Optimization of active compounds production by interaction between nitrate and copper in Sphaeralcea angustifolia cell suspension using Response Surface Methodology

Abstract

Cells in suspension from Sphaeralcea angustifolia produce compounds with anti-arthritic activity (scopoletin, tomentin, and sphaeralcic acid) in Murashige and Skoog (MS) medium with 2.74 mM of total nitrate content. The effect of nitrate and copper contents in the MS medium on the growth of cell suspension and the production of active compounds were tested by means of 2k factorial design (FD) and Central Composite Design (CCD). The growth rate and the duplication time were statically similar and the maximal biomass (9–11 days) was improved by increases of the nitrate concentration. Highest contents of coumarins (4137.00 µg L−1) and sphaeralcic acid (1441.00 µg L−1) were obtained in the culture media by the interaction of 2.74 mM of nitrates and 2 µM of copper at 2 and 4 days; sphaeralcic acid contents were similar to those detected intracellularly after 9 and 11 days of culture. According to the CCD, highest contents of coumarins (4008.00 µg L−1) and sphaeralcic-acid (6107.00 µg L−1) could be obtained with 2.35 µM of copper with 2.42 mM of total nitrate, this condition did not affect the cell growth. The coumarin content is 40-fold and that of the sphaeralcic-acid is 30-fold higher than those detected in biomass. The S. angustifolia cell suspension could be cultured in a stirred tank bioreactor in a batch or in a continuous manner for the production of scopoletin, tomentin, and sphaeralcic-acid.