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Antitumor Principle Constituents of Myrica rubra Var.
acuminata
LING-LING YANG,†,‡ CHIA-CHEN CHANG,†LIH-GEENG CHEN,‡AND
CHING-CHIUNG WANG*,†
Graduate Institute of Pharmacognosy, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110,
Taiwan, Republic of China, and Graduate Institute of Biopharmaceutics, National Chiayi University,
300 University Road, Chiayi 600, Taiwan, Republic of China
Myrica rubra
var.
acuminata
is a native shrub widely distributed and used as folk medicine in Taiwan
for stomach disorders and diarrhea. Column chromatography combined with cytotoxic bioassay-
guided fractionation was performed to isolate the antitumor principles from fresh leaves of
M. rubra
var.
acuminata.
The 20% MeOH eluate fraction of
M. rubra
var.
acuminata
inhibited the viability of
HeLa and P-388 cells in an in vitro assay and an in vivo P-388 tumor-bearing CDF1mouse model.
The percent increase in life span (%ILS) of 20% MeOH eluate fraction was greater than 125%. (-)-
Epigallocatechin 3-
O
-gallate (1) and prodelphinidin A-2,3′-
O
-gallate (2) were isolated from D-20 as
the antitumor principle components. Both compounds can inhibit the growth of HeLa cells, but 1had
lower cytotoxic effects in normal cervical fibroblasts than did 2. Moreover, pretreatment with a
caspase-3 specific inhibitor prevented 1- and 2-induced poly(ADP-ribose) polymerase cleavage. In
view of these results, we suggest that 1and 2can induce apoptosis in HeLa cells and that activation
of caspase-3 may provide a mechanistic explanation for their cytotoxic effects. Therefore, we suggest
that the 20% MeOH eluate fraction extract is good for health and that
M. rubra
var.
acuminata
is an
economically valuable plant.
KEYWORDS:
Myrica rubra
var.
acuminata
; Myricaceae; apoptosis; HeLa cells; P-388 cells; (-)-
epigallocatechin 3-
O
-gallate; prodelphinidin A-2,3′-
O
-gallate
INTRODUCTION
Myrica rubra (Myricaceae) is widely distributed in Taiwan,
and there is one variety, M. rubra Sieb. et Zucc var. acuminata
Nakai (1). The bark of M. rubra has been used locally as an
astringent, an antidote, and an antidiarrheic in Chinese traditional
medicine (2). Previously, several flavonoids, tannins, triterpenes,
and diarylheptanoids were isolated from the bark of M.rubra
(3-8). Pharmacological studies of this medicinal plant have
reported that its methanolic extract showed protective effects
on CCl4- and R-naphthylisothiocyanate-induced liver injury,
whereas the 50% aqueous enthanolic extract and some con-
stituents inhibited melanin biosynthesis and showed antiandro-
genic activity (9). However, the constituents of M. rubra var.
acuminata have not been investigated.
In our previous study, we screened the cytotoxicity effects
of 70% acetone extracts of medicinal plants on HeLa cells using
an 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bro-
mide (MTT) assay (10). The 70% acetone extract of M. rubra
var. acuminata showed greater cytotoxic effects on cervical
tumor cells than on normal cells. In the present study, cytotox-
icity bioassay-guided fractionation was performed to isolate the
antitumor principles from M. rubra var. acuminata, and their
mechanism for inducing the cell death mode in tumor cells was
investigated.
Several chemicals can induce tumor cell death; however,
apoptosis is an efficient strategy for cancer chemotherapy. The
apoptotic mode involves the active participation of affected cells
in a self-destruction cascade that culminates in DNA degradation
via endonuclease activation, nuclear disintegration, and forma-
tion of “apoptotic bodies” that involve the cell remnants. These
apoptotic bodies are rapidly cleaned from local tissues by
macrophages (11). However, little is known about the regulation
and induction of apoptosis by natural products. In the present
study, the human cervical carcinoma cell line (HeLa) and mouse
leukemia cell line (P-388) were differentially susceptible to
apoptosis induced by a natural product. Cell death was detected
and identified using an MTT assay, propidium iodide staining
followed by flow cytometry, DNA electrophoresis, and poly-
(ADP-ribose) polymerase (PARP) proteolysis by Western blot
assay (12).
While many compounds have been shown to inhibit the
proliferation of mammalian cells in culture, only a small
proportion of these demonstrate significant selectivity in vivo
even in the most chemosensitive animal tumor models. There-
* To whom correspondence should be addressed. Tel: +886-2-27361661
ext. 6161. Fax: +886-2-27388351. E-mail: crystal@tmu.edu.tw.
†Taipei Medical University.
‡National Chiayi University.
fore, the antitumor effects of the cytotoxic components were
evaluated using a P-388 tumor-bearing CDF1mouse model (13).
MATERIALS AND METHODS
General. 1H (500 MHz) and 13C NMR (126 MHz) spectra were
measured on a Bruker DRX 500 instrument, and chemical shifts are
given in δ(ppm) values. Values of fast atom bombardment mass
spectrometry (FAB-MS) were obtained on a VG 70-SE mass spec-
trometer using 3-nitrobenyl alcohol containing NaCl as the matrix agent.
Normal phase high-performance liquid chromatography (HPLC) was
conducted on a 250 mm ×4 mm i.d. LiChrospher Si60 column (Merck,
Darmstadt, Germany) using n-hexane-MeOH-tetrahydrofuran (THF)-
HCOOH (47:42:10:1) containing 450 mg/L oxalic acid as the mobile
phase. The flow rate was 1.0 mL/min, with detection at 280 nm.
Reversed-phase HPLC was conducted on a 250 mm ×4 mm i.d.
LiChrospher 100 RP-18e column (Merck) eluted with 0.05 M H3PO4-
0.05 M KH2PO4-CH3CN (44:44:12). The flow rate was 1.0 mL/min,
and detection was at 280 nm. Column chromatography was carried
out on Toyopearl HW-40 (coarse grade) (Tosoh), Diaion HP-20, and
MCI-gel CHP-20P (Mitsubishi Chemical Industry, Japan). All solvents
used for column chromatography were of analytical grade.
Dimethyl sulfoxide (DMSO), MTT, trypan blue, and other chemicals
were purchased from Sigma Industries (St. Louis, MO). Dulbecco’s
modified Eagle medium (DMEM), fetal bovine serum (FBS), anti-
biotics, glutamine, and trypsin-ethylenediaminetetraacetic acid (EDTA)
were purchased from Gibco (Grand Island, NY). Western blotting was
performed using an antibody specific to human PARP (sc-7150),
R-tubulin (sc-8035), antirabbit IgG-AP (sc-2007), and antimouse IgG-
AP (sc-2008), which were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). All other reagents and chemicals were of the highest
purity grade available.
Plant Materials. Fresh leaves of M. rubra var. acuminata were
collected in Taipei, Taiwan, in September 2000 and dried at below 40
°C to yield 5.0 kg of dried leaves. A voucher specimen (MR001) is
deposited in the Graduate Institute of Pharmacognosy Science, Taipei
Medical University.
Isolation. Dried leaves (5.0 kg) were homogenized in 70% aqueous
acetone (50 L) and filtered. The concentrated filtrate (574 g) was
chromatographed over a 45 cm ×9.5 cm i.d. Diaion HP-20 column
(Mitsubishi Chemical Industry) with aqueous MeOH (10% f20% f
40% f60% MeOH) and acetone. The 20% MeOH eluate (51.39 g)
was rechromatographed over an ODS column eluted with 0.05 M KH2-
PO4-0.05 M H3PO4-CH3CN (44: 44: 12) and MCI-gel CHP20P with
aqueous MeOH to yield (-)-epigallocatechin gallate (1, 181.0 mg) and
prodelphinidin A-2,3′-O-gallate (2, 63.2 mg) (Figure 1). Each com-
pound was identified by direct comparison of its spectroscopic data
with authentic samples (4,5,14). Purity tests of the two compounds
were shown to be greater than 95% by normal and reversed phase
HPLC. Retention times for normal and reversed phase HPLC were 11.2
and 12.9 min for 1and 15.3 and 22.8 min for 2, respectively.
(-)-Epigallocatechin Gallate (1). Pale yellow amorphous powder.
FAB-MS: m/z459 [M +H]+.1H NMR (500 MHz, acetone-d6): δ
2.89 (1H, dd, J)2.3, 17.3 Hz, H-4β), 3.02 (1H, dd, J)4.5, 17.3 Hz,
H-4R), 5.05 (1H, s, H-2), 5.54 (1H, br t, J)2.3 Hz, H-3), 6.01 (1H,
d, J)2.4 Hz, H-6), 6.04 (1H, d, J)2.4 Hz, H-8), 6.61 (2H, s, H-2′,H-
6′), 7.01 (2H, s, Gal H-2,H-6). 13C NMR (126 MHz, acetone-d6): δ
78.1 (C-2), 69.2 (C-3), 26.6 (C-4), 157.1 (C-5), 95.8 (C-6), 157.8 (C-
7), 96.5 (C-8), 157.4 (C-8a), 99.0 (C-4a), 130.7 (C-1′), 106.7 (C-2′,
C-6′), 146.2 (C-3′, C-5′), 133.1 (C-4′), 121.9 (Gal C-1), 110.0 (Gal
C-2, C-6), 145.9 (Gal C-3,C-5), 138.8 (Gal C-4), 166.0 (ester carbonyl).
Prodelphinidin A-2,3′-O-gallate (2). Tan amorphous powder; [R]D
-132°(c)1.0, acetone). FAB-MS: m/z761 [M +H]+.1H NMR
(500 MHz, acetone-d6): δ2.95 (1H, d, J)17.6 Hz, H-4′β), 3.13 (1H,
dd, J)4.9, 17.6 Hz, H-4′R), 4.16 (1H, d, J)3.2 Hz, H-3), 4.47 (1H,
d, J)3.2 Hz, H-4), 5.19 (1H, s, H-2′), 5.69 (1H, br d, J)4.9 Hz,
H-3′), 6.12 (1H, s, H-6′), 6.04 (1H, d, J)2.3 Hz, H-6), 6.23 (1H, d,
J)2.3 Hz, H-8), 6.77 (2H, s, B-ring H-2, H-6), 7.12 (2H, s, Gal H-2,
H-6), 6.78 (2H, s, B′-ring H-2, H-6). 13C NMR (126 MHz, acetone-
d6): δ104.0 (C-2), 68.3 (C-3), 27.5 (C-4), 151.4 (C-5), 96.2 (C-6),
152.4 (C-7), 97.7 (C-8), 99.9 (C-4a), 133.7 (B-ring C-1), 107.4 (B-
ring C-2, C-6), 145.7 (B-ring C-3, C-5), 131.7 (B-ring C-4), 80.0 (C-
2′), 67.9 (C-3′), 26.0 (C-4′), 155.8 (C-5′), 96.6 (C-6′), 157.9 (C-7′),
101.5 (C-4′a), 133.9 (B′-ring C-1), 107.1 (B′-ring C-2, C-6), 146.5 (B′-
ring C-3, C-5), 129.3 (B′-ring C-4), 121.7 (Gal C-1), 110.5 (Gal C-2,
C-6), 139.0 (Gal C-4), 145.8 (Gal C-3, C-5), 151.4, 153.8, 157.9 (C-5,
C-7, C-8a), 152.4, 155.8, 156.5 (C-5′, C-7′, C-8′a), 166.3 (ester
carbonyl).
Cell Cultures. The HeLa and P-388 cell lines were obtained from
American Type Cell Culture (ATCC, Rockville, MD) and maintained
in DMEM (Gibco) supplemented with 10% FBS, 100.0 mg/L strep-
tomycin, and 100 IU/mL penicillin (Gibco). All cell cultures were
incubated at 37 °C in a humidified atmosphere of 5% CO2.
Primary Culture. The normal cervical tissue was isolated from the
cervical carcinoma in situ of a Taiwanese woman (45 years) and
identified through the pathology by Dr. Chun-Sen Hsu in Wan-Fang
Hospital, Taipei, Taiwan. Normal fibroblasts were isolated from human
normal cervical tissues. Fresh tissues were rinsed twice with phosphate-
buffered saline (PBS) and then cut into 0.1 cm3pieces. These pieces
were incubated with 0.25% trypsin and 1.5 mg/mL type II collagease
in DMEM for4hat37°C with 5% CO2. Cells were then harvested in
DMEM containing 10% FBS, 100 mg/L streptomycin, and 100 IU/
mL penicillin, centrifuged for 10 min at 1200 rpm, and then seeded in
culture dishes (12). After 1 day, the medium was changed to eliminate
floating cells. Cells were used for experimental protocols from passages
1to3.
Cytotoxicity Assays. A stock solution of test samples (2 ×104µg/
mL) was prepared by dissolving tested samples in DMSO and then
storing it at 4 °C until use. Serial dilutions of the stock solution were
prepared in the culture medium in 96 well microtiter plates. Test
samples at the appropriate concentrations were added to cell cultures
for 48 h without renewal of the medium. The number of surviving
cells was then counted using the tetrazolium (MTT) assay (12). The
cytotoxicity index (CI%) was calculated according to the following
equation: CI% )[1 -(T/C)] ×100%, where Tand Crepresent the
mean optical density of the treated group and vehicle control group,
respectively. In accordance with the CI% of the dose-response curve,
the concentration of the test compound giving 50% of cell growth
inhibition (IC50 value) was estimated.
Agarose Gel Electrophoresis. P-388 cells (1 ×106cells/well) were
treated with D-20 for 24 h, and the extent of DNA fragmentation was
assessed by 1.5% agarose gel electrophoresis (12).
Figure 1. Structures of (−)-epigallocatechin 3-O-gallate, EGCG (1), and
prodelphinidin A-2,3′-O-gallate, PAG (2).
In Vivo Antitumor Assays. P-388 cells (1 ×106cells/mouse) were
transplanted intraperitoneally (ip) into 5 week old CDF1female mice
(DBA male ×BALB/c female) on day 0. D-20 was dissolved in normal
saline. D-20 or normal saline was administered ip once a day on days
0-8. The antitumor effect was defined as the percent increase in life
span (%ILS) calculated according to the following equation: %ILS )
[(T-C)/C]×100%, where Tand Crepresent the mean survival time
(day) of the treated group and of the vehicle control group, respectively.
The body weight of each CDF1mouse was determined every day using
an animal scale. Data are presented as the mean (standard deviation.
Student’s t-test was used for comparison of body weight and survival
time (day) between the test and the blank groups (13).
Flow Cytometry Analysis. After appropriate treatment, HeLa cells
(5 ×105cells/well) were harvested by centrifugation and washed with
PBS. Cells were fixed in ice-cold 80% ethanol, treated with 1.0 mg/
mL RNase A, and stained with 50 µg/mL propidium iodide. Samples
were run through a FACScan (Becton Dickinson, San Jose, CA). Results
are presented as the number of cells vs the amount of DNA as indicated
by the intensity of fluorescence (12).
Western Blot Analysis. HeLa cells (5 ×105cells/well) exposed to
1or 2for 48 h were collected into tubes and then washed with PBS.
Cell pellets were lysed with lysis buffer containing 40 mM Tris-HCl
(pH 7.4), 10 mM EDTA, 120 mM NaCl, 1 mM dithiothreitol, 0.1%
Nonide P-40, and protease inhibitors. Total proteins (50 µg) were used
for Western blot analysis. Western blot analysis was performed using
10% Tris-glycine-sodium dodecyl sulfate-polyacrylamide gels, and
the protein was transferred to a nitrocellulose membrane by electro-
blotting. The membranes were probed with anti-PARP (a rabbit
polyclonal antibody) and visualized using a BCIP/NBT kit (BCIP/NBT,
Gibco) according to the manufacturer’s instructions. As a loading
control, we used anti-R-tubulin (a mouse monoclonal antibody) (15).
RESULTS AND DISCUSSION
The 70% aqueous acetone extract of M.rubra var. acuminata
leaves was strongly cytotoxic to HeLa cells, with on IC50 of
21.69 µg/mL for 48 h. On the basis of the bioassay guide for
fractionation, the aqueous extract was chromatographed on a
Diaion HP-20 column to give five eluted fractions, and their
cytotoxic effects are shown in Figure 2. Of the five fractions,
fraction II (20% MeOH-eluted fraction, D-20) displayed the
strongest cytotoxic effect. Therefore, the antitumor effects of
D-20 were evaluated using murine P-388 leukemia in in vitro
and in vivo models. In an in vitro assay, D-20 induced cell death
and DNA fragmentation in a dose-dependent manner in P-388
cells with an IC50 of 24.8 µg/mL for 24 h (Figure 3). The effect
of D-20 was evaluated for its in vivo antitumor activity against
intraperitoneally implanted P-388 leukemia in CDF1mice. D-20
at 18.75 mg/kg body weight could prolong the life span of P-388
tumor-bearing CDF1mice by more than 125% as compared to
normal saline-treated mice (Table 1). However, the high doses
of D-20 (37.5 and 75.0 mg/kg) caused a toxic reaction in these
mice, D-20-treated group, such that the ILS% was below 100%
(Table 1). The P-388-bearing mice were continuously injected
with D-20 for 9 days, and the body weights of high dose groups
quickly decreased in this period. This situation may be due to
high doses of D-20, which could kill tumor and normal immune
cells and decrease the immune functions. When the P-388-
bearing mice stopped treatment with high doses of D-20, the
tumor cells recurred more quickly than normal cells and body
weights significantly increased. In the 18.75 mg/kg D-20-treated
group, the mean body weight of the mice was significantly lower
Figure 2. Isolation flowchart of M. rubra var. acuminata using bioassay-guided fractionation.
than that of the control group on days 9-18 and ILS% could
be prolonged (Figure 4). The suitable dose D-20 should kill
tumor cells and damage the immune functions less. According
to the above results, D-20 can inhibit the growth of P-388 cells
in vitro and in vivo and is the major antitumor fraction extract
of M.rubra var. acuminata. Therefore, D-20 was rechromato-
graphed on a Toyopearl HW-40(C) column, and the cytotoxic
effects of each developing fraction are shown in Figure 1. The
higher yield and greater cytotoxic effect of the 60% MeOH-
developed fraction were separated and purified using an ODS
column to give 1and 2.
Compounds 1and 2were isolated from leaves of M.rubra
var. acuminata as antitumor principles for the first time, and
yields were 0.0036 and 0.0013%, respectively. The cytotoxic
effects of these compounds exhibited dose-dependent effects
at 5-40 µg/mL in HeLa cells for 24, 48, and 72 h (Figure 5).
Compound 1has fewer cytotoxic effects on primary normal
cervical fibroblasts (NCFs) than on HeLa cells, but that was
not the case for 2(Table 2). Furthermore, the cytotoxic
mechanisms of 1and 2were measured by fluorescence flow
cytometry. DNA fragmentation is a characteristic feature of
apoptosis (16). Figure 6 shows that 1and 2induced DNA
fragmentation at 10-80 µg/mL in HeLa cells for 48 h.
Figure 3. D-20 extract-induced cytotoxicity and DNA fragmentation in
P-388 cells for 24 h. (A) Cytotoxic effects measured by MTT assay (n)
3). (B) DNA fragmentation detected by agarose gel electrophoresis. Data
were used from three separate experiments, the picture for one of which
is shown.
Table 1. Percentage Increase in Life Span (%ILS) of D-20-Treated
P-388-Bearing CDF1Mice
group survival time (day) %ILS
blank (normal saline) 24.3 100.0
D-20 (mg/kg)
18.75 .60.0 .125.0
37.50 22.5 92.8
75.00 17.5 72.2
Figure 4. Effects of D-20 extract on body weights of P-388 tumor-bearing
CDF1mice. The body weight of CDF1mice was evaluated every day
while the mice were alive. Differences in body weights between the 18.75
mg/kg D-20 and the blank groups were significant on days 9−18 (p<
0.05, n)5).
Figure 5. Concentration- and time-dependent cytotoxicity of 1(A) and 2
(B) in HeLa cells by MTT assay (n)3).
Apoptosis produced the typical pattern of apoptotic PARP
cleavage: a catalytically active band of intact PARP at 116 kDa
and an active band at 85 kDa corresponding to the apoptotic
cleavage product of PARP. PARP is proteolytically cleaved
during apoptosis by caspase-3 (17), which reduces PARP’s
enzymatic activity (18), thereby inhibiting DNA repair. Treat-
ment of HeLa cells with 1and 2stimulated proteolytic cleavage
of PARP in a dose-dependent manner. Pretreatment with 100
µM of a caspase-3 specific inhibitor (Ac-Asp-Glu-Val-Asp-
aldehyde) for 2 h inhibited 1- and 2-induced PARP cleavage
(Figure 7). The above results suggest that 1and 2can induce
apoptosis in HeLa cells and that activation of caspase-3 may
provide a mechanistic explanation for their cytotoxicity effects.
Compound 1is well-known as a chemopreventive agent,
which is abundant in tea (19). In the present study, we now
report that leaves of M. rubra var. acuminata contain 1and
that the D-20 extract can inhibit the growth of P-388 cells in
vitro and in vivo. As previously reported (9), the 50% EtOH
extract of leaves of M. rubra inhibited melanin biosynthesis in
vitro and can possibly be used as a whitening agent for the skin.
Therefore, we suggest that the D-20 fraction extract is good
for health and that M. rubra var. acuminata is an economically
valuable plant. In the future, 1will be used as a biosubstance
to control the quality of the D-20 fraction extract.
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