Dye extraction and separation in Rubia tinctorum L. with three solvents in three seasons and its application for cytological studies

In cytology studies, colors are used to differentiate the components of cells that are transparent. Most of the dyes are synthetic and can harm human and environment. Herbal dyes are safe. Since ancient times in Iran, the natural dye of madder (Rubia tinctorum L.) has been used in the textile industry. In this research, the madder root dye was extracted with water, ethanol and acid at the end of spring, summer and autumn and its colors were separated by paper chromatography. Acetoronacin was prepared from the dye extracted from madder. Acetoronacin  was used to stain epidermal cells, stomata guard cells, pollen, and the plant mitosis. The results showed that the best solvent to isolate the dye of madder was water. Chromatography results showed that the type and number of colors (5 colors) in the aqueous extract of madder root in the three seasons were similar, but some of them were higher at the end of the summer. Soluble colors in alcohol (3 colors) were observed in the ethanolic extract at the end of spring. Only one yellow color, probably alizarin, was isolated from the acid extract. Staining the cells with acetoronacin in all cases showed that the color was comparable and even better than acetocarmine. Nuclei and chromosomes of cells were stained brown by staining with acetoronacin in a transparent background. In general, the results showed that this dye can replace the synthetic dyes in the plant cell research laboratories.