CYTOTOXIC COMPOUNDS AGAINST BREAST ADENOCARCINOMA CELLS (MCF-7) FROM PIKUTBENJAKUL

Original Article 71
CYTOTOXIC COMPOUNDS AGAINST BREAST
ADENOCARCINOMA CELLS (MCF-7) FROM PIKUTBENJAKUL
Intouch Sakpakdeejaroen and Arunporn Itharat?
Applied Thai Traditional Medicine Center, Faculty of Medicine, Thammasat University, Khlong Luang,
Pathumthani 12120, Thailand


ABSTRACT: Pikutbenjakul, a Thai Traditional medicine preparation, is composed of five
plants: Piper chaba fruit, Piper sarmentosum root, Piper interruptum stem, Plumbago indica root
and Zingiber officinale rhizome. It is a balanced health preparation in Thai traditional medicine.
From selective interviews of folk doctors in Southern Thailand, it was found that Benjakul was
used as an adaptogen drug for breast cancer patients. It was give them before using cancer
drug. Thus, the objectives of this research were investigating cytotoxic activity against breast
cancer cell (MCF-7) of Pikutbenjakul preparation and its components extracts by using the
SRB assay. The extraction method imitated by folk doctors used by maceration in ethanol and
boiling in water. The results were found that the ethanolic extract of Piper chaba, Zingiber
officinale and Pikutbenjakul showed high cytotoxic activity against breast cancer cell (IC50=
35.17, 31.15 and 33.20 µg/ml, respectively) but water extract showed no cytotoxic activity
against breast cancer cells. Two compounds [piperine and 6-shogaol as 7.48 and 0.54% w/w
of crude extract] were isolated from the ethanolic extract of Pikutbenjakul by bioassay guide
fractionation and were also tested for cytotoxic activity. It was found that piperine and 6-
shogaol had cytotoxicity against MCF7 with IC50 value of 9.80 and 10.18 µg/ml. These results
can support using Pikutbenjakul to treat breast cancer patients of Thai folk doctors.
Keywords: Cytotoxic activity, Breast cancer, MCF7, SRB assay, Thai medicinal plants, Pikutbenjakul,
Piper chaba , Piper sarmentosum, Piper interruptum, Plumbago indica, Zingiber officinale


INTRODUCTION: Cancer has been the first body or increases their immunity. Pikutbenjakul
leading cause of death in Thailand for several also showed no toxicity changes when tested by a
years and the number of people died from cancer sub-chronic toxicity method4). In spite of this
is still increasing every year. For specific types of preparation is commonly used in Thai traditional
cancer occurred only in women, breast and cervix Medicine before the treatment of many diseases,
cancers were the two highest causes of death in there are no reports on testing its pharmacological
Thai women. Plant-based systems have a long activity, such as cytotoxic activity against cancer
history of use in traditional health care1). Sixty cells. Only one record exists of for cytotoxicity
percents of currently used anticancer agents are against cancer cells of the ethanolic extract of
derived in one way or another from natural Piper chaba which showed cytotoxic activity
sources2). Therefore, the usage of ethnopharmaco-
against human lymphocytes, ovarian cells from
logy or traditional use is channel for discovery of Chinese hamster and Dalton’s lymphoma cells
new biologically-active molecules1). Investigation of (IC50 = 0.13,0.145 and 0.3 µg/ml respectively)5). In
indigenous wisdom on cancer treatment of Thai the present study, the five Thai medicinal plant
traditional doctors3) revealed that a Pikutbenjakul, extracts which are ingredients of Pikutbenjakul
which is composed of five Thai medicinal plants formula and the Pikutbenjakul preparation were
(Piper chaba Linn, Piper sarmentosum Roxb, Piper
tested for their cytotoxic activity against breast
interruptum Opiz., Plumbago indica Linn. and adenocarcinoma cell cancer (MCF-7). The isolated
Zingiber officinale Roscoe) has been used as an compounds from the Pikutbenjakul extract were
adaptogen drug for cancer patients. Folk doctors also isolated and tested cytotoxic activity against
would give Pikutbenjakul to treat breast cancer breast cancer cells. These results could also
patients for 2 or 3 weeks before treatment with support the use of these plants by folk doctors to
breast cancer preparation. It is claimed that treat breast cancer patients.
Pikutbenjakul is balances elements in patient’s



?To whom correspondence should be addressed.
E-mail: iarunporn@yahoo.com
Tel: +66 2926 9749 , Fax. +66 2926 9705
J Health Res 2009, 23(2): 71-76

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72 Original Article
MATERIALS AND METHODS:
liquid chromatography (VLC), using silica gel and
a gradient elution of hexane (10x200ml), hexane:
Plant materials
chloroform (10x200ml), chloroform (10x200ml),
The relevant parts of the species, which are chloroform:MeOH
reported to be used against cancer by folk doctors

(1:1) (10x200ml), MeOH
(10x200ml). Drying and evaporation of each
in Thailand, were collected from all parts of fraction yielded residues of 0.22 g, 0.46, 5.61,
Thailand in January to March 2006 (Table 1). 21.72 and 6.26 g, denoted as FA, FB, FC, FD and
Authentication of plant materials was carried out FE respectively. These five fractions were tested
at the herbarium of the Department of Forestry for cytotoxic activity against lung cancer by the
Bangkok, Thailand where the herbarium SRB assay because it was found that
vouchers have been kept. Another voucher has Pikutbenjakul preparation showed the highest
been deposited in the herbarium of Southern cytotoxicity against breast cancer. Thus, the five
Center of Thai Medicinal Plants at Faculty of fractions were also tested against breast cancer
Pharmaceutical Sciences, Prince of Songkhla cell line. It was found that FC showed the highest
University, Songkhla, Thailand (for voucher cytotoxic activity against MCF-7 (% cytotoxic =
numbers see Table1).
82% at concentration 50 µg/ml)
Preparation of plant extracts
An aliquot of the ethanolic extract of
Plant materials were dried at 50°C, powdered Pikutbenjakul was separated by CC (silica gel
and extracted in ways corresponding to those with a gradient of solvents: hexane:EtOAc (8:2);
practised by Thai traditional doctors, i.e. water (350 ml); hexane:EtOAc (7:3) (100ml); hexane:
extraction and ethanolic extraction For the water EtOAc (7:3) (100ml); hexane:EtOAc (6:4) (200 ml);;
extract of each plant, the dried ground plant hexane:EtOAc (1:1) (200ml); EtOAc:hexane (2:8)
material (100 g) was boiled for 30 minutes in (300 ml) , EtOAc (200ml); EtOAc:MeOH (9.5;0.5)
distilled water (300 ml ), filtered and freeze dried. (200ml), EtOAc:MeOH (9:1) 200ml ; EtOAc:MeOH
For the ethanolic extracts dried ground plant (1:1) 200ml and finally MeOH (300 ml). Ten
material (100 g) was percolated with 95 % ethanol milliter fractions were collected for each eluting
and the filtrate concentrated to dryness under solvent and fractions combined, following TLC
reduced pressure. The percentage yields are examination (silica gel/ CHCl3 : MeOH (7:3) and
shown in Table 4. The water extracts were detection with acidic anisaldehyde spray.
dissolved in sterile water and the ethanolic Compound A (158.5 mg, 7.81 % w/w) was
extracts were dissolved in DMSO and all stock isolated as yellow crystals, designated F1, from FC
solution were filtrated by sterile filter paper (0.2 dissolved in chloroform before CC. CC (silica gel)
?m) before testing.
was carried out on the mother liquor with EtOAc:
Isolation and purification of active compounds
hexane (2:8) to get yellow crystals, which were
An aliquot of the ethanolic extract of recrystallized in MeOH. Compound B (9.6mg,
Pikutbenjakul (40 g) was separated by vacuum 0.54 % w/w) as pale yellow oil was isolated from
fractions 66-73 after TLC purification.
Table 1 The summarized data of the investigated plant species as the ingredients of Pikutbenjakul
Plants (Family)
Places for plant collection
Voucher number
Part of used
(Amphor, Province)
Piper chaba Linn
(Piperaceae)
Kaosaming, Chantaburi
SKP 146160301
Fruit
Piper sarmentosum Roxb.
Jombueng, Ratchaburi
SKP
(Piperaceae)
146161901 Root
Piper interruptum Opiz.
(Piperaceae)
Phoopan, Sakonnakhon
SKP 146160901
Stem
Plumbago indica Linn
Talingchan, Bangkok
SKP148160901
Root
(Plumbaginaceae)
Zingiber officinale Roscoe.
(Zingiberaceae)
Khaokho, Petchaboon
SKP206261501
Rhizome
Pikutbenjakul -
-
-
J Health Res 2009, 23(2): 71-76

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Original Article 73
Structure elucidation
Compound B (6-gingerol): C17H26O4 (9.6 mg,
The structure of the isolates (Figure 1) was 0.54%w/w); orange needle crystal solids; EIMS
determined by their NMR data [1H and 13C on a (low resolution) m/z (% relative intensity) 294 (M+,
Varian Unity Inova 500 spectrometer (500 MHz 50), 150 (55), 137 (100). Compound B was
for 1H; 125 MHz for 13C)], UV spectra [ a Hewlett isolated fromthe

ethanolic extract of Pikutbenjakul
Packard 8452A Diode array spectrometer], IR preparation, obtained as orange needle crystal
spectra [Jasco IR-810 spectrometer], EI mass solids. The TLC analysis of this compound was
spectra, Low resolution were obtained from a compared with authentic sample 6-gingerol
JEOL JMS-AX505W spectrometer.
(Wako) by TLC using 3 solvent systems and gave
Compound A (Piperine): C17H19NO3 (158.5 mg, identical behavior. The 1H NMR spectrum,
7.81%w/w); light yellow needle crystal solids; compared with the previous 1H-NMR data of 6-
EIMS (low resolution) m/z (% relative intensity) gingerol, was the same as the spectrum recorded
285 (M+, 75), 201 (100), 173 (19), 143 (17), 115 for 6-gingerol7) (Table 3). Thus, it was strongly
(45). Compound A was the major compound supported that compound B to be 6-gingerol. The
isolated from the ethanolic extract of Pikutbenjakul structure was shown in Figure 2.
preparation. The TLC analysis of this compound
was compared with authentic sample piperine
(Merck) by TLC using 3 solvent systems and gave
identical behavior. The 1H NMR spectrum,
compared with the previous 1H-NMR data of
piperine, was the same as the spectrum recorded

Figure 2 Structure of compound B (6-gingerol)
for piperine6) (see table 2). It was identified as
piperine by comparison on chromatography and
spectral features with an authentic sample
Table 3 1H-NMR spectral data (500 MHz) of BENS3 in CDCl3
purchased from Merck and was the major
Position
?H (mult.,J in Hz)
Position
?H (mult.,J in Hz)
(6-Gingerol)
(Compound B)
product isolated. The structure was shown in
2
6.69 (d, 2.0)
2
6.68 (br.s)
Figure 1.
3-OCH3
3.68 (s; 3H)
3-OCH3
3.87 (s; 3H)
4-OH
-
4-OH
5.51 (s)

5
6.60 (d, 8.4)
5
6.82 (d, 8.4)
6
6.53 (dd, 8.4, 2.0)
6
6.65 (dd, 8.4, 2.1)
1'-2'
2.69 (s; 4H)
1'
2.74 (br.d,2H 7.2)


2'
2.71 (dd, 6.6, 2.1)



2.85 (dd, 6.6, 2.1)
4'
2.44 (dd; 2H, 8.4, 2.0)
4'
2.48 (dd, 17.4, 3.3)



2.58 (dd, 17.4, 8.7)
5'
3.86 (m)
5'
4.04 (m)
6'-9'
1.21–1.32 (m; 8H)
6'-9'
1.25–1.50 (m; 8H)

10'
0.83 (t; 3H)
10'
0.89 (t; 3H, 6.6)
Figure 1 Structure of compound A (piperine)
Note: 6-gingerol from previous report7
Table 2 1H-NMR spectral data (500 MHz) of compound A
isolated from the ethanolic extract of Pikutbenjakul compare
In vitro Assay for Cytotoxic Activity
with piperine in CDCl3 and CD3OD
Human cell lines

Position ?H (mult., J in Hz)
?H (mult., J in Hz) of
The human breast adenocarcinoma cells
of Piperine
Compound A
(MCF-7) (ECACC No:86012803) were obtained
2
3.48 (br.s; 2H)
3.64 (dd; 2H, 11.1, 5.7)
3
1.49 (m; 2H)
1.62 (m; 2H)
from the European Collection of Animal Cell
4
1.56 (m; 2H)
1.71 (m; 2H)
5
1.49 (m; 2H)
1.62 (m; 2H)
Culture (PHLS Center for applied Microbiology
6
3.48 (br.s; 2H)
3.64 (dd; 2H, 11.1, 5.7)
Research, Porton Down, Salisbury, UK). MCF-7
2'
6.36 (d, 14.6)
6.65 (d, 14.7)
3'
7.31 (m)
7.34 (dd, 14.7, 9.6)
cells were cultured in Minimum Essential Media
4'
6.64 (m)
6.88 (dd, 14.7, 9.9)
(MEM) with Earle Salt without glutamine medium
5'
6.65 (m)
6.89 (d, 14.7)
2"
5.86 (s; 2H )
5.98 (s; 2H )
supplement with 10% heated foetal bovine serum,
4"
6.88 (d, 1.6)
7.11 (d, 1.5)
6"
6.79 (dd, 1.6, 8.0)
6.98 (dd, 8.1, 1.5)
1% of 2 mM L-glutamine, 50 IU/ml penicillin and
7"
6.67 (d, 8.0)
6.81 (d, 8.1)
50 ?g/ml streptomycin and 1% non-essential

J Health Res 2009, 23(2): 71-76

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74 Original Article
amino acid and maintained at 37ºC in a 5% CO2 concentrations and 100 ?l of each concentration
atmosphere with 95% humidity8). According to was added to each well of the plates in 6
their growth profiles, the optimal plating densities replicates to obtain final concentrations of 100,
of breast cancer cell line was determined 3x103
50, 25, 10, 5, 1.5, 0.5, 0.1 ?g/ml for extract and
cells/well to ensure exponential growth through-
100, 50, 25, 10, 5, 1.5, 0.5, 0.1, 0.05 nM for
out the experimental period and to ensure a linear vinblastine sulphate (positive control). The final
relationship between absorbance at 492 nm and mixture used for treating the cell contained not
cell number when analyzed by SRB assay.
more than 1% of the solvent, the same as in
solvent control wells. The plates were incubated
Cytotoxicity Assay
for selected exposure times of 72 hours. as
For the assay, cells were washed with indicated. At the end of each exposure time, the
magnesium and calcium free phosphate buffer medium was removed. The wells were then
saline (PBS) (Oxoid Ltd.,UK) PBS were decanted washed with medium, and 200 ?l of fresh
and cells detached with 0.025% trypsin-EDTA medium were added. The plates were incubated
(sigma) PBS were added to a volume of 50 ml .The for recovery period of 6 days and cell number were
cell pellet obtained by centrifugation (1000g , 5 analyzed by SRB assay8,9).
min) were resuspensed in 10 ml of medium to
make single cell suspension and viable cells were Sulphorhodamine B (SRB) assay
counted by trypan blue exclusion in haemocyto-
The anti-proliferative assay, SRB (sulphorhoda-
meter and diluted with medium to give a final mine B) assay,was performed according to method
concentration of 3x103 cells/well. One hundred of Skehan et al9) was used to assess growth
?l/well of these cell suspensions were seeded in inhibition This colorimetric assay estimates cell
96-well microtiter plates and incubated to allow number indirectly by staining total cellular
for cell attachment. After 24h the cells were protein with the dye SRB9). For the procedure of
treated with the extracts and pure compounds. this assay is described in previous report8. The
Each extract was initially dissolved in an amount IC50 values were calculated from the Prism
of DMSO for the ethanolic extracts and sterile program obtained by plotting the percentage of
distilled water for the water extracts and survival versus the concentrations, interpolated
vincristine sulphate (Sigma, Lot No. 34H0 447) by cubic spine. According to National Cancer
was used as the positive control. The extracts Institute guidelines10) extracts with IC50 values <
were diluted in medium to produce 8 20 ?g /ml were considered active.
Table 4 Cytotoxicity activity (IC50 µg/ml ± SEM) of plant extracts and isolated compounds against human breast
adenocarcinoma cell line (MCF7) at exposure time 72 hr (n=3).
Plants
Part of used
Extract
Code
%yield
MCF-7
(IC50 µg/ml ± SEM)[µM]
EtOH
PCE
12.3906
35.17 ± 1.91
Piper chaba Linn
Fruit
Water
PCW
15.8965
>100
Piper sarmentosum Roxb
PSE
1.7449
69.53 ± 9.09

Root
EtOH
Water
PSW
8.5603
>100
Piper interruptum Opiz. Stem EtOH
PIE
0.6596
62.35 ± 5.23
Water
PIW
5.0254
>100
5.0071
40.81 ± 3.62
Plumbago indica Linn Root
EtOH
PLE
Water
PLW
20.3808
>100
Zingiber officinale Roscoe Rhizome EtOH
ZOE
8.5651
31.15 ± 1.40
Water
ZOW
13.7434
>100
EtOH
BENE
7.7332
33.20 ± 0.80
Pikutbenjakul
-
Water
BENW
16.2897
>100
Compound A (Piperine)
-
-
-
7.81
10.18 ± 1.29 [35.72]
Compound B (6-gingerol)
-
-
-
0.54
9.80 ± 2.37 [33.33]
J Health Res 2009, 23(2): 71-76

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Original Article 75
RESULTS AND DISCUSSION: The summarized CONCLUSION: In summary, Pikutbenjakul as a
data of the investigated plant species showed in Thai traditional medicine which was normally
Table 1. The results of cytotoxicity evaluation of used to be adaptogen for cancer treatment
all plant extracts as IC50 (?g/ml) were shown in showed cytotoxic against breast cancer cell. Two
Table 4. This data showed that the ethanolic compounds, piperine and 6-gingerol, were isolated
extract of Piper chaba, Zingiber officinale and from the

ethanolic extract of Pikutbenjakul
Pikutbenjakul were the extracts possessing the preparation which also showed cytotoxic against
highest activity against the MCF7 cell line (IC50 = breast cancer cells. Therefore, the present study
35.17, 31.15 and 33.20 µg/ml, respectively). supports the use of Benjakul to treat breast
Piperine (Compound A) and 6-gingerol (Compound cancer by Thai traditional doctors. Further
B) were cytotoxic compounds against MCF7 that studies involved the investigation about cytotoxic
isolated from ethanolic extract of Pikutbenjakul acitivity against other human cancer cell lines
(Table 4). Piperine is a type of alkaloid which and molecular mechanism of isolated compound
showed the highest percentage of yield of againstbreast
comparative with Pikutbenja-

cancer
Benjakul extraction (7.81%) and was isolated kul extract
from Piper longum11) and many species of Piper. ACKNOWLEDGEMENT: We are grateful to the
Piperine showed cytotoxic activity against MCF-7 Thailand Research Fund (TRF) and Faculty of
(IC50 = 35.72?M).The previous report showed that Medicine, Thammasat University for financial
piperine inhibited the solid tumor development in support for this research.
mice induced with DLA cells and increased the life
span of mice bearing Ehrlich ascites carcinoma REFERENCES:
1. Houghton, P. 1995 The role of plants in
tumor to 58.8%12). Although piperine is a well-
traditional medicine and current therapy. J
defined compound, its cytotoxic mechanisms on
Altern Complem Med 1(3): 131-143.
cancer cells especially breast cancer cells have not
2. Cragg, M.G. and Newman, D.J. 2001 Natural
yet been examined. 6-Gingerol was isolated from
Product Drugs Discovery in Next Millennium
Zingiber officinale7), which is one of the five
National Cancer Institute, Frederick, USA.
components of Pikutbenjakul Preparation. It
3. Itharat, A., Singchangchai, P., Ratana suwan,
P. 1998. Wisdom of Southern Thai traditional
showed cytotoxic against breast cancer cells (IC50
doctors Prince of Songkla University, Songkla,
= 33.3 µM). This result is consistent with the
p.1-6.
other previous report of 6-gingerol. It was found
4. Chaovalitthamrong, P., Attawit, A., Rugsa-
that it showed cytotoxic against human cancer
mun, P. and Junpen, P. 1996 Subchronic
cells (HL-60 cells)13) and inhibits cell adhesion,
Toxicity of Thai Traditional medicine call
invasion, motility and activities of MMP-2 and
Benjakul Thai J Pharm Sci 20(1): 39-51.
5. Unnikrishnan,M.C. and Kuttan, R. 1988.
MMP-9 in MDA-MB-231 human breast cancer
Cytotoxicity of extracts of spices to cultured cells,
cell lines14). However there is no report on its
Nutr cancer 11: 251-257.
cytotoxicity against MCF-7. The results revealed
6. Araujo-Junior, J.X.D., Da-Cunha, E.V.L.,
that Pikutbenjakul prepara-tion possessed high
Chaves, M.C.D.O. and Gray, A.I. 1996.
cytotoxic activity against breast cancer cells. Its
Piperdardine, A Piperidine Alkaloid from Piper
ethanolic extract should be promoted for industry.
tuberculatum. Phytochemistry 44(3): 559-561.
7. Kim, J.S., Lee, S.I., Park, H.W., Yang, J.H.,
Piperine and 6-gingerol should be used as
Shin, T.Y., Kim, Y.C., Baek,N.I., Kim, S.H. Choi,
markers for standardization of chemical and
S.U., Kwon, B.M., Leem, K.H. Jung, M.Y. and
biological fingerprints because piperine was
Kim, D.K. 2008. Cytotoxic Components from the
present in high amount and high cytotoxic
Dried Rhizomes of Zingiber officinale Roscoe. Arch
activity.
Pharm Res 31(4): 415-418.
8. Itharat, A.,.Houghton P.J., Eno-Ammguaye,
E.,.Burke P.J, Sampson J.H. and Raman A. 2004
In vitro cytotoxic activity of Thai medicinal plants
J Health Res 2009, 23(2): 71-76

---

76 Original Article
used traditionallyto
countercurrent chromatography and reversed-

treat cancer. J Ethnopharma-
col 90: 33-38.
phase liquid chromatography. J Chromatogr A
9. Skehan, P., Storeng, R., Scudiero, D., Monks,
1040(2): 193-204.
A., McMahon, I., Vistica, D., Waren, JT.,
12. Sunila ES, Kuttan G. 2004 Immunomo-
Bokesch, H., Kenney, S., Boyd, MR.., 1990. New
dulatory and antitumor activity of Piper longum
colorimetric cytotoxicity assay for anticancer drug
Linn. and piperine. J Ethnopharmacol 90(2-3):
screening. J Natl Cancer Inst 82: 1107-1112.
339-46.
10. Boyed, MR., 1997. The NCI in vitro
13. Lee E, Surh YJ. 1998 Induction of apoptosis
anticancer drug discovery screen. In: Teicher,
in HL-60 cells by pungent vanilloids, [6]-gingerol
B.(Ed.) Anticancer drug development guide;
and [6]-paradol. Cancer Lett. 134(2): 163-8.
preclinical screening, clinical trials and approval.
14. Lee HS, Seo EY, Kang NE, Kim WK. 2008
Humana Press, Totowa, p. 30.
[6]-Gingerol inhibits metastasis of MDA-MB-231
11. Wu S, Sun C, Pei S, Lu Y, Pan Y. 2004 :
human breast cancer cells. J Nutr Biochem 19(5):
Preparative isolation and purification of amides
313-9.
from the fruits of Piper longum L. by upright










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