| Glycyrrhiza uralensis (G.uralensis) has been recognized as one of the most famous medicinal plants in traditional Chinese medicine for thousands of years. Due to recent years’ excessive harvest, wild resources of Guralensis were forbidden to collection. The content of glycyrrhizic acid in most of G.uralensis fails to meet the requirement of Chinese pharmacopoeia. Therefore, it is essential to improve the quality of G.uralensis cultivars. In the recent studies, people tried to explain the content accumulation mechanism of glycyrrhizic acid through origin, cultivation technique and genetics researches. There was little study about inner relation between secondary metabolites in G.uralensis. According to extensive studies, we found that the secondary metabolites in the plant were not isolated existence, and they formed as networks through nodes. Based on those researches, we posit that there is a chemical composition core control network in G.uralensis which can regulate the content of glycyrrhizic acid. Any synthesis pathway which has intermediate intersection with the glycyrrhizic acid pathway is likely to affect the synthesis of glycyrrhizic acid content.In order to study the interaction relationship between secondary metabolites in Glycyrrhiza uralensis, and find out which secondary metabolite was significantly related to the content of glycyrrhizic acid, high content components modeling methods was used to establish high content environment. Ultrahigh-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) and High-performance liquid chromatography (HPLC) methods were used to analyse the content of glycyrrhizic acid, liquiritin, liquiritigenin, isoliquiritigenin, gibberellic acid (GA3), abscisic acid (ABA), jasmonic acid (JA), indole-3-acetic acid (IAA) and salicylic acid (SA). After trend and statistical analysis, most relevant factors were tured out. ABA and liquiritin were used to artificial applying in order to confirm the interaction relationship between glycyrrhizic acid and other secondary metabolic products. In that case, glycyrrhizic acid, liquiritin and ABA were all influence each other. It would be helpful for confirming the exsistance of chemical composition core control network in Guralensis which can regulate the content of glycyrrhizic acid.The following conclusions were drawn in this paper:(1) An efficient simplified method was developed to determine multiple classes of phytohormones simultaneously in the medicinal plant G.uralensis. UPLC-ESI-MS/MS with multiple reaction monitoring (MRM) in negative mode was used for quantification. Only 100 mg of fresh leaves was needed, with onepurification step based on C18 solid-phase extraction (SPE). Cinnamic acid (CA) was chosen as the internal standard instead of isotope-labeled internal standards. Under the optimized conditions, the five phytohormones with internal standard were separated within 4 min, with good linearities and high sensitivity. HPLC method was used to analyse the content of glycyrrhizic acid, liquiritin, liquiritigenin, isoliquiritigenin. Both of them obtained the favourable proof results.(2) It was feasible to establish high glycyrrhizic acid environment by 72 h glycyrrhizic acid root soaking or 45 d glycyrrhizic acid root spraying in the concentration of 1.0 mmol·L-This method was verified by ABA and liquiritin spraying in G.uralensis. It could save the manpower and material resources on naturally selecting plants which had high content. It also provides a new way to improve the content of glycyrrhizic acid and breed good varieties of G.uralensis.(3) The change of secondary metabolites was analyzed within 72 h and 45 d after glycyrrhizic acid stimulation, while correlation statistical software analyzing the correlation of glycyrrhizic acid and other compositions. After high concentrations of glycyrrhizic acid (1.0 mmol·L-1) stimulating, the content of liquiritigenin up 79.3% whith 72 h while ABA up 293% during 48 to 72h. When it came to 45 d glycyrrhizic acid (1.0 mmol·L-1) stimulating, the content of liquiritigenin up 185% whith 45 d while ABA up 369% whith 15 d. It turned out that there was significant positive correlation between glycyrrhizic acid-liquiritin, glycyrrhizic acid-liquiritigenin, glycyrrhizic acid-isoliquiritigenin and glycyrrhizic acid ABA. The most relevant factor was ABA for phytohormone group and liquiritin for chemical component group.(4) By using different concentrations of ABA spraying on leaves, the change of the secondary metabolites was analyzed within 45 d after ABA stimulation. It turned out that in some sense the content of glycyrrhizic acid and liquiritin had improved within 45 d, especially for liquiritin. After high concentrations of ABA (3.96 mg·L-1) stimulating, the content of glycyrrhizic acid up 52% while liquiritin up 392% within 30 d. There was significant positive correlation between ABA-glycyrrhizic acid and ABA-liquiritin, which verified the interaction relationship between glycyrrhizic acid, ABA and liquiritin.When using liquiritin spraying on roots, the most obvious impacts were glycyrrhizic acid ABA and liquiritigenin. After high concentrations of liquiritin (0.05 mmol·L-1) stimulating, the content of glycyrrhizic acid up 22% while ABA up 327% within 7 d. The content of liquiritigenin showed a decline within 45 d, even after the low concentrations of liquiritin (0.01 mmol·L-1) stimulating decreased 45% within 45d, more than after high concentrations stimulating decreased 36%. There was significant positive correlation between liquiritin-glycyrrhizic acid and liquiritin-ABA, which also verified the interaction relationship between glycyrrhizic acid, ABA and liquiritin.(5) After high concentrations of ABA (3.96 mg·L-1) stimulating, the content of a* up 29.7% while b* up 27.7% within 45 d. When it came to high concentrations of liquiritin (0.05 mmol·L-1) stimulating, the content of a* up 14.6% while b* up 15.3% within 45 d. Color index values of a* and * were all higher than the control group within 45 d, significantly, which meant the color of powders turned toward red and yellow. The conclusion was that ABA (3.96 mg·L-1) or liquiritin (0.05 mmol·L-1) stimulating not only could improve the quality in the traditional sense through the color of G. uralensis, but also in the modern sense by improving the content of glycyrrhizic acid and liquiritin.Based on the high glycyrrhizic acid content modeling methods, this study confirmed that glycyrrhizic acid, ABA and liquiritin were positively correlated, which further verified the hypothesis. There was a chemical composition core control network in G. uralensis. It would lay theoretical basis for improving the content of glycyrrhizic acid by applied ABA or liquiritin on G. uralensis. |
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