A few reports examine the effects of the administration of rikkunshito on normal mice to increase appetite and acylated ghrelin production. Matsumura et al. measured blood acylated ghrelin concentration and gastric gene expression at 0.7–1.4% drinking water administration of rikkunshito to normal mice and showed a significant increase in the rikkunshito group compared with the saline-treated group (33). However, in our preliminary study, the amount of acylated ghrelin in the blood tended to increase 180 min after the administration of rikkunshito 1 g/kg by gavage (distilled water administration group, 55.7 ± 9.2 fmol/mL, vs. rikkunshito administration group, 72.3 ± 5.9 fmol/mL), and des-acyl concentration decreased (distilled water administration group, 729.6 ± 74.1 fmol/mL, vs. rikkunshito administration group, 407.1 ± 30 fmol/mL). It is considered that the difference in this result depends on the difference in administration period and administration method. Additionally, Matsumura et al. demonstrated that administration of rikkunshito 7.5 g/day to 21 healthy volunteers for 2 weeks significantly increased blood acylated ghrelin levels compared to that before administration (33). It is considered that the maintenance of physiological homeostasis related to the secretion of appetite-related hormones is strictly controlled. Therefore, the effect of a single dose of rikkunshito on healthy subjects may be limited. To affect normal ghrelin secretion function, rikkunshito may require continuous administration.

Table 1 summarizes the major indexes and ghrelin-promoting effects of rikkunshito on anticancer drug administration, organ removal, and cancer cachexia. Administering cisplatin to rodents results in reduced food intake. Peritoneal administration of cisplatin at 2 mg/kg significantly reduces rat feeding under fasting and free fed conditions up to 24 h. Gavage oral administration at a dose of 1 g/kg of rikkunshito was observed to significantly suppress the reduction in food intake due to cisplatin administration compared to saline-administered rats (11–13, 34, 35). Intraperitoneal administration of cisplatin 2 mg/kg decreases food intake and decreases blood acylated ghrelin levels and hypothalamic acylated ghrelin release 2 h after administration. Rikkunshito has been proven to abolish these declines (11, 12, 35). Similarly, rikkunshito suppressed the decrease in food intake in rats given intraperitoneally at a higher dose of cisplatin (6 mg/kg) (36). Thus, the effect of rikkunshito on ghrelin is very reproducible, and it has been proven that rikkunshito inhibits feeding reduction and an acylated ghrelin secretion-promoting effect in cisplatin-administered rats. Moreover, in cancer-bearing animals, experimental exposure to cancer cells induces cachexia, depending on the type of cancer and the duration of the experiment, resulting in a marked decrease in food intake and body weight. Additionally, a significant reduction in food intake and body weight is also observed in gastrectomized rats (37). An extreme decline in feeding leads to increased starvation and induction of signal abnormalities in peripheral appetite-promoting peptides, despite the lack of appetite in these animal models. Particularly, peripheral acylated ghrelin increases, and exogenous acylated ghrelin reactivity also decreases the so-called ghrelin resistance (13, 38). Rikkunshito administration to cancer cachexia rats is shown to improve the decrease in food intake and prolong life. However, rikkunshito did not further increase blood acylated ghrelin. Rikkunshito not only increases peripheral ghrelin but also stimulates ghrelin receptor signaling and stimulates feeding (13, 38). This mechanism of action of rikkunshito will be focused on in detail at the bottom.

In gastric cancer patients (39–41) and uterine cervical or corpus cancer patients (42), the efficacy of rikkunshito for ghrelin concentration and gastrointestinal dysfunction, including feeding, was evaluated. Ohno et al. reported that rikkunshito at a dose of 7.5 g/day suppressed the increase in oral intake and the decrease in acylated ghrelin due to cisplatin administration from the start of the combined administration of S-1 and cisplatin to patients with gastric cancer (39). Similarly, Takiguchi et al. (40) observed an increase in the acylated ghrelin ratio to total ghrelin at 4 weeks after administration of rikkunshito and an improvement in the Dysfunction after Upper Gastrointestinal Surgery for Cancer (DAUGS) and visual analog scale. These results clearly suggest that rikkunshito clinically promoted ghrelin secretion and suppressed feeding-related decline. However, in patients with proximal gastrectomy, weight gain and increase in the Gastrointestinal Symptom Rating Scale (GSRS) were noticed after administering rikkunshito. Still, they did not affect ghrelin levels (41). These findings suggest that rikkunshito improves gastrointestinal symptoms in patients with gastrectomy, but its effect on ghrelin has various results. This may be mediated by the fact that the main production site of ghrelin is the stomach. The conditions for collecting blood samples and the conditions for measuring acylated ghrelin may differ at each facility. Ohnishi et al. (42) found that rikkunshito (7.5 g/day) administration for 2 weeks in patients with cervical cancer improved appetite up to 2–6 days after paclitaxel administration and delayed onset 24–120 h later, and it significantly suppressed nausea and vomiting. However, acylated ghrelin did not change even after administration of an anticancer drug, and no effect was found by rikkunshito. Moreover, it was reported that there was no effect on nausea and vomiting by chemotherapy for lung cancer patients in the group taking rikkunshito 7.5 g/day for 7 days at the same time (43) but for anorexia. The 14-day administration of rikkunshito (7.5 g/day) inhibited the decrease in plasma acylated ghrelin, and the rate of decrease in calorie intake was lower in rikkunshito than in the control course (18 vs. 25%, P = 0.025) (44). Another researcher reported that the median rate of reduction in food intake was significantly lower with rikkunshito than without it (2 vs. 30%; P = 0.02) (45). Median acylated ghrelin increased significantly from day 3 to day 8 in patients on both courses with and without rikkunshito (9.6–15.7 fmol/mL, P < 0.0001; control, 10.2–17.8 fmol/mL, P = 0.0002). The rate of median increase in plasma acylated ghrelin levels between days 3 and 8 tended to be higher in the rikkunshito than in the control course (68 vs. 48%, P = 0.08). For delayed gastric emptying after pancreaticoduodenectomy, the 21-day administration of rikkunshito showed a significantly upregulating in total ghrelin and acylated ghrelin levels compared to preoperative, but no obvious effect on delayed gastric emptying was observed (46). It is believed that the effectiveness of rikkunshito will become clearer in future large-scale studies.

Blood adrenocorticotropic hormone (ACTH) and corticosterone on the HPA axis in the rodent model are used as indicators of the degree of an acute stress response. Previous studies have not investigated in detail whether rikkunshito inhibits the HPA axis. It was confirmed that the administration of rikkunshito to stress-loaded aged mice significantly decreased the increase in ACTH or corticosterone value (55). This indicates that rikkunshito may act in a suppressive manner on stress itself.

Yakabi et al. (32, 47) and Harada (49) demonstrated that the ICV administration of urocortin 1, which has a strong affinity for the CRF receptor, significantly reduces feeding behavior and abnormal movement of the upper gastrointestinal tract. The concentration of acylated ghrelin in the peripheral blood was significantly reduced, simultaneously, and the supplementation of acylated ghrelin to the urocortin-treated rat significantly improved this decrease in food intake. Urocortin-induced reduction of plasma ghrelin and food intake were restored by CRF2 receptor antagonist. Administration of rikkunshito to urocortin-treated rats significantly improved reduced food intake, abnormal gastrointestinal motility, and acylated ghrelin levels (32, 47, 49). The adrenergic α2 receptor was activated in urocortin-administered rats, and it was also found that rikkunshito contained a component with an antagonistic effect on the receptor (32, 49). However, the administration of an adrenergic β1 receptor agonist also improves urocortin-induced ghrelin lowering, but it is not confirmed whether or not rikkunshito contains a β1 agonist-like ingredients component.

Rodents are often bred and managed with 3–5 animals depending on the cage size. Acute stress can be induced by acclimating to this environment for about a week and then transferring to a completely new cage and bedding (28–30, 50). Novel environmental changes can cause mild and transient corticosterone increase and decreased feeding in mice. Additionally, the concentration of acylated ghrelin in the blood decreases at the same time as stress loading (28–30). Rikkunshito significantly reduced the decrease in food intake and reduction in blood acylated ghrelin concentration due to this novel environmental change stress (28–30). Since the decreased food intake and ghrelin secretion in this model are partially canceled by the administration of 5-HT_2BR or 5-HT_2CR antagonists, serotonin is involved in the decreased food intake in the brain and digestive organs (28–30). ICV administration of CRF1R antagonists mediates reductions in food intake and plasma acylated ghrelin secretion (29), suggesting that intracerebral CRF1R activation is the trigger for the onset of this model. Restraint stress is known as classical physical and mental stress. Restraint stress in mice causes dysfunction of the upper gastrointestinal tract motility and a decrease in acylated/des-acyl ghrelin ratio (48). Administration of rikkunshito to stress mice significantly improved gastric motor function abnormalities such as delayed gastric emptying and gastric motility index. It is speculated that these action by rikkunshito may have canceled the decreased feeding and ghrelin secretion deficiency due to stress.

There are few clinical evidences focusing on the efficacy of rikkunshito on stress and mental illness. Tominaga et al. found that taking rikkunshito (7.5 g/day) for 8 weeks in patients with proton pump inhibitor-resistant non-erosive reflux disease (NERD) represents the mental quality of life in patients with a low body mass index. It proved that the mental component summary (MCS) scores of the SF-8 was significantly improved by rikkunshito (2). Additionally, administration of docetaxel/5-FU/CDDP in patients with advanced esophageal cancer for 2 weeks with rikkunshito (7.5 g/day) significantly improved nausea, as well as sleep, mood, volition, daily living activity, and anxiety and greatly improved quality of life scores, including the feeling of anxiety (51). Although the results of these clinical trials do not show the direct anti-stress effect of rikkunshito, it led to the implementation of larger clinical trials and it is expected to find the usefulness of rikkunshito for gastrointestinal disorders and neuropsychiatric parameters due to stress loading.

Ghrelin release in the stomach and hypothalamus is negatively regulated by 5-HT_2BR and _2CR activation (11). Heptamethoxyflavone, hesperetin, nobiletin, tangeretin, and isoliquiritigenin, which are components of rikkunshito, have an antagonistic activity against 5-HT_2BR and _2CR in vitro. Additionally, these components, when administered alone in vivo, suppressed a decrease in blood ghrelin levels (11). Isoliquiritigenin has been confirmed to transfer to the brain after the administration of rikkunshito and may mediate the decrease in ghrelin secretion due to antagonism of 5-HT_2CR localized in the central nervous system (56).

It is suggested that stress-related hypophagia involves abnormalities in ghrelin kinetics mediated by CRF1 and adrenergic receptors, and stimulation of CRF1 and α receptors negatively regulates ghrelin secretion. Nobiletin and isoliquiritigenin antagonize the CRF1 receptor at IC50 values of 0.36 and 0.67 μmol/L, respectively (56). In addition, glycycoumarin has an IC50 value of 5–39 μmol/L for all subtypes (_A,B,C) of the α₂-adrenergic receptor (AR). The IC50 value of 6-shogaol, which is a component of Zingiberis rhizoma, is 25 μmol/L for α_2A-AR, 8-shogaol is 5–6 μmol/L for α_2A, α_2C-AR, and 10-gingerol is has an IC50 value of 5–31 μmol/L for α_2A, α_2B, α_2C-AR (32).

Previous findings have shown that rikkunshito promotes acylated ghrelin secretion, but it does not increase it beyond physiological secretion. Therefore, it was questioned whether this degree of action could improve the reduced food intake. Rikkunshito enhanced the binding to ghrelin in cells expressing GHS-R1a in vitro and further significantly increased [Ca²⁺] influx in the area under the curve by ghrelin. At the same time, it was discovered that the action was caused by the ingredients of rikkunshito, i.e., atractylodin (13). This finding means a new mechanism in which rikkunshito not only increases the blood acylated ghrelin concentration but also enhances the reactivity of acylated ghrelin with GHS-R1a, thereby increasing the ghrelin signal.

Another stimulating effect of the ghrelin signal has been studied and proposed. Rikkunshito mediates the production of cAMP through the inhibition of phosphodiesterase III against the adenylate cyclase-cAMP-PKA system involved in the ghrelin signal inhibitory effect of leptin via the PI3K-PDE pathway and the activation of ghrelin receptors (57). Further detailed research is required to determine how much this effect of rikkunshito affects the ghrelin signal.