**2.2. Structural study on licorice phenolics exploring the diversity of their skeletons**

**1. Introduction**

bacteria.

cussed below.

**2. Findings from our research**

60 Biological Activities and Action Mechanisms of Licorice Ingredients

**2.1. Purification of licorice phenolics**

various types of phenolic constituents [17, 20].

Licorice (liquorice), the underground portion of *Glycyrrhiza* species, has been used as a remedy for various types of stress, inflammatory diseases, digestive organ disorders, and pain in traditional medicine in Asian and European countries [1, 2]. The main constituent, glycyrrhizin, and the associated aglycone, glycyrrhetinic acid, are also used in modern medicine, whereas the phenolic constituents have been implicated in promoting improved health, particularly with regard to stomach ulcers [2]. Therefore, several research groups have investigated the phenolic constituents of licorice [3] and found that it has beneficial effects for health, including antimicrobial properties [4, 5]. In this chapter, we summarize our studies on phenolic constituents and some of their pharmacological effects, including those linked to drug-resistant

Our research on licorice constituents began with an investigation of tannin-like substances in licorice, because tannins and related constituents in medicinal plants have remarkable antioxidant effects, in addition to their fundamental property of binding with proteins, which is related to its various pharmacological effects [6–8]. In fact, licorice extracts of various origins contain tannin-like substances and show protein-binding properties [9]; our additional studies revealed that some phenolic constituents related to flavonoids contribute to this property. Therefore, we investigated these flavonoids and related compounds as dis-

Although classic column chromatography using silica gel has been applied to the separation of phenolic plant constituents, the irreversible adsorption of phenolic constituents (particularly, tannins or tannin-like substances) has limited ability to effectively separate these compounds. Because countercurrent distribution (CCD) does not use solid supports for separation, it can be applied to solve the problem of irreversible adsorption. Thus, centrifugal partition chromatography (CPC) and droplet countercurrent chromatography (DCCC), which were devised as effective methods for CCD, in addition to simple CCD using separatory funnels, were applied to purify the licorice phenolics in our studies. The solvent system chloroform-methanol-water (7:13:8, by volume) was primarily used for the separation of licorice phenolics derived from *Glycyrrhiza inflata* [9, 10] and those derived from *G. uralensis* [11–16] in these CCD processes. Combinations of column chromatography on a silica gel, ODS-gel, and/or polystyrene gel (MCI-gel CHP-20P) with CCD also afforded satisfactory separation [17, 18]. High-performance liquid chromatography (HPLC) was applied for final purification and to establish the purity of the isolated compounds [18, 19]. However, the CCD systems using the solvent systems ethyl acetate-*n*-propanol-water, *n*-hexane-ethanol-water-ethyl acetate, and chloroform-methanol*n*-propanol-water, in addition to chloroform-methanol-water, were also useful for separating Although the structures of aforementioned licorice phenolics were characterized based on the 1 H and 13C nuclear magnetic resonance (NMR) spectra, including various 1D and 2D methods, the following spectroscopy methods were also key in establishing the structures. Electron impact mass spectrometry (EI-MS) is a useful method for obtaining structural information using fragment ions [16]. On the other hand, fast-atom bombardment (FAB) and electrospray ionization mass spectrometry (ESI-MS) are applicable to the ionization of phenolics, including phenolic glycosides. Notably, the high-resolution FAB and ESI-MS have been used to determine their molecular formulae [17]. Ultraviolet-visible (UV-Vis) spectroscopy was useful for discriminating between phenolic skeletons even if the <sup>1</sup> H NMR spectra were quite similar to each other, as was the case for 3-arylcoumarins and the corresponding isoflavones [16]. Electronic circular dichroism (ECD) spectroscopy was effective not only for identifying the configuration of asymmetric carbons (e.g., those in flavanones, isoflavans, and isoflavanones [9, 15, 17]) in the flavonoid skeletons but also for explaining the spatial relationship between the chromophores in acylated flavonoid glycoside molecules [17]. Based on the data obtained by the aforementioned spectroscopy methods, we uncovered new compound structures and identified known ones isolated from licorice, which can be classified into subgroups based on their structural skeletons as shown in **Table 1**.

As shown in **Table 1**, various types of phenolics have been found in licorice, in addition to the major phenolics (liquiritin, isoliquiritin, and related ones) [21], and their pharmacological properties differ depending on their structures. The strength of the order of the effects also differs depending on the properties examined. Especially, their phenolic hydroxyl and prenyl substituents and also their skeletons related to the molecular flexibility should be considered for their respective properties.

#### **2.3. Properties of licorice phenolics in relation to their health effects**

Polyphenols have been linked to antioxidant effects, and some polyphenols such as tannins have protein-binding effects. Interaction of tannins with protein molecules is regarded to be based on hydrophobic interaction and hydrogen bonding and also covalent bonding in some cases [22]. Although some researches focused on the participation of proline residues of proteins in the complexation [23], the modes of complexation are largely dependent on the structures of tannins and proteins/peptides [24–27]. Therefore, further studies using various types of polyphenols should be conducted in order to clarify the complexation. Thus, we examined the binding and antioxidant effects of licorice phenolics.
