International Journal of Phytomedicine 2 (2010) 80-84

Transcription

1 International Journal of Phytomedicine 2 (2010) Research paper Tumor inhibition and Cytotoxicity assay by aqueous extract of onion (Allium cepa) & Garlic (Allium sativum): an in-vitro analysis Shilpa Shrivastava 1, N.Ganesh 1 * ISSN: *Corresponding author: Dr.N.Ganesh 1 In-charge, Deptt. Of Research, Jawaharlal Nehru Cancer Hospital & Research Centre, Idgah Hills, Bhopal, INDIA 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; invitro culture Introduction Indian dietary habits, additive spices and natural flavors are an ancient asset for Asian subcontinents and world through 1. However, many a times it was noticed that because of the presence of sulphur compounds 2 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 more 3. 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 property 6-8. 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: /ijpm arjournals.org, All rights reserved.

2 (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 CO 2 incubater. After the treatment, the toxicity of our drug (A. cepa & A. sativum) was examined on melanoma tumor cell by trypan blue exclusion method 9 using formula Cell viability = Total cells / 4 X Dilution Factor X10 4 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 invitro 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 CO 2 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.

3 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 CONCENTRATION OF EXTRACT TOTAL VIABLE NON VIABLE Control mg NG mg NG mg GG mg GG mg CG mg CG mg SI G mg SI G mg NO mg NO mg GO mg GO mg WO mg WO mg SI O mg SI O 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. *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 nontreated 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 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. 82

4 The results obtained were opposite to the reports of Fleischauer etal (2000), who reported that the garlic possesses better tumor inhibition property for other tumors 14. 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. Extract Drug Dosing Total cell Normal Cell Apoptotic Cell Micronucleus Control Docetaxel 100mg/kg Doxorubicin 100mg/kg Nasik Garlic 100mg/kg Green Garlic 100mg/kg South Indian Garlic 100mg/kg Chinese Garlic 100mg/kg Nasik Onion 100mg/kg Green Onion 100mg/kg South Indian 100mg/kg Onion White onion 100mg/kg 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:

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