全 文 ：Received date:2005 02 25
Biographies:JIA Jing-ming(1964 ), male , Fuxin , Province of Liaoning , associate professor , major in the biotechnology of
T raitional Chiense Medicines , Tel.024 23986501 , E mail jmingjia@ sina.com;WU Chun-fu(1959 ), male , Yantai ,
Provicne of Shandong , professor ,major in neuropharmacolo gy.Tel.024 23843567 , E mail w uchunf@21cn.com.
文章编号:1006 2858(2005)06 0444 05
Anti-radiation activity of the tissue culture of
Saussurea involucrata Kar.et Kir.
JIA Jing-ming , WU Chun-fu , YU Hai , HU Gao-sheng
(School of Traditional Chinese Materia Medica , Shenyang Pharmaceutical University , Shenyang　110016 , China)
Abstract:Objective To study the anti-radiation ef fect of the tissue culture of Saussurea
involucrata Methods Anti-radiation ef fect w as investigated in mice on body w eight ,
white blood cell counts , serum superoxide dismutase (SOD)act ivity , survival rate , sur-
vival time and micronucleus rate.Results Compared wi th radiation g roup , the tissue cul-
ture of S .involucrata could obviously increase w hite blood cell counts and serum SOD
activi ty(P<0.01)and decrease micronucleus rate(P <0.01).The tissue culture of S .
involucrata also showed a significant increase in the survival rate and the survival time
(P <0.01)of the animals subjected to radiation.Conclusions The t issue culture of S .
involucrata has protect ive effect on irradiated mice.
Key words:Saussureu involucrata ;tissue culture;anti-radiation
CLC number:S 567;R 96;R 818.05　　　Document code:A
　　Radiation is harmful to the body functions and
causes the declination of the body resistance to dis-
eases and arouses the possibility of mutation and
cancer.To a large deg ree , the radiation damage is
mediated by the production of large amount of free
radicals[ 1] .The free radicals could injure all kinds
of biological macromolecules and membranes , caus-
ing the loss of cellular functions , the turbulence of
cell cy cle and the instability of heredity[ 2 , 3] .In the
studies of looking for the drug s that could prevent
radiation-induced injuries , herbal medicine has at-
tracted much attention of the scientists.Some tradi-
tional Chinese medicinal herbs having ant i-oxida-
tion and anti-radical ef fects have been tested fo r the
prevention of radiation-induced damages[ 4 ,5] .
Saussurea involucrata Kar.et Kir.(Composi-
tae), a rare Chinese medicinal herb on the verge of
ex tinction , g row s in the mountains at heights of
2 800-3 400 m in the Tianshan and A er Tai ar-
eas in China.The f low er and stem have been used
as a herbal medicine in most areas of China fo r sev-
eral centuries[ 6] .A variety of pharmacological activ-
ities of S .involucrata has been repo rted , such as
anti-inf lammation[ 7] , cardiotonic ef fects , terminat-
ing the gestation[ 8 , 9] , ant i-cancer[ 10] , anti-radia-
tion[ 11] , anti-fatigue and protection of immune
function[ 12 ,13] .
The secondary metabolites in wild S.involucrata
are mainly flavonoids , including apigenin ,kaempferide ,
acacetin , luteolin , quercetin , apigenin-5 , 6-dimethoxy-
flavone , apigenin-6-methoxy-flavone , and rutin etc.[ 14]
Other constitutes , such as alkaloids , steroids and lignans
were also reported[ 15] .
S .involucrata g row s very slowly.In recent
years , the w ild resources of S .involucrata de-
creased dramatically due to the exhaustive collec-
tion for the production of pharmaceutical prepara-
tions.In order to conserve the natural resources of
第 22 卷 第 6 期
2 0 0 5 年 1 1 月
沈　阳　药　科　大　学　学　报
Journal of Shenyang Pharmaceutical University
Vol.22 　No.6
Nov.2005 p.444
DOI :10.14066/j.cnki.cn21-1349/r.2005.06.013
S .involucrata , t issue culture of S .involucrata is
developed ,which might be used as a po tential sub-
st itute for wild S .involucrata in pharmaceutical
indust ry .In the present study , the anti-radiation ef-
fect of the product of tissue culture of S .involu-
crata is tested.
1　Materials and methods
1.1　Animals
Female Sw iss-Kunming mice(20-25 g), sup-
plied by the Experimental Animal Center of
Sichuan University , were housed under standard
conditions(22±2)℃,50%±10% relative humid-
ity , 12 h light/dark cycle.Food and water w ere
available ad libitum.
1.2　Plant material
The wild S .involucrata(WSauI)used in this
study was purchased from a local market in Xin-
jiang region(China), and identified by Professor
Yilin Chen(Institute of Botany , The Chinese A-
cademy of Sciences).The specimen of WSauI was
deposited in the China National Herbarium(No.
020805 , Beijing , China).The dried flowers
(2.0 kg) of WSauI w ere ground and ex tracted
w ith 95%ethanol for three times under reflux.The
ex tracts were concentrated under vacuum evapo ra-
tion to affo rd brown ext racts(yield 6.3%).The
content of total f lavonoids in the dried ext ract was
1.07%.
1.3　Tissue culture material
Explants of stems and leaves of WSauI w ere
cultured on MS
[ 16]
medium w ith naphthylacetic
acid 2 mg·L -1 ,6-benzy laminopurine 0.5 mg·L-1 ,
3% sucrose , and 0.65% agar for the inducement of
callus and t issue culture.The culture condi tions
w ere as follow s:temperature ,(24±1)℃;light in-
tensi ty , 58.4 μmol·-2·s-1;light period , 12 h/d.
The calli w ere dried at 60 ℃ and g round through a
40-meshes sieve.The tissue culture of S .involu-
crata(TCSauI)(5.0 kg)was ex tracted w ith 95%
ethanol for three times under reflux.The ext racts
w ere concentrated under vacuum evaporation to af-
ford brow n ext racts(450 g).The content of total
flavonoids of the brown ex t racts w as 2.6%.We in-
spected the quality stability of culture patches by
determining the total f lavonoids of TCSauI by spec-
trophotometric and HPLC methods
[ 17 ,18] .Our
studies on chemical constituents and quality cont rol
of TCSauI showed the different culture patches of
TCSauI had good stability(data not show n).
All doses mentioned in the experiments w ere
weight of the ex tract of the callus tissue.All above
samples were suspended in dist illed water contain-
ing 0.5% carboxymethylcellulose sodium(CMC-
Na)before use.Other reagents were purchased
from commercial channels.
1.4　Anti-radiation effect
Experimental protocol TCSauI was suspended
in distilled water containing 0.5% car-
boxymethy lcellulose sodium(CMC-Na)and w as in-
trag ast rically administered once a day at the doses
of 8.3 ,16.6 ,24.9 mg·kg -1 for 30 days.Same vol-
ume of distilled w ater was intrag ast rically adminis-
tered to the mice in blank control g roup and radia-
tion control g roup.Tw enty day s af ter drug admin-
ist ration , the mice of TCSauI-t reated groups and
radiation control g roup were irradiated by γ-ray of
60Co at the dose of 1.0 Gy once a day for 10 day s.
1.4.1　White blood cell counts
Blood samples were withdrawn form the retro-or-
bital sinus of the animals after irradiation at 7 days.The
white blood cells(WBC)were counted by the method
of Chen et al.[ 19] .The count of the WBC was calcu-
lated according to the following equation:
WBC(1×109·L-1)=(To tal WBC of 4 panes
of per count pool)/4×at tenuant times×109.
1.4.2　SOD activity
The serum SOD act ivity was tested by xan-
thine oxidase method wi th SOD kit
[ 20](Insti tute of
Jiancheng Bioengineering , Nanjing ,China).
1.4.3　Micronucleus test
The micronucleus rate in Poly chromatic Ery-
throcy te(PCE) of mice w as measured by the
method described by Yang et al.[ 21] w ith some
slight modifications.General smear w as dried by
airing , then put into methanol fo r 5-10 min , and
dyed fo r 10-15 min by Giemsa.The micronuclei
were counted under microscope.The micronucleus
445第 6 期 JIA Jing-ming et al:Anti-radiation activity of the tissue culture of Saussurea involucrata Kar.et Kir.　　　
rate w as expressed by permillage.
1.4.4　Survival rate
The survival rate of the animals w as recorded
30 days af ter irradiat ion.The mean survival time
and the mean survival rate w ere recorded af ter the
first radiation.
1.4.5　Statistical analysis
The results were expressed as mean ±s.e.
m ., one-way analysis of variance(ANOVA)fol-
lowed by Dunnet t s test and Chi square test w ere
used to compare the drug ef fects w ith blank control
g roup.
2　Results
2.1 　Effect of TCSauI on radiation-induced de-
crease of peripheral white blood cell counts
A statistically significant decrease in white
blood cell counts w as observed in radiation g roup
compared to blank control group(P <0.01).TC-
SauI , at the doses of 16.6 and 24.9 mg ·kg-1
slightly but significantly raised the w hite blood cell
counts w hich decreased fo r radiation(P <0.05),
(Table 1).
Table 1　Effect of TCSauI and WSauI on white blood cell counts decreaseinduced by radiation in mice
Groups Dose/(mg·kg -1) n White blood cell counts/(1×109·L-1)
Blank control 0.0 12 6.54±1.89
Radiation control 0.0 12 1.34±0.69a
8.3 12 1.57±0.42a
TCSauI 16.6 12 1.69±0.74ab
24.9 12 2.12±1.01ab
WSauI 16.6 12 1.86±0.56ab
　　a— P<0.01;b— P<0.01 as compared with radiation control
2.2 　Effect of TCSauI on radiation-induced de-
crease in serum SOD activity
Radiation induced a significant decrease in
serum SOD act ivity(P <0.01).TCSauI signif i-
cantly inhibited the decrease of serum SOD activity
induced by radiation(P<0.01),(Table 2).
Table 2　Effect of TCSauI and WSauI on radiation-induced decrease inserum SOD activity in mice
Groups Dose/(mg·kg -1) n SOD activity/(NU·mL-1 serum)
Blank control 0.0 11 422.42±60.30
Radiation control 0.0 11 270.18±70.95a
8.3 11 360.55±42.34ab
TCSauI 16.6 11 333.47±48.67ab
24.9 11 348.18±36.35ab
WTCSauI 16.6 11 335.42±43.26ab
　　a— P<0.01;b— P<0.01 as compared with radiation control
2.3　Effect of TCSauI on radiation-induced in-
crease in micronucleus rate
Statistically significant increase in micronucle-
us rate w as observed in radiation g roup compared
with blank control g roup(P <0.01).M icronucleus
rate in PCE of mice in TCSauI-treated groups was
significantly lower than that in radiation control
group(Table 3).
Table 3　Effect of TCSauI and WSauI on micronucleus rate in mice
Groups
Dose/
(mg·kg-1) n n of PCE
n of
micronucleus
M icronucleus
rate/ ‰
Blank control 0.0 10 1 000 2.00±0.94 2.0
Radiation control 0.0 10 1 000 25.80±5.77a 25.8
8.3 10 1 000 18.40±2.32ab 18.4
TCSauI 16.6 10 1 000 9.60±3.34ab 9.6
24.9 10 1 000 7.10±2.77ab 7.1
WSauI 16.6 10 1 000 8.80±2.15ab 8.8
　　a— P<0.01;b— P<0.01 as compared with radiation control
446 　　　沈　阳　药　科　大　学　学　报 第 22卷
2.4　Effect of TCSauI on mean survival time and
survival rate after radiation
The mean survival t ime was obviously shorter
and the 30 d survival rate w as lower in radiat ion
g roup than that in blank control g roup(P <0.01).
TCSauI , at the dose of 8.3 mg·kg-1 , significantly
leng thened the mean survival time compared w ith
the radiation control group(P <0.01).The 30 d
survival rate was slight ly increased in TCSauI
groups at the doses of 8.3 and 24.9 mg ·kg-1
(Table 4).WSauI , at the dose of 16.6 mg·kg-1 ,
showed signif icant ef fect.
Table 4　Effect of TCSauI and WSauI on mean survival time and survivalrate after radiation in mice
Groups Dose/(mg·kg-1) n Mean survival time/d Survival rate/ %
Blank control 0.0 15 <30 100.0
Radiation control 0.0 15 7.60±1.99a 0.0a
8.3 15 16.27±7.54ab 13.3a
TCSauI 16.6 15 8.53±5.19a 0.0a
24.9 12 10.33±7.04a 6.6a
WSauI 16.6 15 9.86±3.37a 13.3a
　　a— P<0.01;b— P<0.01 as compared with radiation control
2.5　Effect of TCSauI on radiation-induced body
weight decrease
Radiation for 10 day s significantly decreased
the body weight of the mice compared w ith blank
control g roup(P <0.01).TCSauI at the doses used
could no t antagonize radiation-induced body weight
decrease in mice(Table 5).
Table 5　Effect of TCSauI and WSauI on body weight decrease induced by radiation in mice
Groups
Dose/
(mg·kg-1)
t/ d
0
n m/ g
15
n m/g
30
n m/g
Blank control 0.0 30 21.73±1.08 30 27.50±2.39 30 30.07±2.88
Radiation control 0.0 30 21.73±1.17 30 27.87±2.84 15 25.67±3.29a
8.3 30 21.70±1.21 30 26.30±2.25 26 23.73±3.62a
TCSauI 16.6 30 21.73±1.23 30 26.50±2.10 16 25.13±2.63a
24.9 30 21.73±1.11 30 26.40±2.01 17 24.53±2.35a
WSauI 16.6 30 22.12±1.15 30 26.35±1.89 17 25.68±2.12a
　　a— P<0.01 as compared with control
3　Discussion
Tissue culture has advantages on resources
conserving and indust rializing po tentials.However ,
the eff icacy of the products of tissue cultures is cru-
cial to using them as the substi tute of the plants in-
terested.The present study , by using dif ferent ani-
mal models , f irst demonst rated that TCSauI pos-
sessed the significant ant i-radiation actions , which
implies a potential usefulness of the tissue culture of
S .involucrata in substituting the w ild one in
pharmaceut ical indust ry .
Previous studies have showed that the water
ex tract of w ild S .involucrata had protective ef-
fects on immune functions in irradiated mice.In the
present study , by observing the peripheral white
blood cell count , the serum SOD activity , micronu-
cleus rate and the survival rate in 30 d of irradiated
mice , it w as show n that the tissue culture of S .in-
volucrata had significant pro tective ef fects on radi-
ation-induced injuries in the body.
The radiation-induced tissue damage involves
the f ree radical reactions.The active oxygen free
radicals are generated during and af ter the radia-
tion[ 22] .Some substances which have free radical
scavenging activi ties may release the symptoms in-
duced by radiation[ 23] .The present study observed
that af ter using TCSaul the SOD activity in serum
was significantly higher than that in radiation con-
trol group , suggesting that TCSaul can activate or
protect the endogenous anti-oxidative defending
sy stem of the body.
447第 6 期 JIA Jing-ming et al:Anti-radiation activity of the tissue culture of Saussurea involucrata Kar.et Kir.　　　
The flavonoids contained in S .involucrata ,
such as apigenin , kaempferide , acacetin , luteolin ,
quercetin , apigenin-5 , 6-dinethoxy-flavone , api-
genin-6-methoxy l-flavone , and rutin , are repo rted to
have anti-oxidative activities[ 24 ,25] .These sub-
stances might contribute to the anti-irradiat ion ac-
tivi ties of TCSaul in the present study.Not only the
flavonoids , it is suggested that polysaccharides f rom
w ild S .involucrata also have scavenging act ivit ies
on supero xide anion
[ 26] .The flavonoids constituents
isolated and identif ied f rom TCSauI such as pran-
ferin , chry soeriol-7-O-β-D-rutinoside , quercetin-3-
O-β-D-glucoside , luteolin-7-O-β-D-glucoside , api-
genin-7-O-β-D-g lucoside , and luteolin-4′-O-β-D-
g lucoside w eren t isolated fo rm WSauI.The differ-
ence in ef ficacy and/or potency betw een TCSauI
and WSauI may be due to the dif ferent parts used
in ext raction since the ext ract of WSauI were from
flow ers , and that of TCSauI w as from stems and
leaves , which might lead to the different levels of
flavonoids they contained.
In conclusion , the present study demonstrated
that TCSauI had the anti-radiation ef fects , which
are also possessed by the w ild one.These results
implied the potential of using tissue culture tech-
nique fo r substituting the exhaustive use of w ild S .
involucrata in pharmaceutical indust ry.
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Study on the safety of garlic oil submicron emulsion
and antifugal effects in vitro
GUO Tao , CHU Xiao-qin , YAN Ming , HE Jin , ZHAO Ming-hong , SUN Xue-hui
(General Hospi tal of Shenyang Military Region , Shenyang　110016 , China)
Abstract:Objective To observe the safety and the antifungal effects of garlic submicron emulsion.Methods
LD50 of acute toxicity w as determined by i.v.in mice.The hemoly tic test w as carried out by using 2%ery-
throcy te suspension ,which w as prepared from rabbit blood.Garlic submicron emulsion or garlic oil inject ion
w as administered w ith intravenous drop infusion in Newzeland rabbit on the lef t ear margin w hile 5%g lu-
cose injection was on the right , and the stimulation vessel were observed.The minimal inhibitory concentra-
tions(M IC)of g arlic submicron emulsion , garlic injection , diflucan injection against Candida albicans ,
Candida tropicalis , Cryptococcus neoformans and Aspergil lus f um igatus were determined by the means of
double dilution method w ith sabourand agar.Results The LD50 of garlic submicron emulsion was
282.0 mg·kg-1 , and that of g arlic injection was 167.2 mg·kg-1.Garlic oil submicron emulsion had no ef-
fects on haemolysis and no vascular irri tation.The minimal inhibitory concentrations(M IC)of garlic oil sub-
micron emulsion against Candida albicans , Candida tropicalis , Cryptococcus neoformans and Aspergil lus
f um igatus were 50 , 25 , 50 , 25 mg·L-1 , and garlic injection were 50 , 50 , 100 , 25 mg·L-1 , respectively.
Conclusions The LD50 of garlic submicron emulsion w as 1.7 times than that of garlic oil injection , and the
safety of i t w as better than that of g arlic oil injection.Garlic submicron emulsion still possessed significant
antifungal effects.
Key words:garlic submicron emulsion;garlic injection;dif lucan injection;drug safety ;antifungal activi ty
(上接第 448页)
新疆雪莲组织培养物的抗辐射作用
贾景明 , 吴春福 , 于　海 , 胡高升
(沈阳药科大学 中药学院 , 辽宁 沈阳 110016)
摘要:目的 探讨并比较新疆雪莲培养物(TCSauI)乙醇提取物的抗辐射损伤作用。方法 小鼠灌胃
给予 TCSauI 乙醇提取物 ,剂量 8.3 、16.6、24.9 mg·kg-1 ,每天灌胃 1 次共 30 d , 于灌胃后第 20 天
将对照组与试验组用60Co γ-ray 照射 , 剂量 1.0 Gy ,每天 1 次共 10 次。观察小鼠体质量 、外周白细
胞数 、SOD活性 、骨髓嗜多染红细胞微核率和平均存活时间的变化。结果 与空白给药组相比 , TC-
SauI醇提物能使受照射小鼠的外周白细胞数 、SOD 活性和平均存活时间显著增加(P <0.01), 骨
髓嗜多染红细胞微核率显著降低(P<0.01)。结论 新疆雪莲培养物乙醇提取物具有抗辐射作用。
关键词:新疆雪莲;组织培养物;抗辐射作用
462 　　　沈　阳　药　科　大　学　学　报 第 22卷