**2.** *In vitro* **fertilization of mouse sperm by licorice crude extract and fraction**

herb medicines in traditional Chinese medicine (TCM) for the treatment and prophylaxis of sore throats (as an antitussive), coughs and bronchial catarrh (as an expectorant), inflammation, allergic reactions, rheumatism and arthritis, liver disease, tuberculosis and adrenocorticoid insufficiency [1–5]. Nearly 500 components have been identified in licorice. Among them, glycyrrhizin is controlled at a concentration of more than 2.0% as a standardization marker component because the phamacological properties of licorice might be depend upon glycyr‐ rhizin, and furthermore a flavonoid liquiritin has a quantitative limitation in Japanese pharmacopoeia [1]. Glycyrrhizin is a protein‐kinase inhibitor with anti‐ulcer and anti‐viral activities [6]. In Japan and China, nowadays, glycyrrhizin is used for the treatment of hepatitis [4, 7–9]. It has been reported that glycyrrhizin has interferon‐inducing activity and anti‐HIV‐ 1 activity [10–12]. Moreover, glycyrrhizin is well known as a natural sweetener and used in food additives and cosmetics [13]. On the other hand, flavonoids have also various pharma‐ cological activities such as anti‐hepatotoxic, anti‐inflammatory, anti‐ulcer, anti‐allergenic and anti‐viral as well as cardioprotective activities [2]. **Figure 1** indicates major components in

108 Biological Activities and Action Mechanisms of Licorice Ingredients

In order to control the quality of licorice, we investigated and succeeded the preparation of monoclonal antibody (MAb) against glycyrrhizin and set up an enzyme‐linked immunosorb‐ ent assay (ELISA) as quick, simple and reproducible assay system [14]. Furthermore, a new staining system, eastern blotting was developed [15] and immunoaffinity isolation of glycyr‐ rhizin resulting in glycyrrhizin‐knockout extract, which can be used to survey the real activity of glycyrrhizin in licorice [16, 17]. Regarding flavonoid, anti‐liquiritin MAb [18] and eastern blotting [19] was developed. Since the above knowledge of components in licorice have been accumulated and also Tanaka et al. have been investigating about proteins [20–22] and mitochondria [23] related to fertilization using reproducing assay system, we have started to

survey *in vitro* fertilization (IVF) active components in licorice.

licorice.

**Figure 1.** Major components in licorice.

Although previous studies have investigated whether licorice can increase pregnancy rates or not, the clear result has not been found yet [24]. However, since it is known that glycyrrhizin acts as the modulator of 11β‐hydroxysteroid dehydrogenase (Type 1 and 2) which are the enzyme related to steroidal hormone [25], a speculation of relationship between licorice and testosterone and/or estrogen comes out. In fact Hajirahimkhan et al. searched three licorice species, *G. uralensis*, *G. glabra* and *G. inflata*, and found that isoliquiritigenin indicated strong estrogen‐like activity, suggesting that this compound may be cyclized to liquiritigenin, which is an active flavonoid, under physiological conditions [26].

The sperm from wild‐type C57BL/6 mice has high capacity for fertilization when cultured in standard medium (e.g., HTF) supplemented with bovine serum albumin for IVF or with polyvinyl alcohol (PVA) and methyl β‐cyclodextrin (MBCD). On the other hand, IVF efficiency using the sperm of aged BALB/cA mice (>48 weeks of age) is low, albeit fluctuated between mice [27]. However, we found that the licorice crude extract improved the fertilizing ability of BALB/cA mouse sperm *in vitro* as indicated in **Figure 2** and that the fertilized eggs developed normally [28]. We performed five separate experiments using five different BALB/cA mice. The fertilization rate for HTF medium containing PVA plus MBCD and licorice extract (0.12 mg/ml) was between 15.6 and 84.0% (average: 43.0 ± 11.0%). On the other hand, the fertilization rate was between 0.0 and 43.5% (average: 15.0 ± 7.8%) when licorice extract‐free medium was used. The optimal concentration of licorice extract in HTF medium was 0.3 mg/ml.

**Figure 2.** Influence of licorice extract on the IVF. Sperm from BALB/cA mice was preincubated in conditioned medium with or without licorice extract (0.12 mg/mL). Two‐cell embryos were effectively obtained with preincubation medium containing licorice extract. The extract had a significant effect on IVF (*n* = 5, *P* < 0.05).

Park et al. reported that licorice extract increased cyclophosphamide teratogenicity and upregulated the mRNA expression of cytochrome P‐450 2B in rats [29]. The results suggest 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, can improve the IVF rates without damaging fertilized eggs.

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

**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 EtOAc fraction containing flavonoids.
