全 文 ：中国组织工程研究与临床康复 第 12 卷 第 53 期 2008–12–30 出版
Journal of Clinical Rehabilitative Tissue Engineering Research December 30, 2008 Vol.12, No.53
ISSN 1673-8225 CN 21-1539/R CODEN: ZLKHAH 10579
Department of
Pharmacology, Guilin
Medical College,
Guilin 541001,
Guangxi Zhuang
Autonomous Region,
China

Li Yong-wen☆,
Studying for
doctorate, Associate
professor,
Department of
Pharmacology, Guilin
Medical College,
Guilin 541001,
Guangxi Zhuang
Autonomous Region,
China
liyongwen99@
163.com

Received:2008-05-13
Accepted:2008-06-23
(54200806130013/H)

Li YW, Li L.
Immune-regulating
effects of securidaca
inappendiculata
hassk in rats.
Zhongguo Zuzhi
Gongcheng Yanjiu yu
Linchuang Kangfu
2008;12(53):
10579-10582(China)

[http://www.crter.cn
http://en.zglckf.com]

Immune-regulating effects of securidaca inappendiculata
hassk in rats☆
Li Yong-wen, Li Li

Abstract
BACKGROUND: Many kinds of immunosuppressive drugs, such as cyclosporine A, are commonly used against rejection
in organ transplantation, but are lack of selectivity and specificity. They have potent effective on primary immune response
rather than on secondary immune response.
OBJECTIVE: To develop cell models with low immunity by adding cyclosporine A into splenic lymphocytes for observing
the immune-regulating effects of rat serum containing securidaca inappendiculata hassk on lymphocytes.
DESIGN: Randomized and controlled Chinese medicine serum pharmacological experiment.
TIME AND SETTING: Performed at the Laboratory Center for Medical Science, Guangxi Medical University between
June and September 2007.
MATERIALS: Forty healthy adult Wistar rats were selected for preparation of serum containing securidaca inappendiculata
hassk, and the splenic lymphocytes originated from eight healthy adult Kunming mouse were used for testing immunological
function. Securidaca inappendiculata hassk decoction (Lot No. 20061220) was kindly gifted by Yulin Pharmaceutical
Factory, China. Concanavalin A [ConA, Fluka, Switzerland Lot No.10578] was the product of Fluka Company, Switzerland;
Cyclosporine A (100 mg/L, Lot No. 20070108) was purchased from the North China Drugs Manufacture Group, China.
METHODS: Rats were intragastrically administered securidaca inappendiculata hassk 10.0, 5.0, 2.5 g/kg/d for a total of 7
days, to prepare rat serum containing different levels of securidaca inappendiculata hassk. 20 mg/LConA (50 μL/well),
serum containing securidaca inappendiculata hassk (50 μL/well), cyclosporine A (100 μL/well), and splenic lymphocyte
suspension (100 μL/well) were added into 96-well plate. Serum without medicine was taken as control.
MAIN OUTCOME MEASURES: 96-well plate was inoculated for 68 hours after sample adding and for another 4 hours
after application of methyl thiazolyl tetrazolium (MTT). Absorbance (A) value was determined using enzyme reader.
RESULTS: A value was significantly higher in the non-medicine blood serum + Con A group than in the non-medicine
blood serum (P < 0.01).The medicine blood serum +Con A group had significantly higher A value than non-medicine blood
serum +Con A group ( P < 0.01). A value was significantly lower in the non-medicine blood serum+ Con A+ cyclosporine A
group than in the non-medicine blood serum+ Con A group ( P < 0.01) but was higher than in the non-medicine blood serum
group ( P < 0.05). High- and middle-dose medicine serum + Con A+ cyclosporine A group had higher A value than the
non-medicine blood serum + cyclosporine A group (P < 0.05).
CONCLUSION: Securidaca inappendiculata hassk can improve the immune function of normal mice and elevate the lower
immune function induced by cyclosporine A, which indicates that securidaca inappendiculata hassk has a positive regulatory
effect.


INTRODUCTION

At present, immunosuppressive drugs commonly
used in clinics such as cyclosprine A share the
following common shortcomings: ①Most
immunosuppressives can inhibit both normal and
abnormal immune response of the body due to its
lack of selectivity and specificity. That is the reason
why these drugs can induce infection, increase
incidence of cancer and affect the function of
reproductive system due to its effects of reducing
normal immunity of the body. ②Most
immunosuppressives have strong action to primary
immune response and weak action to secondary
immune response. Chinese medicine is rich in
resources and used for a long time. Searching for
effective, low-toxicity, inexpensive and new
immunosuppressives to prevent rejection and
complication and overcome severe adverse reaction
caused by long-term application of
immunosuooressuves is a hot spot in the study of
Chinese medicine.
Securidaca inappendiculata hassk belongs to the
polygalaceae, the aspiration race of cicada wing
cane, which distributes in India, Burma, Vietnam,
Indonesia and Malaysia. While in our country it
widely grows in Guangdong, Hainan, Guangxi and
Yunnan provinces [1].
So far, there are seven types of chemical
constituents from the vine plants confirmed in
securidaca inappendiculata hassk, such as
flavonoids, xanthone, and terpene, steroidal
glycosides and its glycosides, sugar and
glycosides, alkaloids, organic acid and its
derivatives[2-4]. It is reported that its
pharmacological effects include anti-HIV,
anti-allergic and anti-leukemia, anti-cancer,
antibacterial and other anti-inflammatory
factors [5-8]. In order to further investigate the
mechanism of action of immune regulating effects
of securidaca inappendiculata hassk, we studied
how securidaca inappendiculata hassk enhance the
immune function of normal mouse, resist
cyclosprine A-caused low immunity, and
up-regulate immunity, thereby providing evidence
for clinical application.

MATERIALS AND METHODS

Design: Randomized and controlled Chinese
medicine serum pharmacological experiment.
Basic Medicine

Li YW, et al, Immune-regulating effects securidaca inappendiculata hassk in rats
P.O. Box 1200, Shenyang 110004 cn.zglckf.com 10580
www.CRTER.org
Time and setting
Performed in the Laboratory Center for Medical Science,
Guangxi Medical University between June and September
2007.

Materials: Eight Kunming mice of SPF grade, same number of
male and female, aged 8-10 weeks old, weighing（20±2）g, were
provided by the Laboratory Animal Center, Guangxi Medical
University [permission No. (gui)-2003-0001]. Forty healthy adult
Wistar rats of SPF grade, same number of male and female, aged 3
months old, weighing (250±20) g were included [Manufacture
permission No. SCXKG (gui)-2003-0003; application permission
No. SYKG (gui) 2003-0005].This study was approved by the
Animal Ethic Committee of Guangxi Medical University.

































Methods
Preparation of drug-containing serum
Serum pharmacology method was used. Wistar rats were
administered securidaca inappendiculata hassk (10.0, 5.0 ,
2.5 g/kg/d)and physiological saline for 7 days. Two hours
after the last intragastric administration (prior to intragastric
administration, water but not food was available) and
subsequent to ethyl ether anesthesia, rat blood was taken
from the abdominal aorta, and serum was isolated by
low-speed centrifugation (2 500 r/min, 15 minutes). Next,
the serum was inactivated for 30 minutes at 56 ℃ and
filtered using 0.45 μm millipore filter and 0.22 μm
millipore filter, and finally preserved at –20 ℃.
Detection of splenic lymphocyte proliferation
Mouse splenic lymphocytes were collected. Total number of
cells was obtained, and simultaneously RPMI 1640 (10% fetal
bovine serum volume was calculated to adjust cell
concentration. 20 μg/mL ConA (50 μL/well), serum
containing securidaca inappendiculata hassk (50 μL/well),
cyclosporine A (100 μL/well), and splenic lymphocyte
suspension (100 μL/well). 96-well plate was cultured at 37℃
in a 5% CO2-containing incubator for 68 hours after sample
adding and for another 4 hours after application of methyl
thiazolyl tetrazolium (MTT). Next, 100 μL of hydrochloric
acid/isopropyl alcohol solution (1:300) was added.
Absorbance (A) was determined using an enzyme reader.
Cyclosprine A inhibition rate (%) = (1-A570 value in the
experimental group/A570 value in the blank control group)
×100;Stimulation index = (average A value for the ConA
stimulation well/average A value for the control well)-1.

Design, enforcement and evaluation: Li Yong-wen
designed this study. Li Li and Li Yong-wen performed the
experiments. Professor Huang Ren-bin, doctor’s tutor, who
was from the Department of Pharmacology, Guangxi Medical
University, participated in the evaluation.

Main outcome measures: Splenic lymphocyte
proliferation.

Statistical analysis: Analysis of variance was performed
using SPSS 10.0 software. All experimental data were
expressed as Mean ± SD.

RESULTS

Effect of securidaca inappendiculata
hassk-containing rat serum on splenic lymphocyte
proliferation (Table 1)
























Reagent, medicine
and instrument


Source
RBMI 1640 cell nutrient fluid Gibco Corporation, USA; Cat No.
13200-035
Concanavalin A(ConA) Fluka, Switzerland
Cyclosporine A (100 μg/mL) North China Drugs Manufacture
Group, China
Aseptic Hanks fluid East Saint biotechnology
developmental Co.,Ltd., China
Methyl thiazolyl tetrazolium
(MTT)
Sigma, USA
Hydrochloric acid Third Tianjin Chemical Reagent
Factory, China
Isopropyl alcohol Tianjin Tianxin Fine Chemical
Industry Development Center,
China
Fetal bovine serum Gibco Corporation, USA; Cat No.
16000-036
Physiological saline Tianjin Anling Pharmaceutical
Co.,Ltd.,China
Ethanol Tianjin Chemical Reagent
Co.,Ltd.,China
Ultra-clean working bench Shanghai Pudong Yuexin
Scientific Instrument Factory,
China; pattern No. SZX
Inverted microscope Nanjing Jiangnan Electro-Optic
Group Limited Company,
China; pattern No. XD-101
CO2 incubator Thermo Forma, USA; pattern No.
Mode 1311
Centrifuge Shanghai Anting Scientific
Instrument Factory, pattern No.
TDL-5
Enzyme reader Bio-Rad, USA; pattern No.
TDL-5
Securidaca inappendiculata
hassk decoction
Gifted by Yulin Pharmaceutical
Factory, China and identified as
S. inappendiculata by Professor
Li Mei, who was from the
Department of Medicinal
Plants of Guangxi Medical
University.
Table 1 Effect of securidaca inappendiculata hassk-containing rat
serum on splenic lymphocyte proliferation (x
_
±s, n=8)
Group

A value

Stimulation
index

Cyclosprine A
inhibition rate (%)
Non-medicine
blood serum
0.205±0.047
Non-medicine blood
serum +Con A
0.304±0.068a 0.480±0.194
High dose drug-
containing
serum+Con A
0.585±0.064ac 1.853±0.312
Middle dose drug-
containing
serum+Con A
0.460±0.052ac 1.240±0.372
Low dose drug-
containing
serum+Con A
0.416±0.069ac 1.029±0.312
Non-medicine blood
serum+Con A+CysA
0.227±0.043bc 0.106±0.201 0.290±0.101
High dose drug-
containing serum+
Con A+CysA
0.397±0.068bd 0.937±0.424 0.233±0.102
Middle dose drug-
containing
serum+Con A+Cys A
0.300±0.037bd 0.461±0.224 0.272±0.132
Low dose drug-
containing
serum+Con A+Cys A
0.225±0.035 0.098±0.196 0.262±0.152
Con A: concanavalin A; CysA: cyclosporine A; aP < 0.01, bP < 0.05, vs.
non-medicine blood serum group; cP < 0.01, vs. non-medicine blood serum
+Con A; dP < 0.05, vs. non-medicine blood serum+Con A+CysA group

Li YW, et al, Immune-regulating effects securidaca inappendiculata hassk in rats
ISSN 1673-8225 CN 21-1539/R CODEN: ZLKHAH 10581
www.CRTER.org

Table 1 shows that A value was significantly higher in the
non-medicine blood serum + Con A group than in the
non-medicine blood serum (P < 0.01). A value was
significantly higher in the medicine blood serum + Con A
group than in the non-medicine blood serum+ Con A group
(P < 0.01). This indicates that securidaca inappendiculata
hassk can enhance immune function. A value was
significantly lower in the non-medicine blood serum+ Con
A+ cyclosporine A group than in the non-medicine blood
serum+ Con A group ( P < 0.01) but was higher compared
with the non-medicine blood serum group ( P < 0.05).
High- and middle-dose medicine serum + Con A+
cyclosporine A group had higher A value than the
non-medicine blood serum + cyclosporine A group (P <
0.05). This indicates that securidaca inappendiculata hassk
can resist the low immune function induced by
cyclosporine A, which indicates that securidaca
inappendiculata hassk has a positive regulatory effect.
Factors that may affect the outcomes of study
Carelessness may lead to pollution during the whole
operation. The primary measures in preventing pollution
are as follows: ① Equipment and solution should be
sterilized before use. ② Operators should wear mask and
hat. ③The mouth of culture flask should be sterilized with
75% ethanol. ④Normal operation.

DISCUSSION

Rats, rabbits, and guinea pigs are mostly used to prepare
drug-containing serum in the pharmacological experiments.
The animals used for serum preparation should be
consistent with those used for ex vivo preparation of organs,
tissue, and cells. Thus, the physiological and chemical
differences between animal serums or between animal
serum and human serum can be reduced or avoided, thereby
reducing the immune reactions caused by species
differences and enhancing the reliability of experimental
results.
In the present study, rats were used for preparing
drug-containing serum because they could provide enough
serum to satisfy the experimental requirements.
The present study used drug-containing rat serum to
observe the effects of securidaca inappendiculata hassk on
mouse splenic lymphocyte transformation rate. To better
study the mechanism of action of securidaca
inappendiculata hassk on enhancing mouse immunity, cell
models with low immunity were established by adding
cyclosporine A into splenic lymphocytes to observe the
immune-regulating effects of rat serum containing
securidaca inappendiculata hassk on lymphocytes. Results
demonstrated that securidaca inappendiculata hassk can
improve the immune function of normal mice and elevate
the low immune function induced by cyclosporine A, which
indicates that securidaca inappendiculata hassk, in
particular at high dose, has a positive regulatory effect.

REFERENCES

1 Kunming Institute of Botany, Chinese Academy of Sciences. Flora of
Yun Nnan. Beijing:Science Press 1983:261
2 Zhang LJ, Zhang L, Xu LZ, et al. A new benzophenone from the root
of Securidaca inappendiculata. Yao Xue Xue Bao 2005;40(6):536-538
3 Shi L, Kang WY. Study on chemical constituents in rhizome of
Securidaca inappendiculata. Zhongguo Zhong Yao Za Zhi 2008;
33(7):780-782
4 Zhang LJ, Zhang L, Xu LZ, et al. A new benzophenone from the root
of Securidaca inappendiculata. Yao Xue Xue Bao 2005;40(6):536-538
5 Cao Y, Xia QH, Meng H, et al. Pharmacological effects of serum
containing chinese medicine Bushen Huayu Jiedu Compound Recipe
in lung cancer drug-resistance cells. Chin J Integr Med 2008;14(1):
46-50
6 Bochu W, Liancai Z, Qi C. Primary study on the application of Serum
Pharmacology in Chinese traditional medicine. Colloids Surf B
Biointerfaces 2005;43(3-4):194-197
7 Tang X, Tian X, Ci L. Preparation and evaluation of pH-dependent
gradient-release pellets for TCM.Drug Dev Ind Pharm
2004;30(10):1079-1087
8 Yui S, Ubukata K, Hodono K, et al. Macrophage-oriented cytotoxic
activity of novel triterpene saponins extracted from roots of
Securidaca inappendiculata. Int Immunopharmacol 2001;1(11):
1989-2000






















Li YW, et al, Immune-regulating effects securidaca inappendiculata hassk in rats
P.O. Box 1200, Shenyang 110004 cn.zglckf.com 10582
www.CRTER.org

蝉翼藤对大鼠免疫功能的调节作用☆

李勇文，李 丽(桂林医学院药理学教研室，广西壮族自治区桂林市 541001)

李勇文☆，男，1969 年生，广西壮族自治区
桂林市人，汉族，广西医科大学在读博士，
副教授，主要从事中药药理学的研究。
摘要
背景：临床以环抱素A为代表的免疫抑制药，
多数应用于器官移植后抗排斥反应时缺乏选
择性和特异性，对初次免疫应答反应的抑制
作用较强，对再次免疫应答反应抑制作用较
弱。
目的：采用环抱素 A 免疫抑制剂加入脾淋巴
细胞中建立免疫功能低下细胞模型，观察中
药含蝉翼藤的大鼠血清对免疫抑制淋巴细胞
免疫功能的调节效应。
设计：随机对照中药血清药理学实验。
时间及地点：实验于 2007-06/09 在广西医科
大学医学科学实验中心完成。
材料：健康成年 Wistar 大鼠 40 只制备含蝉
翼藤血清，健康成年昆明小鼠脾细胞为受药
对象。蝉翼藤水煎液由玉林制药厂惠赠，水
煎煮浓缩备用。刀豆蛋白为瑞士 Fluka 公司
产品，环孢素 A 100 mg/L 为华北制药集团产
品。
方法：将 Wistar 大鼠随机分为 4 组，每组 10
只，分别灌胃高中低剂量蝉翼藤 10.0，5.0，
2.5 g/（kg·d）和等量的生理盐水（空白对
照组），共 7 d，制备含蝉翼藤大鼠血清。向
96 孔培养板中分别加入 20 mg/ L 刀豆蛋白
(50μL/孔)、蝉翼藤药物血清(50μL/孔)、环
孢素 A (10μL/孔)、脾细胞悬液(100μL/孔)。
设未加药血清为对照组。
主要观察指标：96 孔板加样后孵育 68 h，加
入 MTT 再孵育 4 h 后，用酶标仪测定样本吸
光度值（A 值）。
结果：空白血清+刀豆蛋白组较空白血清组 A
值高（P < 0.01），含药血清＋刀豆蛋白组比
空白血清+刀豆蛋白组的 A 值高（P < 0.01），
高剂量含药血清组最为显著。空白血清+刀
豆蛋白+环孢素 A 组较空白血清+刀豆蛋白
组 A 值低（P < 0.01），但比空白血清组的 A
值高（P < 0.05），高中剂量含药血清＋刀豆
蛋白＋环孢素A组较空白血清＋刀豆蛋白＋
环孢素 A 的 A 值高（P < 0.05）。
结论：蝉翼藤可以增强正常小鼠的免疫功能，
拮抗环抱素 A 引起的免疫功能低下，起到免疫
正向调节作用，且以高剂量含药血清组为著。
关键词：蝉翼藤；淋巴细胞转化；正向调节
中图分类号: R617 文献标识码: B
文章编号: 1673-8225(2008)53-10579-04
李勇文，李丽.蝉翼藤对大鼠免疫功能的调节
作用 [J].中国组织工程研究与临床康复，
2008，12(53):10579-10582
[http://www.crter.org http://cn.zglckf.com]
(Edited by Yu XP/Song LP/Wang L)























































































○ SCI 引文库收录 CRTER 已发表的
文章 137 篇。

○ 美国《化学文摘》(CA)收录 CRTER
杂志2006年发表的文章1 543篇，2007
年发表的文章 1 425 篇。

○ 荷兰《医学文摘库 /医学文摘》
(EM) 及 Scopus 数所库收录 CRTER
2007 年发表的文章 2 752 篇。2008
年上半年发表的文章 1 488 篇。
○ 上述数据说明 CRTER 出版的文章
已经反映了中国组织工程研究的前沿
学术水平，并得到了国际重要数据库
的关注和学科专家的认可，因此，在
CRTER发表文章可有较大机会实现单
篇文章被国际著名数据库收录及引用
的愿望，提升了稿件的学术价值和国
际传播率及显示度。

○ CRTER 欢迎各类基金项目或重点
实验室课题资助文章以及博士、硕士
研究生的优秀课题文章投稿。


在线投稿：
www.crter.org (右上角)
cn.zglckf.com (右上角)
咨询电话：024-23384352

CRTER 发表文章被 SCI 引文库，CA，EM 数据库收录的最新数据
ISSN 1673-8225 CN 21-1539/R 2008 年版权归《中国组织工程研究与临床康复》杂志社所有