Shikonin production by extractive cultivation in transformed-suspension and hairy root cultures of Lithospermum erythrorhizon

Effects of in situ extraction, fungal elicitation, a permeabilizing agent, and the oxygen transfer rate on shikonin production in transformed suspension and hairy root cultures of Lithospermum erythrorhizon were studied. Shikonin production with in situ extraction in transformed cell and hairy root cultures by n-hexadecane was 7.6 and 3 times higher than those of the control culture. Shikonin production of transformed L. erythrorhizon increased with the enhanced gas exchange, and in situ extraction also increased sucrose consumption and shikonin production. The optimal volume of n-hexadecane in the hairy root culture was similar to that in the transformed cell cultures. In situ extraction at an earlier stage significantly enhanced shikonin production both in transformed cell and hairy root cultures. Dimethylsulfoxide used as a permeabilizing agent was harmful to cell growth and shikonin production, and permeabilizing was unnecessary when in situ extraction was applied. This occurred because with the solvent addition, most shikonin was spontaneously released from the cells and was dissolved in the solvent layer. The combined addition of n-hexadecane of the extract of the fungus Penicillium as an elicitor seemed to result in a higher production of shikonin both in cell suspensions and transformed root cultures. However, an increase of shikonin induction by fungal elicitation in a hairy root culture was not significant in comparison with that of normal cell cultures.