正交法优选调控秦艽次生代谢产物含量变化的条件

Optimum conditions for regulating content change of secondary metabolites of Gentiana macrophylla by orthogonal method

关键词:  正交试验, MVA途径, MEP途径, 洛伐他汀, 膦胺霉素, 环烯醚萜

Key words:  orthogonal test, MVA pathway, MEP pathway, lovastatin, fosmidomycin, secoiridiod glycosides

摘要:

为优选可抑制秦艽次生代谢产物含量变化的条件,该文采用3因素4水平正交试验设计方法,共设计16组处理,研究洛伐他汀(MVA途径抑制剂)、膦胺霉素(MEP途径抑制剂)和取样天数对秦艽中马钱苷酸、獐牙菜苷、獐牙菜苦苷、龙胆苦苷4种主要环烯醚萜类化合物含量的影响。结果表明:(1)4种环烯醚萜类化合物含量变化受取样天数影响最大,其次为膦胺霉素浓度,次之为洛伐他汀浓度。(2)以最佳抑制条件处理后,马钱苷酸、獐芽菜苦苷、龙胆苦苷和獐芽菜苷含量分别下降了69%、36%、33%和4%。基于正交法优选的抑制条件,对4种化合物均可抑制。综上所述,可确定调控秦艽次生代谢产物含量变化的最佳抑制条件为膦胺霉素400 μmol·L^-1,洛伐他汀50 μmol·L^-1,取样天数6 d,该条件为进一步研究MEP和MVA途径在环烯醚萜类化合物代谢合成中的调控机制奠定基础。

Abstract:

Gentiana macrophylla is a traditional Chinese herbal medicine for treating jaundice, hepatitis, and stomachic and choleretic ailments, and its main secondary metabolites are secoiridiod glycosides represented by gentiopicroside. The biosynthetic pathway of gentiopicroside is via methylerythritol phosphate pathway(MEP)and mevalonate pathway(MVA)pathways. Fosmidomycin and lovastatin are specific inhibitors of 1-deoxy-D-xylulose-5-phosphate reductoisomerase(DXR)of the MEP pathway and hydroxymethylglutaryl-CoA reductase(HMGR)of the MVA pathway, and are widely used in the research of the biosynthesis pathway and regulation mechanism of secondary metabolites. The article aims to test and search for the optimal condition for decreasing the contents of four secoiridiod glycosides compounds(loganic acid, sweroside, swertiamarin and gentiopicroside)in G. macrophylla by using the orthogonal test designs. In this study, the concentration of fosmidomycin(A), concentration of lovastatin(B)and sampling days(C)were taken as three factors, and each factor was taken as four levels to design an orthogonal test, with 16 groups of treatment. HPLC was used to determine the contents of four secoiridiod glycosides, and the data were statistically analyzed. The results were as follows:(1)The contents of the four secoiridiod glycosides compounds in G. macrophylla were most affected by sampling time, followed by fosmidomycin concentration and then lovastatin concentration.(2)After treatment with the optimal inhibitory conditions, the contents of loganic acid, swertiamarin, gentiopicroside and sweroside decreased by 69%, 36%, 33% and 4% respectively. In conclusion, optimal inhibition conditions are determined to be fosmidomycin 400 μmol·L^-1, lovastatin 50 μmol·L^-1, samples are harvested on six days, which provides the reference for further research on the regulatory mechanism of MEP and MVA pathways in the metabolism and synthesis of secoiridiod glycosides.