丹参无菌培养体系构建及有效成分含量的HPLC分析

Construction of aseptic cultural system of Salvia miltiorrhiza and determination of active components by HPLC analysis

目的构建丹参无菌培养体系并进行无菌材料有效成分含量测定,为丹参快繁及次生代谢调控研究提供基础。方法利用正交实验筛选丹参叶片诱导愈伤组织的激素配比,并进行芽、根的诱导,建立组织培养体系;HPLC法分析有效成分含量。结果丹参愈伤组织诱导最佳培养基为MS+6-BA(2.0 mg/L)+NAA(1.0 mg/L)+2,4-D(0.5 mg/L),最适生芽培养基为MS+6-BA(2.0 mg/L)+NAA(1.0 mg/L),生根培养基为1/2 MS+NAA(0.5 mg/L)。丹参7种有效成分在无菌苗、愈伤组织和再生苗中均可检测到,含量有显著差异(P<0.05),其中迷迭香酸与丹酚酸B含量较高。结论成功建立了丹参愈伤组织培养体系并获得生长旺盛的再生苗;3种无菌材料有效成分积累存在差异,为丹参进一步研究奠定基础。

Objective The regeneration system of Salvia miltiorrhiza was constructed and contents of active components of the sterile materials were determined, so as to provide the basis for the rapid propagation and secondary metabolism regulation of S. miltiorrhiza. Methods The optimum hormone ratio for inducing callus was screened by orthogonal test. The buds and roots induction were conducted to estabilish the tissue culture system. The contents of effective components were evaluated by HPLC analysis. Results The suitable medium for callus induction was MS+6-BA(2.0 mg/L)+NAA(1.0 mg/L)+2,4-D(0.5 mg/L). The preferred enrichment medium of adventitious bud induction was MS+6-BA(2.0 mg/L)+NAA(1.0 mg/L). And rooting medium was 1/2 MS+NAA(0.5 mg/L). Seven active components in aseptic seedlings, callus, and regenerated seedlings could be detected with significant differences in different aseptic materials(P < 0.05). The contents of rosmarinic acid and salvianolic acid B were higher than the others. Conclusion The culture system of S. miltiorrhiza was successfully established, and vigor regenerated seedlings were also obtained. The accumulation of active components in the three sterile materials showed difference, laying a foundation for further study in S. miltiorrhiza.