**3. Survey of active components in licorice**

The above active EtOAc fraction was bioassay‐guided separated by silica gel column chroma‐ tography with a CHCl3‐MeOH solvent system as an eluent to give five fractions (1–5). Fraction 2 was further purified using a reversed‐phase C18 column eluting with MeOH‐H2O to yield active compound **1**. Fraction 4 was also purified by the same way of fraction 2 to give active compound **2**.

Compound **1** was a yellow powder indicating an ion peak at *m*/*z* 257 [M+H]+ in mass spec‐ trometry having strong yellow fluorescence. From these evidences, compound **1** was supposed to be a non‐conjugated flavonoid like a chalcone derivative, which has strong fluorescence in general. Finally, compound **1** was determined to be isoliquiritigenin (**Figure 4**) [31] by 13C nuclear magnetic resonance (NMR), 1‐dimensional (1D; 1 H and 13C) and 2D correlation spectroscopy (COSY), heteronuclear multiple quantum coherence (HMQC) and heteronuclear multiple‐bond coherence (HMBC) spectroscopies.

that licorice root contains various bioactive components. However, they may not affect gene expression because transcription rates in spermatozoa are low [30]. Our findings indicate that components in licorice other than glycyrrhizin, or together other components and glycyrrhizin,

In order to confirm the property of active component in licorice, the crude extract was fractionated with EtOAc and *n*‐BuOH successively. **Figure 3** showed the fertilization level indicated by the percentage of two‐cell embryos describing that the EtOAc fraction was more effective compared to that of *n*‐BuOH fraction. From this result, active components might be not conjugated compounds like saponins including glycyrrhizin but free compound like

**Figure 3.** Active fraction of licorice extract and glycyrrhizin against IVF. *n*‐BuOH (1.6 mg/mL) and EtOAc (1 mg/mL) fractions of licorice and glycyrrhizin (1.6 mg/mL) were added to the conditioned medium. Activity was observed in the

The above active EtOAc fraction was bioassay‐guided separated by silica gel column chroma‐ tography with a CHCl3‐MeOH solvent system as an eluent to give five fractions (1–5). Fraction 2 was further purified using a reversed‐phase C18 column eluting with MeOH‐H2O to yield active compound **1**. Fraction 4 was also purified by the same way of fraction 2 to give active

trometry having strong yellow fluorescence. From these evidences, compound **1** was supposed to be a non‐conjugated flavonoid like a chalcone derivative, which has strong fluorescence in general. Finally, compound **1** was determined to be isoliquiritigenin (**Figure 4**) [31] by 13C

in mass spec‐

Compound **1** was a yellow powder indicating an ion peak at *m*/*z* 257 [M+H]+

can improve the IVF rates without damaging fertilized eggs.

110 Biological Activities and Action Mechanisms of Licorice Ingredients

flavonoids as indicated in **Figure 1** depending on their solubility.

EtOAc fraction containing flavonoids.

compound **2**.

**3. Survey of active components in licorice**

**Figure 4.** The structures of isoliquiritingenin with key COSY (bold lines) and HMBC correlations (dash arrows).

Compound **2** was a pale yellow powder indicating an ion peak at *m*/*z* 268 [M+H]+ that the mass fragment pattern resembled to a flavonoid compound having a methoxyl group in a molecule. 13C NMR showed a typical lower‐shifted C‐3 carbon (124.9 *m*/*z*) suggesting that compound **2** might be an isoflavone. The spectra of 1 H, 13CNMR and 2D (COSY, HMQC and HMBC) confirmed that compound **2** was formononetin [32] (**Figure 5**).

**Figure 5.** The structures of formononetin with key COSY (bold lines) and HMBC correlations (dash arrows).

#### **4. Confirmation of IVF activity for two isolated components**

The HTF medium containing PVA and MBCD with isoliquiritigenin or formononetin at a concentration range from 0 to 0.04 mg/ml was tested for IVF [33]. The optimal concentration of isoliquiritigenin was found at 0.02 mg/ml resulting in 47.2 ± 16.8% of IVF ratio. In the case of forononetin, the ratio was slightly higher (50.2 ± 9.5%) but not significantly, than that of isoliquiritigenin as indicated in **Figure 6**.

**Figure 6.** Optimal concentration of licorice extract for IVF. Black and gray bars indicated isoliquiritigenin and formono‐ netin, respectively.

We examined the viability of embryos treated with isoliquiritigenin (**Figure 7A**) or formono‐ netin (**Figure 7B**) to confirm that both embryos were morphologically normal similar to the previous observations using licorice crude extract [28].

**Figure 7.** Morphology of blastocysts incubated with isoliquiritigenin or formononetin.
