CYTOTOXICITY OF WALNUT OIL ON ORAL CANCER CELL LINES

Aim: To assay cytotoxic activity of walnut oil on oral cancer cell line Objective: This study is to analyse the cytotoxicity of walnut oil on oral cancer cell line by DNA fragmentation.The cytotoxicity of walnut oil on oral cancer cell line is to be studied. Background: Walnut is unique because they have a perfect balance of n polyunsaturated fatty acids. The increasing market demand of walnut lipids results in the large amount of the oil extraction residue. The walnut residue is rich in nutritional proteins, and the uneconomic use of the by-product discouraged the development of walnut industry. Anticancer peptides have recently received attention as alternative chemotherapeutic agents that overcome the limits of current drugs. Reasons: Oral cancer as become very prevalent nowadays.This research was done to experiment if walnut oil can kill oral cancer cell line.


INTRODUCTION
Cytotoxicity is the quality of being toxic to cells.Types of venom are some examples of toxic agents or immune cell.Chemotherapy as a treatment of cancer relies on the ability of toxic agents to damage cells or to killce reproducing; this preferentially targets rapidly dividing c cells (8). Antibody dependent cell mediated cytotoxicity describes the cell killing ability of certain lymphocytes, which requires which required the target cells being marked by the antibody. Cytotoxicity assays are widely used by the pharmaceutical industry to screen for cytotoxicity in compound libraries.Cytotoxicity can also be measured by sulforhodamineB assay, WST assay, etc (9).
Walnut oil is an oil extracted from walnut Each 100g of oil contains 63.3g of polyunsaturated fatty 22.8g monounsaturated fatty acid and 9.1g saturated fatty acid. It contains no cholesterol .Unlike most nuts that are high in monounsaturated fatty acid.Walnut oil has large amount of polyunsaturated fatty acid. Particularly, alpha linolenic acids (1).
There are two types of walnut oil they are cold pressed and refined.Cold pressed walnut oil is actually more expensive due to the loss of higher percentage of oil.Refined expeller pressed and saturated with the solvent to e highest percentage of the oil available in the nut meal. Walnut oil is very much rich in vitamins and minerals. (2) OF WALNUT OIL ON ORAL CANCER CELL LINES Induja M.P., Gayathri R and Vishnupriya V Saveetha Dental College and Hospitals, Poonamalle High Road, Chennai

A B S T R A C T
Aim: To assay cytotoxic activity of walnut oil on oral cancer cell line Objective: This study is to analyse the cytotoxicity of walnut oil on oral cancer cell line by DNA fragmentation.The cytotoxicity of walnut oil on oral cancer cell line is to be studied. Background: Walnut is unique because they have a perfect balance of n polyunsaturated fatty acids. The increasing market demand of walnut lipids results in the large amount of the oil extraction residue. The walnut residue is rich in nutritional proteins, and the uneconomic use of the by-product discouraged the development of walnut industry. Anticancer peptides have recently received attention as alternative chemotherapeutic agents that overcome the limits of current drugs. Reasons: Oral cancer as become very prevalent nowadays.This research was done to experiment if walnut oil can kill oral cancer cell line. toxic to cells.Types of venom are some examples of toxic agents or immune cell.Chemotherapy as a treatment of cancer relies on the ability of toxic agents to damage cells or to killcells which are preferentially targets rapidly dividing cancer Antibody dependent cell mediated cytotoxicity of certain lymphocytes, which requires which required the target cells being marked by the assays are widely used by the industry to screen for cytotoxicity in compound libraries.Cytotoxicity can also be measured by (juglans regina). Each 100g of oil contains 63.3g of polyunsaturated fatty acid, 22.8g monounsaturated fatty acid and 9.1g saturated fatty acid. It contains no cholesterol .Unlike most nuts that are high in monounsaturated fatty acid.Walnut oil has large amount of alpha-linolenic acids, There are two types of walnut oil they are cold pressed and refined.Cold pressed walnut oil is actually more expensive due to the loss of higher percentage of oil.Refined walnut oil saturated with the solvent to extract the highest percentage of the oil available in the nut meal. Walnut oil is very much rich in vitamins and minerals. (2)

Procurement of oil
The walnut oil was procured from Cyrus India Ltd. And further analysis was done using this oil.

Maintainence of kb cell line:
lines where procured from national centre for cell sciences (NCCS), Pune.The Oral cancer cells were seeded in 24 Welles plate and kept in CO2 incubator.

Treatment of kb cell lines with drug
The cells were treated with walnut oil in three different concentrations (100μl, 200 μl, 300 hours.

Isolation of Genomic DNA
The Cells were placed in a 37°C water bath. It was continuously until the medium thawed. Then it was centrifuged at 1000rpm for 5 minutes at room temperature The supernatant was discarded and cells were washed with fresh medium to remove residual DMSO(Dimethyl Sulphoxide) which is an important polar aprotic solvent that dissolves both polar and non-polar compounds and is miscible in a wide range of organic solvents as well as water.The cell pellet was re-suspended in 3ml of of DMEM(Dulbecco's Modified Eagle's Medium: a composition that helps in maintaining mammalian cell culture) with 10% FBS Bovine Serum which helps in easier coagulation of cells) (6). It was then incubated in a CO2 incubator at a humidified 37°C. The medium was changed every 2 indicator (e.g. Phenol red) in medium changed colour. The culture was kept in a medium with 10% FBS until cell

OF WALNUT OIL ON ORAL CANCER CELL LINES
Hospitals, Poonamalle High Road, Chennai of walnut oil on oral cancer cell line : This study is to analyse the cytotoxicity of walnut oil on oral cancer cell line by DNA fragmentation.The cytotoxicity of walnut oil on oral cancer cell line is to be studied.
: Walnut is unique because they have a perfect balance of n-6 and n-3 polyunsaturated fatty acids. The increasing market demand of walnut lipids results in the residue. The walnut residue is rich in nutritional proteins, product discouraged the development of walnut industry. Anticancer peptides have recently received attention as alternative chemotherapeutic agents Oral cancer as become very prevalent nowadays.This research was done to

METHODS
The walnut oil was procured from Cyrus India Ltd. And further analysis was done using this oil.

Maintainence of kb cell line:
The vial containing KB cell lines where procured from national centre for cell sciences Pune.The Oral cancer cells were seeded in 24 Welles plate and kept in CO2 incubator.

Treatment of kb cell lines with drug (walnut oil)
The cells were treated with walnut oil in three different l, 300μl) and left along for 24 The Cells were placed in a 37°C water bath. It was continuously until the medium thawed. Then it was centrifuged at 1000rpm for 5 minutes at room temperature (5). The supernatant was discarded and cells were washed with esh medium to remove residual DMSO(Dimethyl Sulphoxide) which is an important polar aprotic solvent that polar compounds and is miscible in a wide range of organic solvents as well as water.The cell 3ml of of DMEM(Dulbecco's Modified Eagle's Medium: a composition that helps in maintaining mammalian cell culture) with 10% FBS (Fetal Bovine Serum which helps in easier coagulation of cells) (6). It was then incubated in a CO2 incubator at a humidified °C. The medium was changed every 2-3 days or when ph indicator (e.g. Phenol red) in medium changed colour. The culture was kept in a medium with 10% FBS until cell

This is an open access article distributed under the Creative Commons Attribution License, which permits
line were re-established. The treated cells were then subjected to DNA fragmentation. (4)

MTT Assay
The MTT assay (Mossman, 1983) 6 is based on the ability of live but not dead cells to reduce a yellow tetrazolium dye to a purple formazan product. Cells were maintained in DMEM medium, supplemented with 10% Fetal Bovine Serum, at 37 o C in humidified atmosphere with 5% CO 2 . The cells were plated in 96 well flat bottom tissue culture plates at a density of approximately 1.2X10 4 cells /well and allowed to attach overnight at 37 o C. The medium was then discarded and cells were incubated with different concentrations of the samples (100, 200 & 300 µg) for 24 hours. After the incubation, medium was discarded and 100µl fresh medium was added with 10µl of MTT (5mg/ml). After 4 hours, the medium was discarded and 100µl of DMSO was added to dissolve the formazan crystals. Then, the absorbance was read at 570nm in a microtitre plate reader. Cyclophosphamide was used as a positive control.

RESULTS AND DISCUSSION
Cytotoxicity analysis using various concentrations of walnut oil (100, 200, 300 micrograms) was performed. The viability of the KB cell lines shows a gradual decrease as the concentration of the walnut oil is increased. This exhibits the Cytotoxicity of walnut oil extract with increasing concentration. (10) Oral cancer is one of the major worldwide threats to public health.It is associated with severe morbidity and long-term survival is less than 50% despite advances in the treatment (surgery, radiation, and chemotherapy.The survival of the patients remains very low, mainly due to their high risk of developing a second primary cancer. Therefore, the early detection and prevention of oral cancer and pre malignancy are quite important. The use of synthetic drugs and radiation not only destroy cancer cells but they also cause damage to other cells thereby causing delayed wound healing. Therefore in near future new approaches can be initiated by using walnut oil tempered with other natural compounds may be of great promise in finding a sure cure for cancer patients and can be used to create further scope in the discovery of chemopreventive drugs(7).

CONCLUSION
From the above experiment and research it is proven that walnut oil has the potential to treat oral cancer. walnut oil is the most commonly available product of Juglans regia and is easily available in the market. Though research is still proceeding in various parts of the world to make use of this plant extract to treat cancer, oral-cancer in specific, there is less awareness among the masses(3).In near future the phytochemical properties of walnut oil may be used to design anti-cancer drugs. Also the medicinal property of the various natural herbs should be explored because than the other chemotherapeutic drugs, they don't affect the normal and healthy cells and they don't cause any side effects.