全 文 ：Ng et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2014 15(8):692-700

692




Piper betle leaf extract enhances the cytotoxicity effect of
5-fluorouracil in inhibiting the growth of
HT29 and HCT116 colon cancer cells*

Pek Leng NG1, Nor Fadilah RAJAB1, Sue Mian THEN2,3, Yasmin Anum MOHD YUSOF4,
Wan Zurinah WAN NGAH2,4, Kar Yong PIN5, Mee Lee LOOI†‡6
(1Department of Biomedical Sciences, Faculty of Allied Health, Universiti Kebangsaan Malaysia (UKM),
Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia)
(2UKM Medical Molecular Biology Institute (UMBI), Universiti Kebangsaan Malaysia (UKM), Jalan Yaacob Latiff,
Bandar Tun Razak, Cheras, 56000 Kuala Lumpur, Malaysia)
(3School of Biomedical Science, University of Nottingham Malaysia Campus, 43500 Semenyih, Selangor, Malaysia)
(4Department of Biochemistry, Faculty of Medicine, Universiti Kebangsaan Malaysia (UKM),
Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia)
(5Forest Research Institute Malaysia (FRIM), 52109 Kuala Lumpur, Kepong, Malaysia)
(6School of Biosciences, Taylor’s University, Lakeside Campus, 47500 Subang Jaya, Selangor, Malaysia)
†E-mail: MeeLee.Looi@taylors.edu.my
Received Nov. 26, 2013; Revision accepted Apr. 15, 2014; Crosschecked July 11, 2014

Abstract: Objective: The combination effect of Piper betle (PB) and 5-fluorouracil (5-FU) in enhancing the cytotoxic
potential of 5-FU in inhibiting the growth of colon cancer cells was investigated. Methods: HT29 and HCT116 cells
were subjected to 5-FU or PB treatment. 5-FU and PB were then combined and their effects on both cell lines were
observed after 24 h of treatment. PB-5-FU interaction was elucidated by isobologram analysis. Apoptosis features of
the treated cells were revealed by annexin V/PI stain. High-performance liquid chromatography (HPLC) was performed to
exclude any possible chemical interaction between the compounds. Results: In the presence of PB extract, the cy-
totoxicity of 5-FU was observed at a lower dose (IC50 12.5 µmol/L) and a shorter time (24 h) in both cell lines. Both cell
lines treated with 5-FU or PB alone induced a greater apoptosis effect compared with the combination treatment.
Isobologram analysis indicated that PB and 5-FU interacted synergistically and antagonistically in inhibiting the growth
of HT29 and HCT116 cells, respectively. Conclusions: In the presence of PB, a lower dosage of 5-FU is required to
achieve the maximum drug effect in inhibiting the growth of HT29 cells. However, PB did not significantly reduce 5-FU
dosage in HCT116 cells. Our result showed that this interaction may not solely contribute to the apoptosis pathway.

Key words: Piperaceae, Piper betle L., 5-Fluorouracil, Isobologram analysis, Herb-drug interaction
doi:10.1631/jzus.B1300303 Document code: A CLC number: R979.1


1 Introduction

5-Fluorouracil (5-FU) is an antimetabolite-based
chemotherapeutic drug commonly used in treating
solid human tumors, especially colorectal cancer.
Cytotoxicity of 5-FU is attributed to its ability to
inhibit thymidylate synthase activity and interrupt
DNA and RNA syntheses (Kinsella et al., 1997;
Noordhuis et al., 2004). However, its use is limited
because of the rapid development of acquired re-
sistance and its short half-life (Mader et al., 1998; Jin
et al., 2002). Different treatment approaches have
been designed to increase the efficacy of 5-FU such as
optimizing the administration schedule (Pizzorno

Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology)
ISSN 1673-1581 (Print); ISSN 1862-1783 (Online)
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E-mail: jzus@zju.edu.cn


‡ Corresponding author
* Project supported by the Ministry of Higher Education, Malaysia
(No. UKM-JJ-03-FRGS0044-2010) and Universiti Kebangsaan Ma-
laysia (No. UKM-DPP-2014-131)
© Zhejiang University and Springer-Verlag Berlin Heidelberg 2014
Ng et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2014 15(8):692-700

693
et al., 2009) and using a combination treatment with
leucovorin and oxaliplatin (FOLFOX) (Klampfer et
al., 2005). Nevertheless, drug resistance has also been
noted with increasing 5-FU dosage (Mader et al.,
1998; Pizzorno et al., 2009) and in combination
therapy (Pizzorno et al., 2009). 5-FU resistance is
conferred by the alteration in drug metabolism, drug
transport mechanism, and expression of targeted
proteins such as p53; as a result, cancer cells fail to
undergo cell cycle arrest and apoptosis (Mader et al.,
1998). p53 is the tumor-suppresser protein that helps
in coordinating the response towards 5-FU treatment.
About 30%–50% of colorectal cancer cases have been
found to have mutated p53 protein and hence loss of
sensitivity towards 5-FU (Mader et al., 1998; Bunz et
al., 1999). Thus, there is a need for strategies to
overcome drug resistance and hence enhance drug
delivery to the target site of cancer.
Combination anticancer treatments using the
active compounds from plant extracts are becoming a
new focus of drug discovery. Natural plant extracts
enriched with flavanoids, phenolic compounds, and
other phytochemicals have fewer side-effects com-
pared with chemotherapy drugs, and thus play im-
portant roles as anticancer agents (Pourcel et al.,
2007). There are numerous studies (Tang et al., 2007;
Wang and Yuan, 2008; Fishbein et al., 2009) showing
synergistic drug-herb interactions in enhancing anti-
cancer treatment with minimum cytotoxicity to the
host cells. Fishbein et al. (2009) showed that an anti-
proliferative effect of Red ginsenoside enhanced
5-FU activity in human colon carcinoma, HCT116
cells. Mice treated with combined triptolide and 5-FU
showed a greater tumor growth inhibition in primary
tumor xenografts (Tang et al., 2007).
Piper betle (PB) is a well-known ethnomedicinal
plant in the Asian region (Kumar et al., 2010). PB
leaves have been known for centuries for their cura-
tive properties in, for example, preventing halitosis,
and in the treatment of boils, abscesses, as well as
throat and lung diseases (Guha, 2006). An active
compound of PB, hydroxychavicol (HC), has been
recognized as an antioxidant, anti-proliferative, and
anticancer agent (Young et al., 2006; Fathilah et al.,
2010). Previous research has revealed that pre-
treatment of oral KB carcinoma cells with HC inhib-
ited the attachment of KB cells to type I collagen and
fibronectin, and subsequently resulted in cell cycle
arrest in S and G2/M phases (Chang et al., 2002).
Chakraborty et al. (2012) showed that HC-induced
reactive oxygen species (ROS)-mediated phosphor-
ylation of c-Jun N-terminal kinase (JNK), which, in
turn, phosphorylates endothelial nitric oxide synthase
to produce nitric oxide and mediate cell death in
chronic myeloid leukaemia without depleting gluta-
thione levels. Chakraborty et al. (2012) also showed
that multiple compounds present in crude extracts of
PB may exert better anticancer effects compared with
a single active compound. Combination treatment of
the anticancer drug, cisplatin, with PB increased the
sensitivity of liver cancer cells to cisplatin (Young et
al., 2006). In this present study, we attempt to inves-
tigate the effect of PB on colon cancer cells and its
interaction with the anticancer drug, 5-FU.
Drugs when given in combination may produce
a greater or lesser effect compared with a single drug.
Drug interaction occurs when the pharmacokinetics
and/or pharmacodynamics of a drug is altered by the
presence of another drug, food, or herbs (Hussin,
2001; Renuka et al., 2011). Pharmacodynamic inter-
actions are usually analyzed by the isobologram,
which can distinguish the additive interaction from
synergistic and antagonistic interactions (Tallarida
and Raffa, 1996; Chou, 2006). Synergism is achieved
when either or both respective drugs require a lower
concentration to reach the same effect as in the indi-
vidual treatment respectively with combination index
(CI) <1. Antagonism is defined when a higher con-
centration of either or both drugs achieves the same
effect as the individual treatment (Chou, 2006). In this
study, we found a synergistic interaction of 5-FU and
PB on HT29 cells. This justified further investigation
of the major active compound of PB, HC, on HT29
cells.
Apart from causing a systemic drug interaction
effect, drug-drug or herb-drug interaction could re-
duce or enhance the bioavailability of co-administered
drug (Cabrera et al., 2011). This is known as ‘phar-
maceutical interaction’, in which two substances
interact by direct chemical reaction, either before
ingestion or while mixed together in the stomach and
intestine (Renuka et al., 2011). For example, combi-
nation treatment of ampicillin with tannins forms an
insoluble complex; this bioavailability impairment
reduces the gastrointestinal absorption of ampicillin
(Esimone, 2011). In our current study, we also at-
tempt to identify any pharmaceutical interaction be-
tween PB and 5-FU.
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694
2 Materials and methods
2.1 Aqueous extraction of PB leaves
PB leaves were collected from Bentong, Pahang,
Malaysia. The fresh specimen was sent to the Botany
Department Herbarium, Universiti Kebangsaan Ma-
laysia (UKM), Bangi, Malaysia for identification.
The specimen was then certified with a voucher
number Herbarium UKMB-29852.
The leaves were dried and ground prior to the
extraction. Extraction was conducted using a Soxhlet
extractor coupled with a cooling system. The extrac-
tion method was standardized in collaboration with
the Forest Research Institute of Malaysia, according
to the method of Pin et al. (2011). The ratio of solvent
(distilled water) to raw material was 10 L/kg and the
mixture was stirred at 60 °C for 2 h. The aqueous
extract was filtered with a muslin cloth and filter
paper, and then dried in a Virtis freeze-drier (SP
Scientific, USA).
2.2 2,2-Diphenyl-1-picrylhydrazyl (DPPH) total
free radical scavenging assay
Total free radical scavenging activities of PB
leaf aqueous extract and 4-allylpyrocatechol were
determined by performing DPPH radical assay as
previously described (Molyneux, 2004). Ascorbic acid
(Sigma Chemical, USA) was used as a positive control.
Samples were mixed with methanolic 0.5 mmol/L
DPPH solution in a 1:1 ratio for 30 min in the dark. A
total of 200 μl of the reaction mixture was then
transferred to a 96-well plate and the absorbance was
read at 517 nm. The scavenging activity was calcu-
lated with the equation: radical scavenging activity=
[1−(absorbance of sample/absorbance of positive
control)]×100%. The antioxidant activity of the
sample is expressed as IC50, which is defined as the
concentration of sample required to inhibit the for-
mation of DPPH radicals by 50%.
2.3 Cell cultures and treatments
Human colon cancer cell lines, HT29 and
HCT116 cells (American Tissue Culture Center),
were grown in complete McCoy’s 5A medium with
L-glutamine (Invitrogen, USA). The medium was
supplemented with 10% heat-inactivated (30 min,
56 °C), filter-sterilized fetal bovine serum (Gibco,
USA). The cells were maintained in a 37 °C humid
incubator with 5% CO2. The experiments consisted of
four groups for each cell line: (1) control (cancer cells
without treatment); (2) cancer cells treated with 5-FU;
(3) cancer cells treated with PB leaf extract (range
100.0 to 500.0 μg/ml); (4) cancer cells treated with
5-FU and PB leaf extract. HT29 cells were also
treated with (1) 4-allylpyrocatechol (synthetic HC)
(62.5 to 1000.0 μmol/L) and (2) with 5-FU in com-
bination with 4-allylpyrocatechol. Serial concentra-
tions of PB or 4-allylpyrocatechol, which gave 10% to
90% cell growth inhibition, were combined with dif-
ferent concentrations of 5-FU.
2.4 Cell proliferation assay
Cells were seeded 24 h prior to treatment in a
96-well plate at plating densities of 10 000 cells/well
in order to obtain semi-confluent culture. After re-
spective treatments at every 12-h interval from 12 to
72 h, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymeth-
oxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)
solution (20 μl) was added to each well and the plates
were incubated at 37 °C for 1 h. MTS product was
measured at absorbance 490 nm and cell viability was
calculated using the equation: cell viability=(absorbance
of sample/absorbance of control)×100%.
2.5 Combination index (CI) analysis
Drug combination effect was determined by the
Chou and Talalay equation (Chou, 2006): CI=(dA/DA)+
(dB/DB). dA and dB are the doses of individual
drugs alone, i.e., the concentration of 5-FU or PB/
4-allylpyrocatechol, respectively, that gives 50%
inhibition. DA and DB are the doses of 5-FU and PB/
4-allylpyrocatechol in combination that inhibits 50%
of cell growth. CI>1, CI=1, and CI<1 indicate antago-
nistic, additive, and synergistic effects, respectively.
2.6 Cell apoptosis analysis
Control and treated cancer cells were trypsinized
and cell pellets collected. Pellets were resuspended in
100 μl of 1× annexin binding buffer (100 mmol/L
2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic
acid (HEPES), pH 7.5, 1.4 mol/L NaCl, and 25 mmol/L
CaCl2). Five microlitres of annexin V conjugates
(Invitrogen, USA) and 1 μl of 100 μg/ml propidium
iodide (Sigma, USA) were then added to the mixture
and incubated at room temperature for 15 min. At the
end of incubation, 400 μl of annexin binding buffer
was added and the cells were analyzed within 1 h using a
Facs Arial II flow cytometer (BD Biosciences, Canada).
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2.7 High-performance liquid chromatography
(HPLC) analysis

HPLC analysis was performed on a Prominence
LC (Shimadzu Scientific Intruments, Japan) coupled
with SPD-20A/20AV, UV-VIS detector. A C18 100A
column (250 mm×4.6 mm, 5 μm particle size) was
used as stationary phase. Elution was performed using
a gradient that consisted of a mixture of 0.1% ortho-
phosphoric acid in Milli-Q water and 100% acetoni-
trile, in accordance with the method of Pin et al. (2011).
The flow rate of the mobile phase was 1 ml/min at room
temperature. Chromatograms were acquired at 200
and 260 nm for the detection of PB/4-allylpyrocatechol
and 5-FU, respectively (Ahmad et al., 2011; Pin et al.,
2011).


3 Results

A higher concentration (200.0 μg/ml) of PB ex-
tract was needed to reduce DPPH radicals by 50%
compared with ascorbic acid (60.0 μg/ml).
The IC50 values of 5-FU-treated HT29 and
HCT116 cells were 130.0 and 12.5 µmol/L, respec-
tively, at 72 h. The IC50 values of PB-treated HT29






















and HCT116 cells were 200.0 and 187.5 µg/ml,
respectively, after 36 h of treatment. HT29 cells
treated with 4-allylpyrocatechol showed a lower IC50
(15.0 µg/ml) after 24 h of treatment. In the presence
of PB extract or 4-allylpyrocatechol, the cytotoxic
effect of 5-FU against HT29 cells was observed at a
lower dose (<12.5 µmol/L) and at a shorter time (24 h)
(Figs. 1a–1c). HT29 and HCT116 cells treated with 5-FU
and PB alone induced greater apoptosis effects com-
pared with the combination treatment (Figs. 2a and 2b).
CI analysis indicated PB and 5-FU interacted
synergistically (as shown by CI<1) in HT29 cells, but
antagonistically in HCT116 cells (CI>1) (Fig. 3a).
Combination treatment of 4-allylpyrocatechol and 5-FU
interacted antagonistically in HT29 cells (Fig. 3b).
Interaction of 5-FU and PB/4-allylpyrocatechol
was evaluated by HPLC. Retention time of PB and
5-FU was observed at 4.0 min (200 nm) and 3.0 min
(260 nm), respectively, with a noticeable shift of
peak-height as the concentration varied. The mixture
was analyzed at 200 and 260 nm, respectively. Peak
intensities of 5-FU and PB/4-allylpyrocatechol from
the mixture were the same as when they were ana-
lyzed individually. The mixture of 5-FU and PB/
4-allylpyrocatechol did not show additional com-
pounds by HPLC separation.





















Fig. 1 Effect of PB/4-allylpyrocatechol on the sensitiv-
ity of cells to the cytotoxicity of 5-FU
(a, b) HT29 and HCT116 cells were treated with the indi-
cated concentrations of 5-FU for 24 h in the presence of PB
extract, respectively. (c) HT29 cells were also treated with
5-FU for 24 h in the presence 4-allylpyrocatechol
Data are expressed as mean±standard deviation (n=3).
* P<0.05, compared with control/untreated cells
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Fig. 2 Apoptosis assessment of PB and 5-FU either in combination or alone in HT29 (a) and HCT116 (b) cells
Annexin V-positive/PI negative cells are in early stages of apoptosis and double positive cells are in late stages of apop-
tosis. Annexin V-negative/PI positive cells are necrotic
(a)
(b)
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4 Discussion

In this study, we utilized two different colon
cancer cell lines, i.e., HT29 and HCT116, each with
specific characteristics, to test whether these cancer
cells were resistant to the treatment of PB leaf extract
and the chemotherapy drug 5-FU alone and whether
the combination therapy would enhance the inhibitory
effect of 5-FU. HT29 and HCT116 cells have different
gene profiles. HT29 cells are known to have the mu-
tated tumor-suppressor gene, p53. This is a determi-
nant factor for the success of 5-FU treatment (Bunz et
al., 1999). Our results showed that HCT116 cells with
a functional p53 are more sensitive to 5-FU than
HT29 cells. Lack of functional p53 protein in HT29
cells leads to reduced expression of pro-apoptosis
protein, which then renders the cells resistant to
apoptosis (Boyer et al., 2004). Thus, higher dosage of
5-FU was needed to induce cell death in HT29 cells.
Previous studies suggested that combination
treatment is more effective in treating cancer; it tar-
gets different mechanisms and reduces the chances of
developing resistance (Hiss et al., 2007; Felth et al.,
2009; Majumdar et al., 2009; Tong et al., 2011;
Hörmann et al., 2012; Kumar et al., 2012; Slovin,
2012). Tunicamycin is an antibiotic which can reduce
the effective dosage of doxorubicin, epirubicin, vin-
cristine, and cis-diamine dichloroplatinum in multi-
resistant UWOV2 ovarian cancer cells, thus enhancing



















their cytotoxic effect (Hiss et al., 2007). Majumdar
et al. (2009) found the combination of curcumin and
resveratrol was effective in inhibiting the growth of
colon cancer cells. Low doses of genistein from
soybean increased the sensitivity of prostate cancer
initiating cells to drugs such as docetaxel and cyclo-
pamine (Hörmann et al., 2012; Kumar et al., 2012).
Earlier studies showed that 5-FU interacts synergis-
tically with either triptolide or dichloroacetate in
killing HT29 cells (Tang et al., 2007; Tong et al.,
2011). Combination treatment of 5-FU with dichlo-
roacetate enhanced the 5-FU cytotoxicity at reducing
5-FU concentration from 798.4 to 80.0 µmol/L (Tang
et al., 2007). PB exhibited potent anticancer proper-
ties (Pin et al., 2010), but it is seldom tested in the
combination treatment. Here, we investigated its
combination treatment effect with 5-FU in colorectal
cancer cells.
In our study, PB extract enhanced the cytotoxi-
city of 5-FU by reducing its dosage and treatment
time in HT29 cells (Fig. 1a). However, combination
treatment did not show an obvious apoptosis hallmark
in HT29 cells. Therefore, the cell death observed in
the combination treatment group is unlikely to be
triggered by the apoptosis pathway and we suggest
that PB causes cell death by mediating cell cycle
arrest. On the other hand, we observed that the com-
bination of 5-FU and PB did not mitigate the 5-FU
dosage in HCT116 cells, but necrosis was prominent
Fig. 3 Isobologram at 50% effect level of simultaneous exposure of HT29 and HCT116 cells to 5-FU and PB/
4-allylpyrocatechol
The solid line (CI=1) indicates the alignment of theoretical values of an additive interaction between two substances.
Values above the solid line represent antagonistic interaction; values below the line represent synergism. (a) Concurrent
24 h exposure of HT29 cells and HCT116 cells to 5-FU and PB exhibits synergistic and antagonistic treatment effect
respectively. (b) Simultaneous treatment of 5-FU and 4-allylpyrocatechol treatment inhibits HT29 cell growth antagonistically
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698
when HCT116 cells were treated by combination.
This may be due to the excessive cytotoxic induction
by the treatment, which leads to early massive apop-
tosis followed by failure of complete phagocytosis as
previously described (Zong and Thompson, 2006).
The synergistic interaction of 5-FU and PB on
HT29 cells justified further investigation of the major
active compound of PB, HC, on HT29 cells. HC is a
major phenolic compound found in PB leaves
(Hemamalini et al., 2012). The presence of two hy-
droxyl (OH) groups in HC increases the antioxidant
and anticancer properties of PB (Pin et al., 2010).
Our DPPH result revealed that PB at a concentration
of 250 µg/ml had the highest scavenging activity
while HC showed lower scavenging activity com-
pared with the crude PB extract. The potent anti-
oxidant capacity exhibited by the crude PB extract
may be due to the presence of other phenolic com-
pounds such as vitamins C and E (Hemamalini et al.,
2012). Our study demonstrated the potential anti-
cancer effects of PB and HC since they exerted a
cytotoxic effect in both colorectal cancer cells at
concentrations of more than 100 µg/ml. The cytotoxic
effect of HC was through the formation of electro-
philic metabolites—quinone, quinone methide, and
imine methide—via the oxidative metabolism of HC
(Chang et al., 2002; Jeng et al., 2002). The formation
of electrophile metabolite conjugates with the re-
duced glutathione and reduced glutathione content
sensitized the cells towards ROS (Nakagawa et al.,
2009). Our results showed that 4-allylpyrocatechol
(synthetic HC) is more potent compared with the
crude PB extract in killing HT29 cells. However,
combination treatment of 4-allylpyrocatechol and
5-FU on HT29 cells showed an antagonistic interac-
tion. This observation suggests that the synergistic
effect of PB extract and 5-FU on HT29 cells may not
solely be due to its major active compound, HC, since
PB extract also contains other phytochemicals such as
chavibetol, chavicol, eugenol, and ursolic acid, which
may be involved in mediating apoptosis in cancer
cells (Chang et al., 2002; Jeng et al., 2004;
Arambewela et al., 2006; Young et al., 2006; Yamai
et al., 2009). These other bioactive compounds may
be present and may have triggered the synergistic
effect observed in our current study. Our finding is in
accordance with the study of Young et al. (2006),
who found that the crude extract of PB exhibited a
stronger cytotoxicity than the bioactive compounds,
HC and eugenol, when acting in combination with
cisplatin.
Incompatibility interaction is an undesired reac-
tion that occurs between a drug and another drug or
solution (Newton, 2009). There are two types of in-
compatibility interaction. Physical reaction usually
refers to either phase separation or precipitation due
to a change of relation between ionization, non-
ionization, and solubility (Newton, 2009). In chemi-
cal incompatibility, drugs are chemically degraded
and can manifest themselves through turbidity, pre-
cipitation, and color change. Chemical incompatibil-
ity can probably be identified with HPLC (Newton,
2009; Cabrera et al., 2011). In our study, a simple
visual examination of the 5-FU and PB mixture did
not show the presence of any precipitation in solution,
suggesting that the interaction of 5-FU/PB and 5-FU/
4-allylpyrocatechol is physically compatible. Based
on our HPLC results, no additional compound was
resolved from the 5-FU and PB/4-allylpyrocatechol
mixture, indicating that there is a compatible and stable
interaction between 5-FU and PB/4-allylpyrocatechol
prior to treatment of the cells.
In conclusion, 4-allylpyrocatechol and PB crude
extract have significantly mitigated the 5-FU dosage
and enhanced the cytotoxicity of the drug in killing
colon cancer cells. However, CI and apoptosis analysis
revealed that PB crude extract and 4-allylpyrocatechol
may exert their cytotoxicity via different mechanisms
other than apoptosis when treated together with 5-FU. An
antagonistic interaction of 5-FU and 4-allylpyrocatechol
suggests that 4-allylpyrocatechol may not be the only
compound responsible for enhancing the 5-FU effect,
and there may be other bioactive compounds in PB
capable of modulating the cytotoxic effect of 5-FU.

Acknowledgements
We gratefully acknowledge Miss Nor Syahida ALI-
AHMAT, from Department of Biochemistry, UKM, Malaysia,
for her help in the PB leaf collection.

Compliance with ethics guidelines
Pek Leng NG, Nor Fadilah RAJAB, Sue Mian THEN,
Yasmin Anum MOHD YUSOF, Wan Zurinah WAN NGAH,
Kar Yong PIN, and Mee Lee LOOI declare that they have no
conflict of interest.
This article does not contain any studies with human or
animal subjects performed by any of the authors.
Ng et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2014 15(8):692-700

699
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中文概要：

本文题目：蒌叶提取物增强 5-氟尿嘧啶对结肠癌细胞 HT29 和 HCT116 的生长抑制作用
Piper betle leaf extract enhances the cytotoxicity effect of 5-fluorouracil in inhibiting the
growth of HT29 and HCT116 colon cancer cells
研究目的：探讨蒌叶（PB）提取物对 5-氟尿嘧啶（5-FU）抑制结肠癌细胞 HT29 和 HCT116 生长的影响。
研究方法：HT29 和 HCT116 细胞分别给予 PB、5-FU 以及两种药物联合治疗 24 小时，应用等效线图法
分析 PB 和 5-FU 的药效学相互作用，Annexin V/PI 染色法检测 HT29 和 HCT116 细胞的凋亡
情况，高效液相色谱法排除 PB 和 5-FU 间任何可能的相互化学作用。
重要结论：联合 PB，低剂量 5-FU 可以在短时间内起到细胞毒作用，而单独应用 PB 或 5-FU 治疗较联合
治疗可以诱导更多细胞发生凋亡。进一步采用等效线图法分析显示 PB 和 5-FU 的联合作用在
抑制结肠癌细胞 HT29 和 HCT116 的生长中分别体现出协同和拮抗作用。因此可以认为在
HT29 细胞中，PB 使得较低剂量 5-FU 发挥最大抑制结肠癌细胞生长效果，然而在 HCT116 细
胞中，PB 没有显著降低 5-FU 的药物浓度，说明 PB 和 5-FU 的相互作用不仅仅体现在诱导细
胞凋亡方面。
关键词组：胡椒科；蒌叶；5-氟尿嘧啶；等效线图法；草药-药物相互作用