高产人参发根系的建立及发根中皂苷Rb_1的分离纯化

利用发根农杆菌A4菌株在2年生人参根外植体上直接诱导产生人参发根。对16个人参发根系比生长速率及人参总皂苷含量进行测定,得到3个具有较高比生长速率和人参皂苷含量的发根系。其中发根系R9923具有最高的比生长速率,在连续培养4周后生长量比最初提高了28.8倍。研究R9923发根系的生长曲线和皂苷含量变化规律发现,随着发根生物量的增加,发根中皂苷含量积累得也越多。利用HPLC法测定了R9923发根系中单体皂苷Rg1、Re、Rb1的含量。生长4周的人参发根总皂苷含量达15.2 mg/g,其中人参皂苷Rb1的含量为68.3%,比3年生栽培人参中Rb1含量高1.3倍,可以用于大规模培养。用硅胶柱层析法从人参发根总皂苷中分离提纯人参皂苷Rb1,当洗脱剂中氯仿∶甲醇=7.5∶2.5(体积比)时洗脱效果较好。用硅胶柱法提取的皂苷Rb1纯度为89.95%,得率为72.7%。

Selection of high-yield hairy root clone of ginseng and separation and purification of ginsenoside Rb1 from ginseng hairy root

Hairy roots of ginseng were directly inoculated from the root explants of two-year-old ginseng by Agro bacterium rhizogene A4.The specific growth rate and the ginsenoside content of sixteen kinds of hairy roots were measured,from which three high-yielding clones were selected.The highest-yielding clone was R9923 whose biomass increased by 28.8 times after four weeks culturing.The growth of the hairy root and the development of ginsenoside of clone R9923 were investigated and it was found that the ginsenoside content increases with the biomass of the hairy root.The contents of monomer ginsenoside Rg_1,Re and Rb_1 in clone R9923 were determined by HPLC method.The total content of ginsenoside in the hairy root after four weeks culturing came up to 15.2 mg/g,in which 68.3% was Rb_1.The content of Rb_1 in the hairy root was 2.3 times of that of three-year-old ginseng.The ginsenoside Rb_1 was separated and purified from the hairy root by silica gel column chromatography,in which the optimal solvent ratio of chloroform to methanol was 7.5∶2.5,the purity of Rb_1 was 89.95% and the yield was 72.7%.