634, 1 625, 1 590 , 1 495, 1 474, 1 440,
1 365, 1 240, 1 200, 1 040;UV (M eOH) λmax 394,
298, 228 nm ( logε 3.53, 4.22, and 4.53);
1
H NMR and
13
C NMR (DMSO-d6) da ta, see Tab le
1;E IM Sm /z (%):254(M +, 100), 239(18), 225
(39), 197(25), 183(26), 168(38), 155(99),
142(29), 126(27);HREIMS m /z 254.053 3[M + ]
(calcd fo rC15H10O4 , 254.057 9).
Cypr itibetquinone B ( 7-hydroxy-2, 10-
dmi ethoxy-1, 4-phenanthraquinone) (2) 　 Red
powde r;mp 260 - 262 ℃;IR (KB r) cm -1:3 400,
2 960, 1 662, 1 635, 1 620, 1 500, 1 474, 1 420,
1 240, 1 200, 1 070, 860, 795;UV (MeOH) λm ax
420, 320, 292, 232 nm ( logε3.55, 3.62, 4.05,
4.43);1H NMR and 13C NMR (DMSO-d6) data, see
Tab le 1;E I-MS m /z (%):284 (M +, 100), 269
(49), 255(17), 238(11), 213(12), 185(42),
168(15), 155 (17), 142 (33);HRE I-MS m /z
284.067 7[M +] (ca lcd forC16H12O5 , 284.068 5).
References
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[ 3] Ta lapa tra B, M ukhopadhyay P, Chaudfury P, et a l.
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nobile L indl [ J] . Ind ian J Chem , 1982, 21B(4):386.
[ 4] Ghosh BB, Ray S, Bha ttacha ryya P, et a l. Cym binodin
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257
LIU Dong, et al:Iso lation and structure determ ina tion o f cypritibe tquinone A and B, tw o new
phenan thraquinone s from Cypriped ium tibeticum