The additive effect of calcium carbonate and phosphate on anthraquinone contents in the roots of Rubia tinctorum L.

Madder (Rubia tinctorum L.) is a perennial and herbaceous plant. Madder's root is known as its most important economic part, and it is rich in anthraquinone derivatives. Madder is a plant with medicinal, nutritional, color and health benefits. Recent pharmaceutical studies have shown that the anthraquinones of the madder root have selective inhibitory effects on cancer cells. In this research, according to the water and soil limitations of the tested area, the use of biodegradable calcium carbonate and phosphate fertilizers to produce madder was suggested. So, considering climate change and water shortage as severe risks to sustainable development, the cheapness of madder production and its multiple uses, the planting and production of this plant are mainly used in semi-arid areas. 
In field conditions in Ardakan, Yazd, this research was tested in factorial form in a randomized complete block design with two factors and four replications. Calcium carbonate agent was supplied at five levels of 0, 20, 50, 100 and 150 kg hˉ1 and phosphate from triple superphosphate fertilizer at four levels of 0, 50, 100 and 150 kg hˉ1. In each four-by-four-meter plot, four rows of seeds were planted, two side rows and two plants from the sides of the remaining lines were removed as marginal effects, and the rest of the plants were considered statistical populations. Irrigation water's electrical conductivity was measured during the growth period, and its average was 11.6 ds mˉ2. The plots were irrigated immediately after planting the seeds. Four days later, irrigation was done again. In the fall of the second and third years, soil was again given to the plants along with the desired treatments. Madder plants were harvested in the third winter year. Root fresh-weight biomass was weighed immediately after harvesting. After drying the roots at 104 °C, the dry weight of the roots was measured. A spectrophotometer also determined the content of anthraquinone and hydrogen peroxide in the roots.
The results of bivariate analysis of variance of the data showed that the simple and reciprocal effect of different calcium carbonate and phosphate treatments on functional indicators of biomass, root fresh weight, root dry weight, anthraquinone content and root hydrogen peroxide in this research was significant at P≥ 0.05. The comparison of averages showed that the highest values of biomass, root wet weight, root dry weight, anthraquinone and hydrogen peroxide were observed in the combined treatment of 20 kg h-1 of calcium carbonate and 100 kg h-1 of phosphate, compared to the control. The lowest value of performance indicators investigated in this research was observed in the combined treatment of 150 kg h-1 of calcium carbonate and 150 kg h-1 of phosphate, compared to the control. The simultaneous use of these two fertilizers had a complementary effect on each other. This was because the application of 20 and 50 kg h-1of calcium carbonate and 100kg h-1 of phosphate had an increasing impact on the anthraquinone and hydrogen peroxide content in madder roots. The comparison of the averages showed that in the combined treatment of 20 kg h-1of calcium carbonate and 100 kg h-1of phosphate, compared to the control, the highest wet weight of the root increased by 71%, the highest dry weight of the root increased by 91%, and the highest anthraquinone content increased by 142% was observed. The comparison of the averages showed that in the combined treatment of 50 kg h-1 of calcium carbonate and 100 kg h-1 of phosphate, compared to the control, the highest biomass index was observed, with an increase of 79%.
The results showed that the functional indices of root fresh weight, root dry weight, anthraquinone content and biomass of madder increased significantly under the combined treatment of 20 kg h-1 of calcium carbonate and 100 kg h-1 of phosphate. It is suggested that madder be cultivated using calcium carbonate and phosphate fertilizers to increase the anthraquinone compounds under the influence of biodegradable and inexpensive external environmental stimuli in regions similar to the climatic conditions studied in this research.