全 文 ：Referances:
[1 ]　 Jiangsu New Medical College. Dict ionary of Chinese Ma teria
Med ica (中药大辞典 ) [M ]. Sh angh ai: Shangh ai Science and
Tehchnology Publish er, 1995.
[2 ]　 Lian W Y. Inv es tigation of Chines e medicinal plants of M en-
ispermaceae [ J] . Acta Phytotaxon S in (植物分类学报 ) ,
1975, 13( 1): 32-52.
[3 ]　 Miller R W , Clard y J, Kozlowski J, et al . Ph ytoecdys teroids
of Diplocl isia glau cescens [ J] . Planta Med , 1985( 1): 40-42.
[4 ]　 Virbala C S, Adolf S D, Noel J S. Ch onemo rphine, st igmas-
terol and ecdysterone: s teroids is olated th rough bioass ay-di-
rected plan t screening prog rams [ J] . Steroids , 1989, 53( 3-
5): 559-565.
[5 ]　 Jayasingh e U L B, Wannigama G P, Macleod J K. Glucur-
onid es of Diplocl isia glaucescens [ J ]. J Chem Soc Pakistan,
1998, 20( 2): 131-137.
[6 ]　 Bandara B M R, Jayas ingh e L, Karunaratne V, et al . Diplo-
clisin, a bidesmosidic tri terpenoid sapinin f rom Dip locli sia
g laucescens [ J ]. Phytochemist ry, 1989, 28( 10): 2783-2785.
[7 ]　 Bandara B M R, Jayasingh e U L B, Karunaratne V, et al .
Trit erpenoidal cons ti tuents of Diplocl isia glau cescens [ J].
Planta Med , 1990, 56: 290-292.
[ 8 ]　 Bandara B M R, Jayasingh e L, Karunaratne V, et al . Ecd ys-
terone f rom s tem of Diploclisia glau cescens [ J] . Phytochem -
istr y, 1989, 28( 4): 1073-1075.
[9 ]　 M unoz O, Piovano M, Garbarino J, et al . Tropane alkaloids
f rom Schizanth us l itoral is [ J ]. Phytochemist ry, 1989, 43
( 3): 709-713.
[10 ]　 Imre S, O·· ztu nc A, U·· k timkin N B. Ziganein and ziganein-1-
meth ylath er: Zw eri neue anthrachinone aus Digitalis schis-
chkin ii [ J] . Phytochemistry , 1974, 13: 681-682.
[ 11 ]　 Imre S, U·· ktimkin N B. Zw eri neu e anthrachinone aus dee
w eu zeln von Dig ital is orientalis [ J ]. Phytoch emistr y, 1975,
14( 11): 2310-2311.
[ 12 ]　 Lu X Z, Hao W , Naoki H. Anth raquinones f rom Salvia
p rz ewalskii [ J] . Phytochemistr y, 1992, 31( 2): 708-709.
[ 13 ]　 Kazmi M H, Malik A, Hameed S, et al . Ananth rquinone
derivative f rom Casia l tal ica [ J] . Phytochemist ry, 1994, 36
( 3): 761-763.
Three coumarins from seed of Cnid ium monnieri and their
multidrug resistance reversal effects
ZHANG Qing-lin, ZHAO Jing-hua, BI Jian-jin, CAO Ju-rong , SON G Jing , WU Zu-ze
⒇
( Beijing Institute o f Radiation Medicine, Beijing 100850, China)
　　 Abstract: Object　 To iso late the activ e compounds on reversing multidrug resistance ( MDR) o f tu-
mo r cell f rom the ethanol ex t ract in the seeds of Cnidium monnieri ( L. ) Cuss. Methods　 The fractionation
directed by bioactivi ty w as ca rried out with si lica g el chromatog raphy and RP-HPLC. Results　 Three ac-
tiv e coumarins w ere obtained: imperatorin (Ⅰ ) , edultin (Ⅱ ) and 3′-isobuty ry lox y-O-acetyl columbionetin
(Ⅲ ) . Thei r sturctures were identi fied by spect roscopic analy sis. Conclusion　 These th ree compounds
have a medium reversing MDR of KBV200 in v itro.
Key words: Cnidium monnieri ( L. ) Cuss. ; couma rin; multidrug resistance ( MDR); KBV 200
蛇床子中 3种逆转肿瘤细胞多药耐药活性香豆素
张庆林 ,赵精华 ,毕建进 ,曹菊荣 ,宋　京 ,吴祖泽
(北京放射医学研究所 ,北京　 100850)
摘　要: 目的　从蛇床子 Cnidium monnier中分离逆转肿瘤细胞多药耐药活性成分。 方法　用生物活性跟踪法 ,经
硅胶柱层析、 RP-HPLC等。结果　分离得到 3种活性成分 ,分别为欧芹属素乙 ( impera to rin) ,爱得尔庭 ( edultin) , 9-
异丁酰氧基 -O-乙酰基哥伦比亚苷元 ( 3′-isobutyr ylox y-O-ace tyl co lumbionetin)。 结论　体外实验表明 3种化合物
对耐药的肿瘤细胞 KBV 200具有明显的逆转作用。
关键词: 蛇床子 ;香豆素 ;多药耐药 ; KBV200
中图分类号: R283. 3　　　文献标识码: A　　　文章编号: 0253 2670( 2003) 02 0104 03
1　 Introduction
One of the ma jo r problem of cancer chemo-
therapy is int rinsic or acquired mul tidrug resistance
( MDR). Many kinds of compounds, such as calci-
·104· 中草药　 Chinese T raditional and Herbal Drug s　第 34卷第 2期 2003年 2月
⒇收稿日期: 2002-10-12
um channel bolckers, calmodulin inhibi to rs and in-
dole alkaloids a re known to rev erse MDR, how ev-
er, they all have no t been recommended to routine
clinical use due to to xici ty
[ 1]
. Fo r example, Vera-
pamil, the most ex tensiv ely studies MDR reversing
agent , induces severe to xici ty at the doses re-
quired. Thus w e need to develop new classes of
MDR reversing agents wi th less toxici ty to the
host.
Many traditional Chinese drug s, alone or com-
bined wi th chemotherapic agents, a re used in clini-
cal cancer t rea tment. They a re proven to increase
cura tive effects and decrease the tox icities of the
chemotherapic agents. Some compounds isolated
f rom tradi tional Chinese herbs w ere reported to
have MDR reversing activi tes[ 2] . We believ ed w e
might find the new classes of MDR reversing a-
gents f rom tradi tional Chinese drugs. In this w ork
w e found the ethanolic ex t racts of Cnidium mon-
nieri ( L. ) Cuss. exhibi ted reversing MDR activ i-
ties. Activi ty-bioguided fractionation using chro-
ma tog raphy w as conducted and led to isolate th ree
activ e compounds.
2　Materials and methods
2. 1 　 General experimental pro cedures. The
1
HNM R and
13
CNMR spectra ( CDCl3 ) w ere ob-
tained wi th JNM-GX 400 o r INOV A-600 inst ru-
ments. FAB-M S was perfo rmed on a Zabspec spec-
trometer. Preparativ e HPLC was carried out wi th
a wa ter system equipped wi th a 600E pump, a 996
PDA detector at 254 nm and a Delta-Pak C18
prepacked radial compression co lumn ( 40 mm×
200 mm , 15μm) , elution wi th M eOH-H2O a t vari-
ous mix tures at f low ra te 15 m L /min.
2. 2　 Plant material. The seeds of Cnidium mon-
nieri ( L. ) Cuss. w ere collected in Jiang su
Province. Identi fica tion of specimens w as carried
out by Professo r Zhang Ming-qing f rom Nanjing
Univ ersi ty o f TCM.
2. 3　 Ex traction and activi ty-directed f ractiona-
tion. The dry seeds ( 5 kg ) w ere macera ted wi th
95% EtO H for three times. The EtO H ex tract wa s
concentra ted to dryness in vacuo and submitted to
liquid-liquid parti tio n wi th CHCl3-H2O. The CHCl3
phase w as par ti tio ned again w ith hexane /90%
M eOH. The active aqueous MeOH phase ( 29. 8 g )
w as mixed w ith silica g el ( 150 g ) and evapo rated
to dry in vacuo and w ashed wi th pet ro leum ether,
pet roleum eth er-CH2 Cl2 mix tures ( 100∶ 1 to 1∶
1) , CH2 Cl2 and MeOH. The bioassay results indi-
cated pet roleum ether f raction ( 12 g ) and
petroleum ether-CH2 Cl2 ( 50∶ 1) f raction ( 1. 5 g )
w ere activ e. The pet roleum ether f raction ( 6 g )
w as subjected to f lash chroma tog raphy on silica
gel, eluting wi th pet roleum ether-EtO Ac ( 10∶ 1
to 1∶ 1) . The activ e fractions combined and fur-
ther puri fied wi th preparativ e RP-HPLC to giv e
edultin Ⅱ , ( 28 mg ) and 3′-isobuty ryolx y-O-
acetylcolumbianetin Ⅲ , ( 31 mg ) , eluting w ith
CH3OH-H2O ( 55∶ 45 ) . The petroleum ether-
CH2 Cl2 ( 50∶ 1) f raction w as chroma tog raphed w ith
preparativ e C18 HPLC to yield impera to rin Ⅰ ( 35
mg ) , eluting w ith CH3OH-H2O ( 55∶ 45) .
2. 4　 Cell cul ture and bioassay. Resistant human
oral epidermoid carcinoma cell line, KBV200 w as
deriv ed f rom the parent sensi tiv e KBS cell Line by
stepw ise expo sure to vincristine ( V CR) , they w ere
kindly gi fted by Pro fessor Wang Yu-zi f rom Bei jing
Insti tute of Radia tion M edicine. KBV 200 cells
w ere maintained in the presence o f 1μg /mL VCR.
KBV 200 and KBS w ere cultured in PRM I-1640
supplement wi th 10% fetal calf serum and w ere
g row n at 37℃ in humidi fied atomsphere wi th 5%
CO2. M TT method w as used for cy to to xici ty as-
says
[3 ] . In 96 w ell plates 2× 104 cells w ere seeded
and treated wi th g raded concentrations o f the ex-
tracts or compounds ( disso lv ed in DM SO ) . The
pla tes w ere incubated fo r 72 hours a t 37℃ ( 100%
humidi ty wi th a 5% CO2 atmosphere in ai r) . M T T
w as added and the plates w ere incubated fo r four
hours, then 120μL DMSO were added to dissolv e
fo rmazam , the abso rbance was measured a t 570
nm, using a microplate reader. Each concentration
w as assayed in t riplicate.
2. 5　 Reversal facto r [4 ] . The ED50 values of V CR
alone and wi th reversal compounds against
KBV 200 w ere obtained and the reversal factor was
calcula ted as fo llowing: Reversal factor= ED50 of
·105·中草药　 Chinese T raditional and Herbal Drug s　第 34卷第 2期 2003年 2月
V CR alone /ED50 of V CR in the presence of a giv en
concentra tion o f rev ersal compound.
3　 Results and discussion
From screening the common Chinese drug fo r
reversing MDR of tumo r cells, w e found the EtO H
ex tract of C . monnieri show ed cytotoxicity o f the
MDR o f KBV200 cell line ( ED50 22. 5μg /mL) in
the presence o f V CR, while exhibited no signi fi-
cant cy to toxicity to KBV200 and the parental KBS
cells in the absence of V CR. Fractionation guided
by cytotoxici ty of KBV 200 cel ls in the presence of
V CR led to isola te three activ e coumarins, impera-
to rin (Ⅰ ) , edul tin (Ⅱ ) and 3′-isobutyrylo xy-O-
acety l columbionetin (Ⅲ ) . The st ructures o f the
three compounds w ere identi fied by comparison of
thei r phy sical and spectro scopic da ta
[5 ] .
To examine the MDR reversing activi ty o fⅠ -
Ⅲ , KBV 200 and KBS cells were t rea ted wi th g rad-
ed concentra tions o f V CR in the presence and ab-
sence of Ⅰ -Ⅲ . All three compounds exhibited
w eak cy to to xic activi ty against both KBV 200 and
KBS cells in the absence of V CR. KBV 200 cells
became more sensi tiv e to VCR in the presence of
Ⅰ -Ⅲ , ED50 values o f VCR decreased 1. 2-8. 2 fold
depending on the concentration ofⅠ -Ⅲ ( show n in
Table 1) . While KBS cells show ed no media ted cy-
to toxic response, but in the presence o fⅠ , KBS
cells show ed slight ly augmented cytotoxic re-
sponse, the reason is unknown. These results
clea rly show ed that Ⅰ -Ⅲ rev ersed the MDR of
KBV200. The reversal facto rs ofⅠ -Ⅲ a re show n
in Table 1.
Table 1　 Reversal factors of Ⅰ -Ⅲ against KBV200
Com pounds
/ (μg· mL- 1 )
ED50 / (μg· mL- 1 )
of VCR w ith compound
Reversal factor
Ⅰ 0　μg /mL 1. 82
2. 5μg /mL 1. 48 1. 23
5. 0μg /mL 1. 10 1. 65
10. 0μg /mL 0. 66 2. 78
Ⅱ 0　μg /mL 1. 20
2. 5μg /mL 0. 71 1. 69
5. 0μg /mL 0. 53 2. 27
10. 0μg /mL 0. 15 8. 27
Ⅲ 0　μg /mL 1. 20
2. 5μg /mL 0. 64 1. 89
5. 0μg /mL 0. 37 3. 22
10. 0μg /mL 0. 22 5. 45
　　 The seeds o f C. monnieri are used as a topical
agent fo r ec zenia and pruritus in China, some anti-
allergic principles w ere iso lated f rom this species.
We iso lated th ree coumarins with rev ersing MDR
activ ity, the interactions o f these compounds w ith
Pgp and reversal activ ities in v ivo will be tested.
Acknowledgement: The author s w ish to thank th e fi-
nancial suppo r t of Na tional Na tural Science Foundation of
China ( 39970892)
References:
[1 ]　 Volm M. Mu ltidrug resis tance and i ts reversal [ J ]. Antican cer
Res , 1998, 18: 2905-2918.
[ 2 ]　 Pan Q C, Tian H. Sev eral natural compounds f rom Chin ese
medicin e rev ers e mult idrug resi stance of tumor cell s [ J] . Chin
Sci Bul l (中国科学通报 ) , 1995, 40: 1901-1904.
[ 3 ]　 Carmichae J, Degraff W G, Gazdar A F, et al . Evaluation of a
tet rzolium -based semiautomated colo rimet ric ass ay: ass ess-
m en t of ch emosensi tivi ty tes ting [ J ]. Cancer Res , 1987, 47:
936-942.
[ 4 ]　 Norman B H. Inhibi tors of M RP1-m ediated m ultidrug resis-
tance [ J] . Drugs Future, 1998, 23( 9): 1001-1013.
[ 5 ]　 Kiyoshi H, Mi tsugi K, Kimiye B. Coumarins f rom Chin ese
crud e drug “ Sh ech uangzi” , the f rui t s of Cnidium sp. and
f rom Cnid ium japonium Miq [ J] . Yakugaku Zassh i , 1972, 92
( 10): 1289-1294.
(上接第 100页 )
需要多方关注、共同参与的目标和任务。
References:
[1 ]　 Zhu G G, Dong Z L. Th e analysis of“ Directive on Tradi tional
Medicines” ( d raf t ) and s tud y on dev eloping st rategy of TCM
in Europe [ J]. Foreign Med Sci- Tradi t Chin Med (国外医学
· 中医中药分册 ) , 2002, 24( 2): 67-72.
[2 ]　 Jia Q, Sun X X. Th e t radi tional market of TCM in Japan and
Korea [ J] . World Sci Tech- Modernizat ion Trad it Chin Med
(世界科学技术 -中药现代化 ) , 1999, 1( 3): 55-57.
[ 3]　 Zhao X L. TCM in Au st ralia [ J] . World Sci Tech- Modern-
iz ation Trad it Chin Med (世界科学技术 - 中药现代化 ) ,
2002, 2( 5): 39-42.
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