The ethanol tinctures of 121 Kampo remedies were obtained from Dr. Peter Lepke (Kronen Apotheke Wuppertal, Germany). Tinctures were made by extracting the plant material in ethanol (1:5 w/v). The tinctures used in this study are listed in Supplementary Table S1. All of the chemicals were purchased from either Sigma–Aldrich (5-HT hydrochloride, gamma-aminobutyric acid, tannic acid, schizandrin, schizandrin B, leonurine, boldine, berberine chloride, liquiritigenin, hesperetin, kaempferol, andrographolide, linderane, rosmarinic acid, 4-hydroxybenzaldehyd, chlorogenic acid, caffeic acid), Carl Roth (rutin) or PhytoLab (atractylenolide III).

The expression plasmid pRc/CMV contained cDNA coding for the human 5-HT_3A protein (Invitrogen; Lankiewicz et al., 1998), and psGEM contained cDNA for the α1, β2, and γ2 GABA_A receptor subunits (Sergeeva et al., 2010). cRNAs were prepared using the AmpliCap T7 high-yield message marker kit (Epicenter, Madison, WI, USA), following the manufacturer’s protocol. Oocytes were obtained as previously described (Sherkheli et al., 2010), and 7–20 ng receptor coding cRNA was injected into the oocytes using an injection setup from WPI (Nanoliter 2000, Micro4). The injected oocytes were then stored in ND 96 (96.0 mM NaCl, 2.0 mM KCl, 1.8 mM CaCl₂, 1.0 mM MgCl₂, 5.0 mM HEPES, pH 7.2, 200 U/ml penicillin, 200 μg/ml streptomycin) at 14°C. Electrophysiological experiments were performed 1–5 days (5-HT_3A) or 2–3 days (GABA_A) after the cRNA-injections.

To investigate the heterologously expressed 5-HT_3A and GABA_A receptors, the two-electrode voltage-clamp technique was used as described previously (Saras et al., 2008). All of the recordings were performed in a normal frog ringer’s (NFR) buffer (115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl₂, 10 mM HEPES, pH 7.2) for the GABA_A receptor. Currents were recorded at a holding potential of approximately -60 mV (5-HT_3A receptor) or -40 mV (GABA_A receptors), using the software Cell Works 6.1.1. (NPI). During the measurements, the oocytes were placed in a chamber with a constant and unidirectional ringer flow, allowing the washout of the tested substances and tinctures. The ringer flow was interrupted for the application of test substances and tinctures until the maximal response was transcended. To exclude solvent effects, the compounds were tested at the maximal applied concentration (0.1 Vol.-%). Neither ethanol nor DMSO exhibited a direct activation or modulatory effect on the 5-HT_3A and GABA_A receptors at the examined concentration (data not shown). In addition, the action of the tinctures on non-injected oocytes was tested (1:1,000 dilution; n = 3). None of the tinctures exhibited a direct activating effect with currents >15 nA (Supplementary Table S2). The observed effects were negligible compared with typical agonist-induced currents (∼2 μA). The direct activating effects of the Kampo tinctures were examined using a 1:1,000 dilution. The diluted tinctures were applied to Xenopus laevis oocytes expressing either the 5-HT_3A or α1β2γ2 GABA_A receptor and compared the evoked responses with the response from 5 μM 5-HT or 100 μM GABA, respectively (n = 3–8). For the investigation of the modulatory effects, the tinctures were applied in a 1:1,000-dilutuion witch contains the native agonists (n = 3–8). Concentrations of 10 μM GABA for the GABA_A receptor and 5 μM 5-HT for the 5-HT_3A receptor were used. To identify the active compounds in tinctures with antagonistic and potentiating effects, the known ingredients of plants with previously identified potential bioactivity were tested at a concentration of 1 mM. This approach was successfully used to identify pharmacologically active plant ingredients in a previous study (Herbrechter et al., 2015).

The currents of the tested substances/tinctures were normalized to the means of the reference compounds 5-HT and GABA for the 5-HT_3A and α1β2γ2 GABA_A receptors, respectively. Sigmoidal regression analysis was performed using the 3- or 4-parameter Hill equation (SigmaPlot 8.0., SPSS) to fit concentration-effect curves and calculate the IC/EC₅₀ values. Deviations are represented by the standard error of the mean (SEM). Significant differences were determined using Student’s t-test (Excel 2010, Microsoft), and multiple comparisons were corrected using the Benjamini–Hochberg-correction (^∗p < 0.05; ^∗∗p < 0.005; ^∗∗∗p < 0.0005).

First, the direct activating effects of the tinctures were investigated. The tincture of Ligusticum striatum (rhizoma) showed the strongest activation of the 5-HT_3A receptor, with more than 30% of the 5-HT-induced current (Figure 1; Supplementary Table S2). The strongest activator of the GABA_A receptor was Panax ginseng (rhizoma; Ginseng white), with a mean current amplitude of more than 40% of the GABA-induced current. The strongest 12 direct activating tinctures are shown in Figure 1.

Next, the modulatory effects of the tinctures at a 1:1,000 dilution was evaluated. In these experiments, agonist concentrations of 10 μM GABA for the GABA_A receptor and 5 μM 5-HT for the 5-HT_3A receptor were used. A variety of tinctures with modulatory action for both receptor types were identified. For the 5-HT_3A receptor, tinctures with significant antagonistic and potentiating effects were identified (13 tinctures with potentiating effects and 22 tinctures with inhibitory potential). The strongest potentiation was observed with Chaenomeles speciosa (fructus), which potentiated the 5-HT-induced response with only 37% (Figure 2). In contrast, eight tinctures [Tetradium ruticarpum (fructus), Magnolia biondii (flos), Lonicera japonica (caulis), Paeonia × suffruticosa (cortex), Coptis chinensis (radix), Epimedium brevicornum (herba), Leonurus japonicus (herba), and Lindera aggregata (radix)) exceeded 50% inhibition of the receptor, with a maximal inhibition of more than 98% by the Lindera aggregata (radix) tincture. In addition, the GABA_A receptors were significantly potentiated by 14 tinctures and inhibited by 24 tinctures. The Syzygium aromaticum (flos), Clematis armandii (caulis), Magnolia officinalis (cortex), Mentha canadensis (herba), Scutellaria baicalensis (radix), and Panax ginseng (rhizoma; Ginseng red) tinctures exhibited the strongest potentiation. In particular, Ginseng red potentiated up to 135% (Figure 3). The most effective inhibitory tinctures were Salvia miltiorrhiza (radix), Caesalpinia sappan (lignum), Terminalia chebula (fructus), Gentiana macrophylla (radix), Saposhnikovia divaricata (radix), and Schisandra chinensis (fructus). The inhibition observed by the Salvia miltiorrhiza (radix) tincture exceeded an inhibition of 80% of the GABA-induced currents.

For several of the active tinctures, the active ingredients have already been identified. However, for many of the Kampo herbs that have been shown to act on the 5-HT_3A or GABA_A receptor, the active ingredients are unknown. The investigated substances and their related plants are listed in Table 1 and Supplementary Table S3.

The modulatory effects of these substances on both receptors are shown in Figure 4. Overall, alkaloid compounds were found to be the most effective antagonists. In particular, boldine [Lindera aggregata (radix)] and leonurine [Leonurus japonicus (herba)] each possessed a higher efficacy for the 5-HT_3A receptor than the GABA_A receptor. These compounds were the most potent 5-HT_3A receptor antagonists in the screening (Figure 4). The alkaloid berberine inhibited both receptor types at approximately 60%. Another identified 5-HT_3A receptor antagonist is the flavonoid (-)-liquiritigenin (Herbrechter et al., 2015), which showed nearly no effect on GABA_A receptors in this screen. All of the tested flavonoids inhibited 5-HT_3A receptors with higher efficacy than GABA_A receptors. However, many of the substances showed inhibitory potential rather than potentiating effects on the receptors, with the exception of schizandrin B, which slightly potentiated the GABA_A receptor. Within the phenolic compound category, tannic acid was identified as an antagonist of both receptor types, with an inhibition rate of more than 80%. Andrographolide and the lignane schizandrin were identified as weak GABA_A receptor antagonists.

To quantify the antagonism of the identified active ingredients, concentration-effect curves were generated (Figure 5; Supplementary Table S4).

For the 5-HT_3A receptor, boldine (IC₅₀ = 0.53 ± 0.15 μM) and leonurine (IC₅₀ = 2.17 ± 0.15 μM) were more potent inhibitors than tannic acid (IC₅₀ = 48.2 ± 4.1 μM) and schizandrin (IC₅₀ = 137 ± 22 μM). These four substances had approximately equal IC₅₀ values for the GABA_A receptor (∼100 μM, Supplementary Table S4), with the exception of schizandrin (IC₅₀ > 600 μM). Andrographolide (IC₅₀ = 66.1 ± 26.8 μM) was found to be the most potent GABA_A receptor antagonist. However, it also acted as a partial antagonist of the GABA_A receptor, as even a concentration of 3 mM reduced the GABA-evoked current to 30% of the GABA reference (Figure 5B). Leonurine shifted the EC₅₀ for 5-HT at the 5-HT_3A receptor from 2.5 μM determined in parallel under the same experimental conditions in our laboratory (Herbrechter et al., 2015) to 14.8 μM. While 30 μM leonurine almost completely blocked the evoked current at lower agonist concentrations, it was nearly ineffective at 5-HT concentrations ≥100 μM, suggesting a competitive antagonism of leonurine at the 5-HT_3A receptors.

During the screening for compounds that are pharmacologically active at the 5-HT_3A receptor, a number of remedies with antagonistic properties were identified. Several of these remedies, such as ginger [Zingiber officinalis (rhizoma)], have well-described antiemetic effects and are used for the treatment of nausea and gastrointestinal disorders (Ernst and Pittler, 2000; Haniadka et al., 2012; Ding et al., 2013). During our screen, rhizoma from Zingiber off. sicc. and Zingiber off. vir. exhibited antagonistic effects, and the effect of Zingiber off. sicc. (desiccated) was superior to that observed with the tincture of fresh rhizoma from Zingiber off. vir. This increased inhibition may be due to the manufacturing process of the tinctures, as a higher amount of dry matter was used for the Zingiber off. sicc. tincture, and this may have resulted in an increase in the concentration of the active ingredients within the tincture. Vanilloids from the gingerol and shogaol group of compounds, pungent substances of ginger, are well-known 5-HT_3A receptor antagonists. This may explain, in part, the observed inhibition of 5-HT-induced currents that have been reported previously (Walstab et al., 2013; Ziemba et al., 2015). In addition, terpenes within the essential oil of ginger, including geraniol, citronellol, linalool and galanolactone, were shown to antagonize the 5-HT_3A receptor (Huang et al., 1991; Ziemba et al., 2015). The antagonistic potential of Panax ginseng (radix) tinctures may be mediated by ginsenosides, which act in the pore region of the 5-HT_3A receptor, and its antiemetic properties have been previously described (Min et al., 2003; Kim et al., 2005; Lee et al., 2007). The Magnolia biondii (flos), Mentha canadensis (herba), Glycyrrhiza uralensis (radix) and Syzygium aromaticum (flos) tinctures, which are also reported to have antiemetic effects (World Health Organization, 2005; Bhowmik et al., 2012; Herbrechter et al., 2015), were among the top 20 inhibitory tinctures in the screening. Syzygium aromaticum (flos) contains the phenolic compounds eugenol, gallic acid, and tannins, as well as the flavonoid quercetin (Atawodi et al., 2011; Bhowmik et al., 2012). Instead of eugenol (the main flavoring ingredient in Syzygium aromaticum), which shows a negligible level of antagonism (Ziemba et al., 2015), tannic acid inhibits 5-HT-evoked currents with an IC₅₀ value of approximately 50 μM (Figure 5). In addition to tannic acid, quercetin may contribute to the antagonistic effect of the clove tincture (Lee et al., 2008). Glycyrrhiza uralensis (radix) contains a structurally related flavonoid, liquiritigenin, which has been shown to antagonize the 5-HT_3A receptor in a previous study. This finding may help us to understand the antagonistic effect of this tincture (Herbrechter et al., 2015). Another flavonoid from licorice, named glabridin, was identified as a partial antagonist of the 5-HT_3A-receptor and a potentiator of GABA_A-receptors (Herbrechter et al., 2015; Hoffmann et al., 2016). The antagonistic effect of Mentha canadensis (herba) may be explained through the action of terpenes, which are known to inhibit the 5-HT_3A receptor. Menthol, a terpene from the essential oil of Mentha canadensis (herba), was recently characterized as a 5-HT₃ receptor antagonist (Walstab et al., 2014). In addition, Coptis chinensis rhizomes (Tjong et al., 2011) and Lonicera japonica (flos; Jung et al., 2014) may be useful for the treatment of gastrointestinal disorders through the action 5-HT₃ receptors, as discussed below.

The most potent inhibitory effects were observed for the tinctures of Lindera aggregata (radix) and Leonurus japonicus (herba; Figure 2; Supplementary Table S2) for which no connection to gastrointestinal disorders is described in the literature. By screening known constituents of these plants, boldine (Lindera) and leonurine (Leonurus) are found as potent inhibitors of the 5-HT_3A receptor with an IC₅₀ between 0.5 and 2.3 μM (Figures 5 and 6). Boldine, which has been shown to ameliorate chemotherapy-induced nausea and vomiting as well as symptoms from irritable bowel syndrome, is an alkaloid from the aporphine class. Boldine has been previously shown to antagonize the 5-HT_3A receptor in a competitive manner in a luminescence-based cell assay (Walstab et al., 2014). The contribution of further ingredients, such as the related alkaloids norboldine, reticuline, and linderegatine, to the antagonistic effect of the Lindera tincture is likely (Han et al., 2008). Leonurine from Leonurus was shown to affect cardiac function via creatine kinase inhibition (Wang et al., 2004) and to also reduce platelet aggregation (Lin et al., 2007). These effects are in accordance with some of the traditional uses of Leonurus. The antagonistic effects of Leonurus and the identification of leonurine as a potent competitive 5-HT_3A receptor antagonist are reported here for the first time. Therefore, this study reveals new insights into the pharmacological action of Leonurus. However, the traditional application of Leonurus is not correlated with the pharmacology of the 5-HT₃ receptor (Shang et al., 2014). Therefore, further studies examining if Leonurus can antagonize the 5-HT_3A receptor in vivo are needed.

The GABA_A receptors are inhibitory receptors and therefore reduce neuronal excitability. A variety of disorders are attributable to an imbalance of the GABAergic system. Therefore, benzodiazepines, such as diazepam, potentiate the GABA-induced responses and are used to treat disorders such as insomnia, restlessness, anxiety, and epilepsy (Calcaterra and Barrow, 2014).

In this screening, the potentiating activity of the tinctures correlated well with the medical use of the plants. For many of the top 10 potentiation tinctures, the active ingredients had been previously identified. The best potentiator, Panax ginseng (rhizoma; Ginseng red) is likely one of the most popular herbal drugs. It is traditionally used for multiple purposes, including the treatment of anxiety and insomnia (Kim et al., 2005; Xiang et al., 2008; Lee et al., 2013). The ginsenosides (steroid glycosides) present in this compound may be responsible for the potentiation and hence the sedative and anxiolytic effects of ginseng, due to their potentiating effect on the GABA_A receptor. The ginsenosides Rg₃ and Rc have been shown to potentiate the heterologously expressed GABA_A receptor, and ginsenoside Rc also possesses agonistic properties toward the GABA_A receptor (Choi et al., 2003; Lee et al., 2013). This may explain the observed direct activating effect of the extract (Figure 1). Two Panax ginseng (radix) tinctures were also tested, which differ in their manufacturing process. Whereas Ginseng red is desiccated directly after harvesting, Ginseng white is peeled and whitened prior to the desiccation process. Despite these manufacturing differences, both compounds were of the best nine potentiating tinctures. However, Ginseng red is superior to Ginseng white due to its increased potentiation (135% instead of 30%; Figure 3; Supplementary Table S2).

The Scutellaria baicalensis (radix) tincture potentiated the GABA_A receptor at a similar level to Panax ginseng (rhizoma; Ginseng red; Figure 3; Supplementary Table S2), and it was also previously linked to GABA_A receptor-related effects. Scutellaria baicalensis (radix) possesses anticonvulsant, sedative and anxiolytic effects, which may be explained by the action of the flavone wogonin (Hui et al., 2002; Park et al., 2007). Wogonin was identified as a ligand for the benzodiazepine site of the GABA_A receptor and was shown to potentiate GABA_A receptors in electrophysiological assays (Hui et al., 2002). The observed anxiolytic effects of wogonin in mice were similar to that of diazepam, emphasizing the potency of wogonin (Hui et al., 2002). The Mentha canadensis (herba) and Magnolia officinalis (cortex) tinctures also exhibited a potentiating effect in the screening (Figure 3; Supplementary Table S2). The active components of Magnolia officinalis (cortex) are the lignans magnolol and its isomer honokiol, whose potentiation of the GABA_A receptor is accountable for the anticonvulsant, antidepressive, and anxiolytic effects of Magnolia bark (Taferner et al., 2011; Alexeev et al., 2012). The potentiation of the GABA_A receptor by the Mentha tincture likely occurs due to the action of menthol and the structurally related monoterpenoids, which were shown to potentiate GABA_A receptors in a previous study (Hall et al., 2004). In addition, extracts of Morus alba displayed anxiolytic effects under different experimental paradigms, suggesting that this compound may also act on the GABA_A receptor (Yadav et al., 2008). Our results support this idea, as Morus alba (folia) extracts belonged to the top 10 most potent GABA_A potentiators. However, the active component in Morus alba (folia) extract is still unknown.

The sixth-strongest potentiation was observed by the clove tincture [Syzygium aromaticum (flos)], which has anti-inflammatory, antimicrobial, antioxidant, and anesthetic effects (Chaieb et al., 2007). In vivo, eugenol, the main ingredient of the clove tincture, exhibits a sedative effect in mice and rats (Guenette et al., 2006; Sharma et al., 2012) and has also been shown to potentiate the effects of the GABA_A receptor in vitro (Aoshima and Hamamoto, 1999). Other potentiators were also identified, including carvacrol and thymol (Priestley et al., 2003; Kessler et al., 2014). In addition to eugenol, Syzygium aromaticum (flos) contains carvacrol and thymol (Priestley et al., 2003; Kessler et al., 2014), and all three are potentiators of the GABA_A receptor. The mutual action of these compounds may explain the sedative effects of Syzygium aromaticum (flos).

Antagonism of the GABA_A receptor is typically accompanied by stimulating effects at low doses but can cause seizures and anxiety at higher concentrations. Kampo remedies with antagonistic effects were detected, including the tinctures of Salvia miltiorrhiza (radix), Terminalia chebula (fructus), and Schisandra chinensis (fructus; Figure 3; Supplementary Table S2). However, we could not find an anxiogenic or seizure-inducing adverse side effect for these antagonistic tinctures in the literature. The Schisandra chinensis (fructus) tincture, which inhibited 50% of the GABA-induced current at a 1:1,000 dilution (Figure 3; Supplementary Table S2), possesses side effects such as restlessness and tension, when given at high doses (Eberhard, 2003). These effects may be explained, at least in part, by its antagonism of the GABA receptor. Nevertheless, Schisandra chinensis (fructus) is traditionally used for its hepatoprotective and sedative effects; the latter is based on the influence of the GABAergic and serotonergic systems, as the levels of their respective neurotransmitters can be altered in the brain (Ip et al., 1996; Wei et al., 2013; Zhang et al., 2014). The lignanoid schizandrin was identified as a weak inhibitor of the GABA_A receptor (Figures 4 and 5; Supplementary Table S4), presumably acting in a non-competitive manner (Supplementary Figure S1). However, its contribution to the effect of the tincture remains hypothetically, with regard to the low potency.

The Andrographis paniculata (herba) tincture exhibited a weak antagonism (Supplementary Table S2). However, the tested labdane diterpenoid andrographolide, which possesses antiphlogistic, anticancer, and neuroprotective effects (Chan et al., 2010; Chun et al., 2010; Kou et al., 2014; Lin et al., 2014), is a partial antagonist of the GABA_A receptor (Figure 5; Supplementary Table S4). Moreover, the action of andrographolide appears to be non-competitive in nature (Supplementary Figure S2). The andrographolide content of the A. paniculata leaves was approximately 1% of the dry weight (Chao and Lin, 2010). Hence, the concentration in the applied tincture can be approximated (≈6 μM). Therefore, the antagonistic effect of the tincture is not solely due to the action of andrographolide.

The Salvia miltiorrhiza (radix) tincture exhibited the strongest antagonistic effect on the GABA_A receptor, exceeding a blocking effect of 80% (Figure 3; Supplementary Table S2). This compound is traditionally used for the treatment of cardiovascular diseases and ischemia, and it inhibits platelet aggregation via the action of tanshinones (Han et al., 2008). One specific tanshinone, miltirone, was identified as a ligand at the benzodiazepine binding site of the GABA_A receptor in the CNS, based on ligand binding assays (Lee et al., 1991). Nevertheless, miltirone failed to modulate the GABA-induced current of the GABA_A receptor in a recombinant expression system (Xenopus oocytes) as well as in cultured rat hippocampal pyramidal cells (Mostallino et al., 2004). Hence, the involvement of miltirone in the observed inhibition of GABA_A receptor responses by the Salvia miltiorrhiza tincture seems unlikely. Additionally, rosmarinic acid, a monoterpenoid from Salvia miltiorrhiza (Adams et al., 2006), only showed a slight inhibition of the GABA_A receptor in the screening (Figure 4; Supplementary Table S3) and therefore does not account for the strong antagonistic effect of the tincture.

Salvia miltiorrhiza and Astragalus propinquus are compounds in a two-ingredient intermixture called myelophil. This mixture has been shown to reduce chemotherapy-correlated adverse side effects, including myelosuppression and anemia, from the cytostatic drug fluorouracil (Shin et al., 2008) and possesses no described side effects itself (Jung et al., 2009). Furthermore, myelophil reduced fatigue in patients suffering from chronic fatigue (Cho et al., 2009) and exhibited anti-amnesic effects in scopolamine-treated mice (Lee et al., 2014). Even if the latter is presumably caused by the increased expression of ERKs and mAChR1 (Lee et al., 2014), a contribution of the inhibition of Salvia miltiorrhiza on the GABA_A receptor to the anti-fatigue effects of myelophil is likely, due to the increased neuronal excitability resulting from GABA_A receptor inhibition. In contrast to Salvia miltiorrhiza, Astragalus propinquus did not affect the GABA_A receptor in this study (Supplementary Table S2).