| Budget Amount *help |
¥5,700,000 (Direct Cost: ¥5,700,000)
Fiscal Year 1987: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1986: ¥4,500,000 (Direct Cost: ¥4,500,000)
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| Research Abstract |
Cell suspension cultures of Thalicrum minus (Ranunculaceae) are capable of producing a large amount of alkaloids (mostly berberine), most of which was found to uniquely be secreted into the medium; cell cultures of other Thalictrum species (T. flavum and T. dipterocarpum) accumulate the berberine produced in vacuoles wichout secreting it into the medium. There was no significant correlation in berberine content between the 17 individual maternal plants of T. minus and the corresponding cell lines, although the amount of berberine released into medium was correlated with the berberine-productivity of the cell line. Cell selection yielded a stable, high berberine-producing strain (830 mg/l).
Cell structures were compared between altween alkaloid-producing and non-producing cell cultures of T. minus and Coptis japonica by electron microscopic observation. Alkaloid-producing cells were found to contain an abundance of cytoplasmic small vesicles (0.5 - 1 <micrn>m in diameter), the role of wh
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ich in berberine secretion, however, remains to be solved. Inhibitor experiments (vanadate and DES) indicate that berberine is secreted through an energy-requiring process located in the plasma membrane.
The T. minus cells grew repidly in Linsmaier-Skoog medium (growth medium) containing 1 <micrn>m 2,4-D without producing berberine, although the berberine productivity was remarkably enhanced by a simultaneous administration of 100 <micrn>M NAA and 10 <micrn>M BA in PO43--limited LS medium (production medium). A two-stage culture method using the above two media was found to be suitable for stable, high berberine production. In addition, a new bioreactor (liquid-gas two phase system) was devised for berberine-secreting Thalictrum cells immobilized in Ca-alginate beads, which were alternately soaked in medium and exposed to air. The highest yield of berberine (50 mg/1/day) was obtained by setting the cycle of medium and air supply for 30 sec and 2 min, respectively, during a long term culture (60 days). Less
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