br Discussion Here we focused on seven

Discussion Here, we focused on seven plant alkaloids extracted from yokukansan. These alkaloids individually inhibited 5-HT-mediated 5-HT3A and 5-HT3AB receptor currents weakly. Simultaneous administration of these alkaloids, however, inhibited the 5-HT3A and 5-HT3AB receptor currents strongly. These data indicate that yokukansan is a 5-HT3 receptor antagonist as a result of the sum of the effects of these alkaloids. This might be one mechanism for the disappearance of drug action after isolation of active ingredients from herbal medicines. Imamura et al. reported quantitative data for seven Uncaria hook-derived alkaloids in blood pp2 after a single oral administration of yokukansan to rats (4.0 g/kg, p.o.). Geissoschizine methyl ether, rhynchophylline, isorhynchophylline, corynoxeine, hirsutine, and hirsuteine were detected in plasma at 9.0 ng/ml (24.6 nM), 10.8 ng/ml (28.1 nM), 0.7 ng/ml (1.8 nM), 14.5 ng/ml (37.9 nM), 1.6 ng/ml (4.3 nM), and 6.0 ng/ml (16.4 nM), respectively (Imamura et al., 2011). The plasma concentration of each alkaloid required to antagonize the 5-HT3 receptor seemed to be much lower than the above levels. However, some 5-HT3 receptor antagonists, tend to accumulate in specific areas (e.g., the intestinal wall) rather than in blood plasma (Chen et al., 2015, Fujiwara, 2011, Looby et al., 1997, Nakamura et al., 2013, Nakamura et al., 2011). Moreover, simultaneous administration of each alkaloid strongly inhibited the 5-HT3 receptor current, while single administration did not. It is, therefore, the total local concentration of the mix of alkaloids that inhibits the 5-HT3 receptor current. We focused only on the identified alkaloid components in Uncaria hook of yokukansan; however, yokukansan is composed of seven herbal medicines, Bupleuri Radix, Atractylodis Lanceae Rhizoma, Hoelen, Angelicae Radix, Cnidii Rhizoma, and Glycyrrhizae Radix in addition to Uncaria hook. These other yokukansan constituents may also contain 5-HT3 receptor inhibitors. Simultaneous administration of these seven alkaloids inhibited the 5-HT-mediated 5-HT3A and 5-HT3AB receptor currents in a similar manner. However, inhibition of the 5-HT3A receptor current was significantly stronger compared with that of the 5-HT3AB receptor current after individual application of isocorynoxeine and isorhynchophylline. In addition, inhibition of the 5-HT3AB receptor current was significantly stronger compared with that of the 5-HT3A receptor current after application of hirsuteine (Fig. 3). The biophysical properties of 5-HT3A and 5-HT3AB receptors are different. The 5-HT3AB receptor exhibits large single-channel conductance, low permeability to Ca2+, and a linear current–voltage relationship (Davies et al., 1999). Expression of the 5-HT3AB receptor might differ among individuals because polymorphisms in the promoter region of the 5-HT3B subunit affect its expression (Tremblay et al., 2003). Some antagonists inhibit 5-HT current differently depending on the receptor subunit composition (Thompson, 2013), which indicates that antagonistic effects may differ depending on the individual (Ho and Gan, 2006). Our results showed that the inhibition of 5-HT3A and 5-HT3AB receptors was almost identical upon simultaneous application of alkaloids. Treatment using a single effective drug might cause inter-individual differences, but a combination of weak-efficacy drugs could reduce the possibility of individual differences. Yokukansan has been approved by the Ministry of Health, Labour and Welfare of Japan as a remedy for neurosis, insomnia, and irritability in children. Safety was evaluated in patients administered yokukansan for >6 months (Okahara et al., 2012). However, the safety of long-term oral dosing of 5-HT3 receptor antagonists has not been ascertained (Olver et al., 2011). Yokukansan might be a new 5-HT3 antagonist with minimal side effects compared with the 5-HT3 receptor antagonists used widely as anti-emetic agents.