Vol.2, No.5, 457-463 (2010) Natural Science
http://dx.doi.org/10.4236/ns.2010.25056
Dicranostigma leptopodum (maxim) fedde induced
apoptosis in SMMC-7721 human hepatoma cells and
inhibited tumor growth in mice
Wen-Hua Zhang1, Ming-Hua Lv1, Jun Hai1, Qin-Pu Wang2, Qin Wang1*
1School of life sciences, Lanzhou University, Lanzhou, China; [e-mail]; *Corresponding Author:
2Biology Department, Tianshui Normal College, Tianshui, China
Received 10 February 2010; revised 9 March 2010; accepted 12 April 2010.
ABSTRACT
Dicranostigma Leptopodum (Maxim) Fedde (DL-
F), which had been previously documented to
suppress oxidative hemolysis of erythrocytes
and enhance immune functions of murine peri-
toneal macrophages, was investigated for its
effect on anti-tumor activity. Of alkaloids ex-
tracted from DLF, five have been identified with
employment of chromatographic analysis. An
antiproliferative role of these alkaloids was de-
termined on SMMC-7721 Human Hepatoma Ce-
lls in an apoptosis-inducing manner, through
MTT assaying, Trypan blue exclusion assaying
and cytometric analysis of cell cycle distribu-
tion. To further examine their inhibitory effects
on tumor progression, murine H22 cells were
inoculated into Kunming mice to determine the
role of these alkaloids of DLF in inhibiting tumor
growth in the tumor-implanted mice. It was
found that these alkaloids of DLF enhanced the
tumor shrinkage effectively wherein its tumor
inhibitory rate and immunohistochemistry stain-
ing of the tumor were determined and profiled,
respectively.
Keywords: Dicranostigma Leptopodum (Maxim)
Fedde; Anti-Tumor Activity; Apoptosis;
Tumor-Growth Inhibition
1. INTRODUCTION
The medicinal use of natural products has a time-hon-
ored history along with the development of human civi-
lization. Throughout human history, enormous range of
natural products-compounds that are derived from natu-
ral sources such as plants, animals or micro-organisms-
have been discovered and put into medical use, the latest
version of the Dictionary of Natural Products (DNP;
http://dnp.chemnetbase.co m) encompasses over 214,000
entries. These were identified as leads of drug through
biological assay and became candidates for drug devel-
opment. More than 60% of the marketed drugs derived
from natural sources [1]. Owing to the diverse biological
activities and medicinal potentials, the importance of
natural products for medicine and health has been re-
portedly enormous with examining the experience and
knowledge accumulated of use of natural products [2].
In light of their matchless resource and biologically-
synergic activities in vivo, they continue to contribute to
the expansion of lead drugs and provide insights for
synthesis of their non-natural analogues. Increasingly,
Traditional Chinese medicine (TCM) is receiving recog-
nition from modern western medicine and 908 compo-
nents from Tradition Chinese Medicine Database were
found structurally similar to those deposited in the Com-
prehensive Medicinal Chemistry database of which 327
agents were further identified as common members of
both databases [3]. Although emphasis on high-through-
put screening of synthetic libraries has in part declined
drug discovery research into natural products during last
two decades, the potential for new discoveries of activi-
ties of natural products in the long term is promising,
given that the number of new natural product-derived
drugs could go to zero [4].
Despite huge conceptual difference between Tradi-
tional Chinese Medicine (TCM) and Modern Western
Medications, the preconception-TCM can’t get them
clinically approved - may be bridgeable with increased
knowledge of molecular mechanisms of TCM-derived
drugs [5]. By comparing 669 anti-tumor, anti-cancer or
anti-neoplastic agents identified from Comprehensive
Medicinal Chemistry database (CMC, containing 8659
clinically used Western drugs) to Traditional Chinese
Medicine Database (TCMD, containing 10458 compo-
nents), 26 pairs were found identical in structure and 20
were validated to be originally isolated from herbs [6].
With rationale borrowed from afore-mentioned discov-

W.-H. Zhang et al. / Natural Science 2 (2010) 457-463
458
eries and previous findings that Dicranostigma leptopo-
dum (maxim.) fedde (DLF) possessed physiological re-
levance of antipyretic and analgesic, detumescence, etc.
(Dictionary of Traditional Chinese Medicine 1986), we
further investigated its activities implicated in the induc-
tion of apoptosis of cancerous cells. The scientific name
of DLF was for the first time coined and collectively
classified by Harvard University Herbaria in 1987
(http://www.gbif.net/o ccurrences/86270582/). Enhanced
effects of DLF on immune-suppression were determined
in vivo [7] and its effect on suppressing oxidative hemo-
lysis of erythrocytes was also validated [8]. Efforts made
to separate and characterize the components of DLF
have identified five crystals from DLF of which three
were isocorydine, corydine and protopine [9] and five
alkaloids isolated were dicranostigmine, isocorydine,
corydine, protopine and sinoacutine [10]. In this study,
we treated SMMC-7721Human Hepatoma Cells with
extracted alkaloids of DLF, aiming to examine effect of
alkaloids of DLF on inducing apoptosis of cancerous
cells. Moreover, we treated H1-implanted Kunming
Mice with alkaloids of DLF to have determined tumor
growth-inhibiting effects of DLF.
2. EXPERIMENTAL MATERIALS AND
METHOD
2.1. Materials
2.1.1. Extraction of Alakoids from DLF
The powdered material of roots, stems and leaves of
DLF (12.5 g) was mixed with 75% alcohol (400 mL) for
1 h in a hermetic glass container and then disrupted con-
tinuously with an ultrasonic purge. The whole material
was filtered in vacuum followed by a distillation process.
Add alcohol to the mixture of filtrate to adjust its alcohol
concentration to 85%. After 24 h, adjust the filtrate to pH
8.0 using NaOH and then filter and distill the filtrate
until the alcohol is deprived. Adjust the filtrate to pH 7.0
using HCL [11].
2.1.2. Determination of Alkaloids of DLF Extracts
20 mg/mL standard DLF extracts were diluted to con-
centrations of 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4
mg/mL, 0.5 mg/mL, 0.6 mg/mL and 0.7 mg/mL, respec-
tively. The standard curve was made with these gradient
concentrations and de-ionized water as control [12]. The
separation of the alkaloids from DLF extracts was per-
formed using Sephadex G-50 chromatograph analysis
during which samples (50 ul) flowed through the Sepha-
dex G-50 columns (0.1 mL) steadily at rate of 1 mL/min.
The eluted proteins were determined at 254 nm wave-
length by WD-9430D UV spectrophotometer.
2.1.3. Animals and Cells
Kunming mice were purchased from the Experimental
Animal Center at Lanzhou University. The use and treat-
ment of mice were in accordance with institutional
guideline for Laboratory Animal Care. Muring H22 and
SMMC-7721 Human Hepatoma cells were obtained
from cell library of Institute of Cancer Biology and Drug
Discovery, Lanzhou University. Cells were grown in
RPMI 1640 medium (Gibco) supplemented with 10%
fetal bovine serum (Lanzhou Minhai Biotechnology), 2
mM l-glutamine, 100 units/mL penicillin and 100 mg/
mL streptomycin and incubated at 37℃ in a humidified
atmosphere of 5% CO2 and 95% air.
2.2. Methods
2.2.1. MTT Assay
SMMC-7721 were plated in 96-well microtiter plates at
a density of 4,000/well for culture and incubated in a
humidified 5% CO2-95% air atmosphere at 37℃ for 24
h. Then cells were treated with different concentrations
of alkaloids of DLF (0-24 mg/mL) and incubated for
additional 24 h, 48 h, and 72 h respectively. Cell viabil-
ity was determined by MTT assay [13] whereby 20 μL
of 5 mg/mL MTT was added to each well and incubated
for another 4 hours at 37℃. The supernatant was subse-
quently removed and 100 μL/well DMSO was added to
dissolve the formazan crystal. After shaking plates for 1
min, the absorbance of each well was read at 570 nm
wavelength with microplate reader (Bio-Rad). The vi-
ability of SMMC-7721 cells was calculated employing
the formula below:
Viability = (A57 0 of treated cells/A570 of untreated cells)
× 100%
2.2.2. Trypan Blue Exclusion Assay
Trypan blue dye, which would be excluded by normal
cells but could diffused into cells with disrupted mem-
brane integrity, was employed to determine the number
of the viable cells after treatment with alkaloids of DLF
[14]. 24 hours after the SMMC-7721cells were treated
with PMS-1077, measurements were conducted after
trypan blue (Sigma) was incubated with SMMC-7721
cells for 5 min at room temperature. At least 500 cells
were counted per sample. The cell viability was calcu-
lated with the following equation:
%100
cells ofnumber Total
cells blue ofNumber - cells ofnumber Total
Viability 
2.2.3. Morphological Analysis
To determine the morphological changes of SMMC-
7721 cells upon treatment with alkaloids of DLF,
SMMC-7721 cells which were treated with alkaloids of
DLF for 24 h were observed under inverted dark field
microscope (Nikon, Japan) and photographed after-
wards.
2.2.4. Single Cell Gel Electrophoresis Assay
The single cell gel electrophoresis (SCGE) assay [15]

W.-H. Zhang et al. / Natural Science 2 (2010) 457-463
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459
commonly referred to as “comet assay” allowed the very
sensitive detection of DNA breakage induced by genoto-
xic agents at single cell level. Thus, this method was
adopted to determine the DNA damaging effects of al-
kaloids of DLF on SMMC-7721 cells. Treated with dif-
ferent concentrations of alkaloids of DLF for 24 h,
SMMC-7721 cells were lysed by alkaline lysis solution
(2.5 M NaCl, 100 mM EDTA•Na2, 10 mM Tris pH 10),
10% DMSO and 1% triton X-100 (Sigma) at 4℃ for 1 h.
Another 20 min was allowed for the DNA to unwind in
electrophoresis running buffer solution (300 mM NaOH,
and 1 mM EDTA•Na2, pH 13). Electrophoresis was per-
formed for 20 minutes at 50 V and 300 mA. After elec-
trophoresis, the slides were gently removed and alkaline
pH neutralized with 0.4 M Tris (pH 7.5). Then Ethidium
bromide (75 mL of a 20 mg/mL solution, Sigma) was
added to each slide and a cover glass was placed on the
gel. DNA migration was analyzed on a fluorescence mi-
croscope (Olympus, Japan) (Filter G-2A) and photo-
graphed afterwards.
2.2.5. Flow Cytometric Analysis
SMMC-7721 cells that were treated with alkaloids of
DLF for 24 h were washed twice with PBS and then
fixed with 70% ethanol at -20℃ for about 12 h. Then
cells were washed again twice with PBS and suspended
with 1 mL 100 μg/mL RNase (Sigma) containing 0.1%
Triton-100 and 50 μg/mL propidium iodide (Sigma).
Cells were stained with DNA dye for 30 min and ana-
lyzed by flow cytometer (EPICS-XL, Beckman).
2.2.6. In Vivo Inhibition of Tumor Growth
Six-week old inbred female Kunming mice were inocu-
lated with murine Hepatoma 2.0 × 107 mg/mL H22 cells
[16]. From the second day after the implantation of H22
cells, 40 tumor-bearing mice were grouped randomly
into five groups as the following: 1) Blank control, 2)
Model control, 3) 5-Fu positive control, 4) High dose
control, 5) Low dose control. 48h after tumor implanta-
tion, these mice were I.P. injected with a daily dose of
0.2 mL of 3.0 mg/mL, 0.2 mL 6.0 mg/mL, 0.2 mL, 2
mg/mL, 0.2 mL physiological saline (0.9%) and 0.2 mL
physiological saline (0.9%) of alkaloids of DLF for High
dose group, Low dose group, 5-Fu positive group, Blank
group and Model group, respectively. Mice were exe-
cuted within 24 h after the last dose of alkaloids of DLF
and their tumor was obtained and analyzed. And Spleen
and thymus indexes were examined. Tumor inhibitory
rate (Ri) and organ index were expressed as the follow-
ing formula, respectively [17]:
%
m
m
tumor
tumor 100)
untreated
treated
1(Ri 
mouse
organ
m
m
IndexOrgan 
2.2.7. Immunohistochemistry Analysis
The tumors of mice were excised and fixed in 4% para-
formaldehyde for 24 h. Paraffin sections were prepared
for immunohistochemical staining and hematoxylin and
cosin (H & E) staining [18,19]. Sections for immuno-
histochemical staining were deparaffinized and then hy-
drated by transferring them through the following solu-
tions xylene bath twice for 5 min, 100% ethanol for 5
min twice, and then 90% ethanol, 80% ethanol, 70%
ethanol, and PBS, for 3 min each. Subsequently, sections
were placed in a microwave oven for 15 min at 100℃ in
sodiumcitrate buffer (0.01 M, pH 5.7) to expose epitopes.
After that, sections were incubated at 37℃ with PCNA
antibody for about 1.5 h followed by the visualization
using immunosystem kit (Santa Cruz, CA).
2.2.8. Statistical Analysis
All experimental data were expressed as mean ± SD, and
statistical analysis was performed using Student’s t-test
to compare the results from the untreated group.
3. RESULTS AND DISCUSSION
3.1. Extraction and Determination of
Alkaloids from DLF
The alkaloids extracted from DLF in roots, leaves and
whole part were 5.31%, 5.91% and 5.57%, respectively.
The average content of alkaloids in whole DLF part was
5.59%. Five main alkaloids were separated using chro-
matography and their contents were determined to be
unevenly distributed, as indicated by the peaks of chro-
matographic profile (Figure 1).
(a)
Growing Time (weeks)
(b)
Figure 1. Extraction and determination alkaloids from DLF. (a)
The comparison of alkaloids content in different organs of DLF
during different growing periods; (b) Separation of alkaloids
from DLF extracts and the determination of their proportion.

W.-H. Zhang et al. / Natural Science 2 (2010) 457-463
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3.2. Anti-Proliferative Effect of Alkaloids of
DLF on SMMC-7721 Cells
Treated with assigned concentrations of alkaloids of
DLF for indicated time, SMMC-7721 cells exhibited
compromised cell viability. A concentration-dependent
anti-proliferative effect of alkaloids of DLF on SMMC-
7721cells was determined by MTT assay and Trypan
Blue Exclusion assay (Figure 2).
3.3. Induction of Apoptosis of SMMC-7721
Cells by Alkaloids of DLF
Upon treatment with 8.5 mg/mL alkaloids of DLF for 24
h, SMMC-7721 cells exhibited the characteristic features
of apoptosis including cell shrinkage, cell detachment
and vesicle formation (Figure 3). Flow cytometric ana-
lysis demonstrated that cell cycles were arrested (Figure
4) and cell cycle distribution analysis manifested that
cells were arrested predominately at G1 phase (Figure
5).
3.4. Alkaloids of DLF-Induced DNA Damage
of SMMC-7721 Cells
Upon treatment with various concentrations of alkaloids
of DLF, SMMC-7721 cells exhibited characteristic fea-
0
10
20
30
40
50
60
70
80
90
100
0 1.5 36912 18 24
Viability
Alkaloids of DLF(mg/ml )
24h
48h
72h
(a)
0
10
20
30
40
50
60
70
80
90
100
01.5369 1218
Viability(%)
Alkaloids of DLF(mg/ml)
SMMC-7721
(b)
Figure 2. Determination of anti-proliferative effect of DLF
on SMMC-7721 cells. (a) MTT Assay: Treated with DLF for
24 h, 48 h and 72 h, respectively, cells viability was inhibited
in a concentration-dependent manner; (b) Trypan Blue Ex-
clusion Assay: Effect of alkaloids of DLF on inhibiting the
cell viability was concentration-dependent.
tures of DNA damage. Examination of DNA damage by
Single cell gel electrophoresis revealed appreciable DNA
damage in terms of its length of migrating tails (Figure
6). It’s also observed that the damage was concentration-
dependent.
3.5. Inhibition of Tumor Growth by Alkaloids
of DLF was Determined in Vivo
Murine Hepatoma H22 cells were inoculated to six-week
old Kunming mice and, from 24 h after the inoculation, a
Figure 3. Morphological analysis of Alkaloids-treated SMMC-
7721 by inverted dark field microscopy (X200). Cells were
treated with different concentrations of alkaloids of DLF as
indicated: (a) control; (b) 3.0 mg/ml; (c) 6.0 mg/ml and (d)
12.0 mg/ml.
Figure 4. Flow cytometric analysis of cell cycle distribution
upon treatment with alkaloids of DLF. (a) 0 mg/ml; (b) 3.0
mg/ml; (c) 6.0 mg/ml; (d) 12.0 mg/ml.

W.-H. Zhang et al. / Natural Science 2 (2010) 457-463
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461
Figure 5. Cell cycle distribution analysis by flow cytometry.
Figure 6. Determination of DNA damage of AlkaloidsDLF-
treated SMCC-7721 cells by Single Cell Gel Electrophoresis.
(a) control; (b) 3.0 mg/ml; (c) 6.0 mg/ml and (d) 12.0 mg/ml.
daily dosage of alkaloids of DLF was I.P. injected into
these tumor-bearing mice. After 12 days, mice were ex-
ecuted and their tumors, spleens and thymus were ex-
cised for analysis. Alkaloids of DLF exerted a role as
potent as 5-Fu in enhancing tumor shrinkage. 5-Fu in-
hibited the tumor growth by 55.75% while 6.0 mg/mL
alkaloids of DLF and 12.0 mg/mL alkaloids of DLF in-
hibited the growth of tumor by 42.50% and 51.00%,
respectively (Table 1). By examining effects of alkaloids
of DLF on the growth of spleen and thymus, it revealed
that DLF exerted significant effect on inhibiting the ab-
errant progression thymus of tumor-bearing mice while
the effects of alkaloids of DLF on the growth of spleen
of tumor-bearing mice were not discernible (Table 2).
3.6. Alkaloids of DLF-Mediated Inhibition of
Tumor Progression Analyzed by HE
Staining and Immunohistochemistry
Histological section of tumors excised from tumor-
bearing mice which were treated with 0.9% physiologi-
cal saline, 5-FU, 6.0 mg/mL DLF and 12.0 mg/mL alka-
loids of DLF were examined with employment of HE
staining and Immunohistochemistry (Figure 7). Both
demonstrated that alkaloids of DLF exerted pronounced
effects on inhibiting of the growth and progression of
tumor.
3.7. Discussion
In response to the recognition that fewer side effects
have been documented in phytotherapy and natural prod-
uct-based therapy and therapeutic potential of natural
products [5,20,21], we further explored to study the anti-
tumor activities of alkaloids of DLF. In this study,
approx. 5.7% of alkaloids were extracted from the whole
part of DLF and five alkaloids were identified using
chromatographic analysis which was in consistence with
the separation of alkaloids from DLF [10]. Anti-prolif-
erative effect was determined for alkaloids of DLF on
SMCC-7721 cells and IC50 of alkaloids of DLF on
SMCC-7721 cells was 8.5 mg/ml. Upon treatment with
DLF, SMCC-7721 cells exhibited apoptotic features as a
rule [11]. Flow cytometric analysis of cell cycle distribu-
tion upon treatment with alkaloids of DLF revealed that
cells were arrested in the G1 phase (Figure 4) during
which DNA damage was determined (Figure 6). With in
vitro anti-tumor activity of alkaloids of DLF validated,
in vivo effect of anti-tumor of alkaloids of DLF was
further explored. Tumor implantation was completed
with inoculation of Muring H22 cells in Kunming mice.
Inhibitory rates of 51% and 42% of tumor growth inhibi-
tion were determined for 12.0 mg/ml alkaloids of DLF
and 6.0 mg/ml, respectively, through examining the tu-
mor weight upon treating tumor-bearing mice with alka-
loids of DLF. In addition, effects of alkaloids of DLF on
inhibiting the progression tumor were determined by HE
staining of histological section of tumors (Figure 7).
Table 1. The inhibition effect of the drug to transplanted tumor
H22. X ± s n = 8.
Group Tumor weight Inhibitory rate
Tumor control 0.40 ± 0.23 0
5-FU control 0.18 ± 0.10* 55.75%
AlkaloidsDLF 6.0 mg/ml 0.20 ± 0.09* 42.00%
AlkaloidsDLF12.0 mg/ml 0.23 ± 0.12* 51.50%
Table 2. The effects of DLF on growth of spleen and thymus
of tumor-bearing miceX ± s n = 8.
Group Spleen index (%) Thymus index (%)
Healthy group 3.48 ± 1.31* 4.26 ± 0.80
Tumor group 6.28 ± 1.08 4.88 ± 0.88
5-FU group 2.48 ± 1.08 0.98 ± 0.40*
AlkaloidsDLF 6.0
mg/ml 11 ± 1.28* 2.84 ± 0.90*
AlkaloidsDLF 12.0 mg/ml6.66 ± 1.37 2.84 ± 0.60*

W.-H. Zhang et al. / Natural Science 2 (2010) 457-463
462
Figure 7. Histological Profile of tumor upon treatment. HE
staining of histological section of tumors: (a) 6.0 mg/ml Alka-
loidsDLF; (b) 12.0 mg/ml AlkaloidsDLF; (c) 5-FU; (d) 0.9%
physiological saline. PCNA assaying of histological section of
tumors; (e) 6.0 mg/ml AlkaloidsDLF; (f) 12.0 mg/ml Alka-
loidsDLF; (g) 5-FU; (h) 0.9% physiological saline.
Taken together, an anti-tumor effect of alkaloids of
DLF was determined by means of both in vitro and in
vivo examinations. However, the elaborate molecular
mechanism underlying its anti-tumor activity remains
elusive for these five alkaloids. Since determining the
biological properties of plants used in traditional medi-
cine is helpful to drug screening, thus, further research is
deserved to isolate the very compounds responsible for
the observed biological activity of alkaloids of DLF.
4. CONCLUSIONS
With promising effects of alkaloids of DLF determined
both in vitro and in vivo on anti-proliferating of SMCC-
7721 cells and enhancing shrinkage of tumor, DLF de-
serves further characterization of its very components
entailing its anti-tumor activities and mechanisms un-
derlying its anti-tumor effect. Moreover, its anti-prolif-
erating effect and anti-tumor activities remains to be
extended to other cancerous cell lines and tumors as well.
A comprehensive understanding of its activities on a
spectrum of cancerous cells and tumors will shed light
on its mechanistic elucidation of anti-tumor effects.
5. ACKNOWLEDGEMENTS
This work was jointly supported by International Cooperation Pro-
grams in Gansu Province (NO. 0708WCGA149) and International
Science and Technology Cooperation Project (NO. 2009DFA30990).
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