Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2012, Article ID 926329, 6 pages
EthanolExtract of AbnormalSavda Munziq,a Herbal
Preparation of Traditional UighurMedicine,InhibitsCaco-2
CellsProliferationvia CellCycleArrest andApoptosis
AbdiryimYusup,1HalmuratUpur,1AnwarUmar,2Benedicte Berke,3andNicholas Moore3
1Faculty of Traditional Uighur Medicine, Xinjiang Medical University, Urumqi 830011, China
2Faculty of Pharmacy, Xinjiang Medical University, Urumqi 830011, China
3Department of Pharmacology, University Victor Segalen Bordeaux 2, 33076 Bordeaux, France
Correspondence should be addressed to Halmurat Upur, email@example.com
Received 9 February 2011; Revised 17 April 2011; Accepted 3 May 2011
Academic Editor: W. Vilegas
Copyright © 2012 Abdiryim Yusup et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Aims. Study the effect of Abnormal Savda Munziq (ASMq) ethanol extract on the proliferation, apoptosis, and correlative gene,
expression in colon cancer cells (Caco-2) to elucidate the molecular mechanisms responsible for the anticancer property of
Abnormal Savda Munziq. Materials and Methods. ASMq ethanol extract was prepared by a professional pharmacist. Caco-2 cells
were treated with different concentration of ASMq ethanol extract (0.5–7.5mg/mL) for different time intervals (48 and 72h).
Antiproliferative effect of ASMq ethanol extract was determined by MTT assay; DNA fragmentation was determined by gel
electrophoresis assay; cell cycle analysis was detected by flow cytometer; apoptosis-related gene expression was detected by RT-
PCR assay. Results. ASMq ethanol extract possesses an inhibition effect on Caco-2 cells proliferation, induction of cell apoptosis,
mechanism of ASMq ethanol extract may be involved in antiproliferation, induction of apoptosis, cell cycle arrest, and regulation
of apoptosis-related gene expression such as bcl-2 and Bax activity pathway.
Cancer is the second leading cause of death worldwide. Can-
cer continues to represent the largest cause of mortality in
the world. China is confronted with an increasing incidence
of cancer and cancer deaths annually. Mortality that results
from the common forms of cancer will be unacceptably high
in the 21th century. Despite many therapeutic advances in
the understanding of the processes in carcinogenesis, overall
mortality statistics are unlikely to change until there is a
reorientation of the concepts for the use of natural products
as new chemopreventive agents [1–4].
Natural products include thousands of compounds that
exist in fruits, vegetables, plants, and herbs, and several
clinical anticancer drugs have been derived from natural
products. Thus, development of compounds with anticancer
effects from natural products has currently become a very
important topic. Natural compounds isolated from medic-
inal plants, as rich sources of novel anticancer drugs, have
been of increasing interest since then [5, 6]. Cancer pre-
vention and treatment using traditional Chinese medicines
have also attracted increasing interest, and the development
of pharmacology and molecular biology makes it possible
to screen effective extracts with anticancer activity from
the Traditional Uighur medicinal herbs and many relevant
Traditional Uighur Medicine, the main part of Tradi-
tional Chinese Medicine, has been used for pharmaceutical
and dietary therapy for several millennia. Traditional Uighur
medicine has its own traditional theory for prevention and
treatment of cancer with prescriptions containing Abnormal
Savda Munziq (ASMq) . To date, the anticancer effects
of ASMq and its potential mechanism of action have been
studied on HepG2 cells, Hela cells, T lymphoma cells, and
2Evidence-Based Complementary and Alternative Medicine
breast cancer cells. Furthermore, the total flavonoids of
ASMq were proved to be the most important anticancer-
active ingredients of ASMq . In the present study, we used
effects of ASMq ethanol extract to assess the potential effect
(DMEM), RPMI1640, foetal calf serum (FCS), ethylenedi-
amine tetraacetic acid (EDTA), ribonuclease A (RNase A),
proteinase K, ethidium bromide, N-Lauroyl sarcosine, pro-
from Sigma-Aldrich (Lyon, France). SV Total RNA Isolation
System, Reverse Transcription System, dNTPs Mixture, Taq
DNA polymerase, BenchTop 1kb DNA Ladder, DNA loading
buffer were all from Promega, France. Olig-onucleotides
used in PCR were from Eurogentec S.A, Britsh. All other
chemicals used were of analytical grade.
2.2. Preparation of ASMq Ethanol Extract. ASMq is com-
posed of 10 kinds of herbs . Herbs used in this study
were obtained from Xinjiang Hospital of Traditional Uighur
Medicine (Urumqi, China). High quality herbs were selected
ethanol extract was prepared by a professional pharmacist
. In average, the yield of ethanol extract was 12.0% (w/w)
from ASMq. ASMq ethanol extract used in the experiments
was dissolved in distilled water as a 100mg/mL (w/v) stock
solution and sterilized by 0.45µm Millipore filter unit for
cell culture medium (DMEM) for cell culture and treatment.
2.3. Cell Culture and Treatment. Caco-2 cells, a human colon
cancer cell line, were obtained from the American Type
concentration (4.5g/L) DMEM medium supplemented with
10% FCS, 1% L-glutamine (200mM), and 1% penicillin-
streptomycin (100IU–100µg/mL) in a humidified atmo-
sphere of 5% CO2-95% air mixture at 37◦C. All data pre-
sented in this report were obtained at least from three
2.4. Cell Viability Assay. The viability of the cells was as-
sessed by MTT (3,4,5-dimethylthiazol-2-yl)-2-5-diphenyl
tetrazolium bromide) assay, which is based on the reduction
in a 96-well plate (Coastar from Corning, NY) and routinely
incubated for 24h at 37◦C prior to use. After 24h they
were treated with different concentrations (0.5–7.5mg/mL)
of ASMq ethanol extract for different time intervals (48
and 72h). After the treatment, media containing ASMq
ethanol extract were carefully removed by aspiration. 100µL
of 0.5mg/mL MTT in cell culture medium was added
to each well and incubated for 2h. 100µL of 10% SDS,
0.01M HCl solution was added to each well to dissolve
the formazon crystals formed. The plates were covered
with aluminum foil and kept in an incubator for 12h for
dissolution of the formed formazan crystals. Amount of for-
mazan was determined measuring the absorbance at 560nm
using a microplate reader.
2.5. DNA Fragmentation Detection. The cells were cultured
(5 × 106cells per mL) in 25cm2flasks, total volume 5mL
of medium per flask, in the presence of ASMq ethanol
extract (0.5–7.5mg/mL) for different time intervals (24
and 48h) at 37◦C. Controls were performed at the same
time with DMEM. After incubation for 24h, the cell layer
was rinsed twice with 5mL of PBS. To extract DNA, the
cells were lysed by incubation for 5min with 1mL of
lysis buffer (1% N-Lauroyl sarcosine, 20mM Tris-HCl pH
8.0, 5mM EDTA), and the cell lysates were collected and
transferred into 15mL corning tubes. Proteins were digested
overnight by incubation with 100µg/mL proteinase K at
chloroform (2:1, v/v) were added to the homogenate. After
centrifugation for 5min at 2200×g at 10◦C, one volume of
SEVAG (3mL) was added to the aqueous supernatant and
the mixture was centrifuged for 5min at 2200×g at 10◦C.
and incubated for 5min at 37◦C to eliminate traces of
chloroform. RNA was digested by incubation with 12µg/mL
RNase A for 30min at 37◦C, and then one volume of SEVAG
the DNA in the aqueous supernatant was precipitated at
−10◦C for 4h with ethanol. The mixture was centrifuged
for 45min at 2200×g at 4◦C, and the supernatant removed.
The pellet was rinsed with 70% ethanol, dried at room
temperature for 2h, and resuspended in 200µL of TE
20–1 (20mM Tris-HCl pH 8.0, 1mM EDTA) for DNA
samples were analysed by electrophoresis on a 1% agarose
gel (1.5h at 80V/30mA) with a TBE running buffer (44mM
Tris–HCl, 44mM boric acid, 50mM EDTA, pH 8.0) .
2.6. Cell Cycle Analysis by Flow Cytometry. For cell cycle
analysis, 5 × 105cells seeded in 3mL total volume in 6-
well multidishes were incubated as described above for 48h.
Flow cytometric analyses were conducted using a FACScan
(Becton Dickinson, France). At the end of incubation, the
cells were rinsed twice with PBS and trypsinized in trypsine-
0.02% EDTA mixture. After centrifugation for 10min at
600×g at 4◦C, the supernatant was removed, the pellet
resuspended in 300µL of PBS, then 700µL of cold methanol
were added and the mixture kept at −20◦C for 30min. After
centrifugation for 5min at 600×g and at 4◦C, the pellet was
treated with 2mg/mL RNase A at 37◦C during 30min and
stained with 50µg/mL propidium iodide containing 0.1%
Triton X-100 and EDTA 0.02mg/mL. The percentage of cells
in each stage of the cell cycle was determined by counting
104cells, using cellquest software (Becton Dickinson, France)
Evidence-Based Complementary and Alternative Medicine3
Concentration of ASMq ethanol extract (mg/mL)
Inhibition rate (%)
Figure 1: Inhibitory effect of ASMq ethanol extract on Caco-2 cell
ethanol extract (0.5–7.5mg/mL) at 37◦C, 5% CO2for 48 or72h.
The cell growth was determined by the MTT assay. Results are given
as mean ± SD from three independent experiments.
2.7. Gene Expression Studies. Expression of apoptosis-related
genes, bcl-2, bax, p21, and p53, was studied using reverse
transcriptase-PCR (RT-PCR). 5 × 105cells seeded in 3mL
total volume in 6-well multidishes were incubated with the
presence of ASMq ethanol extract (0.5–7.5mg/mL) for 48h
at 37◦C. The housekeeping genes GAPDH were used as
control. At the end of incubation, the cells were rinsed
twice with PBS and trypsinized in trypsine-0.02% EDTA
mixture. After centrifugation for 5 min at 500×g at 4◦C, the
supernatant was removed, and the pellet was used for RT-
PCR studies. Total RNA was isolated using SV Total RNA
Isolation System (Promega, France). cDNA was generated
by Reverse Transcription System (Promega, France). 10µL
of cDNA product was used for PCR reaction as templates.
PCR was carried out using the gene-specific upstream and
downstream primers (Table 1). Initial denaturation at 95◦C
for 3min was followed by a PCR cycle of denaturation at
94◦C for 1min, annealing at 55◦C for 1min, and extension
at 72◦C for 2min. PCR products were separated on a 1.5%
agarose gel and stained with ethidium bromide .
2.8. Statistical Analysis. The data are expressed as mean ±
standard deviation (SD) for at least three independent deter-
minations in triplicate or quadruplicated for each exper-
imental point. The statistical differences between treated
groups and control groups were determined by Student’s t-
test, and P < 0.05 was statistically significant difference.
3.1. Inhibition of Cell Growth. Caco-2 cells were used as a
model system to examine the effect of ASMq ethanol extract
on their growth. The growth inhibitory effect of ASMq eth-
Figure 2: Induction of DNA fragmentation by ASMq ethanol
extract in Caco-2 cells. Cells were incubated for 48 h at 37◦C with
different concentrations of ASMq ethanol extract (0.5–7.5mg/mL).
Lane 1–5:0.5, 1.0, 2.5, 5.0, and 7.5mg/mL of ASMq ethanol extract,
respectively. Lane 6: cells with no ASMq ethanol extract treatment.
Lane M: 250bp DNA marker.
anol extract was concentration and time dependent
(Figure 1). The IC50 at 48 and 72h was 5.99mg/mL and
3.2. Induction of DNA Fragmentation. Results showed that
Caco-2 cells treated with ASMq ethanol extract did not
induce any DNA fragmentation at the concentration of 0.5–
2.5mg/mL at 48h of incubation. In contrast, the apoptotic
fragments were clearly detected when the cells were treated
7.5mg/mL, for 48h (Figure 2). These observations exhibited
that ASMq ethanol extract-induced Caco-2 cells death was
possibly mediated through an apoptotic pathway.
3.3. Cell Cycle Analysis by Flow Cytometry. Flow cytometry
analysis performed on Caco-2 cells after 48h of incubation
with ASMq ethanol extract (0.5–7.5mg/mL) indicated an
alteration in the percentage of cells in each stage of the
cell cycle: G0/G1, S and G2/M, as compared to the con-
trol (Table 2). We observed at concentrations higher than
5.0mg/mL an increase in the number of cells in the sub-
G1 phase and a decrease in the G0/G1 phase as compared
to the control cells (P < 0.05). However, the percentage of
cells in the S-phase and G2/M phase remained unchanged as
compared to the controls (P > 0.05). On the other hand,
as shown in Figure 3, the sub-G1 peak was detected in a
concentration-dependent manner. These results suggested
that ASMq ethanol extract had a prominent ability to induce
apoptosis in Caco-2 cells.
3.4. Expression of Apoptotic Genes. As the results shown
in Figure 4, the gene expression of Bax increased and the
expression of Bcl-2 decreased concentration dependently in
Caco-2 cells after ASMq ethanol extracts treatment for 48h.
But there were not any changes in the expressions of p53
and p21 genes. This indicated that the induction of apoptosis
with ASMq ethanol treatment was at least related to the
regulation of Bax and Bcl-2 expression, but no relation to the
expression of p53 and p21 genes.
4 Evidence-Based Complementary and Alternative Medicine
200 400 600
0 200400600 800
0 200400 600 800
200 400 600
Figure 3: Cell cycle analysis on Caco-2 cells after treatment with ASMq ethanol extract for 48h. (a) Control without ASMq ethanol extract
treatment; (b) Cells treated with 0.5mg/mL of ASMq ethanol extract; (c) Cells treated with 1.0mg/mL of ASMq ethanol extract; (d) Cells
treated with 2.5mg/mL of ASMq ethanol extract; (e) Cells treated with 5.0mg/mL of ASMq ethanol extract; (f) Cells treated with 7.5mg/mL
of ASMq ethanol extract.
Cancer causes significant morbidity and mortality and is a
major public health problem worldwide. An effective cancer
incidence of cancer. Herbs, herbal formulations, and herb-
derived compounds are known to have curative potential
[3, 6, 12–15]. Abnormal Savda Munziq (ASMq) is a herbal
formulation used for cancers in traditional Uighur Medicine
and its anticancer activity is well documented [7, 16]. Aque-
ous extract, ethanol extract, ethyl acetate extract, and total
flavonoids of ASMq have been reported to possess inhibitory
effect towards a broad range of cancer cells in vitro [8, 9, 17–
19], and ASMq ethanol extract had chemoprotective effects
on DMH-induced colon carcinogenesis . The acting
ingredients in ASMq that exerted the anticancer effect may
include polyphenols such as flavanoids, which are abundant
in ASMq ethanol extract . The molecular mechanism
underlying the ASMq ethanol extract induced apoptosis in
HepG2 cells has been reported to be associated with increase
of caspase-3 activity, DNA fragmentation and Bax/Bcl-2
dependent pathway [9, 19].
Apoptosis, also called the programmed cell death charac-
terized by several morphological and biochemical events, is
now known as an important type of cell death in response
to cytotoxic treatment. It is a general physiological process to
remove unwanted cells without damaging the neighboring
cells and inducing inflammatory responses. In recent years,
many studies have demonstrated that the dysregulation of
apoptosis process is involved in the development of neo-
plastic transformation and tumor growth. The induction of
apoptosis in tumor cells has been shown to be the most
common anticancer mechanism conjoint by many cancer
therapies. Thus, to find the potential therapeutic antitumor
drugs with potent and selective apoptotic effect would be
Evidence-Based Complementary and Alternative Medicine5
Table 1: Oligonucleotides used in the gene expression studies.
sense: 5?-TGCACCTGACGCCCTTCAC -3?
sense: 5?-AAACCTACCAGGGCAGCTA -3?
Table 2: Effect of ASMq ethanol extracts on cell cycle distribution in Caco-2 cells (mean ± SD).
Percentage of cells in each stage (%)
sub-G1 (M1)G0/G1 (M2)
0.53 ± 0.13
0.36 ± 0.13
0.47 ± 0.18
0.55 ± 0.12
5.74 ± 1.51∗
13.56 ± 0.65∗
∗P < 0.05, as compared with control culture Representative data from three individual analyses.
14.60 ± 1.50
18.38 ± 0.71
16.62 ± 0.97
16.09 ± 0.42
16.23 ± 0.19
15.81 ± 0.13
15.29 ± 1.05
16.21 ± 1.65
17.51 ± 2.59
16.59 ± 1.46
16.09 ± 0.45
16.18 ± 0.53
69.79 ± 0.51
65.35 ± 2.23
65.70 ± 1.77
67.12 ± 0.83
61.73 ± 0.80∗
54.69 ± 3.24∗
Bcl-2 ( bp)
Figure 4: Expression of apoptotic genes in Caco-2 cells treated with
ASMq ethanol extracts for 48h. Column 1, control cells with no
extract; Column 3, cells treated with 2.5mg/mL of ASMq ethanol
extract; Column 4, cells treated with 5.0mg/mL of ASMq ethanol
extract; Column 5, cells treated with 7.5mg/mL of ASMq ethanol
extract; Representative data from three individual analyses.
valuable. The administration of many natural compounds
with anticancer effect has been shown to be capable of
inducing the apoptotic death of cancer cells [21–24].
In the present study, we found that ASMq ethanol extract
displayed a significant inhibitory effect on the proliferation
of Caco-2 cell lines in a dose and time dependently manner.
Furthermore, a high-concentration of ASMq ethanol extract
(more than 5.0mg/mL) resulted in significant induction of
apoptosis in Caco-2 cells, as evidenced by DNA fragmenta-
tion and sub-G1 peak of apoptotic markers detection. More
interestingly, the Caco-2 cells treated with ASMq ethanol
extract in the concentration of 7.5mg/mL for 48h exhibited
a dramatic accumulation of cells in sub-G1 phase (13.56%).
Many genes such as p53, p21, and genes in Bcl-2
family have been demonstrated to play important roles
in deciding the initiation and execution of apoptosis in
tumor cells exposed to radiation or anticancer drugs. It
has been demonstrated that Bcl-2 family members, such
as Bcl-2 itself and Bax, are mediators of apoptosis .
The balance of proapoptotic Bax and antiapoptotic Bcl-
2 is known to be important in determining whether cells
die or survive. Bax/Bcl-2 ratio in a cell acts to regulate its
own susceptibility to apoptosis . In the present study, to
clarify the molecular mechanism of apoptosis mediated by
ASMq ethanol extract, we examined the expression of genes
including p53, p21, Bax, and Bcl-2 by RT-PCR. Our studies
indicated that ASMq ethanol extract-induced apoptosis in
Caco-2 cells accompanied by the dose-dependent down-
regulation of Bcl-2 gene expression and upregulation of Bax
gene expression, while p53 and p21 were not significantly
The present study suggested that the anticancer effect of
ASMq ethanol extract was mediated through multiple path-
ways. ASMq ethanol extract inhibits cell growth and induces
DNA fragmentation and apoptosis in a concentration-de-
pendent manner. Induction of apoptosis is possibly related
with Bcl-2 and Bax dependent pathway, but independent of
p53 and p21 gene expression. In addition, as a herbal medi-
cine, ASMq ethanol extract has its unique properties of low
cost, easy oral consumption, and a long history of use by the
Uighur population, all of which are indicative of its potential
application as an anticancer agent.
6Evidence-Based Complementary and Alternative Medicine Download full-text
This research was supported by the Program for New Cen-
tury Excellent Talents in University (NCET), Ministry of
grant from Science and Technology Department of Xinjiang
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