Recently, the demand for and range of use of the secondary metabolites of plants, which are more available than synthetic compounds, has been gradually increased, in medical treatment. Plant tissue culture is useful method for the production of secondary metabolites and it is possible to control productivity by changing the culture conditions, for example, of light, pH, carbon source, amino acid, precursor, polarity, plant growth regulator etc. 1-5)Cynanchum wilfordii HEMSLEY (Asclepiadaceae) is a perennial herb of which the benefits were previously described. [6][7][8] In Korea, its roots, which are known as backhasuo, have been used in folk medicine for the prevention and treatment of various geriatric diseases including vascular disorders, diabetes mellitus, ischemia-induced diseases, and aging progression. 6)The roots of this plant have been reported to contain some polyoxypregnane glycosides. [8][9][10][11][12] In addition, recently one steroidal alkaloid, gagaminine, was isolated for the first time from this plant.13) Gagaminine has also been isolated from other species in the Cynanchum genus 14,15) and evaluated for its potent inhibitory effects on the activity of aldehyde oxidase, an oxygen radical generating enzyme, and hepatic lipid peroxidation in an in vitro assay.13) The structure of this active compound ( Fig. 1) contains a pregnane skeleton with two ester bonds bound with cinnamic acid at C-12 and nicotinic acid at C-20. Thus the introduction of an ester group at the corresponding carbon positions in the skeleton could not easily be achieved using any chemical approach. However, cell cultures often produce secondary metabolites in significant quantities 16) which would be useful for animal tests and are regarded as genetically more stable. In our previous work, we reported the establishment and cultivation of stem cultures of C. wilfordii and presented the results designed to optimize growth and gagaminine content in these cultures. 17) In this report, we describe the comparative results of the effects of various culture conditions, such as 2,4-dichlorophenoxyacetic acid (2,4-D), known as one of the most effective plant growth regulators 17) ; different sucrose and various initial cell concentrations; light and dark conditions; and different temperatures on cell growth and gagaminine production in suspension culture of C. wilfordii. Gagaminine, a steroidal alkaloid isolated from the roots of Cynanchum wilfordii, exhibited potent inhibitory effects on aldehyde oxidase activity and lipid peroxidation. To determine whether it would be possible to mass produce this active component, which would be useful for animal tests, we tried to synthesize it using in vitro cell culture methods with various growth conditions. In a previous study it was found that calli were easily induced from the stem of this medicinal plant and cultivated effectively on MS medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) 2 mg/l. In this work we attempted to determine the effects of various culture conditions on cell gro...
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