Life Sciences 74 (2004) 2467 – 2478

www.elsevier.com/locate/lifescie

Angiogenesis and anti-angiogenesis activity of

Chinese medicinal herbal extracts
Shanshan Wang a,b, Zhengui Zheng c,*, Yinqi Weng c, Yijun Yu c,
Daifu Zhang a, Weihu Fan b, Ruihong Dai b, Zhibi Hu c
a
Department of Cardiovascular Disease, Dongfang Hospital, Shanghai 200120 China
b
Laboratory of Cardiovascular Disease, Huashan Hospital, Shanghai 200040, China
c
Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
Received 7 November 2002; accepted 24 March 2003

Abstract

The aqueous extracts of 24 herbs traditionally used as curing ischemic heart disease in clinic in China were
screened for their in vitro angiogenic activity, another twenty-four traditionally used as anti-tumor or anti-
inflammatory remedies in China were screened for their in vitro anti-angiogenic activity. The activity of
angiogenesis was determined by quantitation of vessels on chick embryo chorioallantoic membrane (CAM)
model and cell proliferation of cultured bovine aortic endothelial cells (BAECs). Among the herbal extracts
examined, the aqueous extracts of Epimedium sagittatum, Trichosanthes kirilowii and Dalbergia odorifera
showed the strong angiogenetic activity both in CAM and BAECs models; and the aqueous extracts of
Berberis paraspecta, Catharanthus roseus, Coptis chinensis, Taxus chinensis, Scutellaria baicalensis,
Polygonum cuspidatum and Scrophularia ningpoensis elicited significant inhibition at a concentration of 1g
dry herb /ml.
D 2004 Elsevier Inc. All rights reserved.

Keywords: Angiogenesis; Anti-angiogenesis; Chinese medicinal herbs

Introduction

The word angiogenesis is first named by Hertig in 1935 and the mechanism was revealed by Folkman
in studying tumor angiogenesis (Folkman, 1972; Folkman and Klagsbrun, 1987). Angiogenesis refers to

* Corresponding author. Tel.: +86-21-51322108; fax: +86-21-54232519.

E-mail address: zhengzhengui@163.com (Z. Zheng).

0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2003.03.005
2468 S. Wang et al. / Life Sciences 74 (2004) 2467 – 2478

the growth of new capillaries from pre-existing capillaries and post-capillary venules. It is a tightly
controlled process that rarely occurs under normal conditions, except for instances of wound healing,
embryonic development and development of the corpus luteum. Many diseases, however, are driven by
persistent unregulated angiogenesis.
It is generally accepted today that tumor growth is angiogenesis-dependent and that every increment
of tumor growth requires an increment of vascular growth. Tumors lacking angiogenesis remain
dormant indefinitely and rapid logarithmic growth follows the acquisition of blood supply. The tumor
angiogenic switch seems to be activated when the balance of angiogenic inhibitors to stimulators is
shifted toward a proangiogenic milieu (Hanahan and Folkman, 1996). There is great interest in
identifying and modulating antiangiogenic pathways and antiangiogenic drug development for
therapeutic purposes.
On the other hand, induction of therapeutic angiogenesis by various methods has recently been
developed to treat ischemic diseases (Baumgartner et al., 1998; Horvath et al., 1997; Losordo et al.,
1998; Risau, 1997; Rosengart et al., 1999). Research in animal models of ischemia has shown that
administration of angiogenic growth factors, either as a recombinant protein or by gene transfer, can
augment nutrient perfusion through neovascularization to promote the development of supplemental
collateral blood vessels that will constitute endogenous bypass conduits around occluded native
arteries. Innovative gene technologies and advances in animal modeling have enabled research
scientists to develop therapeutic angiogenesis strategies applied in animal models of limb or myocardial
ischemia and in treatment of patients with peripheral vascular obstruction or coronary artery diseases.
Several therapeutic strategies have been proposed and tested even at the clinical level. Some potential
alternative strategy may be the use of drugs with angiogenic activity, available in an oral formulation
and which are currently administered to patients for treatment of different pathologies (Silvestre and
Levy, 2002).
The chick embryo chorioallantoic membrane (CAM) model is an extra-embryonic membrane that is
commonly used in vivo to study both angiogenesis and anti-angiogenesis (Ribatti et al., 2001). An
angiogenic response occurs 72–96 hr after stimulation in the form of increased vessel density around the
implant, with the vessels radially converging toward the center like spokes in a wheel (Ribatti et al.,
1995). Conversely, when an angiostatic compound is tested, the vessels become less dense around the
implant and even disappear (Vacca et al., 1999). Quantitation of vessels in large amount of CAM models
can be used to screen drugs from samples of plant extracts.
Some traditional Chinese medicinal herbs have been shown to be effective in curing ischemic heart
disease and anti-tumor in clinic (Jiangsu New Medical College, 1986), but their function was not
scientifically tested and the mechanism is not known. This report describes the results of our screening
of selected Chinese medicinal herbal extracts on angiogenesis and anti-angiogenesis activities in vitro
and in vivo.

Methods

Preparation of crude plant extracts

Forty-seven dried medicinal herbs analyzed in this study were purchased from a local herbal shop.
Catharanthus roseus were collected from Hainan Island, People’s Republic of China. All herbs were
 S. Wang et al. / Life Sciences 74 (2004) 2467 – 2478 2469

identified by Prof. Zhou Xiujia (Shanghai University of Traditional Chinese Medicine). The specimens
were deposited in the Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese
Medicine.
The dried herb (5g) was extracted with 50 ml boiling distilled water under reflux for 2 hours. After
filtration to remove insoluble debris, the extract was dried using a Freeze Drier (Labconco, USA) and
stored at
20jC until assay. During the assay, 1g dry herb /ml and 0.2g dry herb/ml were prepared using
purified water or DMSO and sterilized with a filter of 0.22 Am.

Chorioallantoic membrane (CAM) assay (modified from Nguyen et al., 1994)

The fertilized Hong Kong Ma Hua chicken eggs used in this study were kept in a humidified
incubator at 37jC with the wide end up. The eggs were moved three times a day. After 3 or 4 days of
incubation, the 7-day-old eggs were observed on a self-made lamp and circled the position of embryo-
head. 0.5–1 ml of albumin was aspirated from eggs with an 18-gauge hypodermic needle through a
small hole drilled at the narrow end of the eggs, allowing the small CAM and yolk sac to drop away
from the shell membrane. The shell covering the embryo air sac was punched out and removed by
forceps and the shell membrane on the floor of the air sac was peeled away. At 8-days-old, a
thermanox coverslip loaded with a sample (10 Al) was applied to the CAM surface. The chick embryo
was then returned to the incubator. Three days later, an appropriate volume of a methanol and acetone
mixture (1:1) was injected using a 33-gauge needle into an 11-day-old embryo chorioallantois. The
CAM was cut out from eggs and the number of vessels was observed and vessels radially converging
toward the center were then counted under a microscope. At least 20 eggs were used for each sample
dose. The percent of increase and inhibition were calculated using the following equation: [(Vessel
Number of CAM treated by herb extract – Vessel number of CAM treated by normal saline) / (Vessel
number of CAM treated by normal saline)]  100% = % increase; [(Vessel Number of CAM treated
by normal saline – Vessel number of CAM treated by herb extract) / (Vessel number of CAM treated
by normal saline)]  100% = % inhibition.

In vitro bovine aortic endothelial cells (BAECs) culture and crystal violet assay

Primary cultures of bovine aortic endothelial cells were prepared from fresh bovine aorta (Shanghai
Zoo), cells were cultured in DMEM (Sigma) containing 10% v/v fetal calf serum (FCS) (Sino-America
Biotechnology Company) supplemented with penicillin (100 units/ml), and streptomycin (100 Ag/ml).
Subculture and identification of BAECs were made according to E Z (1997). Antibody to human factor
VIII/von Willebrand-associated antigen (vWF), collagenase, gelatin, thrombin, bradykinin, and crystal
violet were obtained from Sigma (St. Louis, MO). Endothelial cell growth supplement (ECGS) was
purchased from Upstate Biotechnology (Lake Placid, NY).
For assay, BAECs were cultured in DMEM supplemented with only 5% (angiogenesis) or 10%FCS
(anti-angiogenesis), 50 Ag/ml Endothelial Cell Growth Supplement, and 30 Ag/ml heparin (Elkins-sin,
inc., Cherry Hill, NJ) in 96-well plates (Fisher). 3  104 cells per well were cultured for 24 hours in a
CO2 incubator (Heraeus) at 37.8jC with 5% CO2 concentration, then prepared samples, positive (bFGF or
dexamethasone (Sigma)) and normal (normal saline) control (20 Al each well) were added. Cells were
then incubated for 72 hours before harvesting by staining with Crystal violet (McElwain et al., 1995), and
the living cell number was counted using a blood cell counter under microscope (Olympus). At least 24
2470 S. Wang et al. / Life Sciences 74 (2004) 2467 – 2478

wells were used for each sample dose. The percent of increase and inhibition were calculated using the
following equation:
½ðCell Number treated by herb extract
Cell Number treated by normal salineÞ
=ðCell Number treated by normal salineÞ  100% ¼ % increase;
½ðCell Number treated by normal saline
Cell Number treated by herb extractÞ
=ðCell Number treated by normal salineÞ  100% ¼ % inhibition:

Results

The establishment of Chorioallantoic membrane and Bovine aortic endothelial cells models

When CAM was treated with 10 Al1000AU/ml bFGF, more than 50 vessels were observed, while
about 20 vessels on CAM treated with 10 Al normal saline and only 8 vessels on CAM treated

Fig. 1. The chick embryo chorioallantoic membrane (CAM) models treated with normal saline (N), basic fibroblast growth
factor (P), dexamethasone (A), aqueous extracts (1 g herb D.W./ml) of Epimedium sagittatum stems and leaves (S1) and
Scrophularia ningpoensis roots (S2).
 S. Wang et al. / Life Sciences 74 (2004) 2467 – 2478 2471

Fig. 2. Radial vessels on CAM treated with various concentrations of bFGF, dexamethasone and some herbal extracts. F1: CAM
treated with 10 Al 2500 AU/ml bFGF. F2: CAM treated with 10 Al 1000 AU/ml bFGF. C: CAM treated with 10 Al normal saline
as control. D1: CAM treated with 10 Al 10
5 mol/l dexamethasone. D2: CAM treated with 10 Al 10
6 mol/l dexamethasone.
E1: CAM treated with aqueous extracts (1 g herb D.W./ml) of Epimedium sagittatum stems and leaves. E2: CAM treated with
aqueous extracts (1 g herb D.W./ml) of Scrophularia ningpoensis roots.

with10 Al 10
6 mol/l dexamethasone solution. When CAM was treated with 10 Al2500AU/ml
bFGF, the vessels increased 70 and almost no vessels observed on CAM treated with 10 Al 10
5
mol/l dexamethasone solution (Figs. 1 and 2). The difference is large enough for drug screening.
From the figures we also can see that the aqueous extracts of Epimedium sagittatum stems and
leaves and Scrophularia ningpoensis roots has strong effect of pro-angiogenosis and anti-angiogen-
esis, respectively.

Fig. 3. Bovine aortic endothelial cells growth curve in pro-angiogenesis (P) and anti-angiogenesis (A) model system. C1:
BAECs (cultured in medium containing 5% FCS, the same as P1, P2, E1) treated with 20 Al normal saline as control. P1:
BAECs treated with 20 Al 2500 AU/ml bFGF. P2: BAECs treated with 20 Al 1000 AU/ml bFGF. S1: BAECs treated with 20 Al
aqueous extracts (1 g herb D.W./ml) of Epimedium sagittatum stems and leaves. C2: BAECs (cultured in medium containing
10% FCS and 20 Al 1000 AU/ml bFGF, the same as in A1, A2, E2) treated with 20 Al normal saline as control. A1: BAECs
treated with 20 Al 10
5mol/l dexamethasone. A2: BAECs treated with 20 Al 10
6 mol/l dexamethasone. S2: BAECs treated
with 20 Al aqueous extracts (1 g herb D.W./ml) of Scrophularia ningpoensis roots.
2472 S. Wang et al. / Life Sciences 74 (2004) 2467 – 2478

Fig. 3 shows the time course of bovine aortic endothelial cells proliferation cultured in vitro
containing 5% v/v fetal calf serum (FCS). From the figure we can see that the cells proliferate very
slow (C1) when cultured in DMEM medium with 5% v/v fetal calf serum but proliferate quickly when
treated with various concentration of basic fibroblast growth factor (P1, P2) and the aqueous extracts (1g
herb D.W./ml) of Epimedium sagittatum stems and leaves (S1) can strongly stimulate cells proliferation.
This BAECs culture system was used as model to screen pro-angiogenic extracts. In order to screen
antiangiogenic extracts, the BAECs cultures with 10% v/v fetal calf serum and bFGF were used as
control (C2). When cells were treated with various concentrations of dexamethasone (Lansink et al.,
1998) and aqueous extracts (1 g herb D.W./ml) of Scrophularia ningpoensis roots (S2), the living cell
number (A1, A2, S2) decreased and the difference is large enough for drug screening.

Screening of crude extracts on pro-angiogenesis activity from traditional Chinese medicinal herbs

The extracts of 24 species of traditional Chinese medicinal herbs were tested for their
angiogenesis activity using CAM and BAECs models. The screening test was carried out at a

Table 1
Screening of crude extracts on angiogenesis activity from traditional Chinese medicinal herbs using CAM model
Name Used part % Increase (0.2 g herb/ml) % Increase (1 g herb/ml)
Aconitum carmichaeli Root tuber 38.10 F 9.15 89.52 F 17.69
Angelica sinensis Root 44.05 F 29.30 107.62 F 21.08
Astragalus membranaceus Root 19.52 F 2.55 95.24 F 14.37
Camellia sinensis Root 14.29 F 3.38 57.14 F 10.45
Carthamus tinctorius Flower 47.62 F 6.27 79.29 F 10.88
Corydalis yanhusuo Stem tuber 4.76 F 1.34 61.90 F 6.98
Crataegus pinnatifida var. major Fruit 28.57 F 4.39 87.57 F 15.43
Cynanchum paniculatum Root and rhizome 38.10 F 5.06 99.05 F 19.66
Dalbergia odorifera Root heartwood 95.24 F 11.28 169.05 F 30.28
Epimedium sagittatum Stem and leaf 102.38 F 23.65 175.86 F 25.69
Leonurus heterophyllus Whole plant 52.38 F 8.54 82.38 F 12.37
Ligusticum chuanxiong Rhizome 33.33 F 6.20 76.19 F 10.42
Ligustrum lucidum Fruit 19.05 F 3.15 95.24 F 16.58
Paeonia lactiflora Root 90.48 F 15.32 174.29 F 32.64
Panax pseudo-ginseng var notoginseng Root 52.38 F 6.55 136.67 F 21.35
Patrinia villosa Whole plant 108.52 F 17.65 148.39 F 12.17
Peucedanum praeruptorum Root 47.62 F 5.25 104.76 F 14.62
Polygonatum odoratum var pluriflorum Rhizome 71.43 F 9.30 147.62 F 19.08
Pueraria lobata Root 85.71 F 8.78 161.90 F 24.62
Rhodiola sacra Root and rhizome 124.29 F 18.62 173.33 F 27.59
Stephania tetrandra Root 33.33 F 5.21 93.52 F 15.28
Trichosanthes kirilowii Fruit wall 114.29 F 22.39 183.81 F 29.56
Typha angustata Pollen 23.81 F 4.82 61.90 F 8.37
Viscum coloratum Branch and leaf 19.05 F 3.08 90.48 F 12.48
bFGF (positive control) 156.19 F 28.55* 211.43 F 35.65+
Normal saline (normal control) 0 0
Each CAM was treated with 10 Al extract. Values are means F S.E.M., n> = 20.
* 1000 AU/ml bFGF.
+
2500 AU/ml bFGF.
 S. Wang et al. / Life Sciences 74 (2004) 2467 – 2478 2473

concentration between 0.2 g herb/ml and 1 g herb/ml. At a concentration of 1 g herb/ml, 11

extracts were found to increase vessels on CAM by more than 100%. Among them, the extracts of
Epimedium sagittatum stem and leaves and Trichosanthes kirilowii fruit walls showed the highest
angiogenesis activity (>180% increase). At a lower concentration (0.2 g herb/ml), 10 extracts
caused more than 50% increase of vessels on CAM and 4 of them got to 100%. The highest
increase activity was revealed the underground part of Rhodiola sacra (Table 1). When screened
with BAECs model, at a concentration of 1 g herb/ml, 5 extracts were found to increase living cell
numbers by more than 50% and the extract of Trichosanthes kirilowii fruit wall was higher than
100%. Even at a lower concentration of 0.2 g herb/ml, extracts of Angelica sinensis, Dalbergia
odorifera, Epimedium sagittatum, Patrinia villosa and Trichosanthes kirilowii still caused more than
30% increase (Table 2). Compared the result of Tables 1 and 2, we found that the extracts having

Table 2
Screening of crude extracts on angiogenesis activity from traditional Chinese medicinal herbs using in vitro bovine aortic
endothelial cells culture model
Name Used part % Increase (0.2 g herb/ml) % Increase (1 g herb/ml)
Aconitum carmichaeli Root tuber 7.39 F 2.18 9.27 F 1.16
Angelica sinensis Root 32.97 F 5.45 46.49 F 6.58
Astragalus membranaceus Root 2.58 F 0.07 4.77 F 0.62
Camellia sinensis Root 2.16 F 0.38 3.91 F 0.55
Carthamus tinctorius Flower 5.75 F 1.42 13.35 F 2.88
Corydalis yanhusuo Stem tuber –a 1.29 F 0.04
Crataegus pinnatifida var major Fruit 1.25 F 0.13 8.56 F 1.08
Cynanchum paniculatum Root and rhizome 10.26 F 1.95 13.09 F 1.50
Dalbergia odorifera Core of root 37.05 F 3.39 50.35 F 7.11
Epimedium sagittatum Stem and leaf 47.35 F 3.26 75.68 F 9.24
Leonurus heterophyllus Whole plant 7.68 F 1.25 14.66 F 3.25
Ligusticum chuanxiong Rhizome 5.46 F 0.92 7.25 F 1.27
Ligustrum lucidum Fruit 3.56 F 0.35 10.06 F 1.74
Paeonia lactiflora Root 25.47 F 2.32 46.58 F 3.95
Panax pseudo-ginseng var notoginseng Root 10.26 F 1.34 21.37 F 4.29
Patrinia villosa Whole plant 38.75 F 7.48 59.76 F 8.34
Peucedanum praeruptorum Root 6.29 F 0.54 8.35 F 1.20
Polygonatum odoratum var pluriflorum Rhizome 5.35 F 1.66 8.05 F 1.39
Pueraria lobata Root 26.64 F 4.28 51.65 F 6.52
Rhodiola sacra Root and rhizome +b +b
Stephania tetrandra Root –a 7.46 F 2.18
Trichosanthes kirilowii Fruit wall 56.47 F 7.69 102.65 F 11.52
Typha angustata Pollen –a –a
Viscum coloratum Branchlet and leaf 2.43 F 0.05 3.75 F 0.56
bFGF (positive control) 161.39 F 41.68* 216.92 F 19.57+
Normal saline (normal control) 0 0
Cells were cultured in 96-well plate; each well was treated with 20 Al extract. Values are means F S.E.M., n = 24.
a
No increase.
b
Cells formed aggregates after treated and can’t count.
* 1000 AU/ml bFGF.
+
2500 AU/ml bFGF.
2474 S. Wang et al. / Life Sciences 74 (2004) 2467 – 2478

strong pro-angiogenesis activity were similar between two screening models except the extract of
Rhodiola sacra.

Screening of crude extracts on anti-angiogenesis activity from traditional Chinese medicinal herbs

Table 3 and Table 4 were the results of anti-angiogenesis activity from 24 traditional
Chinese medicinal herbs usually used to cure tumor and anti-inflammatory activity in clinics in
China. Like the screening of pro-angiogenesis activity, the test was carried out at two concen-
trations. At a concentration of 1 g herb/ml, 6 extracts were revealed to inhibit angiogenesis on
CAM model by more than 30% and 5 exceed 20% at a lower concentration (0.2 g herb/ml)
extracts were used. Among them, the extract of Scrophularia ningpoensis was the most effect-
ive (Table 3).

Table 3
Screening of crude extracts on anti-angiogenesis activity from traditional Chinese medicinal herbs using CAM model
Name Used part % Inhibition (0.2 g herb/ml) % Inhibition (1 g herb/ml)
Akebia quinata Fruit 5.77 F 1.25 17.31 F 1.50
Aristolochia mollissima Whole plant –a –a
Berberis paraspecta Root 25.00 F 3.18 34.13 F 2.37
Brucea javanica Fruit –a 1.44 F 1.05
Catharanthus roseus Leaf 26.92 F 3.56 33.65 F 1.62
Clematis terniflora Fruit 2.40 F 0.44 10.58 F 0.88
Coptis chinensis Rhizome 25.48 F 4.05 36.54 F 3.72
Dendrobium Chrysanthum Stem 7.69 F 1.15 15.87 F 2.15
Euchresta horfieldii Root 0.48 F 0.06 8.17 F 1.28
Gentiana scabra Root and Rhizome 1.92 F 0.09 11.06 F 0.92
Hedyotis diffusa Whole plant –a 10.10 F 2.07
Lithospermum erythrorhizon Whole plant –a 7.69 F 1.33
Lobelia chinesis Whole plant –a 14.42 F 2.10
Paeonia suffruticosa Root bark –a –a
Paris polyphylla Whole plant –a –a
Physalis pubescens Whole plant 4.81 F 0.58 15.87 F 4.05
Polygonum cuspidatum Whole plant 19.23 F 2.49 39.90 F 6.62
Prunella vulgaris Whole plant 6.25 F 1.46 13.46 F 1.97
Scrophularia ningpoensis Root 20.67 F 3.27 35.10 F 4.68
Scutellaria baicalensis Root 26.92 F 5.15 39.42 F 3.25
Scutellaria barbata Whole plant –a –a
Solanum nigrum Whole plant 3.37 F 0.66 12.50 F 2.11
Sophora flavescens Root –a –a
Taxus chinensis Bark 10.58 F 1.59 25.96 F 4.53
Dexamethasone (positive control) 26.11 F 5.02* 43.37 F 4.56+
Normal saline (normal control) 0 0
Each CAM was treated with 10 Al extract. Values are means F S.E.M., n> = 20.
a
No inhibition.
* 10
6 mol/l dexamethasone.
+
10
5 mol/l dexamethasone.
 S. Wang et al. / Life Sciences 74 (2004) 2467 – 2478 2475

Table 4
Screening of crude extracts on anti-angiogenesis activity from traditional Chinese medicinal herbs using in vitro bovine aortic
endothelial cells culture model
Name Used part % Inhibition(a) (0.2 g herb/ml) % Inhibition (1 g herb/ml)
Akebia quinata Fruit 18.65 F 3.06 39.54 F 4.25
Aristolochia mollissima Whole plant –a –a
Berberis paraspecta Root 37.69 F 6.55 42.38 F 5.49
Brucea javanica Fruit –a 5.59 F 0.75
Catharanthus roseus Leaf 30.05 F 4.35 56.27 F 6.58
Clematis terniflora Fruit 7.52 F 1.15 12.07 F 1.26
Coptis chinensis Rhizome 37.15 F 3.69 54.66 F 4.38
Dendrobium Chrysanthum Stem 10.95 F 2.17 17.84 F 2.92
Euchresta horfieldii Root 2.01 F 0.36 5.83 F 0.72
Gentiana scabra Root and Rhizome 4.75 F 0.47 6.68 F 1.06
Hedyotis diffusa Whole plant 4.25 F 0.81 12.39 F 2.15
Lithospermum erythrorhizon Whole plant –a –a
Lobelia chinesis Whole plant 3.74 F 0.28 4.95 F 0.75
Paeonia suffruticosa Root bark –a –a
Paris polyphylla Whole plant 2.77 F 0.52 5.64 F 0.45
Physalis pubescens Whole plant 8.13 F 0.59 21.05 F 2.08
Polygonum cuspidatum Whole plant 27.82 F 3.35 42.35 F 4.72
Prunella vulgaris Whole plant 10.76 F 1.19 18.54 F 1.57
Scrophularia ningpoensis Root 34.18 F 2.68 45.92 F 7.26
Scutellaria baicalensis Root 41.03 F 7.05 58.75 F 6.43
Scutellaria barbata Whole plant –a –a
Solanum nigrum Whole plant 3.37 F 0.66 12.50 F 2.11
Sophora flavescens Root –a –a
Taxus chinensis Bark 26.34 F 3.16 39.85 F 5.57
Dexamethasone (positive control) 34.19 F 1.85* 55.36 F 4.26+
Normal saline (normal control) 0 0
Cells were cultured in 96-well plate; each well was treated with 20 Al extract. Values are means F S.E.M., n = 24.
a
No inhibition.
* 10
6 mol/l dexamethasone.
+
10
5 mol/l dexamethasone.

In BAECs model, also 6 extracts were found to inhibit angiogenesis by more than 40% at a high
concentration (1 g herb/ml) and 5 extracts exceed 20% at a lower concentration (0.2 g herb/ml). Extract
of Scutellaria barbata were found to have the highest inhibition (Table 4).

Discussion

Angiogenesis is a strictly controlled process in normal human body and regulated by a variety of
endogenous angiogenic and angiostatic factors (Folkman and Klagsbrun, 1987). Pathological angio-
genesis occurs, for example, in cancer, chronic inflammation, or atherosclerosis. Angiogenesis inhibitors
are able to interfere with various steps of angiogenesis, on the other hand, angiogenesis promoters can
stimulate angiogenesis occur basement destruction of blood vessels, proliferation and migration of
endothelial cells.
2476 S. Wang et al. / Life Sciences 74 (2004) 2467 – 2478

In the past fifteen years, investigators have been looking for angiogenesis inhibitors and
promoters from plants. Genistein (diphenolic isoflavonoids and lignans) (Fotsis et al., 1993), two
saponins from Red ginseng, 20(R)- and 20(S)-ginsenoside-Rg3 (Mochizuki et al., 1995), isoliquiritin
from licorice root extract (Kobayashi et al., 1995), shikonin from Lithospermum erythrorhizon (Hisa
et al., 1998), Viscum album coloratum extract (Yoon et al., 1995), ether fraction of water-soluble
extract of Populus nigra leaves (Glinkowska et al., 1997), Soybean phytochemicals (Zhou et al.,
1999), Chrysobalanus icaco methanol extract (Alves De Paulo et al., 2000), an extract of the fern
Polypodium leucotomos (Gonzalez et al., 2000), torilin from Torilis japonica (Kim et al., 2000),
Cassia garrettiana heartwood extract (Kimura et al., 2000), Agaricus blazei extract (Takaku et al.,
2001), Deoxypodophyllotoxin from Pulsatilla koreana (Kim et al., 2002), epigallocatechin gallate
from green tea (Jung and Ellis, 2001) resveratrol from grapes (Brakenhielm et al., 2001) and
ginseng saponins and some related triterpenoid compounds (Shibata, 2001) et al. had anti-angiogenic
activity in vitro or in vivo. On the other hand, saponin from Ginseng Radix rubra (Morisaki et al.,
1995), asiaticoside isolated from Centella asiatica (Shukla et al., 1999), ginkgo-biloba extracts
(Juarez et al., 2000), beta-sitosterol from Aloe vera (Choi et al., 2002) enhanced angiogenesis in
vivo.
We screened 24 species of Chinese medicinal herbs usually used to cure ischemic disease, from
them, boiling water extracts of Epimedium sagittatum stem and leaves, Trichosanthes kirilowii fruit
walls and Dalbergia odorifera root heartwood showed the strong promoting angiogenesis activity both
in vitro and in vivo (CAM). Almost all 24 herbs screened in our experiments display vasodilation
effect (Jiangsu New Medical College, 1986), and it was thought to be the main reason of curing
ischemic disease. We found during these herbs, some of them also have angiogenesis effect, and
interestingly, the extracts of Epimedium sagittatum stem and leaves and Dalbergia odorifera root
heartwood can only stimulate tiny blood vessels, while bFGF and the extracts of Trichosanthes
kirilowii fruit walls can stimulate relatively bigger vessels (Fig. 1). As these herbs all had saponins
(Jiangsu New Medical College, 1986), The angiogenesis activity might be mainly acted by saponins
like Ginseng Radix rubra (Morisaki et al., 1995). The whole plant extract of Rhodiola sacra, in CAM
model, showed the highest activity of angiogenesis, but the cells aggregated when tested using BAECs
model. As this famous traditional herb has anti-aging activity (Ohsugi et al., 1999), its activity needs
further study.
The herbs we selected to screen anti-angiogenesis have been used to anticancer or anti-inflamma-
tory. The results indicated that the boiling water extracts of Berberis paraspecta, Catharanthus roseus,
Coptis chinensis, Taxus chinensis, Scutellaria barbata Polygonum cuspidatum, Scrophularia ning-
poensis having strong anti-angiogenesis activity. Except that Catharanthus roseus and Taxus chinensis
have anti-tumor compound, vincristine and taxol respectively, Polygonum cuspidatum have Resver-
atrol which were revealed as an inhibitor of angiogenesis (Kimura and Okuda, 2001; Brakenhielm et
al., 2001), the anti-angiogenesis activity of Berberis paraspecta, Coptis chinensis, Scutellaria barbata
and Scrophularia ningpoensis extracts was first revealed in this paper. Further purification and
characterization of the extracts is being actively pursued in our institute. We think there might be some
anti-angiogenic chemicals in these herbs. Hisa et al. (1998) reported that shikonin, an ingredient of
Lithospermum erythrorhizon had anti-angiogenic activity in vitro and in vivo, but in our experiments,
the aqueous extracts of the whole plant revealed no anti-angiogenic activity, which indicates that
herbal extracts are complicated and many kinds of fractions extracted by different solvents is needed
in future screens.
 S. Wang et al. / Life Sciences 74 (2004) 2467 – 2478 2477

Acknowledgements

This work was partly funded by Ministry of Education of China (No. 200038) and we gratefully
acknowledge their support.

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