3-epi-18ß-glycyrrhetinic acid or its glucuronide, the metabolites of glycyrrhizinic acid with individual differences, correlated with diagnostic maker for licorice-induced pseudoaldosteronism in humans.

Licorice is a crude drug that is used in traditional Japanese Kampo medicine and is also used as a sweetener. Occasionally, it causes pseudoaldosteronism (PsA) as a side effect. The major symptoms include hypokalemia, hypertension, edema, and low plasma aldosterone levels. PsA might be caused by the metabolites of glycyrrhizinic acid (GL), a component of licorice. The development of PsA markedly varies among individuals; however, the factors that cause these individual differences remain unknown. In this study, 78 patients who consumed Kampo medicines containing licorice were enrolled, and their laboratory data, including serum potassium levels, plasma aldosterone concentrations (PAC), and the concentrations of GL metabolites in the residual blood and/or urine samples were evaluated. Of the 78 participants, 18ß-glycyrrhetinic acid (GA), 3-epi-GA, 3-oxo-GA, 18ß-glycyrrhetinyl-30-O-glucuronide (GA30G), and 3-epi-GA30G were detected in the serum samples of 65, 47, 63, 62, and 3 participants, respectively. Of the 29 urine samples collected, GA30G and 3-epi-GA30G were detected in 27 and 19 samples. 3-epi-GA30G is a newly found GL metabolite. Moreover, 3-epi-GA, 3-oxo-GA, and 3-epi-GA30G were identified in human samples for the first time. High individual differences were found in the appearances of 3-epi-GA in serum and 3-epi-GA30G in urine, and the concentrations of these metabolites were correlated with serum PsA markers. The inhibitory titers of 3-epi-GA, 3-oxo-GA, GA30G, and 3-epi-GA30G on human 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) were almost similar. These findings suggest that 3-epi-GA and/or 3-epi-GA30G are associated with individual differences in the development of PsA. Significance Statement In this study, we detected 3-epi-GA in human serum for the first time. We also identified 3-epi-GA30G as a novel GL metabolite in human urine. These GL metabolite levels showed correlations with markers of PsA. Additionally, there are individual differences in whether or not they appear in the serum/urine. In conclusion, 3-epi-GA/3-epi-GA30G correlates with individual differences in the development of PsA.

Corrigendum: Identification of an alternative glycyrrhizin metabolite causing liquorice-induced pseudohyperaldosteronism and the development of ELISA system to detect the predictive biomarker.

[This corrects the article DOI: 10.3389/fphar.2021.688508.].

Identification of an Alternative Glycyrrhizin Metabolite Causing Liquorice-Induced Pseudohyperaldosteronism and the Development of ELISA System to Detect the Predictive Biomarker.

Liquorice is usually used as crude drug in traditional Japanese Kampo medicine and traditional Chinese medicine. Liquorice-containing glycyrrhizin (GL) can cause pseudohyperaldosteronism as a side effect. Previously, we identified 18ß-glycyrrhetyl-3-O-sulfate (3) as a GL metabolite in Eisai hyperbilirubinuria rats (EHBRs) with the dysfunction of multidrug resistance-related protein (Mrp2). We speculated that 3 was associated with the onset of liquorice-induced pseudohyperaldosteronism, because it was mainly detected in serum of patients with suspected to have this condition. However, it is predicted that other metabolites might exist in the urine of EHBRs orally treated with glycyrrhetinic acid (GA). We explored other metabolites in the urine of EHBRs, and investigated the pharmacokinetic profiles of the new metabolite in EHBRs and normal Sprague-Dawley rats. We further analyzed the serum concentrations of the new metabolite in the patients of pseudohyperaldosteronism. Finally, we developed the analyzing method of these metabolites as a preventive biomarker for the onset of pseudohyperaldosteronism using an enzyme-linked immunosorbent assay (ELISA). We isolated a new GL metabolite, 18ß-glycyrrhetyl-3-O-sulfate-30-O-glucuronide (4). Compound 4 significantly inhibited rat type-2 11ß-hydroxysteroid dehydrogenase (11ß-HSD2) and was a substrate of both organic anion transporter (OAT) 1 and OAT3. Compound 4 was also detected in the serum of patients with suspected pseudohyperaldosteronism at an approximately 10-fold lower concentrations than 3, and these concentrations were positively correlated. Compound 4 showed a lower serum concentration and weaker inhibitory titer on 11ß-HSD2 than 3. We developed an enzyme-linked immunosorbent assay system using an anti-18ß-glycyrrhetyl-3-O-glucuronide (3MGA) monoclonal antibody to measure the serum concentration of 3 to facilitate the measurement of biomarkers to predict the onset of pseudohyperaldosteronism. Although we found 4 as the secondary candidate causative agent, 3 could be the main potent preventive biomarker of liquorice-induced pseudohyperaldosteronism. Compound 3 was detected in serum at a higher concentration than GA and 4, implying that 3 may be a pharmacologically active ingredient mediating not only the development of pseudohyperaldosteronism but anti-inflammatory effects in humans administered GL or other liquorice-containing preparations.

Discrimination of prediction models between cold-heat and deficiency-excess patterns.

OBJECTIVE: The purpose of this study was to extract important patient questionnaire items by creating random forest models for predicting pattern diagnosis considering an interaction between deficiency-excess and cold-heat patterns. DESIGN: A multi-centre prospective observational study. SETTING: Participants visiting six Kampo speciality clinics in Japan from 2012 to 2015. MAIN OUTCOME MEASURE: Deficiency-excess pattern diagnosis made by board-certified Kampo experts. METHODS: We used 153 items as independent variables including, age, sex, body mass index, systolic and diastolic blood pressures, and 148 subjective symptoms recorded through a questionnaire. We sampled training data with an equal number of the different patterns from a 2 × 2 factorial combination of deficiency-excess and cold-heat patterns. We constructed the prediction models of deficiency-excess and cold-heat patterns using the random forest algorithm, extracted the top 10 essential items, and calculated the discriminant ratio using this prediction model. RESULTS: BMI and blood pressure, and subjective symptoms of cold or heat sensations were the most important items in the prediction models of deficiency-excess pattern and of cold-heat patterns, respectively. The discriminant ratio was not inferior compared with the result ignoring the interaction between the diagnoses. CONCLUSIONS: We revised deficiency-excess and cold-heat pattern prediction models, based on balanced training sample data obtained from six Kampo speciality clinics in Japan. The revised important items for diagnosing a deficiency-excess pattern and cold-heat pattern were compatible with the definition in the 11th version of international classification of diseases.

Identification of glycyrrhizin metabolites in humans and of a potential biomarker of liquorice-induced pseudoaldosteronism: a multi-centre cross-sectional study.

Liquorice [main ingredient, glycyrrhizin (GL)] is widely used as a food sweetener and herbal medicine. Occasionally, liquorice consumption causes pseudoaldosteronism as a side effect which causes oedema, hypokalaemia, and hypertension due to hyperactivity of mineral corticoid receptor. We aimed to detect GL metabolites in human blood and urine samples and to determine the pathological relationship between GL metabolites and pseudoaldosteronism. For this multi-centre, retrospective, cross-sectional study, we recruited patients who had visited Center for Kampo Medicine in Keio University Hospital, Department of Japanese Oriental (Kampo) Medicine in Chiba University Hospital, Clinic of Japanese Oriental (Kampo) Medicine in Kanazawa University Hospital, and Department of Oriental Medicine in Kameda Medical Center from November 2011 to July 2018. We collected laboratory data including concentration of serum potassium, plasma activity of renin and aldosterone, and residual blood and/or urine samples of participants who had experienced symptoms/signs of pseudoaldosteronism in the form of increase in blood pressure and occurrence or aggregation of oedema while taking liquorice-containing herbal preparations, and measured GL metabolites using a highly selective liquid chromatography tandem mass spectrometer system. We registered 97 participants (mean age 60 ± 15 years; male:female 14:83). 18ß-glycyrrhetinic acid (GA) was detected in 67 serum samples (median 122 nM, range 5 nM-1.8 µM) and 18ß-glycyrrhetyl-3-O-sulfate (compound 3) in 68 samples (median 239 nM, range 2 nM-4.2 µM). 3-Monoglucuronyl 18ß-glycyrrhetinic acid, 22α-hydroxy-18ß-glycyrrhetyl-3-O-sulfate-30-glucuronide, 22α-hydroxy-18ß-glycyrrhetyl-3-O-sulfate, and GL itself were not or rarely detected. We could not find any correlation between blood pressure or peripheral oedema and serum concentration of GL metabolites. Sulfotransferase 2A1 catalysed the metabolic reaction of GA to compound 3, a major GL metabolite in human blood. High serum concentration of compound 3 was related to lower renin, aldosterone, and potassium levels, suggesting a pathological relationship between compound 3 and liquorice-induced pseudoaldosteronism. This is the first study to identify the association between a novel metabolite, compound 3, and the incidence of pseudoaldosteronism, highlighting it as a promising biomarker.

Prediction of deficiency-excess pattern in Japanese Kampo medicine: Multi-centre data collection.

OBJECTIVE: The purpose of the present study was to compare important patient questionnaire items by creating a random forest model for predicting deficiency-excess pattern diagnosis in six Kampo specialty clinics. DESIGN: A multi-centre prospective observational study. SETTING: Participants who visited six Kampo specialty clinics in Japan from 2012 to 2015. MAIN OUTCOME MEASURE: Deficiency-excess pattern diagnosis made by board-certified Kampo experts. METHODS: To predict the deficiency-excess pattern diagnosis by Kampo experts, we used 153 items as independent variables, namely, age, sex, body mass index, systolic and diastolic blood pressures, and 148 subjective symptoms recorded through a questionnaire. We extracted the 30 most important items in each clinic's random forest model and selected items that were common among the clinics. We integrated participating clinics' data to construct a prediction model in the same manner. We calculated the discriminant ratio using this prediction model for the total six clinics' data and each clinic's independent data. RESULTS: Fifteen items were commonly listed in top 30 items in each random forest model. The discriminant ratio of the total six clinics' data was 82.3%; moreover, with the exception of one clinic, the independent discriminant ratio of each clinic was approximately 80% each. CONCLUSIONS: We identified common important items in diagnosing a deficiency-excess pattern among six Japanese Kampo clinics. We constructed the integrated prediction model of deficiency-excess pattern.

18ß-glycyrrhetyl-3-O-sulfate would be a causative agent of licorice-induced pseudoaldosteronism.

Licorice-induced pseudoaldosteronism is a common adverse effect in traditional Japanese Kampo medicine, and 3-monoglucuronyl glycyrrhetinic acid (3MGA) was considered as a causative agent of it. Previously, we found 22α-hydroxy-18ß-glycyrrhetyl-3-O-sulfate-30-glucuronide (1), one of the metabolites of glycyrrhizin (GL) in the urine of Eisai hyperbilirubinuria rats (EHBRs) treated with glycyrrhetinic acid (GA), and suggested that it is also a possible causative agent of pseudoaldosteronism. The discovery of 1 also suggested that there might be other metabolites of GA as causal candidates. In this study, we found 22α-hydroxy-18ß-glycyrrhetyl-3-O-sulfate (2) and 18ß-glycyrrhetyl-3-O-sulfate (3) in EHBRs' urine. 2 and 3 more strongly inhibited rat type 2 11ß-hydroxysteroid dehydrogenase than 1 did in vitro. When EHBRs were orally treated with GA, GA and 1-3 in plasma and 1-3 in urine were detected; the levels of 3MGA were quite low. 2 and 3 were shown to be the substrates of organic anion transporter (OAT) 1 and OAT3. In the plasma of a patient suffering from pseudoaldosteronism with rhabdomyolysis due to licorice, we found 8.6 µM of 3, 1.3 µM of GA, and 87 nM of 2, but 1, GL, and 3MGA were not detected. These findings suggest that 18ß-glycyrrhetyl-3-O-sulfate (3) is an alternative causative agent of pseudoaldosteronism, rather than 3MGA and 1.

Effect of d-pseudoephedrine on cough reflex and its mode of action in guinea pigs.

d-Pseudoephedrine (PSE) is one of the main ingredients of Ephedrae herba. Although PSE is widely applied for patients with a common cold and upper respiratory inflammation as a decongestant, the effects of PSE on cough have never been reported. In this study, we investigated the antitussive effects of intraperitoneal injection of PSE on the cough reflex induced by microinjection of citric acid into the larynx of guinea pigs. PSE decreased the number of cough reflexes dose-dependently (-18.3 +/- 5.0% at 20 mg/kg, P<0.05; -41.1 +/- 7.2% at 60 mg/kg, P<0.01). Furthermore, PSE (60 mg/kg) increased the threshold intensity for inducing fictive cough by electrical micro-stimulation of the nucleus tractus solitarius (+72.7 +/- 8.4%, P<0.01). On the afferent discharge of the superior laryngeal nerve, PSE suppressed the increases of amplitude and frequency when stimulated by citric acid at laryngeal mucosa. These results demonstrate that PSE possesses an antitussive effect that might be derived from both central and peripheral actions.

Effects of eppikahangeto, a Kampo formula, and Ephedrae herba against citric acid-induced laryngeal cough in guinea pigs.

To evaluate the efficacy of three common antitussive Kampo formulas, eppikahangeto (EPP), bakumondoto (BAK), and shoseiryutogomakyokansekito (SGM), a new cough model of guinea pig was used, which could specifically induce a laryngeal cough by microinjection of citric acid solution into the larynx. Kampo extract was dissolved in water and the animals were given access ad libitum for 3 days, and then the number of coughs during 10 min was counted. EPP extract decreased the number of coughs dose-dependently (0.3% extract, -22.9 +/- 6.6%, P<0.01; 1.0% extract, -32.4 +/- 5.5%, P<0.01). BAK extract and SGM extract had no significant effect. Intraperitoneal injection of codeine (60 mg/kg) also decreased the number of coughs (-36.1 +/- 9.1%, P<0.05). Furthermore, Ephedrae herba (EH) extract reduced the number of coughs (-18.3 +/- 6.0%, P<0.05), but the extract of EPP without EH did not. These results suggest that EPP has an antitussive effect against laryngeally-induced cough in guinea pigs, and the crucial herbal medicine is EH.