Endogenous Inducer of secondary Metabolism in Lithospermum erythrorhizon Cell Cultures

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Cultured cells of Lithospermum erythrorhizon(Boraginaceae)in Linsmaier-Skoog(LS)liquid medium do not synthesize the red naphthoquinone pigment,shikonin,but start to produce it when transferred the LS liquid medium supplemented with certain acidic polysaccharides (agar,agaro-pectin or polygalacturonide).These findings suggested that certain endogenous polysaccharides play an important role in inducing shikonin biosynthesis in Lithospermum cells. The polysaccharide fraction obtained from the cell wall fragments of the shikonin-producing cells cultured in M-9 medium was fractionated by ion-exchange(QAE-Sephadex A-25,0.1-2MKHCO_3) and gel filtration(Sephadex GL6B,0.1MKHCO_3)chromatography,successively.The shikonin-inducing activity was detected by an assay as follows: an aliquot of each fraction was added to the LS liquid medium in which shikonin was not produced without the active principles and shikonin produced was analyzed by HPLC. The most active component was found to induce shikonin biosynthesis by the administration of 30mug/ml medium whereas the crude extract needs 300mug/ml. Methylation analysis and NMR data revealed that the active principle consists of alpha-1, 4-linked galacturonic acid(96-97%) and 1,2-linked rhamnose(3-4%). The polymerization degree was calculated to be ca 18 based on the ratio of reducing-end groups to total sugar. Each oligogalacturonide (polymerization degree of 1-15) was obtained from the partial hydrolysate of the polygalacturonic acids by gel filtration chromatography. Oligogalacturonide with polymerization degree of over 12 showed the inducing activity. The is the first report that an endogenous polysaccharides are capable of inducing normal secondary metabolism in plant cells.