全 文 ：这些结果提示肿瘤发生使机体红细胞膜收缩蛋白、
锚蛋白和带 3蛋白发生交联变构而引起带 3蛋白含
量的下降, 进而导致红细胞膜脂流动性的降低[ 7, 8]。
红细胞膜脂流动性的降低又引起 GPA (血型糖蛋
白A,富含 SA) 等多种重要膜蛋白构象改变, SA 含
量大幅减少,引起膜表面的电负性降低,红细胞聚集
性增加而无法正常识别和清除肿瘤细胞进而导致肿
瘤细胞血行转移。而本实验中两种 AP 中、高剂量
组均可显著降低膜蛋白聚集性而提高带 3蛋白含
量,使 S180小鼠膜脂流动性升高,红细胞膜生理功能
趋于正常, 这可能是 AP 促进红细胞免疫功能发挥
抗肿瘤作用的主要机制之一。
此外, 实验中还发现 AP 在 100 mg / kg 剂量下
作用最佳, 当剂量达到 200 mg/ kg 时对实验中的各
项指标的作用反而有所下降。这一现象表明 AP 存
在作用的最佳剂量。超过这一数值,剂量再增加, 作
用反而下降,这与以往对于其他多糖类成分活性研
究的结论相一致。
References:
[ 1] Ji Y B, Zou X, Gao S Y. Research on aloe polysaccharide [ J]1
J Harbin Univ Commerce ) Nat S ci Edi t (哈尔滨商业大学学
报#自然科学版) , 2003, 19( 4) : 377- 3811
[ 2] Zou X, Ji C F, Gao S Y. Study on comparison on antitumor ef-
fects of three kinds of aloe polysaccharide [ J]1 H arbin Univ
Commerce ) Nat S ci Edi t ( 哈尔滨商业大学学报#自然科学
版) , 2004, 20( 1) : 14-171
[ 3] Zhang X J, Ji Y B, Qu Z Y, et al . Inf luence of Chaihu Capsule
on RBC membrane fun ction of H22 mice [ J ]1 Chin Tr ad it Pat
Med (中成药) , 2003, 25( 4) : 332- 3331
[ 4] Chen Q. Methodology in Phar macological S tudy on Chinese
Mate ria Medica (中药药理研究方法学 ) [ M ]1 Beijing: Peo-
ple. s Medical Publishing House, 19931
[ 5] Fu G H, Jiang X S, Wang X M , et al . Purification and charac-
terization of C1 peptide released from Band 3 protein [ J]1 Chin
J Pathophysiol (中国病理生理杂志) , 1999, 15( 3) : 271- 2741
[ 6] Jia S H, Zhang X J, Peng H S, et al . Ef fect of polysaccharides
of A loe ver a L. on immune funct ion of red blood cell in S180[ J]1
J Harbin Univ Commerce ) Nat S ci Edi t (哈尔滨商业大学学
报#自然科学版) , 2002, 18( 6) : 607- 6091
[ 7] Cui X B, Fu G H , Jiang X S, et al . The changes of Band 3
protein and membrane f luidity in pathologic condit ion [ J]1 J
Harbin Med Univ (哈尔滨医科大学学报) , 2000, 34 ( 6 ) :
396-3981
[ 8] Sun D G, Shi Y, Chen K, et al . Effect s of membrane proteins
cros-s linking on biomechanical propert ies of the erythrocyte
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2000, 19( 2) : 96-1001
Antilipid peroxidation of polyamines from pilose antler
CHEN Xiao-guang1, JIN Shu-li2, DI Lin1, L IU Xin-yu1, ZHANG Xiao-yu1
*
( 1. Department of Pharmacology , Academy of T raditional Chinese Medicine and Materia Medica of Jilin Province,
Changchun 130021, China; 2. Hospital of Changchun Public T raffic Company, Changchun 130021, China)
Abstract: Object To invest ig ate the antioxidant act iv ity of polyamines isolated f rom pilose ant ler
( PAIPA) . Methods The ef fects of PAIPA on the lipid peroxidation ( MDA format ion) in microsomes of rat
brain, liver, and kidney induced by NADPH-Vitamine C ( Vc) and ferrous-cysteine systems in vit ro, the super-
ox ide anion radical product ion ( reduced cytochrome C format ion) in xanthine-xanthine ox idase system in vit ro,
and the CCl4-and ethano-l induced MDA format ion in mice liver in v ivo were evaluated. Results PAIPA could
signif icantly inhibit the lipid perox idat ion ( MDA format ion) in microsomes of rat brain, liver, and kidney in-
duced by NADPH-Vc and ferrous-cyctein, the superoxide anion radical product ion ( format ion of reduced cy-
tochrome C) in xanthine-xanthine oxidase system in vit ro, and the CCl4- and ethano-l induced MDA format ion
in mice liver in vivo. Conclusion PAIPA exhibits an ant iox idant act iv ity.
Key words: pilose ant loer; polyam ines; lipid perox idat ion; superoxide anion radical
鹿茸多胺的抗脂质过氧化作用
陈晓光1,金淑莉2,邸 琳1, 刘新宇1,张晓宇1
( 11 吉林省中医中药研究院 药理室,吉林 长春 130021; 21 长春公交医院,吉林 长春 130021)
摘 要:目的 研究鹿茸多胺的抗氧化作用。方法 测定鹿茸多胺在体外对 NADPH-维生素 C 和 Fe2+-半胱氨酸
系统诱发的微粒体脂质过氧化反应 ( MDA 形成) 的影响, 对黄嘌呤-黄嘌呤氧化酶系统超氧阴离子自由基 ( OA2 )
#901#中草药 Chinese T raditional and Herbal Drugs 第 35 卷第 8 期 2004年 8月
* 收稿日期: 2003-11- 19作者简介:陈晓光( 1964 ) ) ,男,吉林安图人,助理研究员,硕士, 1991 ) 1993年在日本北里研究所附属东洋医学研究所生化室进修, 主要从事中药生物化学及生化药理研究。T el: ( 0431) 6816836 E-mail: xg chen@ 163. com
产生 (还原型细胞色素 C 形成 ) 的影响, 在体内对 CCl4和乙醇诱发的小鼠肝脂质过氧化反应 ( MDA 形成 ) 的影
响。结果 鹿茸多胺在体外能明显抑制 NADPH-维生素 C 和 Fe2+-半胱氨酸系统诱发的大鼠脑、肝、肾微粒体脂
质过氧化反应 ( MDA 形成) ,及黄嘌呤-黄嘌呤氧化酶系统 O A2 的产生 (还原型细胞色素 C 形成)。在体内能抑制
CCl4和乙醇诱发的小鼠肝脂质过氧化反应 ( MDA 形成)。结论 鹿茸多胺具有抗氧化作用。
关键词:鹿茸; 多胺; 脂质过氧化; 超氧阴离子自由基
中图分类号: R2851 5 文献标识码: A 文章编号 : 0253 2670( 2004) 08 0901 04
The unossif ied pilose antler of Cerv us nipp on
T emminck var. mantchuricus Sw inhoe is one of the
most famous Chinese t radit ional medicines, and is
used for the t reatment of aging syndrome, anemia,
neurosis, impotence, sem inal emission and premature
ejaculat ion. In the prev ious papers, that the ext ract
of pilose ant ler significant ly improved age-related bio-
chem ical factors in aged m ice and show ed obv ious in-
hibition on MAO-B act ivity
[ 1- 3]
was reported. The
polyamines isolated from pilose ant ler ( PAIPA) could
increase the synthesies of protein and RNA, and the
act ivity of RNA polymerase Ò in mice liver cell[ 4, 5] .
How ever, the ant iox idant propert ies of PAIPA have
not yet been clarified. The present paper describes
the ant ilipid peroxidation of PAIPA in vi tro and in
vivo .
1 Materials and methods
111 Animals: M ale Kunming m ice ( 20 ? 2) g and
male Wistar rats ( 220 ? 20) g were obtained from
w riter. s institute animal center and housed in f ree
condit ion w ith food and water supply adlibitum .
T he number of animal eligibility w as 980101018 and
980101017, respect ively.
112 Medicine: The unossified pilose antler of C .
nip p on var. mantchur icus Sw inhoe w as suppled by
Antu Pharmaceut ical Factory of Jilin Province,
ident ified by Prof. GUO Cha-i yu f rom Laboratory of
T radit ional Chinese Materia Medica in writer. s inst-i
tute. The PAIPA was supplied by the Laboratory of
T radit ional Chinese Medicine Formula in w riter. s in-
st itute, composed of 7019% putrescine, 2613%
spermine, and 218% sperm idine af ter HPLC analy-
sis.
1. 3 Chemical reagents: Nicot inamide adenine dinu-
cleot ide phosphate ( NADPH) , superox ide dismutase
( SOD, 3 000 U/ mg) , x anthine ox idase ( XOD, 10
U /mL ) , xanthine ( XAN ) , thiobarbituric acid
( TBA ) , 1, 1, 3, 3- tet ramethoxypropane and bovin
serum albumin w ere the products of Sigma Chem ical
Co. All the chemicals used are of analy tical grade.
1. 4 Preparat ion of microsome: The t issues of Wistar
rat ( fasted for 24 hours before exper-i ments) brain,
liver, and kidney were homogenized w ith the four
volume of TM S buf fer ( 0105 mol/ L Tris-HCl, 012
mol/ L sucrose, 3 mmol/ L MgCl2, pH 715) at 4 e ,
respect ively. The homogenate w as centrifuged at 10
000 r/ m in for 20 m in, and the supernatant w as fur-
ther centrifuged at 105 000 r/ min for 90 m in. T he
pellet of microsome fract ion of brain, liver, and kid-
ney w as resuspended w ith TM S buffer. T he protein
content w as determ ined by the method of Low ry
[ 6]
.
The proteins of microsomal suspension f rom rat brain
( 3 mg/ mL) , liver ( 15 mg/ L ) , and kidney ( 5 mg/
mL) w ere used for this experiment .
1. 5 Measurement of lipid perox idat ion of microsome
induced by NADPH-Vc: The reaction system con-
taining 011 mL of brain, liver, or kidney microsomal
suspension, respect ively, PAIPA 0101 mL at differ-
ent concentrat ions, NADPH 011 mL ( 118 mmol/
L) , Vc 01005 mL ( 5 mmol/ L ) and PBS 018 mL
( 011 mol/ L KH2PO4, 0114 mol/ L NaCl, pH 714)
w as incubated at 37 e for 15 min. After addit ion of
the TBA solut ion ( 0167%) to the system, the con-
tent of malondialdehyde ( MDA) f rom lipid peroxida-
t ion w as measured by TBA method
[ 7]
.
1. 6 Measurement of lipid perox idat ion of microsome
induced by ferrous-cysteine: A reaction system of 1
mL containing 011 mL of brain, liver, or kidney m-i
crosomal suspension, respect ively, PAIPA 0101 mL
at different concentrations, cysteine 0102 mL ( 0101
mol/L ) , ferrous sulfate 0105 mL ( 1 mmol/ L ) and
PBS
[ 7]
was incubated. The MDA format ion was de-
tected as described above.
1. 7 Detect ion of superox ide anion radical product ion
in xanthine-xanthine ox idase system: The superoxide
anion product ion w as detected by the method of cy-
#902# 中草药 Chinese T raditional and Herbal Drugs 第 35 卷第 8 期 2004年 8月
tochrome C reduct ion[ 8] and the format ion of the re-
duced cy tochrome C w as used to indicate the produc-
ing superox ide anion radical in x anthine-xanthine ox-i
dase system indirectly.
1. 8 Measurement of lipid perox idat ion of m ice liver
induced by CCl4 after PAIPA treatment in v ivo : All
the mice w ere randomly divided into four groups,
each consisted of 10 mice, cont rol, CCl4, PAIPA 10
and 20 mg/ kg. PAIPA was iv given to the two
PAIPA groups at dose of 10 and 20 mg/ kg , respec-
t ively, and the saline w as iv given to the control and
CCl4 group at dose of 10 mL/ kg , for three days. One
hour after the last administation, 011% CCl4 ( dis-
solved w ith bean oil) w as ip given to the CCl4 and two
PAIPA groups at dose of 10 mL/ kg, the control
g roup w as only ip given w ith bean oil at the same
dose. Tw o hours later, all mice w ere killed by decap-i
tat ion and protions of the liver were rapidly sampled
to measure the MDA content by the T BA method as
described above.
1. 9 Measurement of lipid perox idat ion of m ice liver
induced by ethanol af ter PAIPA treatment in vivo:
M ice w ere divided into four groups, control, ethanol,
PAIPA 10 and 20 mg / kg. PAIPA was iv given to
tw o PAIPA g roups at doses of 10 and 20 mg/ kg, re-
spect ively, and the saline was iv g iven to the control
and ethanol group at dose of 10 mL/ kg , for three
days. After the last adm inist rat ion, all mice were
fasted for eight hours, then 50% ethanol w as ig g iven
to the ethanol group, and two PAIPA groups at dose
of 15 mL/ kg. Twelve hours later, m ice w ere decap-i
tated and the liver w as dissected out to measure the
MDA content as described above.
1. 10 Statist ical analysis: Data presented w ere x ? s
and stat ist ically evaluated by Student. s t-test.
2 Results
2. 1 Effect of PAIPA on lipid perox idat ion of micro-
somes f rom rat brain, liver, and kidney induced by
NADPH-Vc: As shown in Table 1, PAIPA
could significant ly inhibit the lipid peroxidation
Table 1 Effect of PAIPA on NADPH-Vc induced
MDA formation in microsomes of rat
tissues ( x ? s, n= 4)
Groups
Dose
/ (Lg#mL- 1)
M DA/ ( Lmol#g- 1)
Brain Liver Kidn ey
cont rol - 616 ? 012 1115 ? 015 519 ? 015
PAIPA 1 613 ? 013 1111 ? 015 515 ? 014
10 514 ? 015* 916 ? 017* 416 ? 014*
100 419 ? 016* * 816 ? 016* * 410 ? 013* *
* P< 0105 * * P < 0101 v s control group
( MDA format ion) in microsomes of the rat brain,
liver, and kidney induced by NADPH-Vc at concen-
trat ions of 10 ) 100 Lg/ L, and it show ed the obvious
concentration-effect relat ionship.
2. 2 Effect of PAIPA on lipid perox idat ion of micro-
somes from rat brain, liver, and kidney induced by
ferrous-cysteine: As shown in T able 2, PAIPA could
obviously inhibit the lipid perox idat ion in microsomes
of the rat brain, liver, and kidney induced by ferrous-
cysteine at the concentrat ion of 100 Lg / L , and it also
show ed the obvious concentration-effect relationship.
Table 2 Effect of PAIPA on ferrous-cysteine induced
MDA formation in microsomes of rat
tissues ( x ? s, n= 4)
Groups
Dose
/ (Lg#mL- 1)
M DA/ ( Lmol#g- 1)
Brain Liver Kidn ey
cont rol - 515 ? 015 614 ? 012 517 ? 013
PAIPA 1 512 ? 015 611 ? 013 513 ? 015
10 419 ? 016 517 ? 014* 511 ? 015
100 416 ? 015* 417 ? 016* * 415 ? 013*
* P< 0105 * * P < 0101 v s control group
2. 3 Effect of PAIPA on superox ide anion radical
production in xanthine-xanthine ox idase system: As
shown in T able 3, PAIPA could signif icant ly inhibit
the formation of reduced cytochrome C ( superoxide
anion radical product ion) in xanthinexanthine oxidase
system at a concentration of 100 Lg/ L .
2. 4 Effect of PAIPA on lipid perox idat ion of mice
liver induced by CCl4 in vivo: As shown in Table 4,
PAIPA could signif icantly inhibit the lipid peroxida-
t ion in m ice liver induced by CCl4 at doses of 10 and
20 mg/ kg, and it show ed the obvious dose-ef fect re-
lat ionship.
2. 5 Effect of PAIPA on lipid perox idat ion of mice
liver induced by ethanol in vivo : In Table 5 , the
#903#中草药 Chinese T raditional and Herbal Drugs 第 35 卷第 8 期 2004年 8月
Table 3 Effect of PAIPA on formation of reduced
cytochrome C in xanthine-xanthine
oxidase system ( x ? s, n= 4)
Groups
Dose
/ (Lg#mL- 1)
Reduced cytochrome C
/ ( nmol#mg- 1#min- 1)
cont rol - 1618 ? 113
PAIPA 1 1611 ? 116
10 1519 ? 214
100 1318 ? 211*
* P < 0105 v s control group
Table 4 Effect of PAIPA on CCl4-induced MDA for-
mation in mice liver in vivo ( x ? s, n= 4)
Groups Dose/ ( mg#kg- 1) MDA/ (Lmol#g- 1)
cont rol - 4213 ? 612* *
CCl 4 - 13212 ? 3011
CCl 4+ PAIPA 20 7213 ? 1212* * *
10 10213 ? 1812* *
* * P < 0101 * * * P < 01001 v s CCl4 group
Table 5 Effect of PAIPA on ethano-l induced MDA for-
mation in mice liver in vivo ( x ? s, n= 10)
Groups Dose/ ( mg#kg- 1) MDA/ ( nmol#g- 1)
cont rol - 4415 ? 1216* * *
ethanol - 9811 ? 3011
ethanol+ PAIPA 20 4817 ? 1315* * *
10 5516 ? 2415* *
* * P < 0101 * * * P < 01001 v s ethanol group
lipid perox idat ion in mice liver induced by ethanol w as
signif icantly reduced by PAIPA at doses of 10 and 20
mg/kg, and it show ed the obvious dose-effect rela-
t ionship.
3 Discussion
Lipid perox idat ion by free radicals are involved in
many physiological and pathological processes, such
as the tox ic injury, reperfusion injury, ag ing and car-
cinogenesis. Superox ide anion radical ( OA2 ) could be
generated in the NADPH-Vc induced m icrosome lipid
peroxidation systems, hydroxyl radical ( #OH ) and
superoxide anion radical ( OA2 ) could be generated in
the ferrous-cysteine systems. In the experiments of
NADPH-Vc and ferrous-cysteine init iated lipid perox-
idation of microsomes from the rat brain, liver, and
kidney, PAIPA show ed an ant ilipid peroxidation ac-
t ion in vi tro . It was known that CCl4 could be me-
tabolized to generate the radical (#CCl3) evoking lipid
perox idat ion after cytochrome activat ion in liver cell.
Ethnol also could init iate liver lipid perox idat ion. In
this experiment , PAIPA could significant ly inhibit
the CCl4- and ethano-l induced lipid peroxidat ion in
mice liver in vivo . In order to elucidate mechanisms
of the antioxidant action of PAIPA, the inhibit ion of
OA2 radical product ion ( formation of reduced cy-
tochrome C) in xanthine-xanthine oxidase system was
detected. T he results show ed that PAIPA could sig-
nificant ly inhibit the production of ( OA2 ) radical. It
w as suggested that one of the mechanisms of ant iox-i
dant action of PAIPA possibly be related to PAIPA. s
inhibit ing the product ion of OA2 radical. Further-
more, the molecular mechanism of PAIPA scavenging
oxygen radicals direct ly merits further studies.
Acknowledgement: Mr. Song Ha-i peng ( Max-
Planck-Institute for Polymer Research, Germany)
for assisting in this research should be grateful to.
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#904# 中草药 Chinese T raditional and Herbal Drugs 第 35 卷第 8 期 2004年 8月