Endogenous Inducer of secondary Metabolism in Lithospermum erythrorhizon Cell Cultures

紫草细胞在Linsmaier-Skoog(LS)液体培养基中不能合成紫草素，但在含有一定酸性多糖(琼脂、琼脂、多聚半乳糖醛酸苷)的LS液体培养基中开始产生紫草素。这些结果表明某些内源多糖在紫草素生物合成中起重要作用。通过离子交换(QAE-Sephadex A-25，0.1-2MKHCO3)和凝胶过滤(Sephadex GL6B，0.1MKHCO3)层析，从M-9培养的紫草素产生细胞的细胞壁碎片中分离得到紫草素产生多糖组分，测定其诱导紫草素活性的方法如下：在不含有效成分的LS液体培养基中加入等量紫草素，用高效液相色谱法分析紫草素的产生。对紫草素的活性成分进行甲基化分析和核磁共振谱分析表明，紫草素的活性成分主要由α-1，4-半乳糖醛酸(96-97%)和1，2-鼠李糖(3-4%)组成。根据还原端基与总糖的比例计算出聚合度约为18。聚半乳糖醛酸的部分水解物通过凝胶过滤层析得到聚合度为1~15的低聚半乳糖醛酸内酯。聚合度大于12的低聚半乳糖醛酸内酯具有诱导活性。这是首次报道内源多糖能够诱导植物细胞正常的次生代谢。

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.