**4.3.3 Mechanism underlying the inhibitory effect of the extract on melanosis**

The mushroom hot water ME remarkably inhibited mushroom PPO. Commercial L-ERT had the same effect on mushroom PPO. Mushroom PPO activity remarkably declined with increase in the concentrations of L-ERT (Fig. 14). PPO inhibition by the ME was also depended on L-ERT concentration. These results suggest that extracts containing ERT as one of their active compounds potently inhibit mushroom PPO activity.

*In vitro* experiments performed by Encarnacion et al. (2010) also showed that the transcript expression of the proPO genes in the HLS was lower than that in the L-ERT- and *p*amidinophenyl methanesulfonyl fluoride hydrochloride (*p*-APMSF)-treated HLSs (Fig. 15). The PPO activity in the L-ERT-treated HLS was also remarkably low. ERT could have been involved in inhibiting transcriptional factors in the cascade system, leading to a decrease in proPO gene expression. Maeda et al. (2007) reported that in human melanosis, hydroperoxy traxastane-type triterpene decreased the protein levels of PPO and its related proteins in B16 melanoma cells because of inhibition of the transcription factor melanocyte-type isoform of the microphthalmia-associated transcription factor, leading to a decrease in the PPO gene and its related genes. In the case of crustaceans, it is generally believed that the enzyme responsible for the activation of proPO to PPO is a serine protease, or commonly called as proPO-activating enzyme (PPAE). This enzyme is believed to be involved in the final step in

Fig. 14. Inhibitory effects of commercial L-ergothioneine (a), mushroom (*F. velutipes*) extract (b), and residues of diets used in the feeding trial (c) on the activity of mushroom polyphenoloxidase. Results are presented in terms of mean ± standard deviation (*n=3*). The values with different superscript letters represent significant difference (*p*<0.05) at the end of the reaction period (Encarnacion et al., 2010).

Fig. 15. Inhibition of the activation of the proPO system in hemocyte lysate supernatant by commercial L-ergothioneine (L-ERT) and *p*-amidinophenyl methanesulfonyl fluoride hydrochloride (*p*-APMSF). Polyphenoloxidase activity (a) and peptidase activity (b). 7 amino-4-methylcoumarin (AMC) was used as the standard solution in the peptidase activity assay. The final concentration of each inhibitor in the reaction system was 1 mM. Results are presented in terms of mean ± standard deviation (n = 3). The values with different superscript letters represent significant difference between groups for different storage periods (*p* < 0.05) (Encarnacion et al., 2010).

the proPO cascade leading to PPO activation and is itself tightly regulated (Buda & Shafer, 2005). The results of the peptidase activity assay indicated that L-ERT inhibited serine protease activity but that this effect was not as strong as that of *p*-APMSF (Fig. 16b). However, the presence of L-ERT could possibly affect the overall activation of the proPO system because it inhibited PPO activity (Fig.16a). At least 4 mechanisms underlying PPAE regulation have been recognized: gene induction, activation by another protease, requirement for noncatalytic serine protease homologues as cofactors, and inactivation by serine protease inhibitors (Wang & Jiang, 2004). Thus, other proPO activating factors such as

Fig. 16. Gene expression analysis of proPO transcripts in hemocyte lysate supernatant treated with commercial L-ergothioneine (L-ERT) and *p*-amidinophenyl methanesulfonyl fluoride hydrochloride (*p*-APMSF). Representative gel (a) and quantitative analyses of band intensities obtained using the ImageJ software (b). Elongation factor1-α gene (EF1-α) was used as the standard for computing the relative gene expression level for each sample band. The final concentration of the inhibitor in the reaction system was 1mM (Encarnacion et al., 2010).

serine protease homologues and other proteases could have been inhibited by L-ERT. The inhibition of PPO activity in the hemolymphs of the supplement-fed shrimp supports this phenomenon. Moreover, accumulation of ERT in the shrimp muscles by feeding or in the hemolymph by the immersion technique could directly inhibit PPO activity in the carapace during the postharvest period. The thiol (SH) group, present in compounds such as ERT, is a powerful nucleophile, that tends to chelate Zn2+ and Cu2+ (Park et al., 2006). The latent PPO activity of hemocyanin, a copper-binding protein, in whiteleg shrimp *Penaeus vannamei* has been shown to be involved in postmortem melanosis. The mechanism underlying the inhibitory effect of ERT could also be attributed to its Cu2+ chelating activity, leading to the inhibition of melanosis in ME-fed and ME-immersed shrimps and crabs.
