**2. Findings from our research**

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 discussed below.

#### **2.1. Purification of licorice phenolics**

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 various types of phenolic constituents [17, 20].
