Approximately one-fifth of the general population complains of dyspeptic symptoms, such bloating, anorexia, early satiety, and epigastric discomfort (Ford et al., 2015). Eighty percent of them cannot be explained either structurally or organically, and these symptoms are referred to as functional dyspepsia (FD) (Ford et al., 2020). FD accounts for 11–29.2% of the global prevalence of these symptoms, with differences among countries (Mahadeva and Goh, 2006). The economic burden of FD is estimated to be over 18 billion dollars per year in the United States (Lacy et al., 2013).

FD is diagnosed by symptom-based criteria, and the Rome IV criteria were most recently revised in 2016 (Stanghellini et al., 2016). FD is generally divided into three subtypes depending on the main symptoms: postprandial fullness and early satiety (postprandial distress syndrome, PDS), epigastric pain/burning symptoms (epigastric pain syndrome, EPS), and a combination of these symptoms (Asano et al., 2016). In Asia, the PDS subtype is known to be more prevalent than the EPS or combination type, especially in Korea and Japan (Lee and Chua, 2012).

The main causes of FD generally include abnormal gastrointestinal (GI) motility, visceral hypersensitivity, oversecretion of gastric acid, and Helicobacter pylori infection (Enck et al., 2017). Based on these etiological factors, prokinetic drugs, such as ghrelin receptor agonists, serotonin (5-hydroxytryptamine, 5-HT) receptor agonists, muscarinic receptor antagonists, proton pump inhibitors (PPIs), and H. pylori eradicating drugs are used to treat patients with FD (Ford et al., 2020). However, these treatments have clinical limitations, such as a high recurrence rate after cessation-, for example, the recurrence rate after cessation of acotiamide, a prokinetic, therapy, is a half (Shinozaki et al., 2020), and unexpected adverse effects, such as an increased risk of myocardial infraction and risk of ventricular arrhythmias after long-term use of PPIs or cisapride (a 5-HT receptor agonist) (De Maeyer et al., 2008; Shah et al., 2015).

On the other hand, herbal medicine has been used as a treatment option for patients with GI disorders. One study reported that approximately one-third of surveyed patients with functional GI disorders used herbal medicines (Lahner et al., 2013). Many patients with FD have chosen herbal products from traditional Korean medicine (TKM) and traditional Chinese medicine (TCM). Many studies have shown the therapeutic effects of multiherbal decoctions in clinical trials (Kim et al., 2014; Kim et al., 2021) and animal experiments (Jeon et al., 2019; Tan et al., 2020). In particular, the pharmacological theories of TKM and TCM emphasize the synergistic actions of multiherbal combinations for the treatment of multifactorial disorders, such as FD (Liu et al., 2013; Kim et al., 2021).

Yeokwisan, a standardized Korean herbal formula, is composed by six herbs including Poncirus trifoliata Rafinesque (P. trifoliata), Scutellaria baicalensis Georgi (S. baicalensis), Glycyrrhiza uralensis Fischer (G. uralensis), Massa medicata Fermentata, Phyllostachys bambusoides Sieb. et Zucc (P. bambusoides), and Ostrea gigas Thunberg (O. gigas). It has been prescribed for patients suffering from refractory FD including gastroesophageal reflux disease (GERD) in clinic since 2016. However, data about the mechanism of action this formula are yet not available. Accordingly, we aimed to evaluate and explore the effects and underlying mechanisms of this formula in vivo using a loperamide-induced FD mouse model.

To investigate the pharmaceutical potential of Yeokwisan in the treatment of FD and its underlying mechanisms, we herein used a loperamide-induced FD mouse model. Loperamide, a μ2-opioid receptor agonist, suppresses the activity of the GI myenteric plexus, which decreases the tone of the circular and longitudinal smooth muscles of the GI tract (Katzung et al., 2004; Chen et al., 2012). Clinically, loperamide is used to treat diarrhea, and its adverse effects include abdominal pain, nausea, dyspepsia, and constipation (Hanauer, 2008). Thus, high doses of loperamide have been used to establish animal models of FD and constipation in preclinical studies (Jeon et al., 2019; Li et al., 2021).

As expected, our study showed that a single dose of loperamide injection (10 mg/kg, peritoneally) notably delayed both gastric emptying and intestinal transit (Figures 2, 3). Moreover, the administration of Yeokwisan (especially 200 and 400 mg/kg Yeokwisan) significantly attenuated this delayed gastric emptying, as evidenced by stomach weight, extended stomach volume and quantification of the amount of phenol red retained in the stomach (Figures 2A–E). These effects were very similar to those of mosapride, a positive control agent in the present study. 5-HT and its receptors are involved in the regulation of smooth muscle contraction, and 5-HT₄R agonists such as mosapride are the main options for the treatment of functional disorders with impaired GI motility (Yang et al., 2017). In our results, both Yeokwisan and mosapride significantly restored the loperamide-induced downregulation of 5-HT₄R gene expression in stomach tissue (Figure 5C). Abnormally suppressed gastric motility is one of the main causes of FD, and it has a negative impact, particularly on the PDS type of FD compared to the EPS type (Tack et al., 1998; Tack et al., 2002). Approximately 30% of all FD patients and 66% of all PDS patients showed delayed gastric emptying that was related to symptoms of postprandial fullness and early satiety and sometimes to gastroparesis-like symptoms, including vomiting and nausea (Talley et al., 2001; Sarnelli et al., 2003).

On the other hand, ghrelin, called a “hunger hormone”, has attracted attention as a key player in GI motility and as a therapeutic target for FD treatment (Yagi et al., 2013). Several studies found that ghrelin levels were significantly lower in patients with FD than in healthy volunteers (Takamori et al., 2007; Lee et al., 2009b). Both clinical studies (Murray et al., 2005; Binn et al., 2006) and animal studies (Trudel et al., 2002; Qiu et al., 2008) have shown that ghrelin binds to its receptor, GHSR, which consequently leads to the promotion of gastric peristalsis and passage of a meal. Ghrelin cells (G cells) of the stomach produce ghrelin in the form of des-acyl ghrelin, which is then converted into the active form (acyl-ghrelin) by ghrelin O-acyltransferase (GOAT) before being released into the blood (Yagi et al., 2013). As expected, loperamide injection drastically lowered the plasma level of acyl-ghrelin, while the reduction of acyl-ghrelin was significantly normalized by Yeokwisan (especially 400 mg/kg) (Figure 6A). This effect was also supported by the expression levels of genes related to the production of ghrelin (Figure 6B) and its acylation enzyme (GOAT) in stomach tissue (Figure 6C). The production and secretion of ghrelin is stimulated by various hormones, such adrenaline and somatostatin, which bind to receptors of the G cell membrane (Engelstoft et al., 2013). We confirmed that Yeokwisan activated a representative excitatory receptor (ADRB1) but suppressed an inhibitory receptor (SSTR) related to ghrelin production (Figure 6C).

It is well-known that GI motility is a continuous and repetitive process of segmental and peristaltic contractions and relaxations, in which interstitial cells of Cajal (ICCs) play a central role as pacemakers that generate electrical slow waves then transfer the these slow wave to the smooth muscle cells (Sanders et al., 2014). Some clinical studies have suggested that dysfunction or loss of ICCs causes abnormal GI motility-related diseases, including FD (Forster et al., 2005; Farrugia, 2008). To explore the involvement of ICCs in the gastric emptying effect of Yeokwisan, we evaluated the protein expression level of C-kit, a representative parameter of ICCs, in stomach tissue. As expected, administration of Yeokwisan significantly normalized the loperamide-induced loss of C-kit signal as shown by immunohistological findings and protein assays (Figures 4A–D). In fact, ghrelin binds to its receptor (GHSR) on ICCs and sequentially activates ICCs-derived electrical slow waves, as evidenced by in vivo and in vitro experiments (Yang et al., 2014; Kim and Kim, 2019). We also found that Yeokwisan upregulated the gene expression of GHSR in stomach tissue (Figure 6C). Slow wave generation and delivery by activated ICCs are regulated by several key molecules; ANO1 and RYR3 increase Ca²⁺ concentrations inside ICCs and generate intracellular Ca²⁺ waves, respectively (Sanders et al., 2006; Sanders et al., 2012). Then, the generated Ca²⁺ waves deliver electric signals to neighboring smooth muscle cells via gap junctions, which causes the contraction of smooth muscle by active smMLCK (Garcia et al., 1997). Yeokwisan administration attenuated the loperamide-induced changes in the expression of above-mentioned genes in stomach tissue (Figure 5C). In addition, regarding the relaxation process of smooth muscle in the GI tract, the nNOS-mediated production of nitric oxide (NO) is vitally important, as NO is retrograde neurotransmitter in synapses of vagus nerves (Terauchi et al., 2005). In clinical and animal studies, the administration of the NOS inhibitor, N^G-nitro-L-arginine methyl ester, delayed gastric emptying, and nNOS^−/- mice also exhibited delayed gastric emptying (Plourde et al., 1994; Tack et al., 2002; Micci et al., 2005). In our study, Yeokwisan treatment significantly restored the loperamide-induced downregulation of nNOS protein expression in stomach tissue (Figures 5A,B). These results suggest that the pharmacological function of Yeokwisan is mainly associated with the modulation of ghrelin and ICCs activity in stomach tissue.

On the other hand, Yeokwisan did not improve intestinal transit as different from stomach (Figures 3A,B). These effects of Yeokwisan was very intriguing because prokinetics, such as mosapride, cisapride and acotiamide, generally work on the whole GI tract, both stomach and intestine (Tack et al., 2019). These results might indicate the not involvement of μ2-opioid receptor as the main mechanisms of Yeokwisan, because loperamide is an agonist of μ2-opioid receptor, suppressing the motility both in intestine and stomach (Chen et al., 2012). In the present study, we found Yeokwisan only promotes gastric motility via ghrelin-related pathway, but not intestinal transit (Figures 2, 3, 6). So, we propose the μ2-opioid receptor might not be markedly changed by Yeokwisan. In our study, mosapride accelerated the transit in the stomach (Figure 2) and small intestine (Figure 3). In our previous study, an herbal formula, Banha-sasim-tang, also promoted motility in both the stomach and small intestine (Jeon et al., 2019). Nevertheless, Yeokwisan changed potential GI motility-related proteins and genes as a different pattern between stomach and intestine. Briefly, Yeokwisan did not recover the GI motility-related proteins (C-kit and nNOS, Figures 4E–H, 5D,E) and genes (5-HT₄R, ANO1, RYR3, and smMLCK, Figure 5F) expression, ghrelin-related genes (GHSR and GOAT, Figure 6D) expression in intestine, rather than stomach. As we known, G cells dominantly exist in stomach (Sakata and Sakai, 2010). Our data also showed that Yeokwisan significantly elevated ghrelin gene expression in stomach as compared to loperamide-treated group (Figure 6B). Therefore, it was proposed that ghrelin might be a key modulator on prokinetic effects in stomach by Yeokwisan. From the perspective of diarrhea-inducing adverse effects or clinical limitations of prokinetics (Quigley, 2015; Tack et al., 2019), these pharmacological characteristics of Yeokwisan may indicate the proper applicable spectrum of FD patients. Both FD and irritable bowel syndrome (IBS) are functional GI disorders (FGIDs) that are frequently comorbid. According to one study, 32% of patients with FD had overlapping IBS, and 37% of patients with IBS had FD symptoms (Suzuki and Hibi, 2011). IBS includes two major subtypes, the constipation-dominant or diarrhea-dominant type, and 16.6% of patients with diarrhea-dominant type (but 11% of patients with the constipation type) also had FD (Choi et al., 2017). In present study, Yeokwisan promote the gastric motility via modulation of GI-motility-related molecules (Figures 2, 4A–D, 5A–C), not in the small intestine (Figures 3, 4E–H, 5D–F). Because of these gastric-specific properties of Yeokwisan, it may be further beneficial for FD patients with overlapping diarrhea-type IBS.

Due to the clinical limitations of currently available drugs for FD including the relatively high recurrence rate after cessation of 51% (Shinozaki et al., 2020), herbal resources have received attention in drug development (Tan et al., 2020). We herein proved the anti-FD activity of Yeokwisan, a standardized formula consisting of a six-herbal mixture including P. trifoliata, S. baicalensis, G. uralensis, Massa medicata fermentata, P. bambusoides, O. gigas (Table 1). Previous animal studies reported beneficial effects of two composing herbs, P. trifoliata, and S. baicalensis, on dysfunction of GI motility in atropine-induced (Lee et al., 2005), and ritonavir-induced (Mehendale et al., 2007) conditions, respectively. In particular, G. uralensis significantly improved the symptoms of patients with FD in a clinical study (Raveendra et al., 2012). According to the Korean herbal pharmacopoeia (Food and Adiministration, 2012), Yeokwisan has been standardized using six compounds: baicalein and baicalin from S. baicalensis, naringin and poncirin from P. trifoliata, and glycyrrhizic acid and wogonin from G. uralensis (Figures 1A–C). Among these compounds, baicalin and baicalein showed antidepressive effects in stress-induced depression rodent models (Lee et al., 2013; Liu et al., 2019). The depressive mood or dysregulation of 5-HT is well-known to inhibit peristatic movement in the GI tract (Kheder et al., 2018), while patients with FD complain of high comorbidity of depression (Esterita et al., 2021). Gastroprotective effects of naringin, wogonin and poncirin were also reported in gastric ulcer, mucosal damage, and gastritis rat models (Park et al., 2004; Lee JH. et al., 2009; Emin and Volkan, 2019). In particular, naringin improved delayed GI transit by activating ghrelin receptor in a laparotomy-induced rat model (Jang et al., 2013). These previous data support our beneficial effects of Yeokwisan.

Accordingly, we expected the synergistic effects of Yeokwisan, a mixture of six herbs on gastric emptying, may supporting the clinical relevance of this drug for treatment of patients with dyspepsia and GERD (Figure 7). Although we cannot explain the underlying mechanisms, we propose that the stomach-specific effect on peristaltic movement resulted from the mixture of multiple herbs or compounds, which modulate the expression of ghrelin-related genes (Ghrelin, GHSR, GOAT, ADRB1, and SSTR) only in the stomach (Figures 6B,C) but not in the small intestine (Figure 6D). The lack of identification of active compounds is a limitation of the present study. Further research is needed to address these issues. In addition, we have to further investigate the ideal formulation of these multi-herbal combinations to maximize stomach-specific activity and its underlying mechanisms.

Taken together, our present data showed the clinical effect of Yeokwisan on FD in a loperamide-induced functional dyspepsia mouse model. The main mechanisms corresponding to these effects may involve the modulation of the ghrelin pathway, including activation of ICCs in stomach tissue. In particular, we found that Yeokwisan improves gastric emptying via stomach-specific effects on GI motility.