http://www.arjournals.org/ijop.html
Research paper
Tumor inhibition and Cytotoxicity assay by aqueous extract of
onion (Allium cepa) & Garlic (Allium sativum): an in-vitro analysis
ISSN: 0975-0185
Shilpa Shrivastava
1, N.Ganesh
1*
*Corresponding author:
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
Abstract
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- vitro culture
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
(Allium sativum) namely Nasik Garlic, Green Garlic, Chinese Garlic and South Indian Garlicwere 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
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
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:
*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 garlic. It was found that 100 mg concentration and 50 mg concentration of south Indian Garlic followed by 100 mg & 50 mg concentration of Nasik onion exhibit fairly good tumor cell inhibition property as compared to other varieties of garlic and onion.
Figure 1: Showing Viable and Non- Viable cells in Melanoma Cell line treated with aqueous extract of different varieties of Allium cepa and Allium sativum.
The cellular protection or cytoprotection studies were carried in peripheral blood lymphocyte cultures treated with 100 mg/kg body weight concentration of the extract and compared with chemotherapy drug Doxorubicin and Docetaxel. It was found that White Onion followed by Nasik Garlic have better protection against the damage induced by chemotherapeutic agents.
Figure 2: Showing Normal Cell, Micronucleus Cell and Apoptotic cells Observed in blood sample treated with different varieties of aqueous extract of Allium cepa and Allium sativum.
DIFFERENT CONCENTRATION
OF EXTRACT
TOTAL CELLS
VIABLE CELLS
NON VIABLE
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
100 mg CG 100 86 14
50 mg SI G 100 68 32
100 mg SI G 100 47 53
50 mg NO 100 72 28
100 mg NO 100 79 21
50 mg GO 100 60 40
100 mg GO 100 68 32
50 mg WO 100 47 53
100 mg WO 100 75 25
50 mg SI O 100 76 24
The results obtained were opposite to the reports of Fleischauer etal (2000), who reported that the garlic
possesses better tumor inhibition property for other 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.
Conclusion
It was concluded from viability assay and cytoprotection or cytotoxicity assay that Nasik Onion, White Onion and Green Onion have shown better tumor inhibitory and normal cell protection property. On the other hand, Green Garlic, South Indian Garlic and Nasik Garlic revealed tumor inhibitory and cytoprotective activity. Hence, the result suggested that the dietary food must be added and garnished with White Onion, Green Onion, Nasik Onion, South Indian Onion, Nasik Garlic, Green Garlic, South Indian Garlic in the food habits so that the toxicity induced by free radicals and heavy metals or during the course of cancer therapy can be well managed and taken care by such Indian spices.
Abbreviations
MEM: Minimum essential media
RPMI-1640: Roswell park memorial institute-1640 PBS: Phosphate buffer saline
MN: Micronucleus Apop: Apoptosis NC : Normal Cell V: Viable
NV: Non-viable
References
1. Lawson LD. “Garlic: A review of its medicinal effects and indicated active compounds. In Phytomedicines of Europe: Chemistry and Biological Activity. Edited by Lawson LD & Bauer R. American Chemical Society, Washington, DC.1998:176-209.
Extract
Drug
Dosing
Total
cell
Normal
Cell
Apoptotic
Cell
Micronucleus
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
South Indian Garlic 100mg/kg 100 87 9 4
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 Onion
100mg/kg 100 88 10 2
2. Amagase H., Milner J.A. “Impact of various
sources of garlic and their constituents on 7, 12-dimethyliben (a) anthracene binding to mammary cell DNA.” Carcinogenesis. 1993; 14:1627-1631
3. Donaldson MS “Nutrition and Cancer: a review of the evidence for an anticancer diet” Nutr J 2004; 20:3:19.
4. Ernest E. “The role of complementary and alternative medicine in cancer”. Lancet Oncol; 2001; 176-80
5. Izzo AA, Capasso R, Capasso “F. Eating Garlic & onion; a matter of life and death. Br J Cancer 2004; 91:194
6. Emily A. Wilson, Barbara Demming-Adams: “Antioxidants, anti-inflammatory, and antimicrobial properties of garlic and onions” Nutrition & Food science 2007; 37; 178-183 7. Yangh J, Meyers KJ, Vander Heide J. Liu RH,
“Varietal difference in phenolic content and antioxidant and antiproliferative activities of onions.” J Agric Food Chemistry. 2004; 52(22):6787-6793.
8. Vainio H, Weiderpas E. “Fruit & Vegetables in Cancer prevention”. Nutrition & Cancer 2006; 54: 111-142
9. Shah DK, Naciri M, Clee P, Al-Rubeai M. “NucleoCounter—An efficient technique for the determination of cell number and viability in animal cell culture processes” Cytotechnology 2006; 51, 39-44.
10.Azadi HG, Ghaffari SM, Riazi GH, Ahmadian S, Vahedi F “Antiproliferative activity of chloroformic extract of Persian Shallot, Allium hirtifolium, on tumor cell lines” Cytotechnology 2008; 56, 179-185.
11.Raffaella Corvi,, Silvio Albertini, Thomas Hartung, Sebastian Hoffmann, Daniela Maurici, Stefan Pfuhler, Jan van Benthem and Philippe Vanparys “ECVAM retrospective validation of
in vitro micronucleus test (MNT)” Mutagenesis 2008; 23(4):271-283
12.L. Lagneaux, A. Delforge, etal Blood, “Chronic Lymphocytic Leukemic B Cells But Not Normal B Cells Are Rescued From Apoptosis by Contact With Normal Bone Marrow Stromal Cells” Blood 1998; 91; 2387-2396
13.Fench M, the invtro Micronucleus technique: Mutation Research, 2000; 455: 81-95.
