Rapid Culture And Bioreactor Of Hairy Roots Of Two Salvia Species

Salvia miltiorrhiza Bunge is a kind of perennial herb in the Labiatae family. Its dried roots are highly valued. S. miltiorrhiza is one of the most important traditional Chinese medicinal plants in China. It has been widely used for prevention and treatment of cardiovascular disease including coronary heart disease and angina pectoris. Its active ingredients mainly include water-soluble polyphenolics(danshensu, salvianolic acid B, rosmarinic acid and caffeic acid)and lipophilic tanshinones(tanshinoneⅡA, cryptotanshinone, tanshinoneⅠand dihydrotanshinone Ⅰ). This study set S. miltiorrhiza Bunge(ZHDS) and Salvia castanea Diels f.tomentosa Stib(ZDS) hairy root as material, preliminarily studied its rapid culture as well as bioreactor.1. We studied effects of 7 different liquid media on the growth and the active ingredient accumulation of ZHDS and ZDS hairy roots, such as WP, 6,7-V, SJ-1, B5, N6, improved-White and MS. ZDS hairy roots grew best by B5 medium. Next were WP and 6,7-V. The yield and the content of caffeic acid and tanshinones in ZDS hairy roots were the highest by SJ-1 medium. The yield and the content of rosemary acid and salvianolic acid B in ZDS hairy roots were the highest by improved-White medium. ZHDS hairy roots grew best by MS, next was B5. The yield and the content of caffeic acid in ZHDS hairy roots were the highest by B5. The yield and the content of rosemary acid and salvianolic acid B in ZHDS hairy roots were the highest by improved-White medium. The contents of dihydrotanshinone and cryptotanshinone in ZHDS hairy roots were the highest by SJ-1, next was White medium. The contents of tanshinone Ⅰand tanshinone Ⅱ in ZHDS hairy roots were the highest by improved-White, next was SJ-1.2. We studied effects of 6 different carbon sources on the growth and the active ingradient accumulation of ZHDS and ZDS hairy roots, such as galactose, fructose, lactose, glucose,arabinose and saccharose. It showed that galactose and saccarose were the best for the growth of both ZHDS and ZDS hairy roots. The yield and the content of salvianolic acid in ZDS hairy roots were the highest by galactose. Fructose greatly increased the yield and the content of dihydrotanshinoneⅠ, cryptotanshinone and tanshinoneⅠin ZDS hairy roots.The contents of tanshinone ⅡA in ZHDS hairy roots were the highest by saccharose medium. Therefore,fructose and saccharose media can be used to extract the single ingredient of tanshiones.Galactose was the best for ZHDS hair roots growth, next was frutose. Fructose or glucose clealy improved the accumulation of polyphenolics content, but decreased the danshiones content accumulation. So fructose and glucose can be used to extract the active ingredient of polyphenolics. Lactose and arabinose both greatly increased the danshinones accumulation, so they can be used to extract the active ingredient of danshinones.3. We studied effects of 6 different nitrogen sources on the growth and the active ingredient accumulation of ZHDS and ZDS hairy roots, such as lactoalbumin hydrolysate, peptone, beef extract, casein, yeast extract(YE) and NH4NO3. It showed that NH4NO3 was the best for both ZHDS and ZDS hairy roots growth. Lactoalbumin hydrolysate was the best for the polyphenolics content accumulation, but YE was the best for the danshinones of two kinds of hairy roots.4. We designed and processed the air-lift bioreactor. Its body was made of transpatent glass and was put in the dark room. The temprature was 25±1 ℃ by air-conditioner, continuously applying air through air feed pump and controling the speed by gas flowmeter. The preliminary study suggested that ZHDS hairy roots grew fastly by using the air-lift bioreactor. The fresh weight increased 26.4 times. When culturing ZHDS hairy roots in 100 ml erlenmeyer flask, their fresh weight generally grew by 10-15 times. However, its polyphenolics and danshinones ingredients were much lower than those in 100 ml erlenmeyer flask. For example, salvianolic acid B content was less than 30 % and tanshinone ⅡA content was less than 5 % of those in erlenmeyer flask. It suggested that although ZHDS hairy roots grew fastly by air-lift bioreactor,their active ingredients accumulated quite slowly. How improving the active ingredients of hair roots by bioreactor needs further study. Besides, this reactor culturing parameter needs to be optimized.