Kampo Medicine: Evaluation of the Pharmacological Activity of 121 Herbal Drugs on GABAA and 5-HT3A Receptors

Abstract

Kampo medicine is a form of Japanese phytotherapy originating from traditional Chinese medicine (TCM). During the last several decades, much attention has been paid to the pharmacological effects of these medical plants and their constituents. However, in many cases, a systematic screening of Kampo remedies to determine pharmacologically relevant targets is still lacking. In this study, a broad screening of Kampo remedies was performed to look for pharmacologically relevant 5-HT3A and GABAA receptor ligands. Several of the Kampo remedies are currently used for symptoms such as nausea, emesis, gastrointestinal motility disorders, anxiety, restlessness, or insomnia. Therefore, the pharmacological effects of 121 herbal drugs from Kampo medicine were analyzed as ethanol tinctures on heterologously expressed 5-HT3A and GABAA receptors, due to the involvement of these receptors in such pathophysiological processes. The tinctures of Lindera aggregata (radix) and Leonurus japonicus (herba) were the most effective inhibitory compounds on the 5-HT3A receptor. Further investigation of known ingredients in these compounds led to the identification of leonurine from Leonurus as a new natural 5-HT3A receptor antagonist. Several potentiating herbs (e.g., Magnolia officinalis (cortex), Syzygium aromaticum (flos), and Panax ginseng (radix)) were also identified for the GABAA receptor, which are all traditionally used for their sedative or anxiolytic effects. A variety of tinctures with antagonistic effects Salvia miltiorrhiza (radix) were also detected. Therefore, this study reveals new insights into the pharmacological action of a broad spectrum of herbal drugs from Kampo, allowing for a better understanding of their physiological effects and clinical applications.

Keywords: 5-HT3A receptor; GABAA receptor; Kampo; Leonurus japonicus (herba); Panax ginseng (radix); Salvia miltiorrhiza (radix); andrographolide; leonurine.

FIGURE 1

The strongest 12 direct activating tinctures for the 5-HT_3A(A) and GABA_A receptors (B). The 121 tinctures were made from Kampo remedies via ethanol extraction (see section Tinctures and substances). A 1:1,000-dilution was applied to the oocytes and compared with agonist induced currents (5 μM 5-HT, 100 μM GABA). Error bars represent the SEM. Statistical significance was calculated based on the current evoked by ethanol (0.1 Vol.-%; ^∗p < 0.05, ^∗∗p < 0.005; n = 3–5).

FIGURE 2

The modulatory action of the strongest inhibitory and potentiating Kampo tinctures on the 5-HT_3A receptor. (A) The tinctures with the 10 strongest antagonistic (left) and potentiating (right) effects are shown (5 μM 5-HT). Bars exceeding the dotted line represent potentiating effects. Statistical significance was calculated based on the reference solution containing 0.1 Vol.-% ethanol (equal to the max. ethanol concentration in the tinctures; ^∗p < 0.05, ^∗∗p < 0.005, ^∗∗∗p < 0.0005; n = 3–8). (B) Original registrations of the modulation observed by Leonurus japonicus (herba) and Cannabis sativa (semen) using a 1:1,000 dilution are shown. The effects were reversible following a 150-s washout with ringer’s solution.

FIGURE 3

The modulatory action of the strongest inhibitory and potentiating Kampo tinctures on the GABA_A receptors. (A) The tinctures with the 10 strongest antagonistic (left) and potentiating (right) effects are shown (10 μM GABA). Statistical significance was calculated based on the reference solution containing 0.1 Vol.-% ethanol (equal to the max. ethanol concentration in the tinctures; ^∗p < 0.05, ^∗∗p < 0.005, ^∗∗∗p < 0.0005; n = 3–8). (B) Original registrations of the modulation observed via Saposhnikovia divaricata (herba) and Magnolia officinalis (cortex) with a 1:1,000 dilution are shown. The effects were reversible following a 150-s washout with ringer’s solution.

FIGURE 4

Modulatory effects of the investigated ingredients on the 5-HT_3A and GABA_A receptors. (A) The bars represent the normalized current amplitude for the modulation of the 5-HT_3A (gray bars) and GABA_A (white bars) receptors. With the exception of schizandrin B, which was tested at 100 μM, all of the compounds were evaluated at a concentration of 1 mM. Bars exceeding the dotted line represent substances with potentiating effects. The substances are arranged based on their affiliation to classes of plant metabolites, as indicated by the vertical lines. Statistical differences were calculated based on the reference agonist, which contained an equal concentration of the solvent used to dissolve the test compounds (^∗p < 0.05, ^∗∗p < 0.005, ^∗∗∗p < 0.0005; n = 4–7). (B) Original registrations of the inhibition of the 5-HT_3A receptor by tannic acid and leonurine. (C) Original registrations of the inhibition of the GABA_A receptor by tannic acid and leonurine.

FIGURE 5

Concentration-inhibition curves of select plant ingredients for the 5-HT_3A(A) and GABA_A(B) receptor, as well as the original registrations of leonurine for the 5-HT_3A receptor (C) and the 5-HT concentration-effect curve in the presence of leonurine (D). (A,B) The agonist concentrations used in the experiment were 5 μM (5-HT) or 3 μM (GABA). Error bars represent the SEM (A: n = 5–7, B: n = 4–7). Andrographolide acts as a partial antagonist of the GABA_A receptor. (C) Overlayed 5-HT_3A receptor responses, modulated by different concentrations of leonurine (0.1–10 μM) are shown. (D) The concentration-effect curve of 5-HT at the 5-HT_3A receptor in the presence of leonurine (30 μM) is shown. The determined EC₅₀ value was found to be 14.8 μM 5-HT (n = 4).

FIGURE 6

Molecular structures of the alkaloids leonurine, boldine, and tannic acid.