Endogenous Inducer of secondary Metabolism in Lithospermum erythrorhizon Cell Cultures

紫草（紫草科）细胞在Linsmaier-Skoog（LS）液体培养基中不合成红色萘醌色素紫草素，但转移到含有酸性多糖（琼脂、琼脂果胶或多聚半乳糖醛酸苷）的LS液体培养基中时，紫草细胞开始合成紫草素。从在M-9培养基中培养的紫草素产生细胞的细胞壁碎片中获得的多糖级分通过离子交换进行分级（QAE-Sephadex A-25，0.1 -2MKHCO_3）凝胶过滤（SephadexGL 6 B，0.1MKHCO_3）柱层析分离纯化，通过如下方法检测紫草素的诱导活性：将每一级分的等分试样加入到LS液体培养基中，其中在没有活性成分的情况下不产生紫草素，并通过HPLC分析所产生的紫草素。在30 μ g/ml的培养基中，发现最具活性的组分诱导紫草素的生物合成，而粗提物需要300 μ g/ml。甲基化分析和NMR数据显示，活性成分由α-1，4-连接的半乳糖醛酸（96-97%）和1，2-连接的鼠李糖（3-4%）组成。根据还原端基与总糖的比例计算聚合度约为18。通过凝胶过滤色谱法从聚半乳糖醛酸的部分水解产物中获得每种寡聚半乳糖醛酸苷（聚合度为1-15）。聚合度大于12的寡聚半乳糖醛酸苷具有诱导活性。这是首次报道植物内源多糖能够诱导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.