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Tumor inhibition and Cytotoxicity assay by aqueous extract of onion (Allium

cepa) & Garlic (Allium sativum): an in-vitro analysis

Article · January 2011

DOI: 10.5138/ijpm.2010.0975.0185.02013

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 International Journal of Phytomedicine 2 (2010) 80-84
http://www.arjournals.org/ijop.html

Research paper
ISSN: 0975-0185

Tumor inhibition and Cytotoxicity assay by aqueous extract of

onion (Allium cepa) & Garlic (Allium sativum): an in-vitro analysis
Shilpa Shrivastava1, N.Ganesh1*

*Corresponding author: Abstract

Dr.N.Ganesh
1
In-charge, Deptt. Of
Research, Jawaharlal Nehru
Cancer Hospital & Research
Centre, Idgah Hills, Bhopal,
INDIA
E-mail:
nganesh_research2@yahoo.c
o.in
shilpa_biotech44@yahoo.co
m vitro culture
The present study was aimed to investigate the effects of aqueous extract of
four different varieties of Allium cepa (Onion) & Allium sativum (Garlic) on
B16F10 Melanoma cell population by ex-vivo. For cytotoxicity assay, cellular
micronucleus, apoptotic assay and cell viability assay were followed. Our
results showed significant activity of garlic and onion as cytoprotective
agents of normal cells and cytotoxicity agents for tumor cells. A significant
decrease in Melanoma B16F10 cell population by crude extract was
observed. Therefore, Allium cepa & Allium sativum might be a candidate for
naturally healing the tumor.
Keywords: Allium cepa; Allium sativum; B16F10 Melanoma; cell lines; in-

Introduction
Indian dietary habits, additive spices and natural
flavors are an ancient asset for Asian subcontinents and
world through1. However, many a times it was noticed
that because of the presence of sulphur compounds2
which gives a pungent aroma, people sometimes dislike
the flavors and taste. During social gathering due to the
pungent odour, the bad breath may over look or over
neglect any individual and this is the reason people
now reduce the intake of onion and garlic as a major
ingredients or additive flavors to their food and diet.
Literatures have shown very strong evidence that
consumption of fruits and vegetables can protect
against a wide variety of cancers. Most fruits and
vegetables are relatively low in calories, have a low
glycemic index and are rich sources of micronutrients,
dietary fiber and non-nutrient substances called
phytochemicals. These phytochemicals are thought to
contribute for their significant protective effect upon
some of the most important diet-related diseases such
as cardiovascular disease, cancer and many more3.
Recent studies also attempted to revealed that the
consumption of vegetables form the allium’s family
such as onion, garlic and leeks will inhibits stomach,
colorectal, and prostate cancers 4, 5. Garlic & Onion
have also shown to possess anti bacterial, antiallergic,
antioxidant and anti-inflammatory property6-8.
The purpose of this study was to evaluate ex-vivo the
tumor inhibition or antitumor property of aqueous
extract of garlic (A. sativum) and onion (A. cepa) by a
B16F10 melanoma cells.
Materials & Method
Preparation of Extract-
Four different varieties of onion (Allium cepa) namely
Nasik Onion, Green Onion, White Onion & South
Indian Onion and four different varieties of Garlic

doi:10.5138/ijpm.2010.0975.0185.02013
©arjournals.org, All rights reserved.
 Shrivastava et al. International Journal of Phytomedicine 2 (2010) 80-84
(Allium sativum) namely Nasik Garlic, Green Garlic,
Chinese Garlic and South Indian Garlic were collected.
The aqueous extract was prepared by using ultra pure
water as solvent. These were grinded and filtered
through gauze pieces. The filtrate was collected in
petridishes and labeled properly. The petriplates were
covered with aluminum foil and kept for drying in hot
air oven. Dried extracts were taken out and collected in
vials. Aqueous extract was prepared and tested for cell
viability and cytotoxicity assay.
Cell Culture and viability assay
The tumor donor mouse was procured from JNCH &
RC and the tumor was dissected from animal in aseptic
condition. The tumor cells were transferred to MEM
media and media was replaced after every 2 days until
the outgrowth had spread to cover at least 50% of the
growth surface. Further, the cells were sub cultured by
enzymatic method using trypsin. The B16F10
melanoma tumor cell line was maintained in MEM
media in 17 different Petri plates. The cultured cell
line was treated with 0.1 ml dose of different aqueous
extracts of A. cepa & A. sativum at 50 mg & 100 mg
concentration, than plates were kept in CO2 incubater.
After the treatment, the toxicity of our drug (A. cepa &
A. sativum) was examined on melanoma tumor cell by
trypan blue exclusion method9 using formula
Cell viability = Total cells / 4 X Dilution Factor X104
cells
The percentage of nonviable cells were counted by
using formula-
Percentage of nonviable cell = Numbers of
nonviable cell /Total cells X 100
The dye exclusion test is used to determine the number
of viable cells present in cell suspension. It is based on
the principal that live cells possess intact cell
membrane that exclude certain dyes such as trypan
blue, eosin or propidium where as dead cells do not.
Cytoprotective assay
Short term culture of blood lymphocytes was done in-
vitro and the cultures were divided in three groups. The
lymphocytes were cultured in RPMI 1640 media in
aseptic condition for 24hr. The first group was treated
as control. The second group consisted of lymphocytes
81
treated with aqueous extract of A. cepa & A. sativum,
at a dose of 100mg/kg body weight with the help of
syringe driven filter. The third group consisted of
lymphocytes treated with chemotherapeutic drugs
(Docetaxel, Doxorubicin) at a dose of 100mg/kg body
weight. Cultures were incubated in CO2 incubator.
After 24 hours, the cultures were centrifuged for 15
minutes at 1000 rpm. The supernatant was discarded
and the pellet was treated with 0.57% KCl (Hypotonic
Solution) and again centrifuged for 15 minutes at 1000
rpm. The pellet was treated with fixative and kept for
2hr. After 2 hr the sample was again centrifuged for 15
min at 1000 rpm. The process was repeated until a
clear pellet was obtained. Slides were prepared by air
drop method and stained with Giemsa. Then the slides
were observed under microscope for micronucleus and
apoptotic assay. `10-14
Results
Cell Viability assay-
The result of cell viability assay showed that the 50 mg
& 100 mg concentration of extracts of different
varieties of onion (A. cepa) and garlic (A. sativum)
have a certain effect on viability of melanoma cells
cultured in-vitro. (Table 1)
We found that 100 mg South Indian garlic extract is
most potential in minimizing the viability of melanoma
cells in-vitro (68% viable and 32% non-viable)
followed by 50 mg concentration of White onion
extract (47% viable and 53 % nonviable) as compared
to the 50 mg and 100mg concentration of other
varieties of onion and garlic.
Cytoprotective assay- In this assay the nature of
extracts of different varieties of onion & Garlic was
analyzed in comparison to the chemotreauptic drugs
Doxorubicin and Docetaxel (Table 2). It was found that
100 mg/kg body weight of White Onion showed most
potent cytoprotective activity followed by other
varieties of garlic and onion. The toxicity of extract
was calculated by the simple micronucleus and
apoptotic assay.
Discussion
The present study has focused on two major biological
dosimetery assays, the crude extract of four different
varieties of garlic and onion were evaluated for cell
viability test against a B16F10 Melanoma cell line by
trypan blue assay.
 Shrivastava et al. International Journal of Phytomedicine 2 (2010) 80-84

Table 1: The percentage of viable and non-viable

cells shown by the melanoma cells treated with 50
mg and 100 mg concentration of aqueous extract of
different varieties of onion and garlic is shown
below:

DIFFERENT NON
TOTAL VIABLE
CONCENTRATION VIABLE
CELLS CELLS
OF EXTRACT CELLS

Control 100 79 21
50 mg NG 100 79 21
100 mg NG 100 67 33
50 mg GG 100 68 32
100 mg GG 100 80 20
50 mg CG 100 57 43 Figure 1: Showing Viable and Non- Viable cells in
100 mg CG 100 86 14 Melanoma Cell line treated with aqueous extract of
50 mg SI G 100 68 32 different varieties of Allium cepa and Allium
100 mg SI G 100 47 53 sativum.
50 mg NO 100 72 28
The cellular protection or cytoprotection studies were
100 mg NO 100 79 21
carried in peripheral blood lymphocyte cultures treated
50 mg GO 100 60 40 with 100 mg/kg body weight concentration of the
100 mg GO 100 68 32 extract and compared with chemotherapy drug
50 mg WO 100 47 53 Doxorubicin and Docetaxel. It was found that White
100 mg WO 100 75 25 Onion followed by Nasik Garlic have better protection
50 mg SI O 100 76 24 against the damage induced by chemotherapeutic
agents.
100 mg SI O 100 55 45

*NG- Nasik Garlic, GG- Green Garlic, CG- Chinese

Garlic, SIG- South Indian Garlic, NO- Nasik Onion,
GO- Green Onion, WO- White Onion, SIO- South
Indian Onion

The cell lines were treated with 50mg & 100mg

aqueous extract of Allium cepa (onion) & Allium
sativum (garlic) separately and the cell viability in the
treated cell lines was compared to that of the non-
treated control cell lines. The data revealed that onion
have shown better tumor cell inhibition property than Figure 2: Showing Normal Cell, Micronucleus Cell
garlic. It was found that 100 mg concentration and 50 and Apoptotic cells Observed in blood sample
mg concentration of south Indian Garlic followed by treated with different varieties of aqueous extract of
100 mg & 50 mg concentration of Nasik onion exhibit Allium cepa and Allium sativum.
fairly good tumor cell inhibition property as compared
to other varieties of garlic and onion.

82
 Shrivastava et al. International Journal of Phytomedicine 2 (2010) 80-84
The results obtained were opposite to the reports of possesses better tumor inhibition property for other
Fleischauer etal (2000), who reported that the garlic tumors14.

Table 2: Total count of micronucleus, apoptosis & normal cell number in Lymphocytes cultured invitro
treated with chemotreauptic drugs and aqueous extract of different varieties A. sativum & A. cepa.

Drug Total Normal Apoptotic

Extract Micronucleus
Dosing cell Cell Cell

Control - 100 91 7 2

Docetaxel 100mg/kg 100 72 21 7

Doxorubicin 100mg/kg 100 77 17 6

Nasik Garlic 100mg/kg 100 88 11 1

Green Garlic 100mg/kg 100 89 8 3

100mg/kg 100 87 9 4
South Indian Garlic
Chinese Garlic 100mg/kg 100 79 17 4

Nasik Onion 100mg/kg 100 92 7 1

Green Onion 100mg/kg 100 92 5 3

South Indian 100mg/kg 100 88 10 2

Onion
White onion 100mg/kg 100 100 - -

Conclusion Abbreviations
It was concluded from viability assay and MEM: Minimum essential media
cytoprotection or cytotoxicity assay that Nasik Onion, RPMI-1640: Roswell park memorial institute-1640
White Onion and Green Onion have shown better PBS: Phosphate buffer saline
tumor inhibitory and normal cell protection property. MN: Micronucleus
On the other hand, Green Garlic, South Indian Garlic Apop: Apoptosis
and Nasik Garlic revealed tumor inhibitory and NC : Normal Cell
cytoprotective activity. Hence, the result suggested that V: Viable
the dietary food must be added and garnished with NV: Non-viable
White Onion, Green Onion, Nasik Onion, South Indian
Onion, Nasik Garlic, Green Garlic, South Indian Garlic References
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