Medicinal herbs, used for centuries all over the world, are commonly believed to have minimal side effects and are considered safer than synthetic drugs for treating various medical and dental infections. Recently, positive results of few in vitro herbal antimicrobial efficacy studies and well known side effects of synthetic drugs encouraged scientists to consider herbs as alternative medicine. In view of aforementioned context, my research on toxicology of herbs hypothesizes that there exists an optimal concentration of commonly used antibacterial herbs which kill the prokaryotes cells but doesn't kill the eukaryotic cells during short-term exposure. This project would significantly help in filling research gaps between traditional knowledge and proven evidence of herbal safety. Furthermore, there is a dearth of studies of highly efficacious medicinal herbs that classified detailed phytochemical and toxicity data which can be immediately used before conducting expensive in vivo clinical trials. Research methodology involves conducting in vitro cytotoxicity assay analysis of various concentrations of herbal extracts along with positive controls against cultured yeast and E. coli k-12 cells to measure cellular metabolic activity as an indicator of cell viability, proliferation and cytotoxicity.
Methodology and Procedure
For this research, I used CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay from Promega Biotechnology Company. This is a homogeneous, colorimetric method for determining the number of viable cells in proliferation, cytotoxicity or chemosensitivity assays. The assay is composed of solutions of a novel tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethyl phenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine methosulfate) PMS. MTS is bio-reduced by cells into a formazan product that is soluble in tissue culture medium. The absorbance of the formazan product at 490 nm can be measured directly from 96-well assay plates without additional processing. The conversion of MTS into the aqueous soluble formazan product is accomplished by dehydrogenase enzymes found in metabolically active cells. The quantity of formazan product as measured by the amount of 490 nm absorbance is directly proportional to the number of living cells in culture.
For this research, baker’s yeast (Saccharomyces Cerevisiae) was used to grow free-floating yeast cell culture in the culture medium (suspension culture). The yeast–peptone–dextrose (YPD) culture medium, composed of 2% glucose, 1% yeast extract and 2% bacto peptone, was kept incubated at an optimum temperature of 30°C for 72 hours to grow cells exponentially. Similarly, cell culture was prepared for E. coli k-12 strains using LB (Luria Broth) broth growth medium consisting of 10g/L tryptone, 10g/L NaCl and 5g/L yeast extract. After growing cells in the starting flask reached the exponential growth phase, contents were mixed well to have a uniform concentration of cells in the flask and for all the wells to have the same starting number of cells.Cell cultures in 50μl volume (culture medium) were carefully pipetted into wells ( washing or cell harvesting not required with this cell assay) and were kept at a concentration of 105 cells/well in a 96-well microplate. The yeast cells were treated with different concentrations of 50μl volume of test plant extracts ( 50, 150, 250 mg/ml) against the positive control (0.25% fluconazole) and the negative control which contained only the medium (distilled water) and incubated for 48 hours at 37°C. The E. coli k-12 cells were treated with different concentrations of 50μl volume of test plant extracts ( 50, 150, 250 mg/ml) against the positive control (0.2% chlorhexidine) and the negative control which contained only the medium(distilled water) and incubated for 48 hours at 37°C. 20μl of the combined MTS/PMS solution was pipetted into each well of the 96 well assay plate containing 100μl of cells in culture medium. Plate was incubated for 1–4 hours at 37°C in a humidified, 5% CO2 atmosphere. Absorbance was recorded at 490 nm using an ELISA plate reader.
Statistical Analysis
A one-way ANOVA test was used to compare means of all the herbal extracts samples along with controls based on 3 trials. For this, the experimental data was expressed as mean ± SD, the significance of difference among the various treated groups and positive and negative control groups was analyzed by means of one-way ANOVA, and the level of significance was set at p < 0.05.
After this, Tukey's post-hoc statistics test will be used to compare means of all the cell treatments. If there was a statistically significant difference of means that was found using a one-way Anova test, then Tukey's post-hoc statistics test was used to exactly find the herbal extracts where those differences lie.