全 文 ：Haemolytic Activities and Adjuvant Effect of Stemucronatoside A on
the Immune Responses to Ovalbumin in Mice
WANG Hu-Gen , YE Yi-Ping＊
Institute of Materia Medica , Zhejiang Academy of Medical Sciences , Hangzhou 310013 , China
【ABSTRACT】　AIM:To evaluate haemolytic activities and adjuvant effect of the C-21 steroidal glycoside-stemucronatoside
A(SA)from the roots of Stephanotis mucronata.METHOD:The adjuvant potentials on the cellular and humoral immune re-
sponses to mice immunized with ovalbumin(OVA)were examined.ICR mice were immunized subcutaneously with OVA 100
μg alone , a mixture of OVA 100μg and aluminoid 200 μg , or mixture of OVA 100μg and SA 25 , 50 or 100μg on the first
day and fifteenth day.Two weeks later(d 28), concanavalin A(Con A)-, lipopolysaccharide(LPS)- and OVA-stimulated
splenocyte proliferation and OVA-specific antibody in serum were investigated.RESULT:SA showed slight haemolytic activi-
ties with its concentration inducing 50% of the maximum haemolysis(HD50)being 331.39±26.16 μg mL.SA significantly
enhanced the Con A-, LPS-, and OVA-stimulated splenocyte proliferation in OVA-immunized mice at the doses of 50 and 100
μg(P<0.05 or P <0.01 or P<0.001).The OVA-specific IgG , IgG1 and IgG2b antibody levels in serum were signifi-
cantly enhanced especially at the concentration of 50μg compared with OVA control group(P<0.01 or P<0.001).CON-
CLUSION:SA possesses immunological adjuvant activities and low-haemolytic effect.
【KEY WORDS】　Stephanotis mucronata;Stemucronatoside A;Haemolysis;Adjuvants;Proliferation;Antibody
【CLC Number】　R967　　【Document code】　A　　【Ariticle ID】　1672-3651(2005)06-0382-04
【Received on】　2005-06-23
【＊ Corresponding author】　Tel:571-88215624 , Fax:+ 86-571-
88869045 , E-mai l:yeyiping2005@163.com
1　Introduction
C21 steroids and their glycosides are of considerable
interest because of their bioactivities such as antitu-
mor
[ 1-3] , antiepilepsy[ 4] , antifertility[ 5] , immunopoten-
tial
[ 6]
and immunomodulating activities
[ 7] .The dried ro-
ots and stems of Stephanotis mucronata (Blanco)Merr.
(Asclepiadaceae)are used for the treatment of rheuma-
toid arthritis and rheumatic aches in Chinese folk medi-
cine.The main constituents of this species have been
shown as C21 steroidal glycosides
[ 7 , 8] .In the previous
paper , we reported the structural elucidation of three
new C21 steroidal glycosides-stemucronatosides A , B and
C , and their immunomodulating activities in vitro[ 7] .
However , until this report , the immunological adjuvant
effects of stemucronatosides A(SA , Fig 1)have not yet
been reported.Thus , haemolytic activity of stemucrona-
toside A(SA)and its effects on the humoral and cellu-
lar immune responses to ovalbumin in mice were exam-
ined in the present study.
Fig 1　Structure of stemucronatoside A(SA).
2　Materials and Methods
2.1　Mice
Male ICRmice(Grade Ⅱ , 5 weeks old)weighing
18 ～ 22 g were purchased from Zhejiang Experimental
Animal Center (Certificate No.22-2001001 , Hang-
zhou , China)and acclimatized for 1 week prior to use.
Rodents were provided with laboratory chow and tap wa-
ter ad libitum and maintained under controlled condi-
tions with a temperature of 24±1 ℃, humidity of 50±
382　　Chin J Nat Med　Nov.2005　Vol.3　No.6
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中国天然药物 　2005年 11月　第 3卷　第 6期
10%, and a 12 12 h light dark cycle.
2.2　Chemicals
Ovalbumin(OVA), 3-(4 ,5-dimethylthiazol-2-yl)-
2 ,5-diphenyltetrazolium bromide (MTT), concanavalin
A(Con A), lipopolysaccharide (LPS)and rabbit anti-
mouse IgG peroxidase conjugate were purchased from
Sigma;RPMI-1640 medium was from Gibco;goat anti-
mouse IgG1 and IgG2b peroxidase conjugate were from
Southern.Biotech.Assoc.;Fetal calf Serum(FCS)was
provided by Hangzhou Sijiqing Corp;aluminum hydrox-
ide gel(Alum)was provided by Zhejiang Wanma Pharm
Co.,Ltd.All other chemicals were of AR grade.
2.3　Preparation of SA
SA (98%)was isolated and prepared as previous-
ly described
[ 7] , and then analyzed by HPLC(ODS 4.6
×250mm , DAD).A stock SA solution with a concen-
tration of 2 mg mL was prepared by dissolving in
0.89% saline.The solution was sterilized by passing it
through a 0.22μm Millipore filter.
2.4　Haemolytic assay
Red blood cells were obtained from healthy New
Zealand rabbit(Zhejiang Experimental Animal Center ,
China).Blood was collected with BD VacutainerTM(NH
143 IU , Belliver Industrial Estate , Plymouth , UK).
Aliquots of 7 mL of blood were washed three times with
sterile saline solution(0.9%w v NaCl , pyrogen free)
by centrifugation at 180×g for 5 min.The cell suspen-
sion was prepared by finally diluting the pellet to 0.5%
in saline solution.A volume of 0.5 mL of the cell sus-
pension was mixed with 0.5 mL diluents containing
7.81 , 15.63 , 31.25 , 62.5 , 125 , 250 , 500 , or 1000
μg mL concentrations of SA in saline solution.The mix-
tures were incubated at 37 ℃ for 30 min , and centri-
fuged at 70×g for 10 min.Free haemoglobin in the su-
pernatants was measured spectrophotometrically at 412
nm
[ 9] .Saline and distilled water were included as min-
imal and maximal haemolytic controls.The haemolytic
percent developed by the saline control was subtracted
from all groups.Each experiment included tetraplicates
at each concentration.
2.5　Immunization
The experimental mice were immunized by the pro-
cedures as described previously
[ 10] .Six-week-old male
ICR mice were divided into six groups , each consisting
of five mice.Animals were immunized subcutaneously
with OVA 100μg alone , a mixture of OVA 100μg and
aluminoid 200 μg , or mixture of OVA 100 μg and SA
25 , 50 or 100 μg.Saline-treated animals were used as
controls.A boosting injection was given 2 weeks later.
Sera and splenocytes were collected 2 weeks after the
second immunization for proliferation assay and mea-
surement of OVA-specific antibody.
2.6　Splenocyte proliferation assay in vivo
Spleen collected from the OVA-immunized ICR
mice under aseptic conditions , in Hank s balanced salt
solution (HBSS , Sigma), was minced using a pair of
scissors and passed through a fine steel mesh to obtain a
homogeneous cell suspension , and the erythrocytes were
lysed with ammonium chloride (0.8%, W V).After
centrifugation(380×g at 4 ℃ for 10 min), the pel-
leted cells were washed three times in PBS and resus-
pended in complete medium [ RPMI 1640 supplemented
with HEPES 12 mmol L (pH 7.1), 2-mercaptoethanol
0.05 mmol L , penicillin 100 IU mL , streptomycin 100
μg mL , and 10% FCS] .Cell numbers were counted
with a haemocytometer by trypan blue dye exclusion
technique.Cell viability exceeded 95%.Splenocyte
proliferation was assayed as previously described
[ 11] .
Splenocytes were seeded into 4-5 wells of a 96-well flat-
bottom microtiter plate (Nunc)at a cell density of 1×
10
10 L in 100 μL complete medium , thereafter Con A
(final concentration 5 μg mL), LPS (final concentra-
tion 10μg mL), OVA(final concentration 20μg mL),
or RPMI-1640medium were added giving a final volume
of 200μL.The plates were incubated at 37 ℃in a hu-
midified atmosphere with 5%CO2.After 44 h , 50 μL
of MTT solution(2 g L)was added to each well and in-
cubated for 4 h.The plates were centrifuged(1400×g ,
5 min)and the untransformed MTT was removed care-
fully by pipet.To each well 200μL of a DMSO working
solution(180μL DMSO with 20μL , 1 mol L HCl)was
added , and the absorbance was evaluated in an ELISA
reader at 570 nm with a 630 nm reference after 15min.
The stimulation index (SI)was calculated based on the
中国天然药物 　2005年 11月　第 3卷　第 6期
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Chin J Nat Med　Nov.2005　Vol.3　No.6　383　
following formula:SI = the absorbance value for mito-
gen-cultures divided by the absorbance value for non-
stimulated cultures.
2.7　Measurement of OVA-specific antibody
OVA-specific IgG , IgG1 and IgG2b antibodies in
serum were detected by an indirect ELISA as described
previously
[ 10] (Sun et al., 2004).In brief , microtiter
plate wells(Nunc)were coated with 100μL OVA solu-
tion(50 , 25 and 50 μg mL for IgG , IgG1 and IgG2b
antibodies , respectively , in carbonate-bicarbonate buff-
er 50 mmol L , pH 9.6)at 4 ℃ for 24 h.The wells
were washed three times with PBS containing 0.05%
(v v)Tween 20 (PBS Tween), and then blocked with
5%FCS PBS at 37 ℃ for 1 h.After three times of
washing , 100 μL of a series of diluted serum sample or
0.5% FCS PBS as control were added to triplicate
wells.The plates were then incubated at 37 ℃ for 1 h ,
followed by 3 times of washing.Aliquots of 100 μL of
rabbit anti-mouse IgG horseradish peroxidase conjugate
diluted 1∶50000 , or goat anti-mouse IgG1 peroxidase
conjugate 1∶16000 or IgG2b peroxidase conjugate 1∶
8000 with 0.5% FCS PBS were added to each plate.
The plates were further incubated at 37 ℃for 1 h.Af-
ter washing , the peroxidase activity was assayed as fol-
lowing:100 μL of substrate solution(10 mg of o-phe-
nylenediamine and 37.5 μL of 30%H2O 2 in 25 mL of
citrate-phosphate buffer 0.1mol L , pH 5.0)was added
to each well.The plate was incubated at 37 ℃ for 10
min , and enzyme reaction was terminated by adding 50
μL per well of H2 SO 4 1 mol L.The optical density
(OD)was measured in an ELISA reader at 490 nm with
a 595 nm reference.Results were expressed as log2 ti-
ters.Where sets of serum samples have been subjected
to within and between group comparisons , ELISA assays
were performed on the same day for all samples.
2.8　Statistical analysis
The data were expressed as x ±s and examined for
their statistical significance of difference with standard
s-test.P-values of less than 0.05 were considered to be
statistically significant.
3　Results
3.1　Haemolytic activities
The haemolytic activity of SA on rabbit red blood
cells was shown in Table 1.Haemolytic percents of red
blood cell treated with SA were less than 6% at concen-
trations of 3.91-62.5 mg L.The HD50 value for SA
was 331.39±26.16 mg L.
Table 1　Haemolytic activities of Stemucronatoside A(SA)
(x±s , n=4)
Group Absorbance value Haemolytic percent(%)
Distilled water 1.291±0.053 100.000±4.28＊＊
Saline 0.083±0.004 0.00±0.33
SA(μg mL)
500 0.941±0.029 70.99±2.40
250 0.439±0.034 29.46±2.85
125 0.220±0.007 11.35±0.62
62.5 0.153±0.014 5.80±1.17
31.25 0.149±0.008 5.41±0.64
15.63 0.125±0.007 3.46±0.59
7.81 0.092±0.007 0.75±0.56
3.91 0.089±0.004 0.48±0.33
HD50=331.39±26.16
Haemolytic percents of saline and dist illed water were included as minimal
and maximal haemolytic controls.
3.2 　Effect of SA on mitogen- and OVA-stimulated
splenocyte proliferation in vivo
The effects of SA on mitogen- and OVA-stimulated
splenocyte proliferation in OVA-immunized mice are
shown in Fig 2.Con A-stimulated splenocyte prolifera-
tion in the mice immunized with OVA SA(25 , 50 , 100
μg)was significantly higher than that in the OVA con-
trol group (P <0.001).Splenocytes isolated from
OVA SA(25 , 50 , 100μg)-immunized mice and stimu-
lated by LPS show a greater proliferative response than
that observed for the mice immunized with OVA alone
(P <0.05 or P <0.01).OVA-induced splenocyte
proliferation in the mice immunized with OVA SA(50 ,
100μg)was significantly higher than that in the OVA
control group(P <0.05 or P <0.01).However , no
significant differences (P >0.05)were observed be-
tween the OVA control group and OVA Alum group.
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中国天然药物 　2005年 11月　第 3卷　第 6期
Fig 2　Effect of Stemucronatoside A(SA)on mitogen- and
OVA-stimulated splenocyte proliferation in vivo.Groups of
five male ICR mice were immunized with OVA 100(g alone
or with OVA 100(g dissolved in saline containing Alum(200
μg), SA(25 , 50 or 100 μg)on Day 1 and 15.Splenocytes
were prepared 2 weeks after the last immunization and cul-
tured with Con A , LPS , or OVA or RPMI-1640 medium for
48 h.Splenocyte proliferation was measured by the MTT
method as described in the materials and methods , and
shown as a stimulation index.The values are presented as x
±s(n=5).Significant differences with OVA control groups
were designated as ＊ P <0.05 , ＊＊ P <0.01 and ＊＊＊ P <
0.001.
3.3　Effect of SA on the OVA-specific serum antibody
response
To investigate the effect of SA on the induction of
humoral immune responses against OVA in mice , groups
of mice were immunized two times by S.C.routes.The
OVA-specific IgG , IgG1 and IgG2b antibody levels in
the sera were measured 2 weeks after the last immuniza-
tion using ELISA(shown in Fig 3).The total IgG level
in the mice immunized by OVA was significantly en-
hanced by OVA Alum and OVA SA(50 and 100μg)as
compared with control group (P <0.01 or P <
0.001).Significant enhancements in serum IgG1 levels
were observed in all groups of SA-immunized mice com-
pared with OVA control group (P <0.01 or P <
0.001).In addition , SA significantly enhanced total
serum IgG2b levels in OVA-immunized mice at the dos-
es of 25 and 50μg (P<0.01 or P<0.001).Howev-
er , there were no significant differences among the total
serum IgG2b levels in mice immunized with OVA alone
and OVA Alum (P >0.05).Thus , findings indicate
that SA could significantly enhance serum antibody pro-
duction in mice immunized with OVA at the suitable
concentration.
Fig 3　Effect of Stemucronatoside A (SA)on OVA-specific
IgG, IgG1 and IgG2b antibody.Groups of five male ICR
mice were immunized with OVA 100 μg alone or with OVA
100 μg dissolved in saline containing Alum (200 μg), SA
(25 , 50 or 100 μg)on Day 1 and 15.Sera were collected 2
weeks after the last immunization.OVA-specific IgG , IgG1
and IgG2b antibodies in the sera were measured by an indi-
rect ELISA method as described in the materials and meth-
ods.The values are presented as x ±s(n=5).Significant
differences with OVA control groups were designated as ＊ P
<0.05 , ＊＊P<0.01 and ＊＊＊P<0.001.
4　Discussion
New generations of vaccines , particularly those
based on recombinant proteins and DNA , are likely to
be less reactogenic and immunogenic than traditional
vaccines.Therefore , there is an urgent need for the de-
velopment of new and improved vaccine adjuvant.Al-
though a variety of adjuvants have been used in experi-
mental vaccines , most of these materials only elicit an
antibody response and or have undesirable side effects
that have limited their potential application in
vaccines
[ 12 , 13] .
SA showed a slight hemolytic effect , and could
significantly enhance the mitogen- and OVA-stimulated
splenocyte proliferation in OVA-immunized mice , espe-
cially at a dose of 50 or 100μg.The total IgG and IgG1
level in OVA-immunized mice was significantly en-
hanced by SA as compared with the control group.
Moreover , SA also significantly enhanced total serum
IgG2b level especially at a dose of 50 μg (Fig 3).
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Chin J Nat Med　Nov.2005　Vol.3　No.6　385　
Thus , it is likely that SA at suitable dose is effective on
Th1 and Th2 cell , as associated sensitively with an en-
hancement of IgG1 and IgG2b levels.These results sug-
gested that SA deserve further investigation as vaccine
adjuvant.
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黑鳗藤新苷A的溶血性及其对卵清白蛋白免疫小鼠的
免疫佐剂作用
王虎根 ,叶益萍＊
浙江省医学科学院药物研究所 ,杭州 310013
【摘　要】　目的:评价从黑鳗藤根中分离得到的 C21甾体苷--黑鳗藤新苷A(stemucronatoside A , SA)的溶血性及免疫佐
剂作用。方法:以分光光度法测定黑鳗藤新苷 A对血红细胞的溶血百分率;以卵清白蛋白(OVA)100μg , OVA 100 μg 加氢
氧化铝 200μg , OVA 100 μg加黑鳗藤新苷 A 25μg、50μg和 100 μg于第 1 d和 15 d 分别免疫 ICR小鼠 ,二免后 14 天 ,用 MTT
法检测刀豆蛋白 A(Con A),脂多糖(LPS)和 OVA诱导脾淋巴细胞增殖反应 , ELISA 检测血清中的抗 OVA抗体效价。结
果:黑鳗藤新苷A 显示轻微的溶血作用 ,其引起兔红细胞50%溶血的浓度(HD50)为 331.39±26.16 μg ml。黑鳗藤新苷A 在
剂量为 50 和100μg时能显著增强 Con A , LPS 和OVA诱导的 OVA受免小鼠脾淋巴细胞增殖反应(P <0.05 or P<0.01 or P
<0.001);50 μg时能极显著提高OVA 受免小鼠血清中OVA 特异性抗体 IgG , IgG1 和 IgG2b 的水平(P<0.01 or P<0.001)。
结论:黑鳗藤新苷 A具有较低的溶血性和显著的免疫佐剂活性 。
【关键词】　黑鳗藤;黑鳗藤新苷 A;溶血性;佐剂活性;增殖;抗体
386　　Chin J Nat Med　Nov.2005　Vol.3　No.6
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中国天然药物 　2005年 11月　第 3卷　第 6期
CHINESE JOURNAL OF NATURAL MEDICINES　　GRAPHIC ACSTRACT　　　November.2005 Vol.3No.6
Chin J Nat Med , 2005, 3(6):361-366
Screening for Ginsenoside Rg1 Nasal Absorption Enhancers Through Pharmacodynamic Study
CHEN Xin-Mei1 , ZHU Jia-Bi1＊ , SUN Wei-Dong2 , ZHANG Li-Jian3 , SUN Yu-Jiao1 , ZHANG Chao-Lei1
1Pharmaceutical Research Institute , China Pharmaceutical University , Nanjing 210009 ;
2Xinjiang Tefeng Pharmaceutical CO., Ltd., Urumqi 830054;
3Hongjiu Biotech Co., Ltd , Tonghua 135118 , China
Gensenoside Rg1 nasal absorption preparation were studied with borneol or and menthol as absorption enhancers and liquid par-
affin or and CMC-Na as viscosity increase.The absorption rate was increased with the elevating gensenside Rg1 at the range of 100
～ 500 mg ml.
Chin J Nat Med , 2005, 3(6):373-376
Fingerprints of Andrographis paniculata by HPLC UV MS
ZHANG Zun-Jian1＊ , DONG Hai-Juan1 , YU Jing2
　1Center for Instrumental Analysis , China Pharmaceutical University , Nanjing
210009 ;
2School of Public Health , Nanjing Medical University , Nanjing 210029 , China
The fingerprint of Andrographis paniculata by HPLC was established and seven
compounds were elucidated by MS.
【Foundation Item】　This project was supported by Innovation Medicines and
Modernization of Traditional Chinese Medicines Items of National 10th Five-Year
Important Science and Technology Subjects(No.2001BA701A56)
Chin J Nat Med , 2005, 3(6):382-386
Haemolytic Activities and Adjuvant Effect of Stemucronatoside A on the
Immune Responses toOvalbumin in Mice
WANG Hu-Gen , YE Yi-Ping＊
Institute of Materia Medica , Zhejiang Academy of Medical Sciences , Hangzhou 310013 ,
China
Stemucronatoside A(SA)could significantly enhanced the Con A-, LPS-, and OVA-
stimulated splenocyte proliferation in OVA-immunized mice.It showed slight haemolytic
activity.
Chin J Nat Med , 2005, 3(6):392-395
Anti-tumor Activity of Brucine on Mice with Transplanted Tumor
DENG Xu-Kun 1, CAI Bao-Chang1＊ , YIN Wu2 , SUN Liang1 , LI Wei-Dong1 , ZHANG Xiao-Chun1
1School of Pharmacy , Nanjing University of Traditional Chinese Medicine , Nanjing 210029 ;
2 State Key Lab of Pharmaceutical Biotechnology , Nanjing University , Nanjing 210093 , China
Brucine could inhibit the growth of transplanted solid tumor in mice and showed no inhibiting effect to immunity system.
【Foundation Item】　This project was supported by the National Natural Science Foundation of China(No.90209052);the Founda-
tion on Helping Doctoral postgraduates Creative study of Jiangsu Province(No.2005098)