Network Pharmacology-Based Strategy for Elucidating the Molecular Basis Forthe Pharmacologic Effects of Licorice (Glycyrrhiza spp.)

Abstract

Licorice (Glycyrrhiza spp.) is used widely in traditional Chinese medicine (TCM) due to its numerous pharmacologic effects. However, the mechanisms of action of the chemical constituents of licorice and their structure-function relationships are not fully understood. To address these points, we analyzed the chemical compounds in licorice listed in the TCM Systems Pharmacology database and TCM Integrated database. Target proteins of the compounds were predicted using Integrative Pharmacology-based Research Platform of TCM v2.0. Information on the pharmacologic effects of licorice was obtained from the 2020 Chinese Pharmacopoeia, and disease-related genes that have been linked to these effects were identified from the Encyclopedia of TCM database. Pathway analyses using the Kyoto Encyclopedia of Genes and Genomes database were carried out for target proteins, and pharmacologic networks were constructed based on drug target-disease-related gene and protein-protein interactions. A total of 451 compounds were analyzed, of which 211 were from the medicinal parts of the licorice plant. The 241 putative targets of 106 bioactive compounds in licorice comprised 52 flavonoids, 47 triterpenoids, and seven coumarins. Four distinct pharmacologic effects of licorice were defined: 61 major hubs were the putative targets of 23 compounds in heat-clearing and detoxifying effects; 68 were targets of six compounds in spleen-invigorating and qi-replenishing effects; 28 were targets of six compounds in phlegm-expulsion and cough-suppressant effects; 25 compounds were targets of six compounds in spasm-relieving and analgesic effects. The major bioactive compounds of licorice were identified by ultra-high-performance liquid chromatography-quadrupole time-of-flight-tandem mass spectrometry. The anti-inflammatory properties of liquiritin apioside, liquiritigenin, glycyrrhizic acid and isoliquiritin apioside were demonstrated by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Liquiritin apioside, liquiritigenin, isoliquiritin, isoliquiritin apioside, kaempferol, and kumatakenin were the main active flavonoids, and 18α- and 18β-glycyrrhetinic acid were the main active triterpenoids of licorice. The former were associated with heat-clearing and detoxifying effects, whereas the latter were implicated in the other three pharmacologic effects. Thus, the compounds in licorice have distinct pharmacologic effects according to their chemical structure. These results provide a reference for investigating the potential of licorice in treatment of various diseases.

Keywords: flavonoid; licorice; network pharmacology; pharmacologic effects; triterpenoid.

FIGURE 1

Interaction network of chemical compounds containing licorice and the corresponding major targets associated with its heat-clearing and detoxifying effects. To investigate the mechanisms underlying the heat-clearing and detoxifying effects of licorice, a network was constructed based on direct interactions between major hubs that was divided into four functional modules. Purple hexagons represent chemical components, red hexagons denote representative the chemical components of licorice, yellow circles denote the core targets related to regulation of balance of inflammation and the immune system, and blue circles represent the core targets related to cellular functions. The pink circle represents the core target related to modulation of the nervous system, the orange circle represents the core target related to regulation of energy production and metabolism.

FIGURE 2

Interaction network of chemical compounds containing licorice and the corresponding major targets associated with spleen-invigorating and qi-replenishing effects. We wished to investigate the mechanisms underlying the spleen-invigorating and qi-replenishing effects of licorice. Hence, a major hub network was constructed based on the direct interactions between major hubs that were divided into four functional modules. Purple hexagons represent chemical components, pink circles denote core targets related to regulation of balance of inflammation and the immune system, blue circles represent core targets related to nutrition and energy production, green circles denote core targets related to regulation of blood circulation, and the orange circle represents the core target related to modulation of the nervous system.

FIGURE 3

Interaction network of chemical compounds containing licorice and the corresponding major targets associated with phlegm-expulsion and cough-suppressant effects. We wished to investigate the mechanistic basis of the phlegm-expulsion and cough-suppressant effects of licorice. A major hub network was constructed based on the direct interactions between major hubs that was divided into three functional modules. Purple hexagons represent chemical components, pink circles denote core targets related to regulation of balance of inflammation and the immune system, light purple circles represent core targets related to modulation of the nervous system, and orange circles denote core targets related to nutrition and energy production.

FIGURE 4

Interaction network of chemical compounds containing licorice and the corresponding major targets associated with spasm-relieving and analgesic effects. We wished to investigate the mechanisms associated with the spasm-relieving and analgesic effects of licorice. Hence, a network was constructed based on the direct interactions between major hubs that were divided into three functional modules. The purple hexagon represents the chemical composition, the pink circle denoted the core target related to analgesic action, the blue circle represents the core target related to nutrition and energy production, and the yellow circle denoted the core target related to regulation of blood circulation.

FIGURE 5

Representative base peak intensity (BPI) chromatograms of licorice derived from UHPLC–QTOF–MS/MS.

FIGURE 6

Two-dimensional chemical structures of the bioactive compounds in licorice.

FIGURE 7

Reduce effects of the main bioactive compounds of licorice on the LPS-induced production of cytokines and chemokines. The supernatants were collected for measuring the levels of IL-1β (A), TNF-α (B) by ELISA. Data are presented as the mean ± standard error of the mean (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001 vs. Con group; #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 vs. LPS group. Liquiritin apioside, LA; Liquiritigenin, LIQ; Isoliquiritin apioside, IA; Glycyrrhizin, GLY; IL, interleukin; LPS, lipopolysaccharide; TNF, tumor necrosis factor.

FIGURE 8

Reduce effects of the main bioactive compounds of licorice on the PI3K/AKT/NFκB signaling pathway. The levels of PI3K, AKT and NF-κB proteins were detected by Western blotting(J). The relative expression of p-PI3K(A), p-AKT1(D), p-NFκB-p65(G) protein and ratio of p-PI3K/PI3K(B), p-AKT1/AKT1(E), p-NFκB-p65/NFκB-p65(H) and total PI3K(C), AKT1(F) and NFκB-p65/NFκB-p65(I) protein were quantified. Data are presented as the mean ± standard error of the mean (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001 vs. Con group; #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 vs. LPS group. Liquiritin apioside, LA; Liquiritigenin, LIQ; Isoliquiritin apioside, IA; Glycyrrhizin, GLY; LPS, lipopolysaccharide.

FIGURE 9

Proportion of different chemical structure types of licorice in various pharmacologic effects.