全 文 ：服制剂的抗心肌缺血作用, 正常状况下, 大鼠在 iv
Pit 后, 心电图有明显的变化, 实验中对照组在 iv
Pit 后心电图发生明显的变化,表现为 T 波升高, P-
R, Q -T 间期延长, 而葛根素口服制剂在给药 6 d
后,大鼠 iv Pit 后 T 波变化不明显, 证明葛根素口
服制剂有抗心肌缺血作用。通过观察葛根素口服制
剂在缺氧条件下对原代培养心肌细胞的作用显示,
缺氧条件下心肌细胞形态发生改变, 胞浆空泡形成,
胞浆颗粒形成,异形心肌细胞数目增加,同时心肌细
胞分泌 CK, LDH, A-HBDH 明显增多,给葛根素口
服制剂后心肌细胞形态未发生明显改变, CK, LDH ,
A-HBDH 未见明显增多,说明该药具有提高大鼠心
肌细胞抗缺氧能力,对心肌细胞有明显的保护作用。
References:
[ 1]　S ong H L, Chen S L . E ffect of pu erarin on act ion and oxy-
gen r adical s and lipid peroxide in mice su ffering from alcoh ol
[ J ] . R es P ract Chin Med (现代中药研究与实践) , 2003, 17
( 3) : 36-37.
[ 2]　Wang B Y, Li Y K. Methodology and T echnology in R e-
search and Dev elop ment of N ew Chinese Mater ia Med ica (中
药新药研制开发技术与方法) [ M ] . S han ghai: S han ghai S ci-
ence and Tech nology Pub lish ers , 2001.
[ 3]　Xu S Y. Method ology in P harmacological Exp eriments (药理
实验方法学) [M ] . Beijin g: People s Medical Pub lish ing
House, 2002.
[ 4]　Chen Q. M ethodology in P harmacolog ical Study on Chinese
Materia Med ica ( 中药药理研究方法学 ) [ M ] . Beijing:
People s Medical Publis hing House, 1993.
[ 5]　David L S, Rodert D G, Leslie A L. Cell A L aboratory
Manual (细胞实验 指南 ) [ M ] . Beijing : S cience Pres s,
2001.
Antithrombotic ef fects of Veratrum nigrum var. ussuriense alkaloids
HAN Guo-zhu
1
, LI Xin-yan
1
, L U¨ L i
1
, LI Wei-ping
1
, ZHAO Wei-jie
X
( 1. Departm ent of Pharmacolog y , Dalian Medical Univer sity , Dalian 116027, China ; 2. Departm ent o f Chemical
Pharmaceutics, Dalian Univ ersity o f Sciences & Techno lo gy , Dalian 116027, China )
　　Abstract: Obj ect　T o investig ate ant ithrombot ic ef fects o f Ver atr um nigr um L. var ussuriense Nakai
alkaloids ( VnA) in rats. Methods　T he elect rically induced r at carot id arter y thrombosis model w as used
to examine the antiarterial thrombosis ef fect of VnA using occlusion t ime ( OT ) of caro t id artery as phar-
maco logic index ; the stasis-induced infer io r vena cava thrombosis model w as used to evaluate antivenous
thr ombosis ef fect of VnA in terms of thrombosis format ion rate and decr ease in thrombus dry w eight . Re-
sults　T he iv administ ration of VnA ( 7. 2—42. 9) Lg / kg to rats resul ted in a do se-dependent effect and
signif icant pro longat ion in OT. VnA at the do se of 30 Lg / kg produced an antiarterial thrombosis ef fect e-
qual to that of LAS ( 18. 0 mg / kg ) . Also, a sing le bolus iv VnA ( 30 Lg/ kg ) increased OT in a time-depen-
dent manner ; the ant iarter ial thrombosis ef fect w as rapid in onset and lasted at least 80 min, and peaked at
15 min postdosing. VnA ( 15—45 Lg / kg iv ) decreased the thrombus dry w eight significant ly and dose-de-
pendent ly . Conclusion VnA has pow erful inhibitory ef fects against both arter ial and venous thrombosis in
rats and acts in a dose-and t ime-dependent manner. It s ef fect ive dose is as low as Lg per kg body w eight of
rats. The f inding of the VnA ant ithrombo tic effeets reveal a bright future of it s R & D.
Key words : thrombosis; Veratrum nigrum L. var ussuriense Nakai; alkaloid; L ilu
乌苏里藜芦碱抗血栓作用的研究
韩国柱1, 李欣燕1, 吕　莉1 ,李卫平1 ,赵伟杰2
( 1. 大连医科大学 药理教研室,辽宁 大连　116027; 2. 大连理工大学 化学制药系, 辽宁 大连　116027)
摘　要: 目的　研究乌苏里藜芦生物碱 ( VnA ) 对大鼠的抗血栓作用。方法　分别应用电刺激诱发大鼠颈总动脉血
栓形成模型和瘀血诱发下腔静脉血栓形成模型评价 VnA 的抗动、静脉血栓形成作用。结果　大鼠 iv 6 种不同剂量
的 VnA ( 7. 2～42. 9 Lg / kg) 导致血管阻塞时间 ( OT ) 显著延长, 且具明显的剂量依赖性, VnA 30 Lg / kg 产生的抗
动脉血栓形成作用与赖氨匹林 18. 0 mg / kg 的作用相当。单次 iv VnA 30 Lg / kg 使 OT 呈时间依赖性延长。VnA
·1107·中草药　Chinese T radit ional and Herbal Drugs　第 34 卷第 12 期 2003年 12 月
X 收稿日期: 2003-04-15基金项目:辽宁省教育委员会资助项目 ( 20272271)作者简介:韩国柱 ( 1943—) ,男,江苏省泰州市人;大连医科大学药理学教授,主要从事抗血栓药理及药动学研究。
T el: ( 0411) 3647531; E-mail: hgzh x2236@ sina. com. cn
抗动脉血栓形成作用起效迅速, 持续至少 80 min, iv 后 15 min 达效应峰值。iv VnA ( 15～45 Lg/ kg ) 显著减少下腔
静脉血栓干质量, 并呈剂量依赖性。结论　VnA 具有强大的抗动、静脉血栓形成作用, 并具明显的剂量依赖性和时
间依赖性。其抗血栓效能高, 作用强度大,在大鼠的有效剂量低至 Lg / kg 水平。VnA 抗血栓活性的发现为其研究开
发展示了新的前景。
关键词: 血栓; 乌苏里藜芦; 生物碱; 藜芦
中图分类号: R286. 3　　　文献标识码: A　　　文章编号: 0253 2670( 2003) 12 1107 04
　　V eratrum nigrum L. v ar ussuriense N akai is a
w el l know n medicinal plant w idely dist ributed in
northeast region of China. As a source of the Chi-
nese cr ude drug “Lilu”, this herbal medicine has
been r ecorded in Chinese medical books and no ted
fo r it s high to xicity . In the past Chinese and fo-
reign scientists did much research on its medicinal
values, but their research w as only fo cused on its
ant ihypertensive ef fects. So far, no info rmat ion
concer ning its antithrombot ic effects has been re-
por ted in published l iterature.
VnA is the to tal alkaloid ex t racted f rom roo t
of V . nigrum var ussuriense o f M ount . Qian in
Liaoning Province, China , and has been shown to
contain at least eleven alkaloids featuring ester -
type isostero idal st ructur e identif ied by modern
analy tical techniques such as HPLC/ M S, NMR,
etc . T he principal components contained in VnA
include verussurien, verabenzoamine, verazine,
germ idine, jerv ine, g ermer ine, 15-O-( methylbuty-
royl ) -germine, v erussurinine, neogermbudine, zy-
gadenine and echinuline
[ 1～4] .
In the present paper ant ithrombot ic ef fects of
VnA in the rat thrombosis models are reported, so
as to prov ide experimental basis for it s clinical use
and new drug development as a highly eff icacious
ant ithr ombot ic agent .
1　Materials and methods
1. 1　Chem icals and instruments: VnA hydrochlo-
ride injection ( 100 Lg/ mL) w as pr epared by De-
partment of Pharmaceut ical Engineering, Dalian
University of Science and T echno logy . It was di-
luted to the r equired concentrat ion w ith sterile
saline so lut ion prior to use. Heparin sodium was a
pr oduct o f Shanghai Bio chem ical Pharmaceut ical
Factory, lot No. 870516; dl-lysine-acetylsalicylate
( LAS) w as purchased from Anbao Pharmaceut ical
Factory, Anhui Prov ince, lot No. 950327. BT 87-3
model experimental in vivo thrombosis inst rument
w as purchased from Cardiovascular Research De-
partment , Baotou M edical College.
1. 2　Animals: Sprague-Daw ley rats of both sexes
( hal f to half ) w eighing 250—300 g w ere supplied
by Animal Center of Dalian M edical Univer sity
( Approval document No . 022, Liaoning Province,
China) .
1. 3　Rat carot id artery thrombosis model : T he
carot id artery thrombosis in r ats w as induced by
the method developed by Hladovec
[ 5]
w ith minor
modif icat ion. Rats w ere randomly div ided into
g roups and anesthetized by ip 20% urathane ( 1 g /
kg ) . T he unilateral cartot id artery w as isolated
surgically , a dir ect curr ent st imulat ing electr ode
and a temperature elect rode w ere placed at heart-
prox imal and heart-remo te ends o f carot id arter y
r espect ively. VnA-tr eated groups of rats received a
sing le bo lus iv administr ation of VnA, with posi-
t ive and negat ive contr ol groups given iv LAS ( 18
mg/ kg ) and equivolume saline so lut ion respective-
ly . The cont inual elect ric st imulat ion ( 1. 6 mA, 7
m in) was started in 15 min po stdose. T he t ime
elapsing from the beginning of st imulat ion to
abrupt fall in temperature of car ot id ar tery surface
w as reco rded by means o f the temper ature elec-
trode of the inst rument . This time represented the
occlusion t ime ( OT ) of carot id ar tery injured elec-
trically, namely , thrombus format ion t ime. T hen,
the percent increase of OT , an index w as used to
evaluate ant iarterial thrombosis effects of the
drugs, V nA-treated g roups w ere calculated in
comparison w ith saline contr ol g roup.
1. 4　Rat inferior vena cava thrombosis model:
T he inferior vena cava thr ombosis w as induced by
venous blood stasis in accordance w ith Reyers
method
[ 6]
. Br ief ly, rats w ere random ly div ided in-
to gr oups and anesthet ized as above. T he m iddle
·1108· 中草药　Chinese T radit ional and Herbal Drugs　第 34 卷第 12 期 2003年 12 月
abdominal w all o f rats w as incised, then inferior
vena cava w as separated surgical ly; dif ferent
gr oups of rats were t reated w ith iv vary ing doses of
VnA ( 15-45 Lg/ kg ) , heparin ( 400 Lg/ kg ) as posi-
tiv e control and equivolume saline as negat ive con-
tr ol respectiv ely . Five minutes later, the inferior
vena cava w as ligated to induce stasis. T hen, ab-
dom inial cav ity w as closed, and reopened tw o
hour s after ligat ion. The blood vessel of inferior
vena cava w as clamped at 2 cm below the lig at ion.
The thrombus clot in the blood vessel was removed
( if any ) and w eighed after drying at 60 ℃ fo r 20
min. T he animal numbers of thrombus fo rmat ion
and thrombus drying w eight , w hich w ere used to
evaluate the ant ivenous thr ombot ic effects o f the
dr ug , w ere reco rded so as to calculate thrombus
fo rmat ion rate ( animal number of thrombosis/ test
animal number ) and percent inhibit ion of thrombus
w eight respectively .
2　Results
2. 1　Effects of VnA on r at carot id artery throm-
bosis: As seen in Fig . 1, iv inject ion VnA resul ted
in a do se-dependent increase in the OT . The OT s
in the VnA-treated gr oups ( n= 6, each) at iv dos-
es of 7. 2, 10. 3, 14. 7, 21. 0, 30. 0 and 42. 9 Lg/ kg
w er e ( 11. 27±1. 47) ( P> 0. 05) , ( 12. 23±1. 43) ,
( 13. 64±2. 68) , ( 15. 43±3. 59) ( P< 0. 05) , ( 18.
47±3. 89) and ( 20. 21±3. 40) ( P< 0. 01) min, re-
spect iv ely , compared w ith ( 10. 32±1. 35) min in
negat ive contr ol gr oup and ( 18. 24±1. 81) m in in
po sit ive contro l g roup. The percent increases of
OT in VnA-tr eated groups relative to the negat ive
control g roup w ere 9. 2%, 18. 5% , 32. 2%, 49.
5% , 78. 9% and 95. 8% repect ively . It w as also
found that iv VnA ( 30. 0 Lg / kg ) produced an ef-
fect equal to that of iv LA S ( 18. 0 mg / kg ) .
　　 Also, a t ime-dependent increase of OT in
VnA-treated group by iv do se o f 30. 0 Lg / kg ( n=
6, at each t ime point ) w as show n in Fig . 2. The
OT at 2, 5, 10, 15, 20, 30, 50, 80 and 120 min
po stdo se w ere ( 11. 31±2. 61) , ( 11. 74±2. 16)
( P> 0. 05) , ( 13. 96±2. 47) ( P< 0. 05) , ( 17. 36±
3. 51) , ( 15. 37±3. 25) ( P < 0. 01) , ( 13. 36±2.
87) , ( 13. 15±2. 40) , ( 12. 04±2. 01) ( P< 0. 05)
Fig. 1　Dose-ef fect curve of VnA in rats
af ter administration
and ( 11. 16±2. 52) ( P> 0. 05) m in respectively vs
( 10. 02±1. 22) min in the negat ive contr ol group,
w hich corr esponded to the per cent OT incr eases of
12. 9% , 17. 2%, 39. 3% , 73. 2%, 53. 4% ,
33. 4%, 31. 2% , 20. 2% and 11. 3% , respect iv ely .
T he t ime to peak ef fect w as found to be 15 m in
postdose.
Fig. 2　Time-ef fect curve of VnA in rats receiving
an iv administration of 30 Lg/ kg
2. 2　Effects of VnA on rat inferior v ena cava
thrombosis: From Table 1 it could be found that
all iv doses of VnA pr oduced a reduct ion in thr om-
bus fo rmat ion rate ( e. g. 8/ 10, 8/ 12 and 6/ 12 at
15, 30 and 45 Lg / kg respect ively vs 10/ 12 in the
negat ive contro l ) , although there w as no stat is-
t ically signif icant dif ference ( P> 0. 05) . How ever,
compared w ith negat ive control gr oup all above
three doses of VnA decreased the dry weight of
thrombus do se-dependent ly and significant ly [ ( 1.
81±1. 40) , ( 1. 50±0. 61) and ( 0. 71±0. 18) mg
in VnA-treated g roup at ascending o rder of doses
v s ( 2. 87±0. 75) mg in negat ive control ) ( P< 0. 05
at middle and large doses) ] . It w as ev ident that
the three doses emplo yed her e produced the effect
·1109·中草药　Chinese T radit ional and Herbal Drugs　第 34 卷第 12 期 2003年 12 月
po tency slight ly low er than that produced by iv
heparin 400 Lg/ kg .
Table 1　Effect of VnA on stasis-induced venous
thrombosis in rats ( x±s)
Drug n
Dose
/ (Lg·kg - 1)
T hrombus
f ormat ion ra te
Dry weight of
t hrombus/mg
Inhibit ion of
dry weight /%
NS 12 　　- 　10/ 12 2. 87±0. 75 -
VnA 10 　　15 8/ 10 1. 81±1. 40 36. 9
12 30 8/ 12 1. 50±0. 61* 47. 7
12 45 6/ 12 0. 71±0. 18* * 73. 9
Heparin 10 400 5/ 10 0. 50±0. 21* * 81. 9
　　* P< 0. 05　* * P< 0. 01 v s N S control g roup
3　Discussion
　　 In this study the ant ithrombo tic ef fects of
VnA were invest ig ated using rat car ot id ar tery
thr ombosis model and rat inferior vena cava throm-
bosis model. In the former model elect ric st imula-
tion of carot id artery led to endothelia injury , fol-
low ed by platelet adhesion, ag gregat ion and forma-
tion of a platelet-rich thr ombus which caused oc-
clusion of the artery . So , the OT was used to ex-
amine the inhibitory ef fect of VnA against arterial
thr ombosis. In the later model inferior vena cava
w as ligated for tw o hours and thus rendered blood
stasis, consequently leading to act ivat ion of blood
clo tt ing system and format ion of thrombus. Above
stated models are simple, r eproducible and r eliable
fo r evaluat ing antithrombot ic ef fects of drugs, in-
cluding A spir in and Heparin, w hich w er e used as
po sit ive controls, and VnA , w hich w as examined
in the present exper iment .
　　T he findings in this study st rongly demon-
st rate that VnA has pow er ful inhibito ry ef fects
against both ar terial and venous thrombosis, mani-
fested by high ant ithrombot ic ef f icacy and high an-
tithrombot ic potency. VnA was found to be ef fec-
tiv e ant ithr ombot ically at an ex tr emely low doses
(Lg/ kg level ) . VnA of ( 30 Lg / kg ) iv administ ra-
tion produced ant iar terial thrombosis ef fect w hich
equals that produced by 18. 0 mg / kg LAS. VnA
( 42. 9 Lg/ kg ) caused percent increases in OT of
95. 8% , VnA ( 45 Lg/ kg ) signif icantly inhibited
venous thrombosis w ith inhibitor y rate of 73. 9%,
compar ed w ith 81. 9% fo r heparin ( 400 Lg / kg) .
　　T he durat ion of ant ithr ombot ic effect of VnA
was found short ( about 80 min) , suggest ing that
r epeated dosing may be required. It w as also found
that the ant ithrombot ic ef fect peaked at 15 m in af-
ter iv administ ration, indicat ing that there w as a
lag phenomenon of ef fect relat ive it s blood concen-
trat ion. The mechanism of this phenomenon is to
be elucidated.
　　In rat venous thrombosis model w hen thr om-
bus format ion rate as an index of effect w as used
there w as no statistically significant dif ference bet-
w een VnA -treated g roup and saline-treated group.
T his is because thrombus format ion rate is a quan-
tal response index , mo re animal numbers are need-
ed to produce signif icant dif ference than dry w eight
o f thrombus, a graded response index .
　　VnA is the total alkaloid ext racted fr om V ni-
gr um, w hich has been used as a sour ce o f t radi-
t ional Chinese medicine “Lilu”and thought to have
potent hypotensive effect . T he pr esent art icle is
the f irst repo rt on the ant ithrombot ic effects of
VnA in the medical literature to date. T his new
f inding provides a experiment support for the use
o f “Lilu”in the tr eatment of apoplex y[ 7] .
References:
[ 1]　Zhao W J , Chen J , Gu o Y T, et al . Chemical research on the
alkaloids f rom V erat rum nigrum L. var. ussu riense Nakai
[ J ] . B ull Chin Mater Med, 1987, 12 ( 10) : 34-35.
[ 2]　Zhao W J , Tezuka Y, Kikuch i T. S tu dies on th e con s-
t ituents of V eratr um nig rum L. var . ussur iense Nakai. ( 1)
s t ructure and 1H and 13C nuclear magnetic res on ance spect ra
of a new alkaloid, verus surinin e, and related alkaloids [ J] .
Chem P harm Bul l , 1991, 39 ( 3) : 549-554.
[ 3]　T ezuka Y, Kikuchi T , Zh ao W J, e t al . ( + ) -Verus sur ine, a
new s teroidal alkaloid f rom the root s an d rh izoma of V era-
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of ( + ) -verabenzoamine [ J] . J N at P rod, 1998, 61 ( 11) :
1397-1399.
[ 4]　Zhao W J , Gu o Y T , T ezuk a Y, et al. Isolat ion and st ru c-
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t rum nig rum L . var. ussuri ense Nak ai [ J] . Ch ina J of Chin
Mater Med , 1991, 16 ( 7) : 425-426.
[ 5]　Hladovec J. Ex perimental arterial th rombosis in rat s w ith
cont inous reg ist rat ion [ J] . T hrom Diath H aemor rhag , 1971,
26: 407-410.
[ 6]　Reyers I. Failure of aspirin at dif f erent doses to modify ex-
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[ 7]　J iangsu New Medical C ol lege. Dict ionary of Chinese Mater ia
Medica [ M ] . S han ghai: Shanghai People s Pub lish ing
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·1110· 中草药　Chinese T radit ional and Herbal Drugs　第 34 卷第 12 期 2003年 12 月