International Journal of Herbal Medicine 2017; 5(5): 110-113

Tiruchirappalli, Tamil Nadu,

India

E-ISSN: 2321-2187
P-ISSN: 2394-0514
IJHM 2017; 5(5): 110 -113
Received: 15-07-2017
Accepted: 16-08-2017

S Deborah
PG & Research Department
of
Biotechnology, National
College
(Autonomous & CPE),
Tiruchirappalli, Tamil Nadu,
India

SP Anand
Assistant Professor, PG &
Research Department of
Botany,
National College
(Autonomous &
CPE), Tiruchirappalli, Tamil
Nadu, India

G Velmurugan
PG & Research Department
of
Botany, National College
(Autonomous & CPE),
Tiruchirappalli, Tamil Nadu,
India

Correspondence
S Deborah
PG & Research Department
of
Biotechnology, National
College
(Autonomous & CPE),
 Keywords: human breast cancer cell line, eagles minimum essential medium, ic 50, tarenna asiatica, wild
edible fruit

1. Introduction
Plants plays essential role in the folklore medicine from ancient cultures. In addition to the
make use of as food and spices, plants have also been utilized as medicines for over 5000 years
[1]
. It is estimated that 85% of the population in developing countries continues to use
traditional medicines even today [2]. Cancer is one of the major chronic human diseases and
Evaluation of causes large suffering and economic loss of world-wide [3, 4]. Various new strategies are being
developed to control and treat several human cancers [5, 6]. Over 60% of anticancer drugs
In vitro available in the market are of natural product. Natural products are also the leads for
anticancer formulation of many drugs [7]. Therefore, the phytochemicals present in several herbal
products and plants may have the potential to act as preventive or therapeutic agents against
activity of various human cancers [5]. The increased popularity of herbal remedies for cancer therapy
perhaps believes that herbal drugs provide benefit over the allopathy medicines while being
Tarenna less toxic [8, 9]. Since the conventional therapies have devastating and destructive side effects,
asiatica (L.) there is a continuous search of new herbal cures of cancer [10]. Tarenna asiatica fruit is used as
an herbal remedy for various ailments, including eye infection, skin problems and abdominal
fruits pain. The parts of plants are traditionally used to promote suppuration, as anthelmintic and
ethanolic antiulcer agent [11, 12, 19]. In the present study in-vitro anticancer studies were performed
against human breast cancer cell line and Eagles Minimum Essential Medium containing 10%
extract fetal bovine serum (FBS) was used to analyze the cell growth inhibition. The results showed
that the ethanol extract of wild edible fruit Tarenna asiatica (L.) possessed a high amount of
against anticancer activity and the IC50 value also high as 237.08 µg/ml.
human breast
2. Materials and Methods
cancer 2.1. Plant collection and identification
Tarenna asiatica, wild edible mature fruits were collected in the month of April to June at
Kolli hills. The collected fruits specimen was authenticated by Botanical survey of India (BSI),
S Deborah, SP Anand Coimbatore, Tamil Nadu, India.
and G Velmurugan
2.2 Preparation of Extract
Abstract The fresh fruits were dried in shade for about 3 weeks and ground using a mixer to a coarse
Tarenna asiatica (L.) is a powder. The powder of dried solvent using a water bath maintaining at 60-80ºC at ambient
medicinal plant from Kolli conditions to get a crude hydro alcoholic extract devoid of solvents. The extract was prepared
hills, Eastern Ghats, India. by using soxhlet and vacuum evaporator method.
Fruit the Tarenna asiatica is
an edible one and it has been ~ 110 ~
used traditionally for
treatment of a number of
diseases. In the present study
the ethanol extract of the
edible fruit of the plant have
been tested for anticancer
activity. The extract was
prepared by soxhlet separation
and vacuum evaporator
method. The in-vitro
anticancer studies were
performed against human
breast cancer cell line and
Eagles Minimum Essential
Medium containing 10% fetal
bovine serum (FBS) was used
to analyze the cell growth
inhibition. The results showed
that the ethanol extract of wild
edible fruit Tarenna asiatica
(L.) possessed a high amount
of anticancer activity and the
IC50 value also high as 237.08
µg/ml. The plant investigated
possesses remarkable
anticancer activity and hence
isolation of the compound
contributing to the activity
may lead to develop at a novel
and natural phytomedicine for
the disease.
International Journal of Herbal Medicine

2.3 In-vitro evaluation of anticancer activity by MTT assay

2.3.1 Cell line
The human breast cancer cell line (MCF 7) was obtained from
National Centre for Cell Science (NCCS), Pune and grown in 2.4 Statistical analysis
Eagles Minimum Essential Medium containing 10% fetal The absorbance values were denoted as mean ± SEM. The
bovine serum (FBS). The cells were maintained at 37 0C, 5% IC50 is half the maximal inhibitory concentration of the toxic
CO2, 95% air and 100% relative humidity. Maintenance compound which results in the reduction of biological activity
cultures were passage weekly, and the culture medium was by 50%. IC50 was determined using Graph Pad Prism
changed twice a week. software.

2.3.2 Cell Treatment 3. Result and discussion

The monolayer cells were detached with trypsin-ethylene 3.1 In vitro anticancer activity
diamine tetraacetic acid (EDTA) to make single cell The results in cell growth inhibition by the ethanolic extract
suspensions and viable cells were counted using a against human breast cancer cell lines for various concentrations
hemocytometer and diluted with medium containing 5% FBS are shown in table-1 and figure-1. The ethanolic extract at 18.75,
to give final density of 1x10 5 cells/ml. One hundred 37.5, 75, 150 and 300 µg/mL had shown dose dependent
microlitres per well of cell suspension were seeded into 96- inhibition of cells. As the concentration increases, there is an
well plates at plating density of 10,000 cells/well and increase in the cell growth inhibition. 300 µg/mL ethanol extract
incubated to allow for cell attachment at 37ºC, 5% CO 2, 95% of Tarenna asiatica inhibited 57.00% of human breast cancer cell
air and 100% relative humidity. After 24 hours the cells were lines. The estimated IC50 of ethanolic extract of Tarenna asiatica
treated with serial concentrations of the test samples. They against human breast cancer cell lines was 237.08 µg/mL
were initially dissolved in neat Dimethyl Sulfoxide (DMSO) respectively. The results showed that an ethanolic extract of
and an aliquot of the sample solution was diluted to twice the Tarenna asiatica has a high anticancer activity using MTT assay
desired final maximum test concentration with serum free and the other extracts had moderate to weak cytotoxic activity on
medium. Additional four serial dilutions were made to both the cell lines. In the study traditional edible fruit extracts in
provide a total of five sample concentrations. Aliquots of 100 ethanol solvent were tested for cytotoxic activity against MCF7
µl of these different sample dilutions were added to the cell lines and extract showed less significant activity against
appropriate wells already containing 100 µl of medium, other cell lines. Agents capable of inhibiting cell proliferation,
resulting in the required final sample concentrations. inducing apotosis or modulating signal transduction are currently
Following sample addition, the plates were incubated for an used for the treatment of cancer [13, 14, 15]. The use of multiple
additional 48 hour at 37 ºC, 5% CO2, 95% air and 100% chemo preventive agents or agents with multiple targets on
relative humidity. The medium containing without samples cancer cells are considered to be more effective in cancer
were served as control and triplicate was maintained for all treatment [16]. Breast cancer is the most common malignancy
concentrations. among women. MCF-7 cell has become a prominent model
system for the study of breast cancer as it relates to the
2.3.3 MTT assay susceptibility of the cells to apoptosis. Despite the fact that many
3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyltetrazolium bromide tumors initially respond to chemotherapy, breast cancer cells can
(MTT) is a yellow water soluble tetrazolium salt. A subsequently survive and gain resistance to the treatment [2, 17]. In
mitochondrial enzyme in living cells, succinate- this research the ethanolic extract of Tarenna asiatica was tested
dehydrogenase, cleaves the tetrazolium ring, converting the for anticancer activity by MTT assay on cell lines MCF-7. The
MTT to an insoluble purple formazan. Therefore, the amount IC50 value was found to be 237.08 μg/ml by MTT assay against
of formazan produced is directly proportional to the number the extract had a high activity against on MCF-7 cell lines. The
of viable cells. anti-proliferative agent on human breast cancer cells (MCF-7)
After 48 h of incubation, 15 µl of MTT (5mg/ml) in which was due to the presence of lignins and flavonoids [18, 19].
phosphate buffered saline (PBS) was added to each well and The present study shows that the ethanolic extract have
incubated at 37ºC for 4h. The medium with MTT was then significantly increased the percentage of cells with condensed
flicked off and the formed formazan crystals were solubilized nuclei when compared to other solvents.
in 100µl of DMSO and then measured the absorbance at 570
nm using micro plate reader. The percentage cell viability was
then calculated with respect to control as follows

Table 1: MCF-7 cells treated with ethanolic fruits extract of Tarenna asiatica
S. No. Concentration of extract (µg/mL) Absorbance Inhibition of cell growth (%) IC50 value μg/ml
1. 18.75 0.033±0.001633 5.02
2. 37.5 0.083±0.001247 12.69
3. 75 0.186±0.000816 28.32 237.08
4. 150 0.264±0.001247 40.30
5. 300 0.373±0.001247 57.00

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International Journal of Herbal Medicine

Fig 1: Inhibiting the activity of Breast Cancer cell

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Cell Sciences (NCCS), Pune, for providing MCF-7 cancer cell Prakashan, New Delhi, India. 1976; 1:283-284,
line and the Administrative authorities of National College 12. Ramarao N, Henry AN. The Ethnobotany of Eastern Ghats
(Autonomous) for their encouragement and support. in Andhra Pradesh, Botanical Survey of India, Kolkata,
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Lampiran

Apa itu MTT assay?

MTT assay merupakan metode yang jamak digunakan dalam penelitian
mengenai agen antikanker. Metode ini digunakan untuk menguji aktivitas
sitotoksik sampel penelitian pada kultur sel yang digunakan.
Bagaimana prinsip MTT assay?
Prinsip metode ini adalah reaksi redoks yang terjadi di dalam sel. MTT (3-
(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) direduksi menjadi
garam formazan oleh enzim suksinat dehidrogenase yang terdapat di dalam
mitokondria sel hidup. Reaksi dibiarkan terjadi selama 4 jam kemudian
ditambahkan reagen stopper. Reagen stopper tersebut akan melisis membran sel
sehingga garam formazan dapat keluar dari sel, serta melarutkan garam
formazan tersebut. Garam formazan yang terbentuk dikuantifikasi dengan
spektrofotometer dan diukur dalam bentuk absorbansi. Semakin tinggi
absorbansi, semakin banyak sel yang hidup (viabilitas sel tinggi)

Kultur jaringan atau biakan jaringan sering disebut kultur in vitro yakni teknik pemeliharaan jaringan atau
bagian dari individu secara buatan yang dilakukan di luar individu yang bersangkutan. In vitro berasal dari
bahasa latin yang artinya di dalam kaca. Jadi kultur in vitro dapat diartikan sebagai bagian jaringan yang
dibiakkan di dalam tabung inkubasi atau cawan petri dari kaca atau material tembus pandang lainnya. Secara
teori teknik kultur jaringan dapat dilakukan untuk semua jaringan, baik dari tumbuhan, hewan, bahkan juga
manusia karena berdasarkan teori Totipotensi sel (Total Genetic Potensial), bahwa setiap sel memiliki potensi
genetic seperti zigot yaitu mampu memperbanyak diri dan berediferensiasi menjadi tanaman lengkap.

Kultur jaringan atau budidaya in vitro adalah suatu metode untuk mengisolasi bagian dari tanaman seperti
protoplasma, sel, jaringan atau organ serta menumbuhkannya pada media nutrisi yang mengandung zat pengatur
tumbuh tanaman di dalam kondisi yang steril, sehingga bagian – bagian tersebut bias memperbanyak diri dan
beregenerasi menjadi tanaman lengkap / sempurna. Prinsip utama dari teknik kultur jaringan adalah perbanyakan
tanaman dengan menggunakan bagian vegetatif tanaman dengan menggunakan media buatan yang dilakukan di
tempat steril.

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