Artificial insemination is an indispensable technology for cattle breeding and is used for treating infertility in humans. Thus, new or improved methods are needed to increase the efficiency of artificial insemination. In vitro fertilization (IVF) has been developed in mice. Although many mouse lines produced using IVF have been preserved by freezing embryos and/or fertilized eggs, the more efficient IVF using freezing preservation or long‐term refrigeration of sperm is expected to preserve mouse lines more easily. In this chapter, we introduce the active compounds in licorice to improve the rate of IVF. We previously reported that the rate of IVF in mice was improved by adding a water extract of licorice (Glycyrrhiza uralensis), but not glycyrrhizin, to the artificial insemination culture medium. Recently, we analyzed the active ethyl acetate fraction containing high levels of flavonoids. This fraction was further purified by bioassay‐guided separation to isolate isoliquiritigenin and formononetin, which contributed to the improved rate of IVF. Isoliquiritigenin and formononetin may be useful therapeutic agents for infertility treatment.
- in vitro fertilization
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 immunosorbent assay (ELISA) as quick, simple and reproducible assay system . Furthermore, a new staining system, eastern blotting was developed  and immunoaffinity isolation of glycyrrhizin 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  and eastern blotting  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  related to fertilization using reproducing assay system, we have started to survey
In vitrofertilization of mouse sperm by licorice crude extract and fraction
Although previous studies have investigated whether licorice can increase pregnancy rates or not, the clear result has not been found yet . 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 , a speculation of relationship between licorice and testosterone and/or estrogen comes out. In fact Hajirahimkhan et al. searched three licorice species,
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 . However, we found that the licorice crude extract improved the fertilizing ability of BALB/cA mouse sperm
Park et al. reported that licorice extract increased cyclophosphamide teratogenicity and upregulated the mRNA expression of cytochrome P‐450 2B in rats . The results suggest that licorice root contains various bioactive components. However, they may not affect gene expression because transcription rates in spermatozoa are low . 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
3. Survey of active components in licorice
The above active EtOAc fraction was bioassay‐guided separated by silica gel column chromatography 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
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 . 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.
We examined the viability of embryos treated with isoliquiritigenin (Figure 7A) or formononetin (Figure 7B) to confirm that both embryos were morphologically normal similar to the previous observations using licorice crude extract .
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 . It became evident that approximately 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 fertilization 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.
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.
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 . The glycyrrhizin‐knockout extract contains all components except glycyrrhizin. Thus, glycyrrhizin‐knockout extract can function for promoting fertilization Figure 9.
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