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Asian Pacic Journal of Cancer Prevention, Vol 20 3771
DOI:10.31557/APJCP.2019.20.12.3771
Fenugreek Seeds Extract
Asian Pac J Cancer Prev, 20 (12), 3771-3776
Introduction
Cancer is a serious health issue that concerns the entire
world. Its treatment protocols depend on chemotherapy,
radiotherapy and surgical intervention (Huang et al.,
2014). Breast cancer stands out as one of the most
threatening diseases to females around the world. In
2012, for example, 1.57 million cases were ofcially
registered. Today, breast cancer is considered the second
life threatening disease for women in the united states
(Farshori et al., 2013; World Cancer Report, 2019).
Chemotherapy is one of the most regularly adopted
protocol in breast cancer treatment (Siegel et al., 2014).
Treatment based on this protocol is usually accompanied
with unfriendly symptoms, extending from sickness to
bone marrow suppression to development of multidrug
resistance (MDR) (Graidist et al., 2015; American Cancer
Society, 2019). Therefore, discovering characteristic
mixes from plants may provide an elective malignant
growth treatment (Goldman et al., 2019).
In Iraq, breast cancer is becoming a major risk to
women lives after cardiovascular diseases and leading to
23% of the overall death toll. Cancer has been threatening
Iraqi women increasingly since 1986 (Alwan, 2010; Iraqi
Cancer Board, 2015). In addition, the recurrence of this
illness was observed in moderately aged women (45 – 50
years old) although the pinnacle age of exposure was
accounted for to be in the range 50 – 55 years (Alwan,
2014; World Health Organization, 2017). Liver cancer is
considered the third reason for disease mortality around
Abstract
This work is about the utilization of fenugreek seed as an antibacterial and anticancer agents. The antibacterial
activity of fenugreek seed extract on six pathological bacteria strains were specied through conventional biochemical
tests using the Vitek2 automated system and diffusion agar method. The anticancer activities of fenugreek seed extract,
on MCF-7 breast cancer cells, liver cancer HCAM cells and the non-cancerous Vero cell lines, were investigated using
colorimetric MTT assay. Results showed that the highest activity of the extract of the seed was found on Staphylococcus
aureus and Pseudomonas aeruginosa (22 mm and 17 mm diameter of inhibition zones respectively). The seed extract
showed proliferative inhibition on MCF-7 cell line at a concentration of 400 µg/ml and 72 h of the incubation period. This
was accompanied by insignicant apoptosis or necrosis. The seed extract showed no anticancer effect on liver and Vero
cell lines. This work emphasizes that fenugreek seed extract is a potential source of antibacterial and anticancer agents.
Keywords: Fenugreek seed- pathogenic bacteria- breast cancer- antibacterial effect
RESEARCH ARTICLE
Antibacterial and Anticancer Activities of Fenugreek Seed
Extract
Lina A Naser Al-Timimi*
the world (Farooq et al., 2013). According to recent study
by WHO (2017) death with liver cancer in Iraq escalated
to 693 representing 0.39% of the total death toll. It is
worth mentioning that death toll in Iraq amounts to 4.25
per 100,000 placing Iraq in the 122nd positing worldwide
(World Health Organization, 2017).
The harmful nature of pathogenic bacteria may be
controlled using antibiotics. Unfortunately, the misuse
of antibiotics is leading to the speedy development of
ineffectiveness of antibiotic strains to bacteria creating an
alarming clinical status in the remedy of infections. Today,
many infections occur because microorganisms defeat
conventional therapy. In fact, bacteria have the genetic
ability to develop resistance to many antibiotics (Fair
and Tor, 2014; Landecker, 2015; Li and Webster, 2018).
Recently, there has been a surge interest in the
antibacterial properties of the plants extract. It is
progressively acceptable that these phytochemicals will
be prescribed by the doctors as antibacterial medications
(Bhalodia et al., 2011). Many investigations were carried
out on the therapeutic applications of various plants
species on different diseases such as fungal, viral, and
bacterial contagion. Nowadays, approximately 33% of
the world population depend on conventional/ therapeutic
plants and their extract to meet the essential needs. At the
same time, the world health organization (WHO) reported
that 80% of individuals worldwide are accustomed to use
manmade medications (Kumar and Reddy, 2012).
Recently, the study of the therapeutic effects of
plants has increased due to their inclusive medicinal and
Editorial Process: Submission:09/01/2019 Acceptance:11/08/2019
Department of Biology, College of Science, University of Basrah, Iraq. *For Correspondence: lina1977_abbas@yahoo.com
Lina A Naser Al-Timimi
Asian Pacic Journal of Cancer Prevention, Vol 20
3772
economical properties and the successful utilization of
some of these plants in treating human diseases. Some
investigations have led to discovery of restorative properties
of fenugreek seed. Fenugreek (Trigonella Foenum-
gracium), also known in Arab countries as “Helba”, is a
plant from the family of Leguminosae. It grows annually
and is being planted in the Mediterranean countries and
Asia. Fenugreek dried seed are known for their valuable
antibacterial, anticancer and anti-inammatory properties
in India, Egypt and some European countries. These
seed are also known to work as anti-oxidants having
reviving properties. Fenugreek is rich with a wide variety
of metabolites such as tannins, alkaloids, avonoids,
terpenoids and glycosides which are known to have
antimicrobial properties (Khorshidian et al., 2016).
In the present work it is aimed to investigate the
antibacterial activity of fenugreek seed extract in general
and the effect of ethanol fenugreek seed extract on breast
cancer. This work is motivated with the fact that only
few studies have been carried out on anticancer materials
especially on MCF-7 cell line.
Materials and Methods
Fenugreek seed were procured from a herbal shop
in Al-Ashar local market in Basrah city. The seed were
initially washed, dried and then grinded using home
mixer. The grinded seed were then mixed with ethanol and
sterilized distilled water. For the biological purposes, 50 g
of fenugreek seed powder was added to 500 ml of absolute
ethanol. Another 50 g of the grinded seed were added to
500 ml of distilled water. The two mixtures were kept in
a rotary shaker for 24 h and ltered with Whatman No.1
lter paper. A micro lter of 0.45 µm was then used in a
rotary evaporator at 50°C for extra ltration. The extracted
materials were stored at 4°C.
Methanol: Dimethyl Sulfoxide (DMSO) in 1:1 V/V
volume ratio was used to prepare different concentrations
of 125, 250, 500 and 1,000 µg/ml crude plant seed ethanolic
and aqueous extract. The nal DMSO concentration did
not exceed 0.1%.
The antibacterial activity of the extract were studied
on six clinically isolated bacteria strains, these are
Staphylococcus aureus, Pseudomonas aeruginosa,
Proteus mirabilis, Salmonella typhi, Escherichia coli,
and Vibrio parahaemolyticus. These strains were obtained
from different sources (stool, wound infections, urine,
skin lesions) of patients admitted to ‘Al-Shafaa hospital’
in Basrah. All the collected samples were processed upon
receipt in laboratory and cultured in appropriate media
(Manandhar et al., 2019).
In order to classify the isolated bacteria to
Gram-negative and Gram-positive groups, staining,
morphological identication of colonies under optical
microscope and conventional biochemical tests using
Vitek2 automated system, were performed . A bacterial
suspension of each isolate was prepared and equalized
to 0.5 McFarland standard and the solution was spread
on the entire surface of Muller Hinton agar using a
sterilized cotton bud (Barrow and Feltham, 2018). After
drying, a 9 mm diameter pore was made in each plate
by using cork-borer with duplicate and control plates.
Approximately, 0.1 ml of each of the plant extract, with
the concentrations mentioned above, were injected to ll
the wells. All inoculated Petri dishes were incubated at
37°C overnight. The inhibition zone was then measured
from the diameter of the clearing zone in millimeters. All
the dishes were examined for the Minimum Inhibitory
Concentration (MIC) that inhibits the bacterial growth
after incubation (Bansode and Chavan, 2013).
All cell lines that used in this study were obtained from
the Iraqi Center of Cancer and Medical Genetics Research
(ICCMGR), Al-Mustansiryia University, including human
breast cancer (MCF-7) and liver cancer (HCAM) in
addition to the control serving non-cancerous Vero (green
African monkey kidney) cell lines.
Different concentrations of ethanol fenugreek seed
extract were determined by using (MTT) tetrazolium
reduction assay (Aslantürk, 2017). For further biological
inspections, the fenugreek seed extract was re-suspended
in DMSO at 10.00 µg/ml stock solution. The concentration
of the fenugreek seed extract, used to treat the cells, was
in the range 400-1,000 µg/ ml. 20 ml of MTT solution was
added to each well after 24, 48 and 72 hours of treatment.
The Acridine Orange/Propidium Iodide (AO/PI)
pigment was used for the phenotypically recognition of
apoptosis, the cells passing were incited by concentrate.
Morphological consideration includes nuclear
fragmentation, membrane blabbing, cytosolic buildup.
In addition, two hundred cells were tallied to each slide.
The cells of untreated MCF-7 were considered a control.
The inhibition rate (IR) of cell growth was calculated
by counting the percentage of proliferation rate (PR).
PR = B/A where A refers to optical thickness of the
untreated wells and B refers to the optical thickness of the
treated wells. IR=100-PR (Gao et al., 2005).
In order to rate the apoptosis, the existence of inter-
nucleosome DNA cleavage was visualized against a DNA
ladder in agarose gel electrophoresis using biochemical
markers. Breast cancer cells line MCF-7 and HCAM
were cultured in two 25 cm cell culture asks for 24 h
before being treated with fenugreek extract. All the cells
were processed with fenugreek extract overnight. Then,
MCF-7 and HCAM were collected, washed with PBS and
ltered using a DNA ltration, DNeasy and Tissue kits.
By using electrophoresis, the DNA was resolved at 80–100
V through 1.8 % of agarose gel. The gel was recolored
using ethidium bromide and imaged through ultra-violate
trans illuminator.
Results
Antibacterial activity
The antibacterial activity of fenugreek seed extract,
evaluated in terms of inhibition zone, was tested on
six pathogenic bacteria. The MIC results are shown in
Figures 1, 2 and Table 1. Results showed that ethanol
extract has prominent effect on Gram positive S. aureus
and Gram negative P. aeruginosa. At the same time
this extract showed moderate activity on the remaining
types of the bacteria. It is worth noting that none of the
prepared concentrations of ethanol extract showed positive
Asian Pacic Journal of Cancer Prevention, Vol 20 3773
DOI:10.31557/APJCP.2019.20.12.3771
Fenugreek Seeds Extract
activities on the bacteria except on Staphylococcus aureus
and E. coli. The concentrations used in MIC for ethanol
extract were in the range 50-500 µg/ml. Most of the
bacteria were inhibited at MIC of 50 µg/ml. These results
are signicantly important due to the little amount of the
fenugreek seed extract needed to inhibit the growth of the
bacteria. On the other hand, none of the MIC values were
exhibited by Vibrio parahaemolyticus in all the extract.
Cytotoxicity assessment
A cytotoxicity assessment of ethanol fenugreek seed
extract using MTT technique was carried out. In this
part of the work, the anticancer activity of the fenugreek
seed extract was measured on MCF-7 and HCAM; the
normal Vero cell line was used as a control. The tested
concentrations for each cell line were 400, 600, 800
and 1,000 µg/ml. The incubation period was 72 h. The
MTT assay revealed that ethanol fenugreek extract had
an inhibitory action on MCF-7 cell line which amounts
to more than half of the inhibition of the MCF-7 cell.
Proliferation stopped when cell line concentration
was equal to 400 µg/ml after 72 h of incubation. On
the other hand, no cytotoxic effect of the extract was
observed on liver cancer or Vero cell lines regardless of
activity on Vibrio parahaemolyticus and E. coli. On the
other hand, the aqueous extract showed low to moderate
Figure 1. Antimicrobial Activity of Different Concentrations of 125, 250, 500 And 1,000 µG/Ml Ethanol and Aqueous
Fenugreek Seed Extract on Bacterial after Incubation For 24h.
Figure 2. Antimicrobial Activity of Ethanol Fenugreek
Seed Extract on Staphylococcus aureus and Pseudomonas
aeruginosa in Muller-Hinton Agar
Figure 3. Vero cell line. A, Control under light
microscope (magnied at 10x); B, After treated with
different concentrations of 400, 600, 800 and 1,000 µg/
ml ethanol fenugreek seed extract after 72 h under light
microscope (magnied at 10x); C, After treated with
different concentrations of 400, 600, 800 and 1,000 µg/
ml ethanol fenugreek seed extract after 72 h pigmented
with the AO/PI. Notice there is no effect on the cell after
treated. Viewed under 690 wavelength of the uorescent
microscope light microscope (magnied at 10x).
Figure 4. A, Genomic DNA isolated from MCF-7 cell
line after being treated with different concentrations of
400, 600,800 and 1000 µg/ml ethanol fenugreek seed
extract after 72 h; B, The ladder used its range from
100-2,000 (bp).
Lina A Naser Al-Timimi
Asian Pacic Journal of Cancer Prevention, Vol 20
3774
the concentration in action. It is worth mentioning that
apoptosis was determined by using AO/PI recoloring and
DNA fragmentation test.
In this study, breast cancer cells treated with fenugreek
seed extract showed that a perfect DNA ladder style was
not clear in a time and concentration-dependent manner.
This may indicate that the cells were inhibited and
no apoptosis or necrosis occurred. Figure 4 shows
genomic DNA isolated from MCF-7 cell line after being
treated with fenugreek seed extract. After staining using
ethidium bromide in agarose gel, no evidence of DNA
fragmentation was observed. With respect to the AO/PI
coloring, the present study showed no death of MCF-7
cell line after being treated with ethanol fenugreek seed
extract. This emphasizes that MCF-7 cells inhibition
occurs without any apoptosis or necrosis (see Figure 5).
Discussion
There is an urgent need to nd new antibacterial
medications with novel characteristics to tackle the
rise of new infectious diseases and due to the misuse of
conventional antibiotics. There is, therefore, interesting
progress in extracting certain chemicals from different
plants. Plants are known to provide wide spectrum of
chemical compounds having various biological activities.
Antibiotics of medicinal plants have contributed to the
elimination of many diseases caused by pathogenic
bacteria. The importance of these plants can be
demonstrated by the fact that these may be designed
as target-oriented materials unlike chemical antibiotics
playing the role of plant versus drug-resistant bacterial
diseases (Amenu, 2014).
Fenugreek is a herbal plant. Its fresh seed, twigs, roots,
and leaves can be used directly and after being dried as
avoring, supplements and spices. Its medical advantages
have been reported in many studies. Infections by
Staphylococcus aureus and Pseudomonas aeruginosa has
become a serious issue in sick and immune-compromised
patients. For this reason, the antibacterial effect of
fenugreek ethanol extract on these strains receives
signicant interest. The serious issue prompting high
mortality lies in the presence of drug-resistant strains
(Amenu, 2014; Bassetti et al., 2018). In the present
investigation both Gram-positive and Gram-negative
bacterial strains exhibited similar response after exposure
to fenugreek seed extract. This agrees with the work of
Bassetti et al. (Alwan et al., 2017). On the other hand,
ethanol extract exhibited higher activity on most of the
bacterial strains except E. coli compared with the aqueous
extract. This is agreeing with (Alwan et al., 2017) but
disagree with (Salah et al., 2010; Sharma et al., 2017)
where they recorded both extraction of ethanol and
aqueous didn’t exhibit any effect on bacterial species.
Breast cancer is one of the most life-threatening
disorder being suffered by Iraqi young women. In
1986, breast cancer was considered the top most serious
malignancy threatening the Iraqi population (International
Agency for Research on Cancer, 2013). In the present
study, fenugreek seed extract showed cytotoxic activity
which inhibits the MCF-7 cell development. Such
effect was not observed on liver cancer cell lines. This
emphasizes that the effect of fenugreek seed extract is cell
type-dependent. These results comply with other results
reported in the literature (Amin et al., 2005; Kyung et
al., 2006). These antithetic results may be attributed to
the specic activity of fenugreek on transformed and
untransformed cells. Nevertheless, our results emphasize
that fenugreek plant can be used in breast cancer
treatment. Kyung et al., (2011) have reported similar
ndings in their work. It is worth mentioning that breast
cancer MCF-7 cells can resist chemotherapy as they
contain the CASP-3 gene which prompts an acquired
insufciency of caspase-3. Caspase-3 usually functions
via death indexes and cleaves a variety of serious cellular
proteins. DNA cleavage and a portion of the particular
morphological features are in charge of apoptotic cells
such as budding and shrinkage (Shapiro et al., 2001;
Sharief and Gani, 2004).
The impact of cytotoxic compounds demonstrates
Figure 5. Human breast cancer cells (MCF7). A
and B, Control viewed under light microscope the
magnication force for A is 10x and 40x for B; C and D,
MCF-7 cells treated with ethanol fenugreek seed extract
at a concentration of 400 µg/ml and 72 h pigmented
with the AO/PI, notice their color green only. Shown by
the 690 wavelength of the uorescent microscope, the
magnication force is 10x for C and 40x for D.
Bacteria MIC of ethanol extract (µg/ml)
500 250 100 50
Staphylococcus aureus + - + -
Pseudomonas aeruginosa + - + -
Salmonella typhi + - - -
Proteus mirabilis + - - -
Escherichia coli + - - -
Vibrio parahaemolyticus - - - -
Table 1. The Minimum Inhibitory Concentration Rate
(MIC) Of Different Concentrations Of 125, 250, 500
And 1,000 µG/Ml Of Fenugreek Seed Ethanolic Extract
Against Bacterial Test Organisms
Asian Pacic Journal of Cancer Prevention, Vol 20 3775
DOI:10.31557/APJCP.2019.20.12.3771
Fenugreek Seeds Extract
the dissemination of cell populations during the cells life
span. Due to the signicance of cell life span in cancer
progression, few researchers have attempted to describe
the cell cycle capture limit to the plant extract in addition
to the importance of isolated compounds (Bassermann et
al., 2014; Ruijtenberg and Heuvel, 2016). In the present
work, no DNA fragmentation occurs as reported by other
researchers (Shapiro et al., 2001; Sharief and Gani, 2004).
When MCF-7 cell lines experience apoptosis with no
evidence of DNA fragmentation this might be attributed to
the absence of caspase-3. In the present work, normal Vero
cells were used for the differentiation between normal and
cancerous cell patterns. Furthermore, ethanol fenugreek
seed extract showed no cytotoxic activity.
In conclusion, fenugreek seed are potential sources
to new antibacterial compounds as emphasized from the
antibacterial activity of their aqueous and ethanol extract
on many pathogenic bacterial strains. Furthermore,
fenugreek seed extract has shown anticancer activity
through inhibition of more than half of the human breast
cancer MCF-7 cell lines. These seed extract inhibited
cancer cell’s proliferation with no apoptosis or necrosis
observed. Further studies are still required to understand
the mechanism (Shapiro et al., 2001; Sharief and Gani,
2004). of cells inhibition. This positive results on MCF-7
cell lines suggest that fenugreek seed extract is a potential
anticancer agent that should be exploited widely for breast
cancer treatment.
Acknowledgments
I would like to thank Prof. Dr. Ali Abdellatif head of
the tissue culture Laboratory, Department of Biology,
College of Education, University of Basrah for his
guidance and help for testing the cancer cell lines. All the
cell lines that used in the present work were obtained from
the Iraqi Center of Cancer and Medical Genetics Research
(ICCMGR), Al-Mustansiryia University, Baghdad. Also,
I would like to thank Assist prof Dr. Eman Mohammed
plant systematics and anatomy, Department of Biology,
College of science, University of Basrah for her help in
identied the seed.
Conflict of interest
All contributing authors declare no conicts of interest.
Source of Funding
Self-funding.
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