Antiproliferative Activity of Vietnamese Medicinal Plants

Abstract

Methanol, methanol-water (1:1) and water extracts were prepared from seventy-seven Vietnamese medicinal plants and tested for their antiproliferative activities against human HT-1080 fibrosarcoma cells. Among them, fifteen extracts including seven methanol extracts of Caesalpinia sappan, Catharanthus roseus, Coscinium fenestratum, Eurycoma longifolia, Hydnophytum formicarum and Streptocaulon juventas (collected at two areas), six methanol-water (1:1) extracts of Cae. sappan, Cat. roseus, Co. fenestratum, H. formicarum and S. juventas (at two areas), and two water extracts of Cae. sappan and S. juventas exhibited antiproliferative activities in a concentration-dependent manner. Their antiproliferative activities against human cervix HeLa adenocarcinoma, human lung A549 adenocarcinoma, murine colon 26-L5 carcinoma, murine Lewis lung carcinoma (LLC) and murine B16-BL6 melanoma cells were then examined. Co. fenestratum showed selective activity against lung carcinoma and/or lung metastatic cell lines, A549, LLC and B16-BL6, while H. formicarum and S. juventas showed selective activity against human tumor cell lines, HeLa and A549. Characteristic morphological change and DNA fragmentation indicated the antiproliferative activity to be due to the induction of apoptosis.

Full text

Cancer is one of the major causes of death in developed
countries, together with cardiac and cerebrovascular
diseases.
1)
Cancer is clinically treated by surgery, radiother-
apy and chemotherapy. After surgical ablation of progressive
cancer, however, metastasized tumor cells continue to
progress, and this is one of the causes making cancer treat-
ment difficult.
2)
Radioactive rays and most anticancer drugs
damage DNA or suppress DNA duplication to kill tumor
cells growing rapidly. At the same time, they also affect nor-
mal cells to cause serious adverse effects, such as bone mar-
row function inhibition, nausea, vomiting and alopecia.
3,4)
Thus, more effective anticancer drugs with high selectivity
against only malignant cells and with ability to repress tumor
metastasis are desired. As candidates for such drugs, cyto-
toxic, antitumor or anticancer natural products have been
often sought, and plant components such as Vinca alkaloids,
taxoids, etoposide and irinotecan are now used in clinical
treatments.
4)
Vietnam has many medicinal resources,
5,6)
but only a few
have been examined chemically except for Vietnamese gin-
seng.
7,8)
In the course of our search for Vietnamese medicinal
plants,
9—12)
we collected plants which have been used as ton-
ics, for treatment of inflammation, cancer and other condi-
tions,
13,14)
at Seven-Mountain area, Angiang province and at
Lamdong province. From 77 of these plants, 231 extracts
were prepared and their antiproliferative activities were
screened against highly metastatic human HT-1080 fibrosar-
coma cells.
To determine the selectivity of their activities, we exam-
ined their antiproliferative activities against five other cell
lines including three highly metastatic cell lines, i.e., human
cervix HeLa adenocarcinoma, human lung A549 adenocarci-
noma, murine colon 26-L5 carcinoma, murine Lewis lung
carcinoma (LLC) and murine B16-BL6 melanoma cells. An
extract of Streptocaulon juventas showed potent antiprolifer-
ative activities with selectivity against human tumor cells.
This activity was concluded to be due to the induction of
apoptosis, based on characteristic morphological changes
and DNA fragmentation.
MATERIALS AND METHODS
Plant Materials Vietnamese medicinal plants used in
this study were collected at Seven-Mountain area, Tinh Bien
district, Angiang province in March 1998, and at Lamdong
province in May 1998. The plants collected at Seven-moun-
tain area were identified by Prof. Le Cong Kiet (Department
of Botany, University of Hochiminh City, Hochiminh, Viet-
nam), while these collected at Lamdong province were iden-
tified by Mr. Nguyen Duy Chinh (Department of Botany,
Faculty of Environment, Dalat University, Dalat, Vietnam).
Their voucher specimens are preserved at the Museum of
Materia Medica, Research Center for Ethnomedicines, Insti-
tute of Natural Medicine, Toyama Medical and Pharmaceuti-
cal University, Toyama, Japan.
Chemicals Eagle’s minimum essential (EME) and RPMI
1640 media and Dulbecco’s phosphate buffered saline (PBS)
were purchased from Nissui Pharmaceutical Co., Ltd.
(Tokyo, Japan). Dulbecco’s modified Eagle’s medium nutri-
ent mixture Ham’s F-12 (1 : 1) (DMEM/F-12) medium and
heat inactivated fetal calf serum (FCS) were obtained from
Gibco BRL Products (Gaithersburg, MD, U.S.A.). Bovine
serum albumin (BSA), penicillin G and streptomycin sulfate
were from Sigma Chemical Co. (St. Louis, MO, U.S.A.). 3-
(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bro-
mide (MTT) and crystal violet were purchased from Aldrich
Chemical Co., Inc. (Milwaukee, WI, U.S.A.) and Nacalai
Tesque, Inc. (Kyoto, Japan), respectively. Cell culture flasks,
6- and 96-well plates were from Corning Inc. (Corning, NY,
U.S.A.). 5-Fluorouracil (5-FU), and doxorubicin hydrochlo-
ride were purchased from Tokyo Kasei Kogyo Co., Ltd.
(Tokyo) and Kyowa Hakko Kogyo Co., Ltd. (Tokyo), respec-
June 2002 Biol. Pharm. Bull. 25(6) 753—760 (2002) 753
∗ To whom correspondence should be addressed. e-mail: kadota@ms.toyama-mpu.ac.jp © 2002 Pharmaceutical Society of Japan
Antiproliferative Activity of Vietnamese Medicinal Plants
Jun-ya UEDA,
a
Yasuhiro TEZUKA,
a
Arjun Hari BANSKOTA,
a
Quan Le TRAN,
a
Qui Kim TRAN,
b
Yuko HARIMAYA,
a
Ikuo SAIKI,
a
and Shigetoshi KADOTA
*
,a
a
Institute of Natural Medicine, Toyama Medical and Pharmaceutical University; 2630 Sugitani, Toyama 930–0194, Japan:
and
b
National University-Hochiminh City; Hochiminh City, Vietnam.
Received January 10, 2002; accepted February 27, 2002
Methanol, methanol–water (1 : 1) and water extracts were prepared from seventy-seven Vietnamese medici-
nal plants and tested for their antiproliferative activities against human HT-1080 fibrosarcoma cells. Among
them, fifteen extracts including seven methanol extracts of Caesalpinia sappan, Catharanthus roseus, Coscinium
fenestratum, Eurycoma longifolia, Hydnophytum formicarum and Streptocaulon juventas (collected at two areas),
six methanol–water (1 : 1) extracts of Cae. sappan, Cat. roseus, Co. fenestratum, H. formicarum and S. juventas (at
two areas), and two water extracts of Cae. sappan and S. juventas exhibited antiproliferative activities in a con-
centration-dependent manner. Their antiproliferative activities against human cervix HeLa adenocarcinoma,
human lung A549 adenocarcinoma, murine colon 26-L5 carcinoma, murine Lewis lung carcinoma (LLC) and
murine B16-BL6 melanoma cells were then examined. Co. fenestratum showed selective activity against lung car-
cinoma and/or lung metastatic cell lines, A549, LLC and B16-BL6, while H. formicarum and S. juventas showed
selective activity against human tumor cell lines, HeLa and A549. Characteristic morphological change and DNA
fragmentation indicated the antiproliferative activity to be due to the induction of apoptosis.
Key words antiproliferative activity; Streptocaulon juventas; Vietnamese plant; apoptosis; human HT-1080 fibrosarcoma;
metastasis

tively. Ribonuclease (RNase) was purchased from Wako Pure
Chemical Industries, Ltd. (Osaka, Japan). The 100 Base-pair
ladder was obtained from Amersham Pharmacia Biotech,
Inc. (Piscataway, NJ, U.S.A.). Other reagents were of the
highest grade available.
Preparation of Samples Each medicinal plant (30—
213 g) was cut into small pieces and extracted successively
with methanol (200—300 ml, reflux, 2 h, 32), methanol–
water (1 : 1, 200—300 ml, reflux, 2 h), and water (200—
300 ml, reflux, 2 h). The methanol solution was evaporated
under reduced pressure to give a methanol extract, while
methanol–water (1 : 1) and water solutions were concentrated
under reduced pressure and lyophilized to give methanol–
water (1 : 1) and water extracts, respectively.
Cells A highly metastatic human HT-1080 fibrosarcoma
cell line (ATCC # CCL-121)
15)
was obtained from American
Type Culture Collection (Rockville, MD, U.S.A.). Human
cervix HeLa adenocarcinoma (RCB0007)
16)
and human lung
A549 adenocarcinoma (RCB0098)
17,18)
cell lines were pur-
chased from Riken Cell Bank (Tsukuba, Japan). Highly liver
metastatic murine colon 26-L5 carcinoma cell line was estab-
lished by one of the authors (I. Saiki).
19)
Highly lung
metastatic murine LLC cell line originated spontaneously
from murine lung
20)
was kindly provided by Dr. K. Takeda
(Juntendo University, Tokyo). Highly liver metastatic murine
B16-BL6 melanoma cell line, obtained by an in vivo selec-
tion procedure for invasion,
21)
was generously provided by
Dr. I. J. Fidler (M.D. Anderson Cancer Center, Houston, TX,
U.S.A.).
HT-1080, HeLa, A549, LLC and B16-BL6 cell lines were
maintained in 75-cm
2
cell culture flasks in EME medium
supplemented with 10% heat inactivated FCS, 2 mML-(1)-
glutamine and 0.1% sodium hydrogen carbonate at 37 °C
under a humidified 5% carbon dioxide. 26-L5 cell line was
maintained in RPMI 1640 medium containing the same sup-
plement under the same conditions.
Antiproliferative Activity Assay Viability of cells other
than LLC, in the presence or absence of experimental ex-
tracts, was determined using the standard MTT assay
22)
as
described.
23)
In brief, exponentially growing cells were har-
vested and 100-
m
l medium per well with 2310
3
cells sus-
pended was plated in a 96-well plate. After 24-h incubation
at 37 °C under a humidified 5% carbon dioxide to allow cell
attachment, the cells were treated with varying concentra-
tions of test specimens in their respective medium (100
m
l)
and incubated for 72 h under the same conditions. After 2 h
of the MTT (0.4—0.5 mg/ml, 100
m
l) addition, the formazan
formed was extracted with dimethyl sulfoxide (DMSO) and
its amount was measured spectrophotometrically at 550 nm
with Perkin-Elmer HTS-7000 Bio Assay Reader (Norwalk,
CT, U.S.A.).
In the case of LLC cells, standard crystal violet staining
assay was used in following the literature procedure.
24)
In
brief, exponentially growing cells were harvested and 100-
m
l
medium per well with 1310
3
cells suspended was plated in a
96-well plate. After 24-h incubation at 37 °C under a humidi-
fied 5% carbon dioxide atmosphere, 100-
m
l medium contain-
ing various concentrations of test specimen was added to
each well and incubated for 72 h under the same conditions.
After fixation with 25% glutaraldehyde solution (20
m
l),
the cells were stained with 0.5% crystal violet in 20%
methanol/water for 30 min. After gentle rinsing with water,
the retained crystal violet was extracted with 30% acetic acid
and measured spectrophotometrically at 590 nm.
Each extract was dissolved in a bit of DMSO and added
PBS, and then diluted with the medium; final concentration
of DMSO was less than 0.25%. 5-FU and doxorubicin were
used as positive controls, and EC
50
values were calculated
from the mean values of data from four wells.
In Vitro Growth Inhibition Test In brief, exponentially
growing HT-1080 cells were harvested and 100-
m
l EME
medium per well with 5310
3
cells suspended was plated in a
96-well plate. After 24-h incubation, the cells were treated
with varying concentrations of test specimens in EME
medium (100
m
l) and incubated for 12 and 24 h. Viability of
cells was determined using the standard MTT assay.
Observation of Morphological Changes Morphologi-
cal changes were observed as described previously.
25,26)
Briefly, exponentially growing HT-1080 cells were harvested
and plated 1310
5
cells per well in a 6-well plate. After 24-h
incubation, the cells were treated with varying concentrations
of test specimens and incubated for 24 h. At the end of incu-
bation, the morphological changes of the cells were recorded
by photomicrography using a phase contrast microscope
(Olympus Optical Co., Ltd., Tokyo).
Detection of DNA Fragmentation DNA was isolated
and detected by the procedure described previously.
25,26)
Briefly, HT-1080 or LLC cells (.2310
6
cells) were preincu-
bated in EME medium for 24 h, and then cultured with vari-
ous concentrations of test specimen in serum free DMEM/F-
12 medium containing 0.1% BSA, 100 IU/ml penicillin G
and 80 IU/ml streptomycin for 24 h. At the end of the incuba-
tion, cells were pelleted and lysed in 600
m
l of lysis buffer
(10 mM Tris-HCl buffer, pH 8.0, 10 mM EDTA and 0.2% Tri-
ton X-100) for 10 min on ice. After the lysate was cen-
trifuged at 14000 rpm for 10 min, the supernatant was ex-
tracted with TE buffer (10 mM Tris–HCl buffer, pH 8.0, 1 mM
EDTA)-saturated phenol, and then centrifuged at 14000 rpm
for 10 min. The upper layer was then extracted with CIAA
solution (chloroform : isoamylalcohol524 : 1), and DNA in
the upper layer (500
m
l) was precipitated with 3 M NaCl
(50
m
l) and cold ethanol (1000
m
l) at 220 °C overnight. After
drying, DNA was dissolved in TE buffer. Contamination of
RNA was eliminated by incubation with 1 mg/ml RNase at
37 °C for 30 min. Following the addition of loading buffer,
fragmented DNA was electrophoresed on 1.5% agarose gel
in TAE (40 mM Tris, 20 mM acetic acid, 1 mM EDTA) at
100 V for 30 min and visualized by ethidium bromide stain-
ing.
RESULTS AND DISCUSSION
From 77 Vietnamese medicinal plants (Table 1), methanol,
methanol–water (1 : 1) and water extracts were prepared, and
their antiproliferative activities were examined against highly
metastatic human HT-1080 fibrosarcoma cells (Table 2). Fif-
teen of the extracts showed antiproliferative activities in a
concentration-dependent manner with EC
50
values less than
20
m
g/ml: methanol extracts of Caesalpinia sappan (EC
50
,
15.8
m
g/ml), Catharanthus roseus (EC
50
, 5.88
m
g/ml), Eu-
rycoma longifolia (EC
50
, 15.8
m
g/ml), Hydnophytum formi-
carum (EC
50
, 9.97
m
g/ml) and Streptocaulon juventas (EC
50
,
754 Vol. 25, No. 6

June 2002 755
Table 1. Vietnamese Medicinal Plants Collected at Seven-Mountain Area, Tinh Bien District, Angiang Province and at Lamdong Province, Their Families,
Parts Used, Local Names, Therapeutic Applications and Voucher Specimen Numbers (TMPW No.)
Plant name Family Part used Local name Therapeutic application TMPW No.
Collected at Seven-Mountain area, Tinh Bien district, Angiang province
Amomum villosum LOUR. Zingiberaceae Leaf Sa nhan Digestive disease, diarrhoea 20449
(5A. repeus SONN.)
Ampelocissus martini PLANCH Vitaceae Root Sam hong Tonic 20451
Angelica dahurica (FISCH. ex HOFFM.) Umbelliferae Aerial part Bach chi Fever, rheumatism, leucorrhea 20415
BENTH. et HOOK. F.
Aquilaria crassna PIERRE ex LACOMTE Thymelaeaceae Wood Tram toc Asthma 20457
Artemisia vulgaris L. Compositae Leaf Ngai diep Antibacteria, fever, leucorrhea 20442
Asparagus cochinchinensis (LOUR.) MERR. Liliacea Root Thien mon, Tuberculosis, inflammation, 20421
Thien dong diabetes, breast cancer
Barleria lupulina LINDL. Acanthaceae Aerial part Kim vang Injury, fever, asthma 20435
Borassus flabellifer L. Arecaceae Flower Thot not bong, Diuretic, anthelminthic, inflammation 20419
Thot not, Thot lot
Bupleurum chinense DC. Umbelliferae Aerial part Sai ho Fever 20458
Caesalpinia sappan L. Caesalpiniaceae Wood To moc, Vang Diarrhoea, inflammation 20454
Catharanthus roseus (L.) G. DON Apocynaceae Aerial part Dua can Cancer (lung, leukemia), diabetes 20427
(5Vinca rosea L.)
Ceiba pentandra (L.) GAERTN. Bombacaceae Fruit Gon Malaria, inflammation, diarrhoea 20428
Cinnamomum iners REINW. ex BLUME Lauraceae Bark Hau phac, Rheumatism, tonic for stomach 20431
Hau phac nam
Combretum quadrangulare KURZ. Combretaceae Aerial part Tram bau Anthelminthic, hepatitis, inflammation 20456
Cupressus funebris ENDL. Cupressaceae Fruit Hoang dan, Fever, physical injury 20422
Huynh dan
Cyperus rotundus L. Cyperaceae Rhizome Co cu, Co gau Menstrual disorder, uterus inflammation, 20423
diarrhoea
Desmodium heterophyllum (WILLD.) DC. Fabaceae Aerial part Hanthe Fever, inflammation 20430
Drynaria quercifolia (L.) J. SM. Polypodiaceae Rhizome Rang bay Tuberculosis, antibacteria 20445
Elsholtzia ciliata (THUNB.) HYLAND. Lamiaceae Aerial part Kinh gioi Fever, inflammation 20436
Euphorbia tirucalli L. Euphorbiaceae Stem Xuong kho Inflammation, antibacteria 20459
Eurycoma longifolia JACK Simaroubaceae Root Ba benh, Leucorrhea, malaria, fever 20411
Bach benh
Eurycoma longifolia JACK Simaroubaceae Aerial part Ba benh, Tonic, diarrhoea 20412
Bach benh
Ficus sagitta VAHL Moraceae Stem Manh trau Tonic 20439
Glycyrrhiza uralensis FISCH. Fabaceae Root Cam thao Tonic, inflammation, diarrhoea, 20420
(5G. glabra L.) Addison’s disease
Hedyotis diffusa WILLD. Rubiaceae Aerial part Luoi ran trang, Inflammation, hepatitis, antitumor 20416
Bach hoa xa
thiet thao
Hydnophytum formicarum JACK Rubiaceae Gall Bi ky nam Hepatitis, rheumatism, diarrhoea 20418
Lasia spinosa (L.) THW. Araceae Whole plant Mop gai, Ray gai Inflammation, rheumatism 20440
Leonurus heterophyllus SWEET Lamiaceae Aerial part Ich mau Menstrual disorder, inflammation, 20433
tonic for women
Lindernia crustacea (L.) F. MUELL. Scrophulariaceae Stem Mau thao, Fever, hepatitis, leucorrhea 20437
Day luoi dong
Luvunga scandens (ROXB.) BUCH.-HAM. Rutaceae Branchlet Than xa huong Rheumatism, fever 20453
Marsilea quadrifolia L. Marsileaceae Aerial part Rau bo, Co bo Hepatitis, malaria, diabetes 20447
Melaleuca leucadendra (L.) L. Myrtaceae Stem and fruit Tram Fever, rheumatism, diarrhoea 20455
Miliusa velutina (DUN.) HOOK. F. et THOMS.Annonaceae Wood Co sen Inflammation, antibacteria 20424
Nauclea officinalis (PIT.) MERR. Rubiaceae Fruit Huynh ba Inflammation 20432
Orthosiphon spiralis (LOUR.) MERR. Lamiaceae Aerial part Rau meo Diuretic, inflammation 20448
(5O. stamineus BENTH.)
Panicum repens L. Poaceae Aerial part Cu co ong, Rheumatism, inflammation, leucorrhea 20425
Cu gung
Paraboea treubii (FORBES) BURTT Gesneriaceae Aerial part Bac thau da Cough, fever 20414
(5Boea treubii FORBES)
Parameria laevigata (JUSS.) MOLDENK Apocynaceae Stem Do trong day Rheumatism, hypertension 20460
Polanisia chelidonii (L. F.) A. DC Capparaceae Aerial part Man ri tia, Fever, inflammation 20438
(5Cleomo chelidonii L. F.) Man man tim
Polypodium subauriculatum BLUME Polypodiaceae Rhizome Bach xa Fever 20417
Rhinacanthus nasutus (L.) KURZ Acanthaceae Aerial part Kien co, Tuberculosis, inflammation, rheumatism, 20434
Bach hac hypertension
Sansevieria cylindrica BOJER Agavaceae Leaf Ngai nga, Inflammation 20443
Nanh heo, Nga Voi
Streptocaulon juventas (LOUR.) MERR. Asclepiadaceae Root Ha thu o trang Tonic, malaria, leucorrhea 20429
Tinospora cordifolia (WILLD.) MIERS Menispermaceae Stem Than thong, Malaria, fever, inflammation 20452
Day than thong
Tinospora crispa MIERS Menispermaceae Stem Day coc Fever, malaria 20426

6.04
m
g/ml) from Seven-Mountain area; methanol extracts of
Coscinium fenestratum (EC
50
, 11.7
m
g/ml) and S. juventas
(EC
50
, 1.15
m
g/ml) from Lamdong province; methanol–water
extracts of Cae. sappan (EC
50
, 13.8
m
g/ml), Cat. roseus
(EC
50
, 8.99
m
g/ml), H. formicarum (EC
50
, 11.3
m
g/ml)
and S. juventas (EC
50
, 12.1
m
g/ml) from Seven-Mountain
area; methanol–water extracts of Co. fenestratum (EC
50
,
18.1
m
g/ml) and S. juventas (EC
50
, 0.886
m
g/ml) from Lam-
dong province; water extracts of Cae. sappan (EC
50
,
17.8
m
g/ml) from Seven-Mountain area and S. juventas
(EC
50
, 4.96
m
g/ml) from Lamdong province.
Next, antiproliferative activities of the 15 extracts were ex-
amined against human cervix HeLa adenocarcinoma, human
lung A549 adenocarcinoma, murine colon 26-L5 carcinoma,
murine Lewis lung carcinoma (LLC) and murine B16-BL6
melanoma cells (Fig. 1, Table 3). These are three human
tumor cells (HT-1080, HeLa and A549) and three murine
tumor cells (26-L5, LLC and B16-BL6), while the four cell
lines (HT-1080, 26-L5, LLC and B16-BL6) are invasive and
metastatic and the LLC cell line is drug-resistant.
27)
The methanol and methanol-water extracts of Co. fenestra-
tum showed strong and selective antiproliferative activities
against two kinds of lung carcinoma cells, A549 and LLC;
methanol extract: EC
50
against LLC cells, 1.65
m
g/ml;
methanol–water extract: EC
50
against A549 and LLC cells,
2.88 and 2.84
m
g/ml, respectively. Lung cancer is one of the
major causes of death by cancer, and lung is one of the tis-
sues where many cancer cells including LLC and B16-BL6
cells metastasize. The methanol and methanol–water extracts
also showed antiproliferative activities against B16-BL6
cells, and berberine, a constituent of Co. fenestratum,
28)
has
recently been reported to inhibit metastasis of LLC cells.
29)
It
is interesting that Co. fenestratum and berberine are effective
and specific against tumors in lung, in spite of their use as
drugs for diseases of the digestive system, i.e., diarrhoea and
dysentery.
All extracts of Cae. sappan and Cat. roseus showed an-
tiproliferative activities against HT-1080 and LLC cells but
not against A549 cells, suggesting the presence of compo-
nents effective for the treatment of drug-resistant tumor. This
may be interesting, because the drug-resistant tumor is a
problem in long-term chemotherapy. In contrast, all extracts
756 Vol. 25, No. 6
Table 1. (continued)
Plant name Family Part used Local name Therapeutic application TMPW No.
Vernonia cinerea (L.) LESS. Compositae Aerial part Bach dau ong Hepatitis, fever, diarrhoea 20413
Collected at Lamdong province
Adenosma glutinosum (L.) DRUCE Scrophulariaceae Aerial part Nhan tran Hepatitis, tonic for women 20616
Ageratum conyzoides L. Compositae Aerial part Cay cut lon Inflammation 20462
Aloe vera L. Aloaceae Leaf Lo hoi Cold, fever 20611
Andrographis paniculata (BURM. F.) NEES Acanthaceae Aerial part Xuyen tam lien Inflammation, hypertension 20626
Artemisia apiacea HANCE ex WALP. Compositae Aerial part Thanh hao Malaria, inflammation 20621
Cassia tora L. Caesalpiniaceae Seed Thao quyet minh Hepatitis, hypertension 20622
Codonopsis javanica (BLUME) HOOK. F. Campanulaceae Root Dang sam Tonic, leukemia, inflammation, hepatitis 20466
Coscinium fenestratum (GAERTN.) COLBER. Menispermaceae Stem Vang dang, Malaria, diarrhoea, inflammation 20606
(5C. usitatum PIERRE) Hoang dang
Datura metal L. Solanaceae Flower Ca doc duoc Asthma, inflammation 20463
Eleutherine bulbosa (MILL.) URB. Iridaceae Bulb Sam dai hanh Cough, inflammation 20618
Eucommia ulmoides OLIV. Eucommiaceae Bark Do trong Hypertension, rheumatism 20468
Gymnopetalum cochinchinensis (LOUR.) Cucurbitaceae Stem and leaf Cut qua Cough, inflammation 20465
KURZ
Heliotropium indicum L. Boraginaceae Aerial part Voi voi Inflammation 20625
Kalanchoe pinnata (LAM.) PERS. Crassulaceae Aerial part Truong sinh, Antibacteria, inflammation 20624
Thuoc bong
Launaea pinnatifida CASS. Compositae Aerial part Sa sam, Galactopoietic, leucorrhea 20617
Sa sam nam
Lonicera japonica THUNB. Caprifoliaceae Flower Kim ngan Inflammation, rheumatism 20609
Luffa cylindrica (L.) ROEM. Cucurbitaceae Fruit Muop gai, Muop Inflammation, cough 20614
Merremia bimbim (GAGNEP.) van Convolvulaceae Seed Bim bim Anthelminthic, diuretic 20461
OOSTSTR. (5Ipomea bimbim GAGNEP.)
Mimosa pudica L. Mimosaceae Aerial part Mac co Inflammation, hepatitis, hypertension 20612
Nelumbo nucifera GAERTN. Nelumbonaceae Kernel Tam sen Hypertension, heart disease 20619
Panax pseudo-ginseng WALL. Araliaceae Root Tam that Tonic, sterility, cancer 20620
Phyllantus amarus SCHUM. et THONN. Euphorbiaceae Aerial part Cho de rang cua, Inflammation, hepatitis 20464
Cho de than xanh
Piper lolot C. DC. Piperaceae Aerial part La lot Rheumatism, diarrhoea 20610
Plantago major L. Plantaginaceae Aerial part Ma de Inflammation 20613
Polygonum multiflorum THUNB. Polygonaceae Root Ha thu o, Tonic, malaria 20469
Ha thu o do
Polyscias fruticosa (L.) HARMS Araliaceae Stem and leaf Dinh lang Tonic, inflammation 20467
Schefflera octophylla (LOUR.) HARMS Araliaceae Bark Ngu gia bi chan Tonic, inflammation 20615
chim, Chan chim
Smilax glabra ROXB. Smilacaceae Rhizome Tho phuc linh Inflammation 20623
Sophora flavescens AIT. Fabaceae Root Kho sam Inflammation, diarrhoea 20608
Streptocaulon juventas (LOUR.) MERR. Asclepiadaceae Root Ha thu o trang Tonic, malaria, leucorrhea 20470
Xanthium strumarium L. Compositae Fruit Ke dau ngua Inflammation, malaria 20607

June 2002 757
Table 2. Yields (%) and Antiproliferative Activities against Human HT-1080 Fibrosarcoma Cells (EC
50
in
m
g/ml) of Each Extract of Vietnamese Medicinal
Plants
Yield (%) EC
50
(
m
g/ml)
Plant name
MeOH ext. MeOH–H
2
O ext. H
2
O ext. MeOH ext. MeOH–H
2
O ext. H
2
O ext.
Collected at Seven-Mountain area, Tinh Bien district, Angiang province
Amomum villosum 32.6 11.6 10.5 81.5 .100 .100
Ampelocissus martini 2.2 4.7 2.4 .100 .100 .100
Angelica dahurica 9.5 4.9 3.2 60.0 .100 .100
Aquilaria crassna 3.2 1.4 0.9 69.5 73.7 .100
Artemisia vulgaris 17.1 11.4 8.6 38.0 82.2 73.6
Asparagus cochinchinensis 1.1 0.4 0.3 66.9 .100 .100
Barleria lupulina 11.5 7.8 4.4 .100 .100 .100
Borassus flabellifer 3.0 5.4 2.8 73.2 .100 100
Bupleurum chinense 9.7 6.7 4.1 .100 .100 .100
Caesalpinia sappan 10.2 1.9 0.3 15.8 13.8 17.8
Catharanthus roseus 22.7 7.6 4.1 5.88 8.99 95.0
Ceiba pentandra 5.8 4.7 4.1 .100 .100 .100
Cinnamomum iners 9.9 4.4 1.3 .100 .100 .100
Combretum quadrangulare 11.5 5.4 4.2 47.9 49.6 46.6
Cupressus funebris 31.6 5.7 3.9 .100 .100 .100
Cyperus rotundus 2.3 3.1 2.0 70.8 .100 .100
Desmodium heterophyllum 8.6 3.6 1.4 .100 .100 .100
Drynaria quercifolia 4.9 2.2 1.9 .100 .100 .100
Elsholtzia ciliata 3.1 2.2 2.6 .100 .100 .100
Euphorbia tirucalli 8.8 5.7 5.1 .100 .100 .100
Eurycoma longifolia (root) 2.1 1.4 0.7 15.8 53.7 .100
Eurycoma longifolia (arial part) 7.4 4.3 2.5 55.8 55.1 84.5
Ficus sagitta 6.0 4.0 1.7 33.0 26.5 .100
Glycyrrhiza uralensis 8.0 3.5 1.0 .100 88.4 .100
Hedyotis diffusa 12.6 6.2 2.4 63.0 54.7 .100
Hydnophytum formicarum 8.4 2.7 1.2 9.97 11.3 22.3
Lasia spinosa 5.4 3.2 2.3 .100 .100 .100
Leonurus heterophyllus 3.9 2.3 2.1 .100 .100 .100
Lindernia crustacea 10.6 4.7 3.8 .100 .100 .100
Luvunga scandens 2.8 1.3 0.8 .100 .100 .100
Marsilea quadrifolia 4.0 4.3 2.7 .100 .100 .100
Melaleuca leucadendra 16.3 4.8 2.4 66.0 .100 .100
Miliusa velutina 4.8 1.5 0.3 60.7 .100 .100
Nauclea officinalis 9.3 15.7 3.4 23.2 30.5 26.7
Orthosiphon spiralis 6.0 5.5 5.8 .100 .100 .100
Panicum repens 9.1 6.2 1.8 80.1 22.9 .100
Paraboea treubii 16.4 7.8 2.6 58.9 79.3 .100
Parameria laevigata 7.7 4.4 1.6 .100 .100 .100
Polanisia chelidonii 11.5 7.3 4.8 .100 .100 .100
Polypodium subauriculatum 4.2 1.3 1.1 .100 .100 .100
Rhinacanthus nasutus 6.9 7.4 4.3 .100 .100 .100
Sansevieria cylindrica 2.7 0.6 1.2 .100 .100 .100
Streptocaulon juventas 10.9 5.8 3.5 6.04 12.1 47.6
Tinospora cordifolia 6.1 3.1 3.3 68.1 .100 .100
Tinospora crispa 2.6 1.4 1.1 50.4 .100 .100
Vernonia cinerea 8.1 4.1 2.2 86.7 .100 .100
Collected at Lamdong province
Adenosma glutinosum 4.3 3.5 4.3 .100 .100 .100
Ageratum conyzoides 3.4 2.8 3.1 .100 .100 .100
Aloe vera 57.6 23.8 5.1 .100 .100 .100
Andrographis paniculata 9.3 4.6 3.5 90.0 .100 .100
Artemisia apiacea 6.0 4.7 1.8 97.8 .100 .100
Cassia tora 5.9 1.9 1.4 85.6 .100 .100
Codonopsis javanica 30.5 10.0 12.5 .100 .100 .100
Coscinium fenestratum 9.4 2.8 0.9 11.7 18.1 76.0
Datura metal 9.5 5.8 5.2 51.6 .100 .100
Eleutherine bulbosa 7.4 3.9 0.9 80.2 .100 .100
Eucommia ulmoides 0.6 0.3 0.5 79.7 .100 .100
Gymnopetalum cochinchinensis 6.2 4.6 3.5 93.0 .100 .100
Heliotropium indicum 8.3 5.5 2.6 .100 .100 .100
Kalanchoe pinnata 6.0 1.8 0.9 .100 .100 .100
Launaea pinnatifida 2.6 4.3 3.3 .100 .100 .100
Lonicera japonica 32.8 4.7 1.7 .100 .100 .100
Luffa cylindrica 5.4 5.1 2.1 .100 .100 .100
Merremia bimbim 4.9 1.1 1.3 49.1 71.4 .100

of H. formicarum and S. juventas showed the potent activities
against A549 cells with EC
50
values less than 4
m
g/ml, but
not against LLC cells. The activities against A549 cells of
the methanol and methanol–water extracts of S. juventas
from Lamdong province (EC
50
, 0.121 and 0.138
m
g/ml, re-
spectively) were stronger than a positive control 5-FU (EC
50
,
0.244
m
g/ml). It should be noted here that H. formicarum and
S. juventas selectively suppressed the proliferation of human
tumor cells, HT-1080, HeLa and A549. Moreover, the
methanol extract of S. juventas from Lamdong province
showed the activity also against 26-L5 cells (EC
50
,
15.3
m
g/ml), in spite of weaker activities of other extracts.
The extract of E. longifolia was antiproliferative against all
cells.
S. juventas extract, which showed selective and the most
potent activity, induced HT-1080 cells in a spindle-shape
when the present at more than 1
m
g/ml and in a multi-bleb-
bing-shape when the present at 4
m
g/ml (Fig. 2), while in-
hibiting their growth in a time- and concentration-dependent
manner (Fig. 3). These are the morphological changes typical
of apoptosis.
30)
Thus, we examined DNA fragmentation to
clarify whether the S. juventas extract induced apoptosis or
not. As can be seen in Fig. 4, the extract induced ladder-like
DNA fragmentation in a concentration-dependent manner
against LLC and HT-1080, and this DNA fragmentation was
in parallel with the growth inhibition. These ladder fragmen-
tations of DNA and characteristic morphological changes in-
dicate that antiproliferation by S. juventas is caused by apop-
tosis. Many anticancer drugs damage DNA or suppress its
duplication, not to kill cells directly but to induce apopto-
sis.
31—33)
The extract of S. juventas induced apoptosis with
selectivity to tumor cells and thus seems to be a desirable
candidate for a clinical drug.
Though there is no report on the antiproliferative activity
of Co. fenestratum, which shows selective activity against
lung-related tumor cells, its constituents, benzylisoquinoline-
type alkaloids such as berberine,
28,34)
were reported to be cy-
totoxic.
35)
The antiproliferative constituents of Cae. sappan
also have not been reported, but the major antioxidative com-
ponent of Cae. sappan, brazilin,
36)
seemed to inhibit growth
of tumor cells, based on a report that antioxidative phenolic
compounds were cytotoxic against tumor cells.
37)
Cat.
758 Vol. 25, No. 6
Table 2. (continued)
Yield (%) EC
50
(
m
g/ml)
Plant name
MeOH ext. MeOH–H
2
O ext. H
2
O ext. MeOH ext. MeOH–H
2
O ext. H
2
O ext.
Mimosa pudica 5.5 1.8 1.8 .100 .100 .100
Nelumbo nucifera 12.3 11.0 6.3 61.7 .100 .100
Panax pseudo-ginseng 5.4 5.4 3.4 .100 .100 .100
Phyllantus amarus 7.5 5.2 3.2 .100 36.9 82.8
Piper lolot 7.9 4.3 4.0 85.6 .100 .100
Plantago major 15.0 5.2 4.3 .100 .100 .100
Polygonum multiflorum 17.0 8.7 1.9 70.8 21.9 .100
Polyscias fruticosa 15.8 4.5 5.5 .100 .100 .100
Schefflera octophylla 2.3 1.4 1.7 .100 .100 .100
Smilax glabra 17.5 4.8 1.3 64.9 87.8 .100
Sophora flavescens 11.3 3.6 2.0 .100 .100 .100
Streptocaulon juventas 3.0 1.3 1.9 1.15 0.886 4.96
Xanthium strumarium 3.0 2.8 3.6 88.0 .100 .100
Table 3. Antiproliferative Activities of the Extracts against Human HT-1080 Fibrosarcoma, Human Cervix HeLa Adenocarcinoma, Human Lung A549
Adenocarcinoma, Murine Colon 26-L5 Carcinoma, Murine Lewis Lung Carcinoma (LLC) and Murine B16-BL6 Melanoma Cells (EC
50
values in
m
g/ml)
Scientific name Extract HT-1080 HeLa A549 26-L5 LLC B16-BL6
Caesalpinia sappan MeOH 15.8 15.5 41.4 23.2 8.20 20.1
MeOH–H
2
O 13.8 27.8 73.8 49.7 16.7 50.6
H
2
O 17.8 23.7 49.8 59.4 16.4 23.0
Catharanthus roseus MeOH 5.88 20.7 .100 .100 4.36 .100
MeOH–H
2
O 8.99 51.6 .100 .100 12.7 .100
Eurycoma longifolia MeOH 15.8 14.2 19.1 15.8 2.29 9.16
Hydnophytum formicarum MeOH 9.97 11.3 1.03 22.6 65.6 50.6
MeOH–H
2
O 11.3 16.3 0.780 87.1 .100 .100
Streptocaulon juventas
a)
MeOH 6.04 13.6 0.790 48.8 69.8 .100
MeOH–H
2
O 12.1 15.1 0.943 .100 .100 .100
Coscinium fenestratum MeOH 11.7 30.1 5.32 79.0 1.65 5.85
MeOH–H
2
O 18.1 49.0 2.88 .100 2.84 8.91
Streptocaulon juventas
b)
MeOH 1.15 4.21 0.121 15.3 48.9 45.3
MeOH–H
2
O 0.886 5.47 0.138 47.0 51.1 66.3
H
2
O 4.96 15.7 0.591 .100 .100 .100
5-FU 0.198 0.0871 0.244 0.0673 0.0276 0.0782
Doxorubicin 0.104 0.195 0.0325 0.0573 0.107 0.0944
a) Collected at Seven-Mountain area. b) Collected at Lamdong province.

roseus, which is used for treatment of cancer and diabetes in
Vietnam,
13,14)
was reported to contain the anticancer indole
alkaloids, vinblastine and vincristine,
38,39)
which are com-
monly called Vinca alkaloids or Catharanthus alkaloids. E.
longifolia, inhibiting proliferation of all cells, was also re-
ported to contain several cytotoxic quassinoids and indole al-
kaloids.
40,41)
H. formicarum and S. juventas, exhibiting selec-
tive activity against human tumor cell lines, are used for
treatments of hepatitis, rheumatism and diarrhoea and for
treatments of malaria and leucorrhea and as tonic, respec-
tively, in Vietnam.
13,14)
But their constituents or biological
and pharmacological activities have not been reported scien-
June 2002 759
Fig. 1. Cellular Viabilities in the Presence of the Methanol Extract of
Streptocaulon juventas Collected at Lamdong Province and the Methanol
Extract of Coscinium fenestratum
After 24-h preincubation at 37 °C, the cells were cultured in medium with each ex-
tract for 72 h under the same conditions. Results are expressed as the mean (% of con-
trol)6S.D. (n54).
Fig. 2. Morphological Changes in Human HT-1080 Fibrosarcoma Cells Treated with the Methanol Extract of Streptocaulon juventas Collected at Lam-
dong Province
After 24-h preincubation, the cells were cultured for 24 h without (A) or with the extract at 1 (B) and at 4
m
g/ml (C). Original magnification: 3100.
Fig. 3. Growth of Human HT-1080 Fibrosarcoma Cells in the Presence of
the Methanol Extract of Streptocaulon juventas Collected at Lamdong
Province
After 24-h preincubation at 37 °C, the cells were cultured in media with each extract
for 12 and 24 h under the same conditions. Results are expressed as the mean (% of
control)6S.D. (n54).
Fig. 4. The Methanol Extract of Streptocaulon juventas from Lamdong
Province Induced DNA Fragmentation in Murine Lewis Lung Carcinoma
(LLC) (A) and Human HT-1080 Fibrosarcoma cells (B)
After the cells were cultured for 24 h with various concentrations of the extract, the
fragmented DNA was isolated, electrophoresed on 1.5% agarose gel, and then visual-
ized by ethidium bromide staining. (A) Lane 1: 100 base-pair ladder marker; lane 2:
normal; lanes 3—7: treated with 1, 3, 10, 30 and 100
m
g/ml of the S. juventas extract,
(B) Lane 1: 100 base-pair ladder marker; lane 2: normal; lanes 3—5: treated with 1, 3
and 10
m
g/ml of the S. juventas extract, respectively.

tifically. The antiproliferative activities of Co. fenestratum,
showing selective activity against lung-related tumor cells, of
H. formicarum, showing selective activity against human
tumor cells, and of S. juventas, showing selective activity
against human tumor cells with induction of apoptosis, are
interesting and thus their constituents are under investigation
and will be reported elsewhere.
Acknowledgement This work was supported in part by
a Grant-in-Aid for International Scientific Reseach (No.
13576027) from the Ministry of Education, Culture, Sports,
Science and Technology, Japan.
REFERENCES
1) World Health Organization, “World Health Statistics Annual 1996,”
World Health Organization, Geneva, 1998.
2) Fidler I. J., Kripke M. L., Science, 197, 893—895 (1977).
3) Kligerman M. M., “Cancer Medicine,” ed. by Holland J. F., Frei E. III,
Lea & Febiger, Philadelphia, 1973, pp. 541—565.
4) Medical Economic, “Physicians’ Desk Reference,” 52nd ed., Medical
Economic, Montvale, 1998.
5) World Health Organization Regional Office for Western Pacific, Insti-
tute of Materia Medica, “Medicinal Plants in Viet Nam,” Science and
Technology Publishing House, Hanoi, 1998.
6) Ito M., Yamane Y., Nguyen D. C., Tran K. Q., Honda G., Natural Med-
icines, 50, 420—425 (1996).
7) Cuong N. M., Taylor W. C., Sung T. V., Phytochemistry, 52, 1711—
1714 (1999).
8) Kim T. H., Thuy N. T., Shin J. H., Baek H. H., Lee H. J., J. Agric. Food
Chem., 48, 2877—2881 (2000).
9) Banskota A. H., Tezuka Y., Tran K. Q., Tanaka K., Saiki I., Kadota S.,
Chem. Pharm. Bull., 48, 496—504 (2000).
10) Tezuka Y., Stampoulis P., Banskota A. H., Awale S., Tran K. Q., Saiki
I., Kadota S., Chem. Pharm. Bull., 48, 1711—1719 (2000).
11) Adnyana I K., Tezuka Y., Banskota A. H., Tran K. Q., Kadota S., J.
Nat. Prod., 64, 360—363 (2001).
12) Tran Q. L., Adnyana I. K., Tezuka Y., Nagaoka T., Tran Q. K., Kadota
S., J. Nat. Prod., 64, 456—461 (2001).
13) Vo V. C., “Dictionary of Vietnamese Medicinal Plants,” Medicine Pub-
lisher, Hochiminh City, 1996.
14) Do T. L., “Vietnamese Medicinal Plants,” Medicine Publisher, Hanoi,
2001.
15) Rasheed S., Nelson-Rees W. A., Toth E. M., Arnstein P., Gardner M.
B., Cancer, 33, 1027—1033 (1974).
16) Gey G. O., Coffman W. D., Kubicek M. T., Cancer Res., 12, 264—265
(1952).
17) Giard D. J., Aaronson S. A., Todaro G. J., Arnstein P., Kersey J. H.,
Dosik H., Parks W. P., J. Natl. Cancer Inst., 51, 1417—1423 (1973).
18) Lieber M., Smith B., Szakal A., Nelson-Rees W., Todaro G., Int. J.
Cancer, 17, 62—70 (1976).
19) Ohnishi Y., Sakamoto T., Fujii H., Kimura F., Murata J., Tazawa K.,
Fujimaki M., Sato Y., Kondo M., Une Y., Uchino J., Saiki I., Tumor
Biol., 18, 113—122 (1997).
20) Aptekman P. M., Lewis M. R., J. Immunol., 66, 361—364 (1951).
21) Hart I. R., Am. J. Pathol., 97, 587—600 (1979).
22) Rubinstein L. V., Shoemaker R. H., Paull K. D., Simon R. M., Tosini
S., Skehan P., Scudiero D. A., Monks A., Boyd M. R., J. Natl. Cancer
Inst., 82, 1113—1118 (1990).
23) Banskota A. H., Tezuka Y., Prasain J. K., Matsushige K., Saiki I.,
Kadota S., J. Nat. Prod., 61, 896—900 (1998).
24) Saito K., Oku T., Ata N., Miyashiro H., Hattori M., Saiki I., Biol.
Pharm. Bull., 20, 345—348 (1997).
25) Suda K., Murakami K., Hasegawa H., Saiki I., J. Trad. Med., 17,
236—244 (2000).
26) Wu W., Murakami K., Koketsu M., Yamada Y., Saiki I., Anticancer
Res., 19, 5375—5382 (1999).
27) Furusawa Eii., Furusawa Eik., Sokugawa L., Chemotherapy, 29, 294—
302 (1983).
28) Siwon J., Verpoorte R., van Essen G. F. A., Svendsen A. B., Planta
Med., 38, 24—32 (1980).
29) Mitani N., Murakami K., Yamaura T., Ikeda T., Saiki I., Cancer Lett.,
165, 35—42 (2001).
30) Wyllie A. H., Kerr J. F. R., Currie A. R., Int. Rev. Cytol., 68, 251—306
(1980).
31) Barry M. A., Behnke C. A., Eastman A., Biochem. Pharm., 40,
2353—2362 (1990).
32) Ling Y.-H., Priebe W., Perez-Soler R., Cancer Res., 53, 1845—1852
(1993).
33) Fisher D. E., Cell, 78, 539—542 (1994).
34) Pinho P. M. M., Pinto M. M. M., Kijjoa A., Pharadai K., Diaz J. G.,
Herz W., Phytochemistry, 31, 1403—1407 (1992).
35) Orfila L., Rodríguez M., Colman T., Hasegawa M., Merentes E.,
Arvelo F., J. Ethnopharmacol., 71, 449—456 (2000).
36) Moon C.-K., Park K.-S., Kim S.-G., Won H.-S., Chung J.-H., Drug
Chem. Toxicol., 15, 81—91 (1992).
37) Mahmoud N. N., Carothers A. M., Grunberger D., Bilinski R. T.,
Churchill M. R., Martucci C., Newmark H. L., Bertagnolli M. M.,
Carcinogenesis, 21, 921—927 (2000).
38) Noble R. L., Beer C. T., Cutts J. H., Ann. N.Y. Acad. Sci., 76, 882—
894 (1958).
39) Adamson R. H., Dixon R. L., Ben M., Crews L., Shohet S. B., Rall D.
P., Arch. Int. Pharmacodyn. Ther., 157, 299—311 (1956).
40) Morita H., Kishi E., Takeya K., Itokawa H., Tanaka O., Chem. Lett.,
1990, 749—752 (1990).
41) Kardono L. B. S., Angerhofer C. K., Tsauri S., Padmawinata K., Pez-
zuto J. M., Kinghorn A. D., J. Nat. Prod., 54, 1360—1367 (1991).
760 Vol. 25, No. 6