Cuminum Cyminum

Journal of Applied Sciences Research, 5(11): 1881-1888, 2009
© 2009, INSInet Publication
Antitumor and Antibacterial Activities of [1-(2-Ethyl, 6-Heptyl) Phenol] from Cuminum
Cyminum Seeds
Amal A.I. Mekawey, Mokhtar M.M. and Rasha M. Farrag
The Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo-Egypt
Abstract: 1-(2-Ethyl, 6-Heptyl) Phenol (EHP), a biologically active compound formerly extracted by
benzene from Cuminum cyminum (cumin) Egyptian seeds and of activity against a number of fungal
pathogens, exhibited antitumor activity against six types of tumor cell lines (HEPG2, HELA, HCT116,
MCF7, HEP2, CACO2). EHP showed no cytotoxicity when its activity was investigated on the normal
fibroblast cell line (BHK). MCF7 was the most sensitive tumor cell line where only 33% of the cells
survived followed by HEPG2 (41 % of the cells survived) and HEP2 (56% of the cells survived) at an
EHP concentration of 10 μg/ml. The percentage of tumor cell survival of CACO2 and HCT116 was 72%
and76% respectively exhibiting much less activity, however EHP activity against HELA was negligible.
EHP activity was also investigated against eight bacterial human pathogens (four Gram-positive and four
Gram-negative ones). Higher activity was observed against Gram-positive bacteria than Gram-negative ones
where Staphylococcus aureus, Streptococcus pneumoniae, Bacillus subtilis and Bacillus thuringiensis were
more sensitive than Salmonella typhi, Escherichia coli, Serratia marcescens and Pseudomonas aeruginosa.
This study directs the attention to the antifungal, antibacterial and antitumor benefits of the cheap, safe,
available and tasteful cumin.
Key words: antitumor agents, SRB, Cuminum cyminum, ELISA, BHK, antibacterial agents
INTRODUCTION
Cancer is a class of diseases or disorders
characterized by uncontrolled division of cells and the
ability of these cells to invade other tissue, either by
direct growth into adjacent tissue through invasion or
by implementation into distant sites by metastasis.
Metastasis is defined as the stage in which cancer cells
are transported through the blood stream or lymphatic
system [15].
The unregulated growth that characterizes cancer
is caused by damage to DNA, resulting in mutations to
genes that encode for proteins controlling cell division
[17]
.
The effect of plant extracts as antitumor agents
were widely studied due to their low toxicity and side
effects. Spices and herbs have been used for thousands
of centuries by many cultures to enhance the flavor
and aroma of foods. Early, cultures also recognized the
value of using spices and herbs in preserving foods and
for their medicinal value. Scientific experiments since
the late 19th century have documented the
antimicrobial properties of some spices, herbs, and their
components [18].
Cuminum cyminum seeds are stomachic, diuretic,
carminative, stimulant, astringent, emmenagogic and
antispasmodic, hypoglycemic, contraceptive in treatment
of sores, burn and slow continuous fever. It is valuable
in dyspepsia, diarrhoea and hoarseness, antimalarial,
laxative, and may relieve flatulence and colic [26].
In the West, it is used mainly in veterinary
medicine, as a carminative, but it remains a traditional
herbal remedy in the East. It is supposed to increase
lactation and reduce nausea in pregnancy. It has been
shown to be effective in treating carpal tunnel
syndrome, as well as diarrhoea, indigestion and
morning sickness [28]. Cumin also shows promise as a
natural way to increase breast size. Used in a poultice,
it relieves swelling of the breast or the testicles. Cumin
stimulates the appetite. The leaf extracts were found to
inhibit the growth of leukemic cells hence it contains
active ingredients against tumor cells [31].
Scientific strategies for the in vitro evaluation of
natural products with biological activity have changed
in the past few years. One recent development is the
highly automated bioassay screening based on
colorimetric methods that quantify the proliferation of
cell cultures [24,10] , these techniques which are
considered quick and inexpensive for the evaluation of
antitumor [5,32] and antiviral activity [41] of a large
number of natural product extracts, have also easily
permitted to guide the isolation and purification of their
biologically active principles [7].
Corresponding Author: Amal A.I. Mekawey, The regional center for mycology and biotechnology- Al-Azhar University,
Cairo-Egypt
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J. Appl. Sci. Res., 5(11): 1881-1888, 2009
The effect of plant extracts on cancer cell was
studied due to their low toxicity and side effects. The
inhibition of ascites tumor cells by garlic extracts was
investigated. Soybean seed extracts showed antitumor
activity due to the presence of trypsin inhibitor [1].
Interest in a large number of traditional natural
products has increased [21,40,39] . It has been suggested
that aqueous and ethanolic extracts from plants used in
allopathic medicine are potential sources of antiviral
and antitumor agents [6,40] . Furthermore, the selection of
crude plant extracts for screening programs has the
potential of being more successful in its initial steps
than the screening of pure compounds isolated from
natural products [22, 4].
The tumor inhibitors of plant origin depend upon
the type of cancer cells and plant species as well as
the extract used. Extract of Allamanda cathartica gave
significant activity against P-388 leukemia in mouse
[13]
. Different plant species growing in Egypt showed
anticancer activity [12]. The principles separated from
plants were also studied such as alkaloids, terpenes,
flavonoids [27,16,11] and chlorophyll [4,33].
Cumin is being studied for its role in cancer
prevention, says Pensiero. “Cumin contains limonene,
a type of phytochemical that is being investigated for
its role in blocking cancers, specifically prostate
cancer”, it may also reduce cholesterol [29]. Cumin's
distinctive flavour and strong warm aroma is due to its
essential oil content. Also, cumin has strong
antimicrobial activities such as, antibacterial activity [26];
antifungal activity [19], and antitumor activity [43].
Cumin (Cuminum cyminum) is a widely used
ingredient in food. It has been used for a very long
time in traditional medicine in the treatment of gastric
disorders. Cumin seeds showed antimicrobial activities
for different microorganisms, including bacterial strains,
yeasts and fungi. In India, many spices included cumin
seeds have been traditionally used since ancient times,
for the preservation of food products as they have been
reported to have antiseptic and disinfectant properties.
In this respect, a pre liminary screening for
antimicrobial activities of cumin has been carried out.
Of the spices surveyed, the results indicate that cumin
has potent antimicrobial activities against the test
organisms Bacillus subtilis, Escherichia coli and
Saccharomyces cerevisiae [8,9].
The essential oils extracted from the seeds of
seven spices,including Cuminum cyminum, have been
studied for antibacterial activity against eight
pathogenic bacteria, causing infections in the human
body. It has been found that the oil of C. cyminum is
very effective against all tested bacteria, this oil is
equally or more effective when compared with standard
antibiotics, at a very low concentration [34].
Essential oils extracted from seeds of Cuminum
cyminum was analyzed by gas chromatography (GC)
and GC-mass spectrometry (MS) [26,19] . The main
components of C. cyminum oil exhibited activity on
many species from Gram-positive and Gram-negative
bacterial. Antibacterial testing showed high activity of
the essential C. cyminum oil against Bacillus subtilis
and Staphylococcus epidermidis as well as, the activity
was particularly high against the genera Clavibacter,
Curtobacterium, Rhodococcus, Erwinia, Xanthomonas,
Ralstonia, and Agrobacterium, which are responsible
for plant or cultivated mushroom diseases worldwide.
In general, a lower activity was observed against
bacteria belonging to the genus Pseudomonas. These
results suggest the potential use of the above essential
oils for the control of bacterial diseases
Previously, extract of Cuminum cyminum (cumin)
seeds possessed antifungal activity in an in vitro study
against ten pathogenic fungal isolates from human hair,
nail and skin [23]. The current study focused on
investigating the 1-(2-Ethyl, 6-Heptyl) Phenol (EHP)
compound as an antitumor and antibacterial agent, its
cytotoxicity against a normal cell line was studied as
well.
MATERIALS AND METHODS
1-(2-Ethyl, 6-Heptyl) Phenol (EHP) formula
structure (figure 1) was described and used in this
study for testing its activity as an antitumor and its
cytotoxicity [23]
Fig. 1: EHP compound
I-Antitumor Activity: Six tumor cell lines and one
normal cell line (normal fibroblast) were provided from
National Cancer Institute (NCI), Cairo University and
listed in table (1).
Table 1: Type of tumor cell lines
Human Tumor Cell Line
HEPG2
HELA
HCT116
CACO
MCF7
HEP2
BHK
1882
Type of Tumor Cell
Liver carcinoma cell line
Cervical carcinoma cell line
Colon carcinoma cell line
Colon carcinoma cell line
Breast carcinoma cell line
Larynx carcinoma cell line
Normal fibroblast cell line
J. Appl. Sci. Res., 5(11): 1881-1888, 2009
Measurement of Potential Antitumor Activity and
Cytotoxicity by SRB Assay: Potential antitumor
activity and cytotoxicity of EHP compound was tested
using SRB technique[36].
Tumor cells were plated in 96 – multi-well plate
(104 cells/well) for 24 hrs before treatment with EHP
to allow attachment of cell to the wall of the plate.
Then, different concentrations of the EHP compound at
(0, 1, 2.5, 5 and 10 µg/ml) were added to the cell
monolayer triplicate wells after prepared for each
individual dose. Monolayer cells were incubated with
the compound for 48 hrs at 37 °C and in atomaosphere
of 5 % CO2.
After 48 hrs. cells were fixed, washed and stained
with Sulfo – Rhodamine – B stain (SRB). Excess of
stain was washed with acetic acid and attached stain
was recovered with Tris EDTA buffer. The color
intensity was measured in an ELISA reader. The
relation betwee n surviving fraction and drug
concentration is plotted to get the survival curve of
each tumor cell line.
II- Antibacterial Activity:
1.Culture Medium: Nutrient agar medium was used
for bacterial growth [Beef Extract, 3.0g; Bacteriological
Peptone, 5.0g; Agar, 20.0g, the pH was adjusted at 6.2
± 0.2 at 25 (±2)°C. The medium was prepared by
dissolving the solid ingredients in 1 liter of cold
distilled water and then heating to 60-70 °C with
stirring. Medium was sterilized by autoclaving at 121°C
(1.5 atm.) for 15-20 minutes [3].
Test Organisms: Eight clinical bacterial strains
employed for this investigation including four Grampositive (Stap hylococc us aureus; Streptoc occu s
pn eu mo ni ae ; Bacillu s su bt il is an d Ba ci ll us
thuringiensis) and four Gram-negative bacteria
(Escher ich ia col i; Sal mon ell a typhi; Serra tia
marcescens and Pseudomonas aeruginosa). All strains
were kindly provided from culture collection of the
Regional Center for Mycology and Biotechnology
(RCMB), AL–Azhar University.
Antimicrobial Assays: Diffusion agar technique,
antibacterial potentiality against several species was
expressed as the measurement of diameter of their
inhibition zone. Hole-plate diffusion method was used;
1 cm diameter of holes were made using sterile cork
borer in Nutrient agar sterile plates (10x 10 cm), which
had previously been seeded with tested bacterial
isolates. Holes were filled with 100 µL of different
concentration of EHP compound (1, 2.5, 5, and 10
µg/ml). Control holes were filled with benzene solvent.
Plates were left in a cooled incubator at 4 (±2)°C for
one hour and then incubated at 37 (±2) °C. Inhibition
zones developed was measured after 24 hours of
incubation time [26] Amoxicilli was used as a standard
antibacterial agent.
RESULTS AND DISCUSSION
Biologically active compounds from plant sources
have had a dramatic impact in medicine including:
quinine for treatment of malaria, reserpine for
controlling hypertension, cocaine as a muscle relaxant
and vincristine for treating children with leukemia. The
main problem in cancer therapy is represented in the
sever toxicity and the side effects of the current drugs
and radiotherapy. Hence, it became crucial to search
for antitumor agents from the natural and safe plant
resources. It should also be noted that regarding
antibiotics there should always be sources for new safe
ones to overcome the problems of microbial resistance,
side effects and long-term therapy.
Cumin has been used as a folk medicinal plant –
schools of natural medicine tout its stimulant and
antimicrobial properties. Cumin does have proven
carminative and antispasmodic qualities, and is
therefore said to be useful in the treatment of diarrhea,
stomachache, and menstrual cramps. Eastern medicine
also recommends it for pregnant women to settle
morning sickness and increase breast milk production
[37]
.
Being of diverse benefits, attention has been
attracted to cumin in the current study to investigate
the effectiveness of EHP, a cumin seed extract, against
six tumor cell lines as well as against four Gramnegative and four Gram-positive bacterial pathogens.
The first step in the current study was to investigate
the cytotoxic effects of EHP against normal cells where
BHK was the chosen normal cell line. Results revealed
negligible cytotoxic effects for EHP; increasing the
concentration up to 10 µg/ml resulted in the survival of
85.6% of the normal cells (Table 2 and Figure 2).
Evaluation of the antitumor effect of the EHP
compound of Cuminum cyminum seeds using six cell
lines (HEPG2; HELA; HCT116; CACO2; MCF7;
HEP2) was performed. It is worth noting that according
to the available research, no previous work has been
reported on the antitumor activity of the Egyptian seeds
of Cuminum cyminum.
Regarding its antitumor activity, the SRB assay of
benzene extract of Cuminum cyminum treated cells
showed that 1 µg/ml concentration of the EHP
compound had activity against MCF7 cell line followed
by HEPG2 where 73% and 84% of surviving cell lines
was obtained respectively. While, the rest of the tumor
cell lines exhibited great resistance to the cumin
compound at the same concentration. An EHP
concentration of 2.5 µg/ml exhibited activity only
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J. Appl. Sci. Res., 5(11): 1881-1888, 2009
against the MCF7 cell line (only 45% of the cells
survived).While, a negligible effect was observed
regarding the other ones (Table 2 and Figure 2).
Table (2) and figure (2) reveal that a concentration
of 5µg/ml of the EHP cumin compound introduced
good activities toward two of the investigated tumor
cell lines (MCF7 and HEP2); 38% of MCF7 tumor
cells survived and 58% of HEP2. However mild
activities were detected in case of CACO2 (72% of
cells survived) and HEPG2 (77% of cells survived).
Negligible effects were observed for the rest of cell
lines.
In case of 10 µg/ml EHP concentration, MCF7 cell
line was the most affected (33% of cells was survived)
followed by HEPG2 and then HEP2 cell lines where
41% and 56% of the cells survived respectively. The
effect of EHP on the rest of the investigated cell lines
was mild to negligible (72% of CACO2 cells survived
while for HCT116 and HELA cells lines only 24% and
15% of cells was affected, respectively).
Antitumor activity has been reported from other
plant extracts; acetone and ethyl acetate extracts of
Stevia rebaudiana showed cytotoxic activity on HEP2
cells. Acetone extracts showed the highest cytotoxic
activity followed by ethyl acetate and chloroform
extracts [33]. Also, the ethanol extract from Annona sp.
exhibited activity on MDBK and HEP2 cells where
CC50 values were 34.5 and 55 mg/ml at 24 hr
respectively, Furthermore the value for the same extract
on HEP2 cells at 72 hr was 49.6x10 -3 mg/ml. The
cytotoxic activity of the Annona, muricata, Annona.
cherimolia and Ruagea membranacea species has been
extensively proven [4].
Moreover, Jacaranda copaia and Tapirira
guianensis extracts had cytotoxic effect on eight human
tumor cell lines (representing lung, breast, colon, and
pancreas) [30]. While, great reduction of tumor cell lines
was exhibited in mice when injected with 10 µg/ml of
root extract of Gossampinus malabaria [42].
The compound of interest was characterized by
possessing aromatic ring and terminal hydroxyl group
and this type of compounds was believed to possess
activity against cancer. This conclusion was confirmed
by many reports that these compounds interact with
DNA by intercalation and act as topoisomerase
inhibitors [2,20].
Further in vivo studies will be conducted using
experimental mice suffering carcinoma they will be fed
cumin to investigate the effect of cumin ingestion on
cancer.
The antibacterial activity of EHP against four
Gram-negative and four Gram-positive bacterial
pathogens is tabulated in table (3) and illustrated in
figure (3). The most sensitive bacterial strain was
Staphylococcus aureus where an inhibition zone of 0.8,
1.6, 2.8, and 3.5 cm was obtained when applying EHP
concentrations of 1, 2.5, 5.0, and 1.0 µg/ml
respectively. Staphylococcus aureus was followed by
Bacillus subtilis , Streptococcus pneumoniae and
Bacillus thuringiensis.
In the present study, the activity of EHP against
Gram-negative bacteria was less than that against
Gram-positive ones where the largest zone of inhibition
in Gram-negative bacteria was observed against E. coli;
1.5 and 1.8 cm inhibition diameters were reported at 5
and 10 µg/ml EHP concentration respectively. It should
also be noted that E. coli was the only Gram-negative
bacteria affected by the lower EHP concentration of 2.5
µg/ml (0.6 cm diameter of inhibition zone). It has been
reported that alcoholic and aqueous extracts of eight
herbs were prepared after proper drying and grinding.
The in vitro activities of the extracts in two
concentrations were evaluated against E. coli. Among
the all spices tested in vitro, the highest degree of antiE.coli activity was exhibited by alcoholic and aqueous
extracts of Cuminum cyminum at a concentration of
500 mg/ml[25].
In the current study, E. coli was followed by
Salmonella typhi where 0.6 and 1.2 inhibition zone
diameters were recorded at 5 and 10 µg/ml EHP
concentration respectively. For Serratia marcescens, an
inhibition zone diameter of 1cm was detected at both
5 and 10 µg/ml. Pseudomonas aeruginosa was the least
affected; 0.5 mm inhibitory zone was recorded at high
EHP concentrations of 5 and 10 µg/ml.
The current antibacterial cumin results agree with
other studies reporting more effect against Grampositive than Gram-negative bacteria. For Gramnegative bacteria, the antibacterial activity of cumin by
disk diffusion was studied [14,19,35] where Cumin essential
oil showed complete zone of inhibition against
Staphylococcus aureus, Staphylococcus epidermidis,
Bacillus cereus and Bacillus subtilis at 2 and 6 µL
levels. Regarding Gram-negative bacteria, earlier
studies reported reduction in bacterial growth due to
the incorporation of cumin seed ingredients in the
medium with Cuminum cyminum essential oil showing
little effect against E. coli, P. aeruginosa and
Salmonella sp.[38]. Alternatively, Rajan and Nagaraj [31]
studied the antimicrobial activity of Cuminum cyminum
against E. coli and S. aureus, its seeds had the least
effect against Staphylococcus aureus, while E. coli was
susceptible to all the spices extracts.
Conclusively, the uses of natural products as drugs
are crucial instead of the synthetic compounds which
possess sever toxicity and side effects besides being
very expensive. Hence, the use of natural products
represents a valuable solution. This study conveys the
use of cumin as a helper in the therapy or the control
of the cancer of liver, breast and larynx directing the
attention to a cheap treasure called cumin. Moreover,
it can be used in the treatment of various pathogenic
bacterial diseases. The antiviral activity of EHP will be
investigated in a further study.
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J. Appl. Sci. Res., 5(11): 1881-1888, 2009
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J. Appl. Sci. Res., 5(11): 1881-1888, 2009
Fig. 2: Cytotoxicity activity of 1-(2-Ethyl, 6-Heptyl) Phenol extracted from the seeds of Cuminum cyminum
Fig. 3: Antibacterial activity of 1-(2-Ethyl, 6-Heptyl) Phenol extracted from the seeds of cumin
Table 2: antitumor activity and cytotoxicity of 1-(2- ethyl, 6- heptyl) phenol compound
Conc. (µg/ml)
Tumor cell line (survival cell)
Normal cell line
MCF7
HEPG2
HEP2
CACO2
HCT116
HELA
BHK
------------------------------------------------------------------------------------------------------------------------------------------------------0
1
1
1
1
1
1
1
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------1
0.73
0.846
0.965
0.96715
0.9826
0.99599
0.98702
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2.5
0.45
0.83
0.8385
0.8958
0.9319
0.93654
0.95053
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------5
0.38
0.774
0.5854
0.72485
0.9
0.8557
0.90542
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------10
0.33
0.416
0.5657
0.72011
0.768
0.85982
0.85644
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------IC50 (µg)
2.21
8.93
IC 50: The dose of compound which reduced 50% of survival tumor cell lines
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J. Appl. Sci. Res., 5(11): 1881-1888, 2009
Table 3: In vitro antibacterial susceptibility of pathogenic bacterial strains to EHP.
Bacterial strains
Concentration (µg/ml)
1
2.5
5
10
--------------------------------------------------------------------------------------------------------------------------------Staphylococcus aureus
0.8
1.6
2.8
3.5
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Streptococcus pneumoniae
0.5
1.7
2
2.7
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Bacillus subtilis
1.2
1.9
2.3
3
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Bacillus thuringiensis
0.5
1.2
1.7
2.3
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Salmonella typhi
0
0
0.6
1.2
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Escherichia coli
0
0.6
1.5
1.8
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Pseudomonas aeruginosa
0
0
0.5
0.5
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Serratia marcescens
0
0
1
1
Data are expressed as mean diameter of inhibition zone (cm)
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