A triterpenoid saponin that is the 3-O-beta-glucuronide of glycyrrhetic acid. It is a metabolite of glycyrrhizin contained in licorice and potentially a causative agent in the pathogenesis of pseudoaldosteronism.

Identification

IUPAC Names

30-hydroxy-11,30-dioxoolean-12-en-3beta-yl beta-D-glucopyranosiduronic acid

Molecular Formula
C36H54O10
Mass
646.818
Monoisotopic Mass
646.37170
Charge
0
InChI
InChI=1S/C36H54O10/c1-31(2)21-8-11-36(7)27(34(21,5)10-9-22(31)45-29-25(40)23(38)24(39)26(46-29)28(41)42)20(37)16-18-19-17-33(4,30(43)44)13-12-32(19,3)14-15-35(18,36)6/h16,19,21-27,29,38-40H,8-15,17H2,1-7H3,(H,41,42)(H,43,44)/t19-,21-,22-,23-,24-,25+,26-,27+,29+,32+,33-,34-,35+,36+/m0/s1
InChIKey
HLDYLAJAWSKPFZ-QDPIGISRSA-N
SMILES
[H][C@@]12C[C@](C)(CC[C@]1(C)CC[C@]1(C)C2=CC(=O)[C@]2([H])[C@@]3(C)CC[C@H](O[C@@H]4O[C@@H]([C@@H](O)[C@H](O)[C@H]4O)C(O)=O)C(C)(C)[C@]3([H])CC[C@@]12C)C(O)=O
Synonyms

(3beta,20beta)-20-carboxy-11-oxo-30-norolean-12-en-3-yl beta-D-glucopyranosiduronic acid

18beta-glycyrrhetinic acid-3-O-beta-D-glucuronide

18beta-glycyrrhetyl-3-O-glucuronide

3-MGA

3-monoglucuronyl glycyrrhetinic acid

3-monoglucuronyl-glycyrrhetinic acid

3-O-beta-D-glucuronopyranosyl-18beta-glycyrrhetinic acid

3-O-glucuronopyranosylglycyrrhetinic acid

3beta-D-(monoglucuronyl)-18beta-glycyrrhetinic acid

3MGA

glycyrrhetic acid 3-O-beta-D-glucuronide

glycyrrhetic acid 3-O-mono-beta-D-glucuronide

glycyrrhetinic acid 3-O-glucuronide

glycyrrhetinic acid 3-O-mono-beta-D-glucuronide

glycyrrhetinic acid 3-O-monoglucuronide

glycyrrhetinic acid acid 3-O-beta-D-glucuronide

glycyrrhetyl 3-monoglucuronide

starrhizin

Species

glycyrrhiza

NCBI:txid4634710.1016/j.synbio.2021.07.001

Europe PubMed Central results


Importance of the neutrophil-lymphocyte ratio and systemic immune-inflammation index in predicting colorectal pathologies in fecal occult blood-positive patients.

Author: Hasirci I, Şahin A.

Abstract: <h4>Background</h4>The fecal occult blood (FOB) test is one of the primary screening methods for colorectal cancer (CRC). In this study, we aimed to investigate the effect of the neutrophil/lymphocyte ratio (NLR) and systemic immune-inflammation index (SII) in predicting CRC and other colorectal pathologies in patients with a positive FOB test.<h4>Methods</h4>This retrospective study included patients with a positive FOB test who underwent colonoscopy for the investigation of the etiology. The optimal cutoff levels of NLR and SII for predicting colorectal pathologies were determined using the receiver operating characteristic analysis.<h4>Results</h4>Of the 157 FOB+ patients, 35% were male and 65% were female, with a median age of 59 years. There were 96 patients in Group 1 and 61 patients in Group 2. The mean age of the patients in Group 2 was significantly higher (p < 0.001). The rate of male patients was significantly higher in Group 2. NLR and SII were significantly higher in Group 2 than in Group 1 (p < 0.001). The area under the curve values of NLR and SII in predicting colorectal pathologies in FOB+ patients were 0.817 and 0.825, respectively. At the cutoff values of 0.689 and 0.795, NLR and SII had a sensitivity of 60.66% and 95.83%, respectively, and a specificity of 95.83% and 100%, respectively.<h4>Conclusion</h4>Neutrophil/lymphocyte ratio and SII can be used as important biomarkers in the early diagnosis of CRC and other colorectal lesions in patients with a positive FOB test.

Anti-allergic activity of 18beta-glycyrrhetinic acid-3-O-beta-D-glucuronide.

Author: Park HY, Park SH, Yoon HK, Han MJ, Kim DH.

Abstract: Glycyrrhizin (18beta-glycyrrhetinic acid-3-O-beta-D-glucuronopyranosyl-(1 --> 2)-beta-D-glucuronide, GL) was transformed to 18beta-glycyrrhetinic acid-3-O-beta-D-glucuronide (GAMG) by Streptococcus LJ-22. The antiallergic activities of GL and GAMG was measured using a RBL cell assay system and contact hypersensitivity model mice. GAMG exhibited anti-allergic activity with IC50 values of 0.28 mM. GAMG, which is sweeter than GL, and 18beta-glycyrrhetinic acid, which is a GAMG metabolite by human intestinal bacteria, also inhibited the passive cutaneous anaphylaxis and skin contact inflammation. In conclusion, GAMG may be useful as a new sweet food additive and an anti-allergic agent.

A possible involvement of 3-monoglucuronyl-glycyrrhetinic acid, a metabolite of glycyrrhizin (GL), in GL-induced pseudoaldosteronism.

Author: Ohtake N, Kido A, Kubota K, Tsuchiya N, Morita T, Kase Y, Takeda S.

Abstract: Glycyrrhizin (GL), a major ingredient of Glycyrrhiza Radix (licorice), is widely used to treat various disorders or as a sweetener. It is also known that GL occasionally induces pseudoaldosteronism. It is conceivable that the active form of GL in pseudoaldosteronism induction is glycyrrhetinic acid (GA). Although it is reported that 3-monoglucuronyl-glycyrrhetinic acid (3MGA) is detectable specifically in the plasma of patients with GL-induced hypokalemia, pharmacokinetics and a hypokalemia induction mode of action for 3MGA have not been clarified. We investigated the toxicokinetics of GL, GA and 3MGA in a single or multiple oral administration of GL. The results suggested that higher blood concentrations of 3MGA were maintained by the multiple administration compared to the single dose, whereas the concentrations of GA and GL showed no difference. We injected 3MGA intravenously and found that it can decrease the plasma potassium level (PPL) in vivo. It is clinically recommended to avoid a combination treatment of GL and furosemide. While treatment with a low dosage of furosemide had no effect on PPL, the multiple administration of GL and furosemide markedly decreased PPL compared to the effect of administering GL alone. In the single dosage regime, there was no difference between PPL after the combination treatment and after administering GL alone. Collectively, these findings suggested that accumulation of 3MGA may be involved in the pathogenesis of pseudoaldosteronism induced by chronic GL treatment.

Down-regulation of a hepatic transporter multidrug resistance-associated protein 2 is involved in alteration of pharmacokinetics of glycyrrhizin and its metabolites in a rat model of chronic liver injury.

Author: Makino T, Ohtake N, Watanabe A, Tsuchiya N, Imamura S, Iizuka S, Inoue M, Mizukami H.

Abstract: Glycyrrhizin (GL) has been used to treat chronic hepatitis in Japan and Europe. It is thought to induce pseudoaldosteronism via inhibition of type 2 11beta-hydroxysteroid dehydrogenase (11beta-HSD2) by glycyrrhetinic acid (GA), a major metabolite of GL. A previous clinical study suggested that 3-monoglucuronyl-glycyrrhetinic acid (3MGA), another metabolite of GL, might play a more important role in the pathogenesis of pseudoaldosteronism. The present study evaluates the pharmacokinetics of GL and its metabolites in rats with chronic liver injury induced by a choline-deficient l-amino acid-defined (CDAA) diet to clarify the relationship between 3MGA and pseudoaldosteronism. In rats fed a CDAA diet, plasma concentrations and urinary eliminations of GL and 3MGA were markedly higher than in the rats fed the control diet; the plasma concentration of GA was unaffected when GL was orally administered. Immunohistochemical analysis revealed the suppression of levels of multidrug resistance-associated protein (Mrp) 2 and its localization in the hepatic tissue of rats fed a CDAA diet. When 3MGA was i.v. injected in rats fed a CDAA diet or injected in Mrp2-dysfunctional Eisai hyperbilirubinemic rats, plasma concentrations of 3MGA were higher, and biliary excretion of 3MGA was lower than in each control group. The results suggested that 3MGA would be excreted to bile via hepatic Mrp2 and that its dysfunction would reduce 3MGA clearance. 3MGA accumulated by liver fibrosis resulted in the increased excretion through renal tubule and might be strongly related to the pathogenesis of pseudoaldosteronism because 11beta-HSD2 is expressed in renal tubular epithelial cells.

Metal containing complexes with anticancer properties.

Author: Nazarov A, Hartinger C.

Abstract: NA

3-Monoglucuronyl-glycyrrhretinic acid is a substrate of organic anion transporters expressed in tubular epithelial cells and plays important roles in licorice-induced pseudoaldosteronism by inhibiting 11β-hydroxysteroid dehydrogenase 2.

Author: Makino T, Okajima K, Uebayashi R, Ohtake N, Inoue K, Mizukami H.

Abstract: Licorice (glycyrrhiza root) has been used as a herbal medicine worldwide with its main active constituent being glycyrrhizin (GL). Licorice sometimes causes adverse effects such as inducing pseudoaldosteronism by inhibiting type 2 11β-hydroxysteroid dehydrogenase (11β-HSD2) caused by glycyrrhetinic acid (GA), a major metabolite of GL. In this study we compared the inhibitory effects of GA, GL, and 3-monoglucuronyl-glycyrrhetinic acid (3MGA), another metabolite of GL, on 11β-HSD2 activity by using microsomes and rat kidney tissue slices. GA, 3MGA, and GL inhibited 11β-HSD2 in rat kidney microsomes, with IC(50) values of 0.32, 0.26, and 2.2 μM, respectively. However, the inhibitory activity of these compounds was reduced markedly, in the slices, in a medium containing 5% bovine serum albumin. Assays using human embryonic kidney 293 cells with transient transformation in transporter genes showed that 3MGA is a substrate of human organic anion transporter (OAT) 1, human OAT3, and human organic anion-transporting peptide 4C1, whereas GA is not. When GA (100 mg/kg/day) was administered orally for 16 days to Eisai hyperbilirubinemic rats, plasma concentrations and urinary excretion of 3MGA were significantly higher, whereas the activity of 11β-HSD2 in kidney microsomes was significantly lower compared with Sprague Dawley rats. These results suggest that 3MGA is actively transported into tubules through OATs, resulting in the inhibition of 11β-HSD2. Because the plasma level of 3MGA depends on the function of hepatic transporters, monitoring 3MGA levels in plasma or urine may be useful for preventing pseudoaldosteronism when licorice or GL is prescribed to patients.

Comparison of the exposure of glycyrrhizin and its metabolites and the pseudoaldosteronism after intravenous administration of alpha- and beta-glycyrrhizin in rat.

Author: Xu R, Xiao Q, Cao Y, Yang J.

Abstract: Glycyrrhizin, the major bioactive component in licorice root extract, exists as 2 isomers, α and β-glycyrrhizin, and is associated with causing pseudoaldosteronism due to its principal metabolites, glycyrrhetinic acid and 3-monoglucuronyl-glycyrrhetinic acid. The aim of this study was to compare (a) the pharmacokinetics of glycyrrhizin and its metabolites in rat after the first and last intravenous doses of either α- or β-glycyrrhizin administered once a day over 6 days, (b) kidney levels of the metabolites at 24 h after the last dose and (c) the urinary cortisol:cortisone ratio (as a biomarker of pseudoaldosteronism) in total urine collected for 24 h after the last dose.After the first dose, the clearance of glycyrrhizin in rats given α-isomer was significantly higher than in those given β-isomer and the AUC0-24 h values of glycyrrhizin and the metabolites were all significantly higher in β group than in α group. After the last dose, the AUC0-24 h values of glycyrrhizin and its metabolites were again significantly higher in rats given β-isomer than those given α-isomer and were all higher than the corresponding values after the first dose. Moreover, only kidney levels of glycyrrhetinic acid were detected in β group. The urinary cortisol:cortisone ratio was higher in rats given β-isomer and the correlation coefficients of the ratios with the AUC0-24 h values of 2 metabolites were 0.81 and 0.89 respectively.The results of the present study indicate that α-glycyrrhizin is a safer drug than β- glycyrrhizin probably due to a lower systemic exposure to the 2 metabolites.

Comparative study for pharmaceutical quality among bland-name drug and generic drugs of compound glycyrrhizin injections in China.

Author: Koga K, Kikuchi H.

Abstract: The physicochemical properties (pH and osmolarity), ingredients, and impurities containing in compound glycyrrhizin injections (eight items) marketed in China were compared with those in bland-name drug (Stronger Neo-Minophagen C injection). Glycyrrhizin (GZ), glycine (Gly), and l-cysteine (CysH) as the ingredients, moreover, glycyrrhetinic acid (GA), 3-monoglucuronyl-glycyrrhetinic acid (MGGA), and l-cystine (CysS) as the impurity were determined by HPLC. The pH and osmolarity were different every each pharmaceutical product, but the variation between batch was very small. On the other hand, although the contents of GZ, Gly, and CysH in bland-name drug were approximately 100% of the label claim, the contents of GZ in generic drugs were the range of 91.8-100.9%, indicating the GZ contents in four products were clearly less than value indicated in label (<97%). The remarkable difference was not accepted by impurities content such as GA and MGGA. The contents of CysH in generic drugs were the range of 79.9-100.4%, and CysS was determined in all generic drugs, suggesting that CysH may decompose to be CysS depending on the pH of injections in generic drug only. Because the variation of the ingredient content was big and products with a little quantity for the ingredients were recognized, establishment of the preparation that can maintain the prescribed ingredient content and the severity of the assay will be required.

3-Monoglucuronyl glycyrrhretinic acid is a possible marker compound related to licorice-induced pseudoaldosteronism.

Author: Makino T.

Abstract: One of the most common adverse effects of traditional Japanese kampo and traditional Chinese medicine is pseudoaldosteronism caused by licorice. In this review, the authors describe the mechanisms of licorice-induced pseudoaldosteronism by the pharmacokinetics of chemical constituents and its metabolites containing licorice. Glycyrrhizin (GL), the main constituent of licorice, is absorbed as glycyrrhetinic acid (GA), which is a metabolite of GL produced by enterobacteria before its release into the circulation. Circulating GA is metabolized in the liver to become 3-monoglucuronyl-glycyrrhetinic acid (3MGA), which is excreted into the bile via multidrug resistance protein 2 (Mrp2). If Mrp2 function is damaged for some reason, 3MGA is secreted from the liver into the circulation, and excreted into the urine via organic anion transporters expressed at the basolateral side of tubular epithelial cells. Circulating GA cannot be excreted into the urine since GA binds highly to serum albumin and thus does not pass through glomerular filtration and is not a substrate of transporters expressed on tubular epithelial cells. Licorice-induced pseudoaldosteronism develops due to the inhibition of type 2 11β-hydrosteroid dehydrogenase (11β-HSD2) which results in the accumulation of cortisol in tubular epithelial cells that activate mineral corticoid receptors to stimulate the excretion of potassium that results in hypokalemia. GA, unlike 3MGA, cannot pass through tubular epithelial cells and cannot inhibit the enzyme in the cells. Therefore, 3MGA may be a genuine causative agent for licorice-induced pseudoaldosteronism. When licorice is used, 3MGA in plasma or urine could function as a marker compound to prevent the adverse effects.

[Scientific Evaluation of Crude Drugs and Kampo Medicines Using the Eastern Blotting Method and Its Application to Biological Metabolic Studies].

Author: Morinaga O.

Abstract:  The scientific evaluation of crude drugs and kampo medicines (KMs) was demonstrated using the eastern blotting method with monoclonal antibodies (MAbs) against bioactive natural compounds. Scutellariae radix is one of the most important crude drugs used in KMs. Part of its pharmaceutical properties is due to the flavone glycoside baicalin (BI). A quantitative analysis method based on eastern blotting was developed for BI using an anti-BI MAb. A rapid, simple, sensitive, specific analytical system was subsequently established for BI with the eastern blotting technique using dot-blot and chemiluminescent methods. This system was useful as a high-throughput analytical method for the determination of BI in KMs as well as HPLC and enzyme-linked immunosorbent assay systems. Furthermore, an eastern blotting method was applied to the biological metabolic study of glycyrrhizic acid (GL), the major active constituent of licorice, for investigation of metabolites of GL such as 3-monoglucuronyl-glycyrrhetinic acid (3MGA) because licorice causes pseudoaldosteronism as a side effect. This approach may make it possible to determine the pathogenic agents of licorice-induced pseudoaldosteronism.

Isolation of a novel glycyrrhizin metabolite as a causal candidate compound for pseudoaldosteronism.

Author: Morinaga O, Ishiuchi K, Ohkita T, Tian C, Hirasawa A, Mitamura M, Maki Y, Yasujima T, Yuasa H, Makino T.

Abstract: Pseudoaldosteronism is a common adverse effect associated with traditional Japanese Kampo medicines. The pathogenesis is mainly caused by 3-monoglucuronyl glycyrrhetinic acid (3MGA), one of the metabolites of glycyrrhizin (GL) contained in licorice. We developed an anti-3MGA monoclonal antibody (MAb) and an ELISA system to easily detect 3MGA in the plasma and urine of the patients. However, we found that some metabolites of GL cross-reacted with this MAb. Mrp2-deficient Eisai Hyperbilirubinemia rats (EHBRs) were administered glycyrrhetinic acid (GA), and we isolated 22α-hydroxy-18β-glycyrrhetyl-3-O-sulfate-30-glucuronide (1) from the pooled urine with the guidance of positive immunostaining of eastern blot as the new metabolite of GL. The IC<sub>50</sub> of 1 for type 2 11β-hydroxysteroid dehydrogenase (11β-HSD2) was 2.0 µM. Similar plasma concentrations of 1 and GA were observed 12 h after oral administration of GA to EHBR. Compound 1 was eliminated via urine, whereas GA was not. In Sprague-Dawley (SD) rats orally treated with GA, compound 1 was absent from both the plasma and the urine. Compound 1 was actively transported into cells via OAT1 and OAT3, whereas GA was not. Compound 1, when produced in Mrp2-deficiency, represents a potential causative agent of pseudoaldosteronism, and might be used as a biomarker to prevent the adverse effect.

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

Author: Ishiuchi K, Morinaga O, Ohkita T, Tian C, Hirasawa A, Mitamura M, Maki Y, Kondo T, Yasujima T, Yuasa H, Minamizawa K, Namiki T, Makino T.

Abstract: 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-18β-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-18β-glycyrrhetyl-3-O-sulfate (2) and 18β-glycyrrhetyl-3-O-sulfate (3) in EHBRs' urine. 2 and 3 more strongly inhibited rat type 2 11β-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 18β-glycyrrhetyl-3-O-sulfate (3) is an alternative causative agent of pseudoaldosteronism, rather than 3MGA and 1.

Functional specialization of UDP-glycosyltransferase 73P12 in licorice to produce a sweet triterpenoid saponin, glycyrrhizin.

Author: Nomura Y, Seki H, Suzuki T, Ohyama K, Mizutani M, Kaku T, Tamura K, Ono E, Horikawa M, Sudo H, Hayashi H, Saito K, Muranaka T.

Abstract: Glycyrrhizin, a sweet triterpenoid saponin found in the roots and stolons of Glycyrrhiza species (licorice), is an important active ingredient in traditional herbal medicine. We previously identified two cytochrome P450 monooxygenases, CYP88D6 and CYP72A154, that produce an aglycone of glycyrrhizin, glycyrrhetinic acid, in Glycyrrhiza uralensis. The sugar moiety of glycyrrhizin, which is composed of two glucuronic acids, makes it sweet and reduces its side-effects. Here, we report that UDP-glycosyltransferase (UGT) 73P12 catalyzes the second glucuronosylation as the final step of glycyrrhizin biosynthesis in G. uralensis; the UGT73P12 produced glycyrrhizin by transferring a glucuronosyl moiety of UDP-glucuronic acid to glycyrrhetinic acid 3-O-monoglucuronide. We also obtained a natural variant of UGT73P12 from a glycyrrhizin-deficient (83-555) strain of G. uralensis. The natural variant showed loss of specificity for UDP-glucuronic acid and resulted in the production of an alternative saponin, glucoglycyrrhizin. These results are consistent with the chemical phenotype of the 83-555 strain, and suggest the contribution of UGT73P12 to glycyrrhizin biosynthesis in planta. Furthermore, we identified Arg32 as the essential residue of UGT73P12 that provides high specificity for UDP-glucuronic acid. These results strongly suggest the existence of an electrostatic interaction between the positively charged Arg32 and the negatively charged carboxy group of UDP-glucuronic acid. The functional arginine residue and resultant specificity for UDP-glucuronic acid are unique to UGT73P12 in the UGT73P subfamily. Our findings demonstrate the functional specialization of UGT73P12 for glycyrrhizin biosynthesis during divergent evolution, and provide mechanistic insights into UDP-sugar selectivity for the rational engineering of sweet triterpenoid saponins.

Glycyrrhetic Acid 3-O-Mono-β-d-glucuronide (GAMG): An Innovative High-Potency Sweetener with Improved Biological Activities.

Author: Guo L, Katiyo W, Lu L, Zhang X, Wang M, Yan J, Ma X, Yang R, Zou L, Zhao W.

Abstract: Glycyrrhetic acid 3-O-mono-β-d-glucuronide (GAMG) is an important derivative of glycyrrhizin (GL) and has attracted considerable attention, especially in the food and pharmaceutical industries, due to its natural high sweetness and strong biological activities. The biotransformation process is becoming an efficient route for GAMG production with the advantages of mild reaction conditions, environmentally friendly process, and high production efficiency. Recent studies showed that several β-glucuronidases (β-GUS) are key GAMG-producing enzymes, displaying a high potential to convert GL directly into the more valuable GAMG and providing new insights into the generation of high-value compounds. This review provides details of the structural properties, health benefits, and potential applications of GAMG. The progress in the development of the biotransformation processes and fermentation strategies to improve the yield of GAMG is also discussed. This work further summarizes recent advances in the enzymatic synthesis of GAMG using β-GUS with emphasis on the physicochemical and biological properties, molecular modifications, and enzymatic strategies to improve β-GUS biocatalytic efficiencies. This information contributes to a better framework to explore production and application of bioactive GAMG.

Exploration for the real causative agents of licorice-induced pseudoaldosteronism.

Author: Makino T.

Abstract: I investigated the causative agents of licorice-induced pseudoaldosteronism, which is a frequent side effect of Japanese traditional Kampo medicines. Glycyrrhizin (GL), the main ingredient of licorice, is absorbed after being metabolized to glycyrrhetinic acid (GA) by intestinal bacteria, and then metabolized in liver to 3-monoglucuronyl-glycyrrhetinic acid (3MGA). In normal condition, 3MGA is excreted into bile via a multidrug resistance-related protein (Mrp) 2, therefore, 3MGA does not appear in blood circulation. However, under the dysfunction of Mrp2, 3MGA appears in the blood circulation and is excreted into the urine by not glomerular filtration but tubular secretion via organic anion transporter (OAT) 1 and 3. At this time, 3MGA inhibits type 2 11β-hydroxysteroid dehydrogenase (11βHSD2) in tubular cells to cause pseudoaldosteronism. Since GA is not the substrates of these transporters, GA cannot inhibit 11βHSD2 in tubular cells. Therefore, it was considered that 3MGA was the causative agents of licorice-induced pseudoaldosteronism. After that, I isolated and identified three other GL metabolites, 22α-hydroxy-18β-glycyrrhetyl-3-O-sulfate-30-glucuronide (1), 22α-hydroxy-18β-glycyrrhetyl-3-O-sulfate (2), and 18β-glycyrrhetyl-3-O-sulfate (3) from the urine of Mrp2-deficient rats orally treated with GA, and found that their blood and urinary concentrations were much higher than 3MGA and that their pharmacokinetic behaviors were similar to 3MGA. 3MGA was not detected in the blood of patients with pseudoaldosteronism who developed rhabdomyolysis due to licorice, and compound 3 was detected at a high concentration. In addition, a multicenter retrospective study was conducted using the serum and urine of 97 patients who took Kampo medicines containing licorice. Of a total of 97 patients, 67 detected GA in the serum (median 122 nM, 5 nM-1.8 µM) and 68 detected compound 3 (median 239 nM, 2 nM-4.2 µM), and there were no cases of detection of GL, 3MGA, compounds 1, and 2. High blood concentrations of compound 3 were associated with low plasma renin activity, plasma aldosterone levels, and serum potassium levels. It is highly probable that compound 3 is the true causative agent of pseudoaldosteronism.

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

Author: Ishiuchi K, Morinaga O, Yoshino T, Mitamura M, Hirasawa A, Maki Y, Tashita Y, Kondo T, Ogawa K, Lian F, Ogawa-Ochiai K, Minamizawa K, Namiki T, Mimura M, Watanabe K, Makino T.

Abstract: 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 18<i>β</i>-glycyrrhetyl-3-<i>O</i>-sulfate (<b>3</b>) as a GL metabolite in Eisai hyperbilirubinuria rats (EHBRs) with the dysfunction of multidrug resistance-related protein (Mrp2). We speculated that <b>3</b> 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, 18<i>β</i>-glycyrrhetyl-3-<i>O</i>-sulfate-30-<i>O</i>-glucuronide (<b>4</b>). Compound <b>4</b> significantly inhibited rat type-2 11<i>β</i>-hydroxysteroid dehydrogenase (11<i>β</i>-HSD2) and was a substrate of both organic anion transporter (OAT) 1 and OAT3. Compound <b>4</b> was also detected in the serum of patients with suspected pseudohyperaldosteronism at an approximately 10-fold lower concentrations than <b>3</b>, and these concentrations were positively correlated. Compound <b>4</b> showed a lower serum concentration and weaker inhibitory titer on 11<i>β</i>-HSD2 than <b>3</b>. We developed an enzyme-linked immunosorbent assay system using an anti-18<i>β</i>-glycyrrhetyl-3-<i>O</i>-glucuronide (3MGA) monoclonal antibody to measure the serum concentration of <b>3</b> to facilitate the measurement of biomarkers to predict the onset of pseudohyperaldosteronism. Although we found <b>4</b> as the secondary candidate causative agent, <b>3</b> could be the main potent preventive biomarker of liquorice-induced pseudohyperaldosteronism. Compound <b>3</b> was detected in serum at a higher concentration than GA and <b>4</b>, implying that <b>3</b> 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.

O-glycosyltransferases from <i>Homo sapiens</i> contributes to the biosynthesis of <b>Glycyrrhetic Acid 3-O-mono-β-D-glucuronide</b> and Glycyrrhizin in <i>Saccharomyces cerevisiae</i>.

Author: Xu K, Zhao YJ, Ahmad N, Wang JN, Lv B, Wang Y, Ge J, Li C.

Abstract: Glycyrrhizin (GL) and Glycyrrhetic Acid 3-O-mono-β-D-glucuronide (GAMG) are the typical triterpenoid glycosides found in the root of licorice, a popular medicinal plant that exhibits diverse physiological effects and pharmacological manifestations. However, only few reports are available on the glycosylation enzymes involved in the biosynthesis of these valuable compounds with low conversion yield so far. In mammals, glycosyltransferases are involved in the phase II metabolism and may provide new solutions for us to engineer microbial strains to produce high valued compounds due to the substrate promiscuity of these glycosyltransferases. In this study, we mined the genomic databases of mammals and evaluated 22 candidate genes of O-glycosyltransferases by analyzing their catalytic potential for O-glycosylation of the native substrate, glycyrrhetinic acid (GA) for its glycodiversification. Out of 22 selected glycosyltransferases, only UGT1A1 exhibited high catalytic performance for biosynthesis of the key licorice compounds GL and GAMG. Molecular docking results proposed that the enzymatic activity of UGT1A1 was likely owing to the stable hydrogen bonding interactions and favorite conformations between the amino acid residues around substrate channels (P82~R85) and substrates. Furthermore, the complete biosynthesis pathway of GL was reconstructed in <i>Saccharomyces cerevisiae</i> for the first time, resulting in the production of 5.98 ± 0.47 mg/L and 2.31 ± 0.21 mg/L of GL and GAMG, respectively.

Concurrent production of glycyrrhetic acid 3-<i>O</i>-mono-β-d-glucuronide and lignocellulolytic enzymes by solid-state fermentation of a plant endophytic <i>Chaetomium globosum</i>.

Author: Gao B, Xiao Y, Zhang Q, Sun J, Zhang Z, Zhu D.

Abstract: Glycyrrhetic acid 3-<i>O</i>-mono-<i>β</i>-d-glucuronide (GAMG) as an important derivative of glycyrrhizin (GL) shows stronger biological activities and higher sweetness than GL. The biotransformation process is considered as an efficient strategy for GAMG production, due to its mild reaction, high production efficiency and environmentally friendly status. In this study, licorice straw was used for the first time as a medium for GAMG and lignocellulosic enzyme production via solid-state fermentation (SSF) of endophytic fungus <i>Chaetomium globosum</i> DX-THS3. The fermentation conditions including particle size, temperature, seed age, inoculum size, and moisture of substrate were optimized. Furthermore, additional nitrogen sources and carbon sources were screened for GAMG production by <i>C. globosum</i> DX-THS3 of SSF. Under optimal fermentation conditions, the percent conversion of glycyrrhizin reached 90% in 15 days, whereas the control needed 35 days to achieve the same result. The productivity of optimization (<i>P</i> = 2.1 mg/g/day) was 2.33-fold that of non-optimization (<i>P</i> = 0.9 mg/g/day). Meanwhile, high activities of filter paper enzyme (FPase) (245.80 U/g), carboxymethyl cellulase (CMCase) (33.67 U/g), xylanase (83.44 U/g), and <i>β</i>-glucuronidase activity (271.42 U/g) were obtained faster than those in the control during SSF. Our study provides a novel and efficient strategy for GAMG production and indicates <i>C. globosum</i> DX-THS3 as a potential producer of lignocellulolytic enzymes.<h4>Supplementary information</h4>The online version contains supplementary material available at 10.1186/s40643-021-00441-y.

Efficient, continuous mutagenesis in human cells using a pseudo-random DNA editor.

Author: Chen H, Liu S, Padula S, Lesman D, Griswold K, Lin A, Zhao T, Marshall JL, Chen F.

Abstract: Here we describe TRACE (T7 polymerase-driven continuous editing), a method that enables continuous, targeted mutagenesis in human cells using a cytidine deaminase fused to T7 RNA polymerase. TRACE induces high rates of mutagenesis over multiple cell generations in genes under the control of a T7 promoter integrated in the genome. We used TRACE in a MEK1 inhibitor-resistance screen, and identified functionally correlated mutations.