**5. Licorice in future**

It is considered that the decrease of population is now a serious problem in advanced countries depending on diversification of course of life (late marriage, unmarried, etc.). Carlsen et al. reported interesting evidence related to human semen [34]. It became evident that approxi‐ mately 30% decrease of sperm number during 40 years from 1950 to 1990 occurred. From this data, it suggested that the number of sperm was half in 2005 compared to that in 1950. This evidence may be deeply related to the decrease of population. In order to increase the fertili‐ zation ratio, the activation of sperm is necessary against the decrease of sperm number and also the mechanism of increase of IVF by isoliquiritigenin or formononetin should be evident.

We investigated the incorporation and distribution of isoliquiritigenin or formononetin in mice sperm in order to obtain some information related to the mechanism. **Figure 8** showed the fluorescence staining in mice sperm using fluorescence microscope. Staining distributed in almost all part of sperm. It seems to be that this phenomenon might be related to activation of sperm although the mechanism is still unknown and should be solved from now.

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

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

It is considered that the decrease of population is now a serious problem in advanced countries depending on diversification of course of life (late marriage, unmarried, etc.). Carlsen et al. reported interesting evidence related to human semen [34]. It became evident that approxi‐ mately 30% decrease of sperm number during 40 years from 1950 to 1990 occurred. From this data, it suggested that the number of sperm was half in 2005 compared to that in 1950. This evidence may be deeply related to the decrease of population. In order to increase the fertili‐ zation ratio, the activation of sperm is necessary against the decrease of sperm number and also the mechanism of increase of IVF by isoliquiritigenin or formononetin should be evident. We investigated the incorporation and distribution of isoliquiritigenin or formononetin in mice sperm in order to obtain some information related to the mechanism. **Figure 8** showed the fluorescence staining in mice sperm using fluorescence microscope. Staining distributed in

previous observations using licorice crude extract [28].

112 Biological Activities and Action Mechanisms of Licorice Ingredients

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

netin, respectively.

**5. Licorice in future**

**Figure 8.** Fluorescence imaging of mice sperm in the preincubation medium with isoliquiritigenin (Iso) and formono‐ netin (For). Signals were observed intensely in postacrosomal region (star) and mid piece (arrow).

Licorice used to be used for approximately 70% of TCM formula. From this current status, the pseudohyperaldosteronism often occurred by over uptake of licorice. It is well known that this phenomenon was occurred by taking of much glycyrrhizin. Glycyrrhizin is hydrolyzed to give glycyrrhetic acid (aglycone) by enteric bacteria, resulting the reabsorption of glycyrrhetic acid. This compound is changed to mono glycoside in liver. Glycyrrhetic acid monoglycoside inhibits 11β‐hydroxysteroid dehydrogenase (HSD), which catalyzes the conversion of cortisol to cortisone resulting in the accumulation of cortisol which reacts to aldosterone receptor.

**Figure 9.** The inhibition of 11β‐hydroxsteroid dehydrogenase (HSD) by glycyrrhetic acid monoglycoside inhibits in liver.

This phenomenon activates the reabsorption of sodium ion, and on the other hand potassium ion is eliminated inducing pseudohyperaldosteronism. Therefore, if we take too much licorice extracts for promotion of fertilization, such disease may be occurred. Previously, we purified glycyrrhizin from the crude extract of licorice using an immunoaffinity column conjugated with anti‐glycyrrhizin monoclonal antibody, and prepared glycyrrhizin‐knockout extract [17]. The glycyrrhizin‐knockout extract contains all components except glycyrrhizin. Thus, glycyr‐ rhizin‐knockout extract can function for promoting fertilization **Figure 9**.
