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顾源远1 陈建伟1,2* 李祥1,3 许惠琴1 王明艳4
（1 南京中医药大学药学院，江苏南京，210046；2 江苏省方剂研究重点实验室，江苏南京，210046；3 江苏省中药炮制重点实验室，江苏南京，210029；
4 南京中医药大学基础医学院，江苏南京，210046）
摘要 目的：研究从明党参果实超临界提取物中分得的两个呋喃香豆素化合物对体外培养的人脐静脉内皮
细胞（HUVEC）氧化应激损伤的保护作用，为其在心血管系统疾病尤其是动脉粥样硬化疾病上的运用提
第九届世界中医药大会
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供理论依据。方法：采用过氧化氢（H2O2）建立体外培养的HUVEC细胞氧化应激损伤模型。细胞种板经
过24h孵育贴壁后，将细胞分为正常对照组、H2O2氧化损伤组、H2O2加药物组，除前2组外，其余各给药
组给予药物预培养24 h，然后加入180 μmol•L-1H2O2，继续培养4 h。MTT法检测细胞存活率；检测各组细
胞中超氧化物歧化酶（SOD）活性水平，一氧化氮合酶（NOS）活性水平，细胞培养液丙二醛（MDA）水
平，一氧化氮（NO）水平；用流式细胞仪检测细胞凋亡水平。结果：珊瑚菜内酯以及5-羟基-8-甲氧基补
骨脂素能明显改善H2O2 （180 μmol•L-1）所致的HUVEC细胞形态学损伤，提高细胞存活率；提高细胞内SOD、
NOS活性，使培养液中NO含量增加，脂质过氧化物MDA生成减少；减少H2O2诱导的细胞凋亡率，恢复血
管内皮细胞增殖。结论：珊瑚菜内酯和5-羟基-8-甲氧基补骨脂素具有较好的内皮细胞保护作用，其机制可
能是与抗脂质过氧化，对氧自由基的清除，以及抗细胞凋亡作用有关。
关键词 明党参；珊瑚菜内酯；5-羟基-8-甲氧基补骨脂素；氧化损伤；人脐静脉内皮细胞；细胞凋亡
Protective Effects of Two Coumarins in Changium smyrnioides Wolff on Human Umbilical Vein
Endothelial Cell Injury Induced by Hydrogen Peroxide
Gu Yuanyuan1 Chen Jianwei1，2* Li Xiang1，3 Xu Huiqin1 Wang Mingyan4
（1College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210046，China；2Jiangsu Key
Laboratory for TCM Formulae Research, Nanjing 210046，China；3Jiangsu Key Laboratory for Chinese Material
Medica Processing，Nanjing 210029，China；4 Basic medical college, Nanjing University of Chinese Medicine,
Nanjing 210046，China）
ABSTRACT Objective: To study the protective effect of two coumarins separated from the fruit of Changium
smyrnioides Wolff. on human umbilical vein endothelial cell (HUVEC) injury induced by H2O2. Hope to provide
a theoretical basis for the clinical application of these two compounds in cardiovascular diseases, especially in
atherosclerotic diseases. Methods: Using H2O2 to establish the injured model of HUVEC cells. After HUVEC
cells being cultured in 96 well plates for 24 h, they were divided into three groups: normal group, H2O2 injured
group and phellopterin, 5-hydroxyl-8-methoxy-psoralen with different concentrations treated group. HUVEC cells
in drug treatment group were pre-cultured with concentrations of phellopterin and 5-hydroxyl-8-methoxy-psoralen
for 24 h, then added 180 μmol•L-1 H2O2 to treat for 4 hours. The Survival rate of HUVECs was determined by
microplate reader. The intracellular activities of superoxide dismutase (SOD), total nitric oxide synthase (NOS),
extracellular nitric oxide (NO) and malondialdehyde (MDA) level were detected by detection kits. The apoptotic
index was detected by flow cytometry. Results: phellopterin and 5-hydroxyl-8-methoxy-psoralen can improve the
morphological damage of HUVEC cells induced by H2O2, promote the viability of injured endothelial cells and
increase the activity of SOD, NOS, while decrease NO and MDA production. The apoptotic index were reduced
by both phellopterin and 5-hydroxyl-8-methoxy-psoralen. Conclusion: Both phellopterin and 5-hydroxyl-
8-methoxy-psoralen have protective effects on endothelial cells injured by H2O2, the mechanism may be related to
their effects of anti-lipid peroxidation, oxygen free radicals scavenging and anti-apoptosis.
Key words Changium smyrnioides Wolff.; phellopterin; 5-hydroxyl-8-methoxy-psoralen; oxidative stress;
human umbilical vein endothelium vascular; cell apoposis
Vascular endothelial cells have a variety of physiological functions, involving blood coagulation, immunology,
material transfer and release of bioactive substances and other important life activities. Oxidative stress, which
mediates endothelial cell injury by producing oxygen free radical, is closely
related
to the pathological process of atherosclerosis[1]. Therefore, the search for new
anti-free radicals and endothelial cells protective drugs is of great significance
for prevention and treatment of atherosclerotic diseases.
Changium smyrnioides Wolff is a unique and single species of Umbelliferae
medicinal plants. The medicine can moisturize lung, dissipate phlegm to stop
coughing, regulate stomach to stop vomit, detoxificate and subside swelling.
In accordance with the views of chemical plant taxonomy, most Umbelliferae
plants contain coumarins. However, the past researches about Changium
smyrnioides Wolff put more emphasis on its beneficial and allergenic
ingredients. Existing researches had neither found its coumarin compositions
nor reported the pharmacological effect of coumarin constituents in Changium
smyrnioides Wolff both at home and abroad. Through in-depth study, our
group seperated two furancoumarins (phellopterin,
O O
OR
O
OCH3
I R= II R=H
I: phellopterin
II: 5-hydroxyl-8-methoxy-psoralen
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5-hydroxyl-8-methoxy-psoralen) from the fruits and roots of Changium smyrnioides Wolff for the first time[2-3].
Modern pharmacological studies have shown that furanocoumarins, which is for clinical use of cardiovascular
diseases and arteriosclerosis, have strong pharmacological activity, including the effects of expansion of coronary
artery, calcium antagonist and vasodilator. Our grouphad found the alcohol extract of Changium smyrnioides
Wolff can raise blood serum SOD and GSH-*** to decrease the increased level of lipid peroxide induced by high
cholesterol, but the physical foundation is not clear[4]. It is reasoned that the two coumarins in Changium
smyrnioides Wolff fruits may protect the injured vascular endothelial cells induced by oxidative stress, thus can
treat cardiovascular diseases and prevent atherosclerosis. In order to explore whether these two compounds have
such protective effects, this study use H2O2 to establish HUVEC oxidative stress injury model in order to observe
protective effects of different concentrations of phellopterin, 5-hydroxyl-8-methoxy-psoralen and its mechanism.
1 Materials and Methods
1.1 Materials phellopterin, 5-hydroxyl-8-methoxy-psoralen (purity ≥ 99%, supplied by our laboratory);
Newborn bovine serum (NBS) (It was purchased from Hangzhou holly leaf Biological Engineering Materials Co.,
Ltd.); DMEM/F12 medium powder (It was purchased from Gibco Company); SOD, NOS, NO, MDA detection kit
(They were purchased from Nanjing Jiancheng Biological Engineering Institute); Annexin V-FITC/PI Apoptosis
Detection Kit (It was purchased from KGI company).
1.2 Methods
1.2.1 Cell culture HUVECs were obtained from the school of Basic Medical in our university. They were
grown in medium DMEM supplemented with 10% fetal bovine serum (FBS), 1 × 105 IU•L-1 penicillin and 100
mg•L-1 streptomycin. Cells were cultured in 50 mL dishes and grown in a 37 ℃, 5% CO2 humidified incubator.
When the cell state is stable, divide cells and continue the next step experiment.
1.2.2 Cell division and MTT test HUVEC cells were seeded in 96 well plates at a density of 5×103 cells per
well, after 24 h, adherent cells were randomly divided into 3 groups: normal control group, H2O2 model group,
phellopterin (0.01 μmol•L-1, 0.1 μmol•L-1, 1 μmol•L-1, 5 μmol•L-1) treated group and
5-hydroxyl-8-methoxy-psoralen (0.01 μmol•L-1, 0.05 μmol• L-1, 0.5 μmol•L-1 ,5 μmol•L-1) treated group. Drug
treated groups were medicated with coumarin compounds for 24 h, then add H2O2 (180 μmol•L-1) for 4h. Remove
the medium, add 1 g•L-1 MTT 100 μL and incubate for 4 h. Finally, move the supernatant, add 150 μL DMSO,
determine the absorbance on microplate reader at 490 nm wavelength after mixing and calculate the cell viability.
1.2.3 Determination of NO in culture medium Cells were divided and treated according to the "1.2.2" method,
then collected the supernatant. According to the method in kit instructions, measure the NO content in the culture
supernatant. All experiments were repeated at least 3 times.
1.2.4 Determination of MDA levels in culture medium Cells were divided and treated according to the "1.2.2"
method, then collected the supernatant. According to the method in kit instructions, detect the MDA content in the
culture supernatant. All experiments were repeated at least 3 times.
1.2.5 Determination of SOD, NOS activity in cells Cells were divided and treated according to the "1.2.2"
method, then collected the supernatant. In accordance with the method described in the kit instructions, determine
the SOD, NOS activity in HUVECs. All experiments were repeated at least 3 times.
1.2.6 Detection of apoptosis by Flow cytometry Cells were seeded in 6 well plate, treated and divided
according to "1.2.2" method, then suspended cells in PBS, collected 1×106 cells. Stain the collected cells by the
method in Annexin V-FITC/PI kit instructions and detect the apoptosis by flow cytometry.
1.2.7 Statistical analysis Use SPSS 11.5 statistical software to analyze the experimental data with x ± s. Use
analysis of variance to make multi-group comparison.
2 Results
2.1 Establishment of H2O2 injury model and effects of drugs on normal cell viability
As shown in Fig.1-2,1-3, incubating cells with 180 μmol•L-1 H2O2 for 4 h allowed survival rate of HUVEC cells
dropped to 50.4%, thus using this method to establish damage model. Experimental results showed that both
phellopterin (concentration 0.01~5 μmol•L-1) and 5-hydroxyl-8-methoxy-psoralen (concentration 0.01~5 μmol•L-1
had no significant cytotoxic effect on HUVECs (Fig.1-4A, B). Therefore, chosing the two drugs over the above
concentration range can exclude the toxicity effect on HUVEC cells, would not affect cell viability and
interference the results. Therefore, the following test chose phellopterin and 5-hydroxyl-8-methoxy-psoralen dose
groups from 0.01~5 μmol•L-1 to do experiments.
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Normal cells Injured cells
Fig.1-2 Micrographs of endothelial cells (×200)
2.2 Effects of drugs on viability of H2O2 injured cell
H2O2 can decrease viability rate of human umbilical vein endothelial. there were significant differences (P <0.01)
comparing with the control group. Tab.1-2A showed that phellopterin can inhibited the damage caused by
hydrogen peroxide significantly (P<0.01) from 0.01 to 5 μmol•L-1. While the drug concentration increased, cell
survival rate increased (Fig.1-4A); Tab.1-2B showed that 5-hydroxyl-8-methoxy-psoralen also had a significant
(P<0.01) inhibition of the damage caused by hydrogen peroxide from 0.01 to 5 μmol•L-1. The effects are also dose
dependent (Fig.1-4B).
2.3 Effects of drugs on NO content and NOS activity of vascular endothelial cell
After endothelial cells were incubated with drugs for 24h and then treated with H2O2 for 4 h, the intracellular NOS
activity dropped from 0.3155 U•ml-1 to 0.2305 U•mL-1, NO content in culture medium was from 43.05 μmol•L-1
down to 24.07 μmol•L-1. Tab.1-3A showed that phellopterin can increase intracellular NOS activity to 0.2759
U•ml-1, increase NO content in culture medium to 32.88 μmol•L-1 from 0.01 to 5 μmol•L-1. It showed a
dose-dependent within the concentration range, while the drug concentration increased, NO content and NOS
activity increased. On the while, Tab.1-3B showed that 5-hydroxyl-8-methoxy-psoralen can increase intracellular
NOS activity to 0.2801 U•ml-1, increase the NO content in culture medium to 37.28 μmol•L-1 from 0.01 to 5
μmol•L-1, it also showed the same dose-dependent as phellopterin.
2.4 Effects of drugs on MDA and SOD levels
H2O2 injury can cause the release of intracellular MDA increasing (P<0.01) and SOD activity significantly
lowering (P <0.01); Tab.1-4A showed that, compared with the normal cells, after injured cells being pretreated by
phellopterin, from 0.01 to 5 μmol•L-1 , the release of MDA decreased, while SOD activity increased. It showed a
dose-dependent within the concentration range. Likewise, Tab.1-4B showed that, after injured cells being
pretreated by 5-hydroxyl-8-methoxy-psoralen, from 0.01 to 5 μmol•L-1, the release of MDA decreased, while SOD
activity increased comparing with the normal cells. It also showed a dose-dependent within the concentration
range.
2.5 Effects of drugs on HUVEC apoptosis
Flow cytometry analysis revealed that the apoptosis rate of H2O2 injured HUVEC cells was 10.7% (The apoptosis
rate of normal cells was 2.2%). After injured cells being pretreated by phellopterin, the early apoptosis rate can be
reduced to 3.8 % at 5 μmol•L-1. It showed a dose-dependent within the concentration range. Likewise, after
injured cells being pretreated by 5-hydroxyl-8-methoxy-psoralen, the early apoptosis rate can be reduced to 2.4 %
at 5 μmol•L-1. It also showed a dose-dependent within the concentration range (Fig.1-6A~J).
0
20
40
60
80
100
120
control
100
150
180
200
300
400
500
600
H2O2/umol
cell survival/% of control
Fig.1-3 The cell viability on H2O2 injured HUVEC
A B
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0
20
40
60
80
100
120
control 0.01 0.1 1 5
phellopterin/umol
cell survival/% of control
0
20
40
60
80
100
120
control 0.01 0.05 0.5 5
5-hydroxyl-8-methoxy-psoralen/umol
cell survival/% of control
Fig.1-4A, B The effect of pellopterin and 5–hydroxyl-8-methoxy-psoralen on normal HUVECs（x ±s, n = 6）
A B
-10
10
30
50
70
90
110
control model 0.01 0.05 0.5 5
5-hydroxyl-8-methoxy-psoralen/umol.L-1
cell survival/% of control
0
20
40
60
80
100
120
control model 0.01 0.1 1 5
Phellopterin/umol.L-1
cell survival/% of control
Fig.1-5A, B The protective of phellopterin and 5-hydroxy-8-methoxy psoralen on H2O2 injured HUVECs（x ±s, n
= 6）
A
B
C
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D
E
F
G
H
I
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J
Fig. 1-6 Effect of phellopterin and 5-hydroxy-8-methoxy psoralen on apoptotic rate in H2O2 induced HUVEC
cells
A: Control group; B: Model group; C: phellopterin 0.01 μmol•L-1 ; D: phellopterin 0.1 μmol•L–1; E: phellopterin 1
μmol•L-1
F: phellopterin 5 μmol•L–1; G: 5-hydroxy-8-methoxy psoralen 0.01 μmol•L–1; H:5-hydroxy-8-methoxy psoralen
0.05 μmol•L–1; I5-hydroxy-8-methoxy psoralen 0.5 μmol•L–1; J:5-hydroxy-8-methoxy psoralen 5 μmol•L–1
Tab. 1-2A Effects of phellopterin and hydrogen peroxide on the viability of HUVEC (%,x ±s, n=6)
Phellopterin/μmol•L-1 H2O2/μmol•L-1 Viability/%
0 0 100±0.06
0 180 54.12±0.01##
0.01 180 71.04±0.04**
0.1 180 73.26±0.02**
1 180 73.84±0.03**
5 180 80.81±0.07**
## P < 0.01 vs normal group , ** P < 0.01 vs H2O2 injured group
Tab.1-2B Effects of 5-hydroxy-8-methoxypsoralen and hydrogen peroxide on the viability of HUVEC (%,x ±s,
n=6)
5-Hydroxy-8-methoxypsoralenPhellopterin /μmol•L – 1 H2O2/μmol•L- 1 Viability/%
0 0 100±0.06
0 180 54.12±0.01##
0.01 180 69.61±0.01**
0.05 180 70.70±0.02**
0.5 180 71.32±0.01**
5 180 74.59±0.02**
## P < 0.01 vs normal group , ** P < 0.01 vs H2O2 injured group
Tab.1-3A Effects of phellopterin on release of nitric oxide and nitric oxide synthase from HUVEC injured by
H2O2 (x ±s, n=3)
Groups NO（μmol•L- 1） NOS（U•mL- 1）
Normal 43.05±0.001 0.3155±0.2
H2O2 injured 24.07±0.002## 0.2305±0.002##
Phellopterin 0.01μmol•L- 1 dose 28.57±0.01 0.2417±0.01
Phellopterin 0.1μmol•L- 1 dose 28.77±0.002* 0.2606±0.004**
Phellopterin 1μmol•L- 1 dose 29.65±0.02* 0.2641±0.003**
Phellopterin 5μmol•L- 1 dose 32.88±0.02* 0.2759±0.01**
## P < 0.01 vs normal group， * P < 0.05 vs H2O2 injured group，** P < 0.01 vs H2O2 injured group
Tab.1-3B Effects of 5-hydroxy-8-methoxypsoralen on release of nitric oxide and nitric oxide synthase from
HUVEC injured by H2O2 (x ±s, n = 3)
Groups NO（μmol•L- 1） NOS（U•mL- 1）
Normal 43.05±0.001 0.3155±0.2
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H2O2 injured 24.07±0.002## 0.2305±0.002#
5-Hydroxy-8-methoxypsoralen 0.01μmol•L- 1 dose 29.65±0.005* 0.2594±0.01
5-Hydroxy-8-methoxypsoralen 0.05μmol•L- 1 dose 33.61±0.02* 0.2677±0.007*
5-Hydroxy-8-methoxypsoralen 0.5μmol•L- 1 dose 34.44±0.001** 0.2712±0.002*
5-Hydroxy-8-methoxypsoralen 5μmol•L- 1 dose 37.28±0.01** 0.2801±0.004*
## P < 0.01 vs normal group， * P < 0.05 vs H2O2 injured group，** P < 0.01 vs H2O2 injured group
Tab.1-4 A Effects of phellopterin on release of MDA and SOD dismutase from HUVEC injured by H2O2
(x ±s, n=3)
Groups MDA（nmol•mL- 1） SOD（U•mL- 1）
Normal 2.07±0.001 13.24±0.02
H2O2 injured 3.65±0.001## 5.645±0.001##
Phellopterin 0.01μmol•L- 1 dose 2.83±0.002 8.689±0.009*
Phellopterin 0.1μmol•L- 1 dose 2.64±0.003* 9.943±0.01
Phellopterin 1μmol•L- 1 dose 2.45±0.003* 10.83±0.004**
Phellopterin 5μmol•L- 1 dose 2.36±0.003* 11.38±0.008**
## P < 0.01 vs normal group， * P < 0.05 vs H2O2 injured group，** P < 0.01 vs H2O2 injured group
Tab.1-4B Effects of 5-hydroxy-8-methoxypsoralen on release of MDA and SOD dismutase from HUVEC injured
by H2O2 (x ±s, n=3)
Groups MDA(nmol•ml- 1) SOD(U•ml- 1)
Normal 2.07±0.001 13.24±0.02
H2O2 injured 3.65±0.001## 5.645±0.001##
5-Hydroxy-8-methoxypsoralen 0.01μmol•L- 1 dose 3.16±0.003* 6.203±0.01
5-Hydroxy-8-methoxypsoralen 0.05μmol•L- 1 dose 2.78±0.004 10.78±0.003**
5-Hydroxy-8-methoxypsoralen 0.5μmol•L- 1 dose 2.58±0.002* 11.38±0.01*
5-Hydroxy-8-methoxypsoralen 5μmol•L- 1 dose 2.22±0.002** 11.85±0.004**
## P < 0.01 vs normal group , * P < 0.05 vs H2O2 injured group, ** P < 0.01 vs H2O2 injured group
3 Discussions
Vascular endothelial cells locate between blood and tissue, and contact blood directly. They are vulnerable to the
pathophysiological changes in the blood and these changes are easy to damage the functional inte***ce of vascular
endothelial cells. When body suffers from the stimulatiom damage and cardiovascular risk, vascular endothelium
cells were the first actor, accompany with endothelial cell dysfunction. In this study, HUVEC cells were treated
with H2O2 so as to simulate the oxidative stress simulation to vascular endothelial cell. The results showed that,
incubating HUVEC cells with H2O2 for 4 h could induce oxidative stress damage to HUVEC cells and the
survival rate decreased significantly (P<0.01). Pretreating cells with different concentrations of phellopterin and
5-hydroxyl-8-methoxy- psoralen from 0.01 to 5 μmol•L-1 for 24 h can enhance the survival rate of HUVEC cells
under oxidative stress. The effects were dose dependent.
Under normal circumstances, the speed of intracellular reactive oxygen species production and clearance keeps
balance. However, when the production of reactive oxygen species exceeds the scavenging capacity of
antioxidants, the bioavailability of reactive oxygen species will increase. It will cause endothelial cell dysfunction,
decrease endothelial endogenous NO content and enhance vasoconstriction. Those will lead to vascular smooth
muscle cell proliferation, macrophage infiltration and inflammation[5-7]. This strdy found that different
concentrations of phellopterin and 5-hydroxyl-8-methoxy-psoralen can enhance intracellular H2O2-induced SOD
activity, reduce MDA formation, increase total NOS activity in cells and increase NO content levels. The results
indicated that these two coumarins in Changium smyrnioides Wolff can increase endogenous oxidative defense
system activity, scavenge free radicals, promote endogenous NO production and release and enhance the ability of
antioxidant to protect cells injured by oxidative stressl.
Oxidative damage induced by the hypertension, ischemia/reperfusion, inflammation can cause apoptosis of
cardiac and vascular endothelial cells, antioxidants and free radical scavengers can partially reverse the apoptosis
process[8]. It was found that the early apoptosis rate of HUVECs injured by H2O2 for 4 h was significantly higher
than that of the normal cells. Both phellopterin and 5-hydroxyl-8-methoxy-psoralen can significantly reduce the
第九届世界中医药大会
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early apoptosis rate. The results indicated that these two coumarins have distinct anti-apoptotic effects, and we
still need further study to find by which way these two compoumds can block the apoptosis cascade reaction.
In this study, we use H2O2 to produce oxidative stress injured model of vascular endothelial cells and observe the
protective effects of phellopterin and 5-hydroxyl-8-methoxy-psoralen on the injured HUVEC cells. It was found
that these two compounds can increase the activity of intracellular antioxidant system, regulate NOS activity of
cells, promote the restoration of normal levels of NO formation, and show a distinct anti-apoptosis activity. These
effects may be the reason why both phellopterin and 5-hydroxyl-8-methoxy-psoralen can play the role of
protecting H2O2 injured HUVEC cells. This results provided a bright prospect for our further study of the potential
cardiovascular prevention and treatment drugs for atherosclerosis related diseases.
Acknowledgement This project was financially supported by Specialized Research Fund for the Doctoral
Program of Higher Education (No. 200803150009)
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（1 南京中医药大学药学院，江苏南京，210046；2 江苏省方剂研究重点实验室，江苏南京，210046；3 江苏省中药炮制重点实验室，江苏南京，210029；
4 南京中医药大学基础医学院，江苏南京，210046）
摘要 目的：研究从明党参果实超临界提取物中分得的两个呋喃香豆素化合物对体外培养的人脐静脉内皮
细胞（HUVEC）氧化应激损伤的保护作用，为其在心血管系统疾病尤其是动脉粥样硬化疾病上的运用提
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供理论依据。方法：采用过氧化氢（H2O2）建立体外培养的HUVEC细胞氧化应激损伤模型。细胞种板经
过24h孵育贴壁后，将细胞分为正常对照组、H2O2氧化损伤组、H2O2加药物组，除前2组外，其余各给药
组给予药物预培养24 h，然后加入180 μmol•L-1H2O2，继续培养4 h。MTT法检测细胞存活率；检测各组细
胞中超氧化物歧化酶（SOD）活性水平，一氧化氮合酶（NOS）活性水平，细胞培养液丙二醛（MDA）水
平，一氧化氮（NO）水平；用流式细胞仪检测细胞凋亡水平。结果：珊瑚菜内酯以及5-羟基-8-甲氧基补
骨脂素能明显改善H2O2 （180 μmol•L-1）所致的HUVEC细胞形态学损伤，提高细胞存活率；提高细胞内SOD、
NOS活性，使培养液中NO含量增加，脂质过氧化物MDA生成减少；减少H2O2诱导的细胞凋亡率，恢复血
管内皮细胞增殖。结论：珊瑚菜内酯和5-羟基-8-甲氧基补骨脂素具有较好的内皮细胞保护作用，其机制可
能是与抗脂质过氧化，对氧自由基的清除，以及抗细胞凋亡作用有关。
关键词 明党参；珊瑚菜内酯；5-羟基-8-甲氧基补骨脂素；氧化损伤；人脐静脉内皮细胞；细胞凋亡
Protective Effects of Two Coumarins in Changium smyrnioides Wolff on Human Umbilical Vein
Endothelial Cell Injury Induced by Hydrogen Peroxide
Gu Yuanyuan1 Chen Jianwei1，2* Li Xiang1，3 Xu Huiqin1 Wang Mingyan4
（1College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210046，China；2Jiangsu Key
Laboratory for TCM Formulae Research, Nanjing 210046，China；3Jiangsu Key Laboratory for Chinese Material
Medica Processing，Nanjing 210029，China；4 Basic medical college, Nanjing University of Chinese Medicine,
Nanjing 210046，China）
ABSTRACT Objective: To study the protective effect of two coumarins separated from the fruit of Changium
smyrnioides Wolff. on human umbilical vein endothelial cell (HUVEC) injury induced by H2O2. Hope to provide
a theoretical basis for the clinical application of these two compounds in cardiovascular diseases, especially in
atherosclerotic diseases. Methods: Using H2O2 to establish the injured model of HUVEC cells. After HUVEC
cells being cultured in 96 well plates for 24 h, they were divided into three groups: normal group, H2O2 injured
group and phellopterin, 5-hydroxyl-8-methoxy-psoralen with different concentrations treated group. HUVEC cells
in drug treatment group were pre-cultured with concentrations of phellopterin and 5-hydroxyl-8-methoxy-psoralen
for 24 h, then added 180 μmol•L-1 H2O2 to treat for 4 hours. The Survival rate of HUVECs was determined by
microplate reader. The intracellular activities of superoxide dismutase (SOD), total nitric oxide synthase (NOS),
extracellular nitric oxide (NO) and malondialdehyde (MDA) level were detected by detection kits. The apoptotic
index was detected by flow cytometry. Results: phellopterin and 5-hydroxyl-8-methoxy-psoralen can improve the
morphological damage of HUVEC cells induced by H2O2, promote the viability of injured endothelial cells and
increase the activity of SOD, NOS, while decrease NO and MDA production. The apoptotic index were reduced
by both phellopterin and 5-hydroxyl-8-methoxy-psoralen. Conclusion: Both phellopterin and 5-hydroxyl-
8-methoxy-psoralen have protective effects on endothelial cells injured by H2O2, the mechanism may be related to
their effects of anti-lipid peroxidation, oxygen free radicals scavenging and anti-apoptosis.
Key words Changium smyrnioides Wolff.; phellopterin; 5-hydroxyl-8-methoxy-psoralen; oxidative stress;
human umbilical vein endothelium vascular; cell apoposis
Vascular endothelial cells have a variety of physiological functions, involving blood coagulation, immunology,
material transfer and release of bioactive substances and other important life activities. Oxidative stress, which
mediates endothelial cell injury by producing oxygen free radical, is closely
related
to the pathological process of atherosclerosis[1]. Therefore, the search for new
anti-free radicals and endothelial cells protective drugs is of great significance
for prevention and treatment of atherosclerotic diseases.
Changium smyrnioides Wolff is a unique and single species of Umbelliferae
medicinal plants. The medicine can moisturize lung, dissipate phlegm to stop
coughing, regulate stomach to stop vomit, detoxificate and subside swelling.
In accordance with the views of chemical plant taxonomy, most Umbelliferae
plants contain coumarins. However, the past researches about Changium
smyrnioides Wolff put more emphasis on its beneficial and allergenic
ingredients. Existing researches had neither found its coumarin compositions
nor reported the pharmacological effect of coumarin constituents in Changium
smyrnioides Wolff both at home and abroad. Through in-depth study, our
group seperated two furancoumarins (phellopterin,
O O
OR
O
OCH3
I R= II R=H
I: phellopterin
II: 5-hydroxyl-8-methoxy-psoralen
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5-hydroxyl-8-methoxy-psoralen) from the fruits and roots of Changium smyrnioides Wolff for the first time[2-3].
Modern pharmacological studies have shown that furanocoumarins, which is for clinical use of cardiovascular
diseases and arteriosclerosis, have strong pharmacological activity, including the effects of expansion of coronary
artery, calcium antagonist and vasodilator. Our grouphad found the alcohol extract of Changium smyrnioides
Wolff can raise blood serum SOD and GSH-*** to decrease the increased level of lipid peroxide induced by high
cholesterol, but the physical foundation is not clear[4]. It is reasoned that the two coumarins in Changium
smyrnioides Wolff fruits may protect the injured vascular endothelial cells induced by oxidative stress, thus can
treat cardiovascular diseases and prevent atherosclerosis. In order to explore whether these two compounds have
such protective effects, this study use H2O2 to establish HUVEC oxidative stress injury model in order to observe
protective effects of different concentrations of phellopterin, 5-hydroxyl-8-methoxy-psoralen and its mechanism.
1 Materials and Methods
1.1 Materials phellopterin, 5-hydroxyl-8-methoxy-psoralen (purity ≥ 99%, supplied by our laboratory);
Newborn bovine serum (NBS) (It was purchased from Hangzhou holly leaf Biological Engineering Materials Co.,
Ltd.); DMEM/F12 medium powder (It was purchased from Gibco Company); SOD, NOS, NO, MDA detection kit
(They were purchased from Nanjing Jiancheng Biological Engineering Institute); Annexin V-FITC/PI Apoptosis
Detection Kit (It was purchased from KGI company).
1.2 Methods
1.2.1 Cell culture HUVECs were obtained from the school of Basic Medical in our university. They were
grown in medium DMEM supplemented with 10% fetal bovine serum (FBS), 1 × 105 IU•L-1 penicillin and 100
mg•L-1 streptomycin. Cells were cultured in 50 mL dishes and grown in a 37 ℃, 5% CO2 humidified incubator.
When the cell state is stable, divide cells and continue the next step experiment.
1.2.2 Cell division and MTT test HUVEC cells were seeded in 96 well plates at a density of 5×103 cells per
well, after 24 h, adherent cells were randomly divided into 3 groups: normal control group, H2O2 model group,
phellopterin (0.01 μmol•L-1, 0.1 μmol•L-1, 1 μmol•L-1, 5 μmol•L-1) treated group and
5-hydroxyl-8-methoxy-psoralen (0.01 μmol•L-1, 0.05 μmol• L-1, 0.5 μmol•L-1 ,5 μmol•L-1) treated group. Drug
treated groups were medicated with coumarin compounds for 24 h, then add H2O2 (180 μmol•L-1) for 4h. Remove
the medium, add 1 g•L-1 MTT 100 μL and incubate for 4 h. Finally, move the supernatant, add 150 μL DMSO,
determine the absorbance on microplate reader at 490 nm wavelength after mixing and calculate the cell viability.
1.2.3 Determination of NO in culture medium Cells were divided and treated according to the "1.2.2" method,
then collected the supernatant. According to the method in kit instructions, measure the NO content in the culture
supernatant. All experiments were repeated at least 3 times.
1.2.4 Determination of MDA levels in culture medium Cells were divided and treated according to the "1.2.2"
method, then collected the supernatant. According to the method in kit instructions, detect the MDA content in the
culture supernatant. All experiments were repeated at least 3 times.
1.2.5 Determination of SOD, NOS activity in cells Cells were divided and treated according to the "1.2.2"
method, then collected the supernatant. In accordance with the method described in the kit instructions, determine
the SOD, NOS activity in HUVECs. All experiments were repeated at least 3 times.
1.2.6 Detection of apoptosis by Flow cytometry Cells were seeded in 6 well plate, treated and divided
according to "1.2.2" method, then suspended cells in PBS, collected 1×106 cells. Stain the collected cells by the
method in Annexin V-FITC/PI kit instructions and detect the apoptosis by flow cytometry.
1.2.7 Statistical analysis Use SPSS 11.5 statistical software to analyze the experimental data with x ± s. Use
analysis of variance to make multi-group comparison.
2 Results
2.1 Establishment of H2O2 injury model and effects of drugs on normal cell viability
As shown in Fig.1-2,1-3, incubating cells with 180 μmol•L-1 H2O2 for 4 h allowed survival rate of HUVEC cells
dropped to 50.4%, thus using this method to establish damage model. Experimental results showed that both
phellopterin (concentration 0.01~5 μmol•L-1) and 5-hydroxyl-8-methoxy-psoralen (concentration 0.01~5 μmol•L-1
had no significant cytotoxic effect on HUVECs (Fig.1-4A, B). Therefore, chosing the two drugs over the above
concentration range can exclude the toxicity effect on HUVEC cells, would not affect cell viability and
interference the results. Therefore, the following test chose phellopterin and 5-hydroxyl-8-methoxy-psoralen dose
groups from 0.01~5 μmol•L-1 to do experiments.
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Normal cells Injured cells
Fig.1-2 Micrographs of endothelial cells (×200)
2.2 Effects of drugs on viability of H2O2 injured cell
H2O2 can decrease viability rate of human umbilical vein endothelial. there were significant differences (P <0.01)
comparing with the control group. Tab.1-2A showed that phellopterin can inhibited the damage caused by
hydrogen peroxide significantly (P<0.01) from 0.01 to 5 μmol•L-1. While the drug concentration increased, cell
survival rate increased (Fig.1-4A); Tab.1-2B showed that 5-hydroxyl-8-methoxy-psoralen also had a significant
(P<0.01) inhibition of the damage caused by hydrogen peroxide from 0.01 to 5 μmol•L-1. The effects are also dose
dependent (Fig.1-4B).
2.3 Effects of drugs on NO content and NOS activity of vascular endothelial cell
After endothelial cells were incubated with drugs for 24h and then treated with H2O2 for 4 h, the intracellular NOS
activity dropped from 0.3155 U•ml-1 to 0.2305 U•mL-1, NO content in culture medium was from 43.05 μmol•L-1
down to 24.07 μmol•L-1. Tab.1-3A showed that phellopterin can increase intracellular NOS activity to 0.2759
U•ml-1, increase NO content in culture medium to 32.88 μmol•L-1 from 0.01 to 5 μmol•L-1. It showed a
dose-dependent within the concentration range, while the drug concentration increased, NO content and NOS
activity increased. On the while, Tab.1-3B showed that 5-hydroxyl-8-methoxy-psoralen can increase intracellular
NOS activity to 0.2801 U•ml-1, increase the NO content in culture medium to 37.28 μmol•L-1 from 0.01 to 5
μmol•L-1, it also showed the same dose-dependent as phellopterin.
2.4 Effects of drugs on MDA and SOD levels
H2O2 injury can cause the release of intracellular MDA increasing (P<0.01) and SOD activity significantly
lowering (P <0.01); Tab.1-4A showed that, compared with the normal cells, after injured cells being pretreated by
phellopterin, from 0.01 to 5 μmol•L-1 , the release of MDA decreased, while SOD activity increased. It showed a
dose-dependent within the concentration range. Likewise, Tab.1-4B showed that, after injured cells being
pretreated by 5-hydroxyl-8-methoxy-psoralen, from 0.01 to 5 μmol•L-1, the release of MDA decreased, while SOD
activity increased comparing with the normal cells. It also showed a dose-dependent within the concentration
range.
2.5 Effects of drugs on HUVEC apoptosis
Flow cytometry analysis revealed that the apoptosis rate of H2O2 injured HUVEC cells was 10.7% (The apoptosis
rate of normal cells was 2.2%). After injured cells being pretreated by phellopterin, the early apoptosis rate can be
reduced to 3.8 % at 5 μmol•L-1. It showed a dose-dependent within the concentration range. Likewise, after
injured cells being pretreated by 5-hydroxyl-8-methoxy-psoralen, the early apoptosis rate can be reduced to 2.4 %
at 5 μmol•L-1. It also showed a dose-dependent within the concentration range (Fig.1-6A~J).
0
20
40
60
80
100
120
control
100
150
180
200
300
400
500
600
H2O2/umol
cell survival/% of control
Fig.1-3 The cell viability on H2O2 injured HUVEC
A B
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0
20
40
60
80
100
120
control 0.01 0.1 1 5
phellopterin/umol
cell survival/% of control
0
20
40
60
80
100
120
control 0.01 0.05 0.5 5
5-hydroxyl-8-methoxy-psoralen/umol
cell survival/% of control
Fig.1-4A, B The effect of pellopterin and 5–hydroxyl-8-methoxy-psoralen on normal HUVECs（x ±s, n = 6）
A B
-10
10
30
50
70
90
110
control model 0.01 0.05 0.5 5
5-hydroxyl-8-methoxy-psoralen/umol.L-1
cell survival/% of control
0
20
40
60
80
100
120
control model 0.01 0.1 1 5
Phellopterin/umol.L-1
cell survival/% of control
Fig.1-5A, B The protective of phellopterin and 5-hydroxy-8-methoxy psoralen on H2O2 injured HUVECs（x ±s, n
= 6）
A
B
C
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D
E
F
G
H
I
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J
Fig. 1-6 Effect of phellopterin and 5-hydroxy-8-methoxy psoralen on apoptotic rate in H2O2 induced HUVEC
cells
A: Control group; B: Model group; C: phellopterin 0.01 μmol•L-1 ; D: phellopterin 0.1 μmol•L–1; E: phellopterin 1
μmol•L-1
F: phellopterin 5 μmol•L–1; G: 5-hydroxy-8-methoxy psoralen 0.01 μmol•L–1; H:5-hydroxy-8-methoxy psoralen
0.05 μmol•L–1; I5-hydroxy-8-methoxy psoralen 0.5 μmol•L–1; J:5-hydroxy-8-methoxy psoralen 5 μmol•L–1
Tab. 1-2A Effects of phellopterin and hydrogen peroxide on the viability of HUVEC (%,x ±s, n=6)
Phellopterin/μmol•L-1 H2O2/μmol•L-1 Viability/%
0 0 100±0.06
0 180 54.12±0.01##
0.01 180 71.04±0.04**
0.1 180 73.26±0.02**
1 180 73.84±0.03**
5 180 80.81±0.07**
## P < 0.01 vs normal group , ** P < 0.01 vs H2O2 injured group
Tab.1-2B Effects of 5-hydroxy-8-methoxypsoralen and hydrogen peroxide on the viability of HUVEC (%,x ±s,
n=6)
5-Hydroxy-8-methoxypsoralenPhellopterin /μmol•L – 1 H2O2/μmol•L- 1 Viability/%
0 0 100±0.06
0 180 54.12±0.01##
0.01 180 69.61±0.01**
0.05 180 70.70±0.02**
0.5 180 71.32±0.01**
5 180 74.59±0.02**
## P < 0.01 vs normal group , ** P < 0.01 vs H2O2 injured group
Tab.1-3A Effects of phellopterin on release of nitric oxide and nitric oxide synthase from HUVEC injured by
H2O2 (x ±s, n=3)
Groups NO（μmol•L- 1） NOS（U•mL- 1）
Normal 43.05±0.001 0.3155±0.2
H2O2 injured 24.07±0.002## 0.2305±0.002##
Phellopterin 0.01μmol•L- 1 dose 28.57±0.01 0.2417±0.01
Phellopterin 0.1μmol•L- 1 dose 28.77±0.002* 0.2606±0.004**
Phellopterin 1μmol•L- 1 dose 29.65±0.02* 0.2641±0.003**
Phellopterin 5μmol•L- 1 dose 32.88±0.02* 0.2759±0.01**
## P < 0.01 vs normal group， * P < 0.05 vs H2O2 injured group，** P < 0.01 vs H2O2 injured group
Tab.1-3B Effects of 5-hydroxy-8-methoxypsoralen on release of nitric oxide and nitric oxide synthase from
HUVEC injured by H2O2 (x ±s, n = 3)
Groups NO（μmol•L- 1） NOS（U•mL- 1）
Normal 43.05±0.001 0.3155±0.2
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H2O2 injured 24.07±0.002## 0.2305±0.002#
5-Hydroxy-8-methoxypsoralen 0.01μmol•L- 1 dose 29.65±0.005* 0.2594±0.01
5-Hydroxy-8-methoxypsoralen 0.05μmol•L- 1 dose 33.61±0.02* 0.2677±0.007*
5-Hydroxy-8-methoxypsoralen 0.5μmol•L- 1 dose 34.44±0.001** 0.2712±0.002*
5-Hydroxy-8-methoxypsoralen 5μmol•L- 1 dose 37.28±0.01** 0.2801±0.004*
## P < 0.01 vs normal group， * P < 0.05 vs H2O2 injured group，** P < 0.01 vs H2O2 injured group
Tab.1-4 A Effects of phellopterin on release of MDA and SOD dismutase from HUVEC injured by H2O2
(x ±s, n=3)
Groups MDA（nmol•mL- 1） SOD（U•mL- 1）
Normal 2.07±0.001 13.24±0.02
H2O2 injured 3.65±0.001## 5.645±0.001##
Phellopterin 0.01μmol•L- 1 dose 2.83±0.002 8.689±0.009*
Phellopterin 0.1μmol•L- 1 dose 2.64±0.003* 9.943±0.01
Phellopterin 1μmol•L- 1 dose 2.45±0.003* 10.83±0.004**
Phellopterin 5μmol•L- 1 dose 2.36±0.003* 11.38±0.008**
## P < 0.01 vs normal group， * P < 0.05 vs H2O2 injured group，** P < 0.01 vs H2O2 injured group
Tab.1-4B Effects of 5-hydroxy-8-methoxypsoralen on release of MDA and SOD dismutase from HUVEC injured
by H2O2 (x ±s, n=3)
Groups MDA(nmol•ml- 1) SOD(U•ml- 1)
Normal 2.07±0.001 13.24±0.02
H2O2 injured 3.65±0.001## 5.645±0.001##
5-Hydroxy-8-methoxypsoralen 0.01μmol•L- 1 dose 3.16±0.003* 6.203±0.01
5-Hydroxy-8-methoxypsoralen 0.05μmol•L- 1 dose 2.78±0.004 10.78±0.003**
5-Hydroxy-8-methoxypsoralen 0.5μmol•L- 1 dose 2.58±0.002* 11.38±0.01*
5-Hydroxy-8-methoxypsoralen 5μmol•L- 1 dose 2.22±0.002** 11.85±0.004**
## P < 0.01 vs normal group , * P < 0.05 vs H2O2 injured group, ** P < 0.01 vs H2O2 injured group
3 Discussions
Vascular endothelial cells locate between blood and tissue, and contact blood directly. They are vulnerable to the
pathophysiological changes in the blood and these changes are easy to damage the functional inte***ce of vascular
endothelial cells. When body suffers from the stimulatiom damage and cardiovascular risk, vascular endothelium
cells were the first actor, accompany with endothelial cell dysfunction. In this study, HUVEC cells were treated
with H2O2 so as to simulate the oxidative stress simulation to vascular endothelial cell. The results showed that,
incubating HUVEC cells with H2O2 for 4 h could induce oxidative stress damage to HUVEC cells and the
survival rate decreased significantly (P<0.01). Pretreating cells with different concentrations of phellopterin and
5-hydroxyl-8-methoxy- psoralen from 0.01 to 5 μmol•L-1 for 24 h can enhance the survival rate of HUVEC cells
under oxidative stress. The effects were dose dependent.
Under normal circumstances, the speed of intracellular reactive oxygen species production and clearance keeps
balance. However, when the production of reactive oxygen species exceeds the scavenging capacity of
antioxidants, the bioavailability of reactive oxygen species will increase. It will cause endothelial cell dysfunction,
decrease endothelial endogenous NO content and enhance vasoconstriction. Those will lead to vascular smooth
muscle cell proliferation, macrophage infiltration and inflammation[5-7]. This strdy found that different
concentrations of phellopterin and 5-hydroxyl-8-methoxy-psoralen can enhance intracellular H2O2-induced SOD
activity, reduce MDA formation, increase total NOS activity in cells and increase NO content levels. The results
indicated that these two coumarins in Changium smyrnioides Wolff can increase endogenous oxidative defense
system activity, scavenge free radicals, promote endogenous NO production and release and enhance the ability of
antioxidant to protect cells injured by oxidative stressl.
Oxidative damage induced by the hypertension, ischemia/reperfusion, inflammation can cause apoptosis of
cardiac and vascular endothelial cells, antioxidants and free radical scavengers can partially reverse the apoptosis
process[8]. It was found that the early apoptosis rate of HUVECs injured by H2O2 for 4 h was significantly higher
than that of the normal cells. Both phellopterin and 5-hydroxyl-8-methoxy-psoralen can significantly reduce the
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early apoptosis rate. The results indicated that these two coumarins have distinct anti-apoptotic effects, and we
still need further study to find by which way these two compoumds can block the apoptosis cascade reaction.
In this study, we use H2O2 to produce oxidative stress injured model of vascular endothelial cells and observe the
protective effects of phellopterin and 5-hydroxyl-8-methoxy-psoralen on the injured HUVEC cells. It was found
that these two compounds can increase the activity of intracellular antioxidant system, regulate NOS activity of
cells, promote the restoration of normal levels of NO formation, and show a distinct anti-apoptosis activity. These
effects may be the reason why both phellopterin and 5-hydroxyl-8-methoxy-psoralen can play the role of
protecting H2O2 injured HUVEC cells. This results provided a bright prospect for our further study of the potential
cardiovascular prevention and treatment drugs for atherosclerosis related diseases.
Acknowledgement This project was financially supported by Specialized Research Fund for the Doctoral
Program of Higher Education (No. 200803150009)
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