Elicitation And Effective Process Strageties For Enhancement Of Tanshinones Production In Salvia Miltiorrhiza Hairy Root

Salvia miltiorrhiza Bunge (Lamiacae), or Danshen in Chinese, is a valuable Chinese herb. There has been renewed research interest over the last few decades in its source and therapeutic utilization following the discovery of its new pharmacological effects. The major obstacle for Danshen as a high-quality plant medicine to enter the international market is the lack of a stable source of the herb with high contents of the bioactive components. A potential bioprocess alternative is to use plant hairy root cultures to produce the active compounds of Danshen. Although there is some reported studies on this culture process, more comprehensive and systematic studies are still needed. The is project is to study the methods and process strategies for the improvement of secondary metabolite production in the S. miltiorrhiza Bunge (Lamiacae) hairy root cultures, and their relative action mechanism were investigated.Elicitation has become one of the most effective methods for enhancing secondary metabolite production in hairy root cultures. In this project, the effects of elicitation on the tanshinones production were tested with a number of biotic and abiotic elicitors. The biotic elicitor yeast extract(YE)and abiotic elicitor Ag+ were selected for further study due to their stronger enhancing effects on the tanshinones production and simplicity in preparation.In order to understand the mechanism of elicitors'effects on tanshinones production, the involvement of the membrane Ca2+ influx was investigated during the elicitation of tanshinones production by various elicitors. Different from the common viewpoint that the plasma membrane Ca2+ influx is essential for enhancing the secondary metabolite production by elicitors, our results suggest that the elicitation of tanshinone accumulation in S. miltiorrhiza hairy roots was independent of the plasma membrane Ca2+ influx.It is fruitful to explore the effective process strategies for enhancing secondary metabolite production in plant tissue cultures. Based on the results of elicitation and in situ adsorption and the self-immobilized characteristic of hairy roots in liquid cultures, the application of a semi-continuous culture process involving repeated medium renewal, elicitor addition and resin replacement was studied. The resultsshow that the integration of multiple elicitation, in situ adsorption and medium renewl of the semi-continuous operation can synergistically enhance tanshinone production in S. miltiorrhiza hairy root cultures. Over a period 60 day of culture, the root biomass was increased to 30.5 g dw/L and the volumetric tanshinone yield to 87.5 mg/L (about 15-fold increase), with 76.5% adsorbed to the resin. The volumetric productivity of total tanshinone reached 1.46 mg/L-d, more than 7.4 times that of the batch culture.A model for the dynamic process of tanshinones biosynthesis and distribution in culture was established considering the synergistic effect of the elicitation and in situ product adsorption. The model reflects favorably the combined effects of two technologies on the tanshinones production and provides reliable predication for further scale-up and research of the process.