Evaluation on the effect of Common Food Additive by Cell Based Assay
K. Mythili, C. Vichitra*, S. Gayatri, K. Chitra
Faculty of Pharmacy, Sri Ramachandra Institute of Higher Education (Deemed to be University), Chennai, TN
*Corresponding Author E-mail: cvichy97@gmail.com
ABSTRACT:
Maltol is a sugar used as common food additive. It is used extensively as a food preservative, antioxidant, and also in cosmetic and pharmaceutical formulations. The aim of the study is to estimate the purity of the drug using quantitative method of analysis, to evaluate the effect of common food additive such as maltol by using MTT assay. By using L6 myoblast cell line, the viability of the cell was determined. Determination of ash values is valuable for the assessment of purity of sample and identified with distinguishing additional earthy matters. Ash values such as total, acid-insoluble and water-soluble ash were worked out as 3.2, 0.965, 3.57 respectively. Extractive values indicate the nature of basic constituents of the sample and it was noticed by alcohol and water. Unnecessary woody material was examined by the method of crude fiber content and was found to be 3.29 and 6.23% of crude fiber. The percentage cell viability was high at the concentration of 62.5µg/ml for the drug maltol by MTT assay.
KEYWORDS: Maltol, MTT assay, cell viability, food preservative, L6 myoblast cell line.
1. INTRODUCTION:
Formula: C6H6O3
Molar mass: 126.11 g/mole
Density: 1.35 g/cm³
Melting point: 161 to 162 °C
Maltol is a sugar and also commonly available organic compound that is used as a food additive12. It is used generally as a food preservative, antioxidant, and in cosmetic and pharmaceutical formulations. The sources of maltol are bark of larch tree, pine needles, and roasted maltol4. It is used to impart sweet aroma. Maltol is used as an aroma enhancer to give a fresh baked smell in breads and cakes.
Preliminary phytochemical screening is valuable in finding out authenticity of drug. Ash values are useful to find out the type of minerals in drug, which provides physicochemical property of drug5. Extractive values provide some detail with reference to the nature of the chemical components. It is also valuable for the assessment of exact component soluble in particular solvent used for extraction.2
Cell based assay are applied for screening variety of compounds to conclude the effect of test molecules on cell viability, cell proliferation and cytotoxicity1. Cell viability assessment method is used to determine the ratio of live and dead cells. Cell proliferation assay method is used to monitor the growth rate of cell populations11.
Cytotoxicity assay is used to test the effects of compounds on the viability cell developed in culture which in turn helps to measure viable cell, dead cells and also identify the mechanism of cell death1.
L6 Myoblast cell line contains skeletal muscle of rat (QUADRICEPS). Exposure to α-difluoro methyl ornithine, an enzyme stimulated irreversible inhibitor of ornithine decarboxylase slow down the insulin which makes separation of L6 myoblast cells1. Yessotoxin (YTX) can induce apoptic events in L6 myoblast from rat through activation of mitochondrial pathway indicating an intercellular response.
Thus, the present assessment aims towards preliminary phytochemical screening using quantitative method of analysis and evaluation of in vitro cytotoxic effects of a selected compound maltol on L6 Myoblast cell line.
AIM AND OBJECTIVE:
· To estimate the purity of the drug using quantitative method of analysis.
· To identify the mechanism of cell death by measuring the number of viable and dead cells grown in culture.
· To assess the effect of common food additive such as maltol by using MTT assay.
MATERIALS AND METHODS:
1. Quantitative Analysis:
Ash values, extractive values are considered as an aid for the purity of drug. The percentage of active constituents or the total compounds (E.g. Total alkaloids) in a drug with reference to a known compound are determined by different analytical methods.
Reagents:
Dilute hydrochloric acid, 90 % ethanol, 10 % v/v nitric acid, 2.5 % v/v sodium hydroxide and distilled water.
1.1 Evaluation of Ash Values:
The sample was ignited and the ash remained after this process was evaluated by means of particular techniques. The total amount of material remained after ignition can be calculated by total ash method.
Acid -insoluble ash can be attained by the reaction of above ash with dilute HCl and the residue was ignited. Water soluble ash is the variation between mass of total ash and mass of total ash after the reaction with water.
a) TOTAL ASH:
About 2gm of drug was correctly weighed and transferred into a tarred crucible. The crucible was heated gently using a flame of 2 cm high until vapors are evolved and then lowered to heat it more strongly to burn the carbon present in it. The residue is then cooled using desiccators. Finally, the residue was weighed.
b) ACID –INSOLUBLE ASH VALUES:
The ash resulted from the above manner was rinsed with 25ml of dilute HCl and transferred to a 100ml glass apparatus. Then the solution was boiled for 5 min. Ash less filter paper was used for the filtration and hot water was used for washing the residue. A clean silica crucible was taken and it was ignited, cooled and weighed. The residue along with the filter paper was placed in a weighed crucible. Then it was heated gently till all the vapors are evolved, and heated continuously to remove all the carbon. Then the residue was weighed after the cooling process.
c) WATER SOLUBLE ASH VALUES:
The ash resulted from the first process was rinsed with 25 ml of water and collected into a 100ml glass apparatus. Then the solution was boiled for 5 min. Ash less filter paper was used for the filtration and hot water was used for washing the residue. A clean silica crucible was taken and it was ignited, cooled and weighed. The residue along with the filter paper was placed in a weighed crucible. Then it was heated gently till all the vapors are evolved, and heated continuously to remove all the carbon. Then the residue was weighed after the cooling process.
1.2 Determination of Solvent Extractive Values:
This technique concludes the number of active components extorted by means of solvents from the known quantity of sample. There are two methods given below that were used for the determination of alcohol and water-soluble extractive values.
a) Alcohol-Soluble Extractive Values:
About 5gm of sample was weighed and transferred in to 250 ml flask. About 100ml of ethanol was measured and poured into the conical flask. The conical flask was then corked and reserve for 24 hrs, with frequent shaking and the solution was filtered. About 25ml of the above solution was transferred into a weighed porcelain dish. Then it was heated to dryness and completely dried in an oven at 100°C. Desiccator was used to cool the residue and finally the residue was weighed.
b) Water Soluble Extractive Values:
It has been identified in a method as same as alcohol soluble extractive value. In this method 100ml of chloroform is used instead of ethanol. About 5gm of sample was weighed and transferred in to conical flask. About 100ml of chloroform was and poured into the conical flask. The conical flask was then corked and reserve for 24 hrs, with frequent shaking and the solution was filtered. About 25ml of the above solution was transferred into a weighed porcelain dish. Then it was heated to dryness and completely dried in an oven at 100°C. Desiccator was used to cool the residue and finally the residue was weighed.
2. MTT Assay for Cytotoxicity:
Cells in the growth phase are depicted to the cytotoxic drug. The period of revelation is assessed as the moment in time needed for maximal damage to take place, but is also controlled by stability of the compound. These cells started proliferating for 2 or 3 doubling times after the exclusion of drug. This helps to differentiate among the cells that stay viable and can proliferate and the cells that stay viable and cannot reproduce.
The quantity of cells which survived are identified indirectly by MTT (3- (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) tetrazolium (yellow tetrazole) dye reduction method. The determination of cell proliferation is done by quantitative colorimetric method that is MTT reduction. Mitochondrial succinic dehydrogenase metabolizes it to yield a purple formazan product in living cells.
The substrate for MTT assay has been prepared from a solution which is physiologically balanced, then it is added to a culture at last concentration of 0.2-0.5 mg/ml at an incubation period of 1 to 4hrs.The viable cells having active metabolism converts the MTT (yellow tetrazole) into purple formazan by reduction. The formazan formed is the only marker for viable cells.
The quantity of formazan can be evaluated by note down the changes in absorbance at 570nm using a plate reading spectrometer.
CELL VIABILITY ASSAY:
Viability assay is to determine the ability of tissues, cells, or organs to maintain or recover viability. Cell production and examination for cytotoxic results of drugs can be measured using this assay method. From the host tissue, primary cell lines can be directly isolated and grown in culture. L6 myoblast culture was purchased from NCCS, Pune and maintained with DMEM high glucose media with 10 % Foetal bovine serum, in an atmosphere of 5 % CO2 at 37° C.
The L6 myoblast was seeded at a density of 4 x 104 cells per well in 48-well plate. After getting 70 % confluency, cells were reacted with concentrations of test drug (1000, 500, 250, 125, 62, 31.25 µg/mL) in serum free media.
After 24 hours of treatment the 50 µL of MTT solution (5 mg /mL in PBS) was put in to each well and the cells were further incubated at 37˚C for a period of 3 hrs. The medium was subsequently aspirated and 150 µL of DMSO was included to the purple colored soluble formazan crystals. The plates were shaken and using the micro plate reader optical density was measured at 570 nm.
RESULTS AND DISCUSSION:
Phytochemical screening is valuable in finding out authenticity of sample. Ash values are useful to find out the type of minerals in drug, which provided physicochemical property of drug. Extractive values provided some detail with reference to the nature of the chemical components. It is also valuable for the assessment of exact component soluble in particular solvent used for extraction.
Cell based assay are applied for screening variety of compounds to conclude the effect of test molecules on cell viability, cell proliferation and cytotoxicity. Cell viability assessment method is used to determine the ratio of live and dead cells. Cell proliferation assay method is used to monitor the growth rate of cell populations.
Cytotoxicity assay is used to test the effects of compounds on the viability cell developed in culture which in turn helps to measure viable cell, dead cells and also identify the mechanism of cell death.
The purity of a crude drug was assessed with ash values by recognizing the overload of earthy materials. The occurrence of ash has been decided as total, acid-insoluble and water-soluble ash. Nature of basic constituents of the crude plant can be identified by solvents like alcohol and water which are represented as extractive values.
Table 1: Ash values and Extractive values
|
S. No. |
Parameters |
Percentage Yield (% w/w) |
|
1 |
Total ash |
3.2 |
|
2 |
Acid - Insoluble ash |
0.965 |
|
3 |
Water - Soluble ash |
3.57 |
|
4 |
Alcohol – Soluble extractive |
3.29 |
|
5 |
Water – Soluble extractive |
6.23 |
Maltol was tested for its cytotoxic activity on L6 myoblast cells to check the response and health of cells by MTT assay. Result showed that the drug maltol possessed significant cytotoxic effect against the L6 myoblast cells.
The time and concentration dependent decrease in the MTT viability index by the drug maltol was observed in this study. Cell viability assay established the drug dependent cytotoxicity on L6 myoblast cells.
The results revealed that the percentage cell viability of maltol was found to be 98.24 ± 5.57 at a concentration of 62.5 µg/ml.
The values of absorbance which is lesser than the control cells determine a fall in the rate of cell proliferation. Inversely the higher absorbance rate determines an increase in cell proliferation. Increase in proliferation may be due to cell death caused by morphological changes.
Table - 2: Percentage of Cell Viability
|
S. No |
Concentration (µg/ml) |
Cell Viability (%W/W) |
|
1. 2. 3. 4. 5. 6. |
31.25 62.5 125 250 500 1000 |
93.21 ± 3.84 98.2 4 ± 5.57 80.1 ± 5.00 76.66 ± 0.99 65.15 ± 6.90 58.67 ± 2.95 |
The purity of drug maltol was estimated using quantitative method of analysis. The percentage of cell viability was predicted from cytotoxic effects and has been found to be nontoxic.
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Received on 16.04.2019 Modified on 18.05.2019
Accepted on 21.06.2019 © RJPT All right reserved
Research J. Pharm. and Tech. 2019; 12(11):5433-5436.
DOI: 10.5958/0974-360X.2019.00942.9