The Development of Immunological Quantification Method for Glycyrrhetinic Acid in Serum to Estimate the Potential Risk for Pseudoaldsteronism.

To evaluate the medicinal properties of a glycyrrhizin (GL)-deficient strain of Glycyrrhiza uralensis, we investigated the anti-allergic effect of the hot water extract obtained from its roots on contact hypersensitivity in mice, and compared it with that of the hot water extract of a commercial crude drug, Glycyrrhiza Radix. The hot water root extract of the GL-deficient strain contained glucoglycyrrhizin (GGL) and rhaoglucoglycyrrhizin (RGL) instead of GL, and it showed anti-allergic activity against contact hypersensitivity in a fashion similar to that of the crude drug extract. We further confirmed the presence of glycyrrhetinic acid (GA), a major metabolite of GL, in mice serum after oral administration of the hot water root extract of a GL-deficient strain. We demonstrated that GGL underwent hydrolysis by intestinal microflora of mice to form GA. These results suggest that a GL-deficient strain of G. uralensis is a useful medicinal resource since the glycosides of GA work in a fashion similar to that of GL when orally administered.

Aim To examine serum glycyrrhetinic acid (GA) concentrations following oral administration of Glycyrrhiza radix (GR) containing Kampo extracts in mice. Methods We prepared 24 types of GR‐containing Kampo extracts which were orally administered to mice at 10 times the human dose. Subsequently, serum GA concentrations were measured using high‐performance liquid chromatography. In addition, glycyrrhizin hydrolysis by cecal contents and small intestinal transport were measured to clarify differences in pharmacokinetic parameters among the Kampo extracts. Results Concerning blood GA concentrations, the prescriptions showed both bimodal and unimodal profiles, which could be attributed to differences in the constituent crude drugs. We further verified that Scutellaria radix inhibited glycyrrhizin (GL) hydrolysis mediated by intestinal flora. These findings suggest that serum GA concentrations should be established for each prescription. There was a good correlation between Cmax and AUC 0‐48 ; furthermore, a strong correlation was observed between the amounts of GR or GL administered and AUC 0‐48 . In contrast, only a weak correlation was found between the amount of GR and the Cmax of GA. Given that GA enters enterohepatic circulation, AUC 0‐48 is proportional to the administered dose. However, it is influenced by factors such as the rate of intestinal absorption, as well as rates of hydrolysis and reabsorption after biliary excretion; therefore, no simple correlation with Cmax was observed. Conclusion Our findings suggest that the total amount of GA absorbed following consumption of a GR‐containing Kampo extract can be estimated from the amount of the crude drug.

Aconiti Lateralis Radix Praeparata (lateral roots of Aconitum carmichaelii Debeaux, Fuzi), is commonly used to treat various cardiovascular diseases, particularly heart failure. However, its strong cardiotoxicity limits its clinical applicability. Glycyrrhizae radix et rhizoma, (the root of Glycyrrhiza uralensis Fisch., Gancao), is known to synergistically increase the cardiotonic effects of Fuzi and alleviate the myocardial injury caused by Fuzi to some extent. However, the detailed mechanism via which the combination of Fuzi and Gancao reduces toxicity and increases or preserves the efficacy of Fuzi requires further investigation. Oxidative stress injury models in H9C2 cells and mice with acute heart failure were established to evaluate the optimal synergistic protective concentration of Fuziline and Glycyrrhetinic acid (GA). A GA probe was then synthesized and used for target fishing using chemical and biological methods. Finally, the target and its function were verified using fluorescence co-localization, Western blotting, protein interaction analysis, molecular docking, and calcium ion imaging. The best pharmacodynamic potential was achieved with a 1:1 or 2:1 ratio of Fuziline and GA concentrations. At these ratios, they regulated the protein levels of the downstream players of the Ca2+ signaling pathway via MDH2 and CALR, thereby balancing Ca2+ homeostasis in the myocardial tissue and mitigating the effects of heart failure. This study aimed to investigate the compatibility of Fuziline and GA, the active metabolites of a traditional Chinese medicine (TCM) pair, in exerting their cardiac effects, identify the direct biological targets and verify the mechanism of compatibility.

Glycyrrhetinic acid (GA) is an ingredient of triterpene saponins found in Gancao (Radix Glycyrrhizae). Here, we investigated the protective effects of GA in H9c2 cells, and explored its possible mechanism of action. Different concentrations of GA were used to treat H9c2 cells under oxygen glucose deprivation. We analyzed cell necrosis and apoptosis using optical microscopy, Hoechst 33342 staining, FITC-annexin V/PI double-staining and lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB) and interleukin (IL)-1β assays. Changes in related pro-apoptosis and anti-apoptosis proteins were detected by Western blot. Optical microscopy showed that GA improved cell morphology, including cell shrinkage, cauliflower-like membrane blebbing, and even some cell debris. Meanwhile, GA also ameliorated cell nuclei characteristics such as nucleus size, chromatin condensation and bright staining from Hoechst 33342 staining. GA also lowered the apoptotic rate and the levels of LDH, CK-MB and IL-1β in a dose-dependent manner. Furthermore, GA treatment increased Bcl-2 protein expression and decreased caspase-8 and Bax protein expression, while elevating the Bcl-2/Bax ratio. GA preconditioning increased p-AKt protein expression; however, after adding LY 294002, the p-AKt expression decreased obviously. Our results demonstrated that GA could protect H9c2 cells from apoptosis in a dose-dependent manner, and the potential mechanism might be related to the PI3K/AKt signaling pathway.

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.

To evaluate the safety and efficacy of Glycyrrhiza uralensis root produced using artificial hydroponic and artificial hydroponic-field hybrid cultivation systems, we investigated the pharmacokinetics of a major metabolite of glycyrrhizin (GL), glycyrrhetinic acid (GA). Hot water extracts obtained from the roots of the artificial hydroponic-field hybrid cultivated Glycyrrhiza uralensis were orally administered at a dose of 100mg/kg as GL in mice and, compared with a commercial crude drug, Glycyrrhizae Radix. The temporal changes in serum GA concentration was found to depend on the GL concentration of the hot-water extracts. When hot-water extracts containing relatively high GL were administered, bimodal peaks appeared. In contrast, a broad single peak was detected when a hot-water extract containing relatively low GL content was administered. These tendencies in the serum GA concentration time course were observed for all samples, regardless of their derivation. Moreover, we compared the pharmacokinetic parameters and found that the Cmax and AUC0-48 values after oral administration of the extracts from Glycyrrhiza uralensis roots produced by the artificial cultivation system are within the range of variation for the commercial crude drugs. These results suggest the possibility that roots of Glycyrrhiza uralensis cultivated by the artificial hydroponic-field hybrid cultivation system can be used in addition to currently available commercial crude drugs produced from wild plant resources.

Glycyrrhizic acid(GA) in Glycyrrhiza uralensis(G.uralensis) is physiologically active.In this study,the total DNA of wild G.uralensis was randomly transformed into Hansenula anomala by implantation of low-energy Ar~+ and N~+,to produce five recombinant yeast strains relating to biological synthesis of the GA or Glycyrrhetinic acid (GAs).After culturing in liquid medium for 96 h,the resultant GA,18α-GAs and 18β-Gas were determined by reversed-phase high performance liquid chromatography(RP-HPLC),and the corresponding concentrations were 114.49,0.56,and 0.81 mg·L~(-1).After one hundred primers were analyzed with random amplified polymorphic DNA (RAPD),the seven different DNA fragments were produced by the N7059 strain of recombined yeasts,and,the polymerase chain reaction(PCR) verified that one of them came from the genome of G.uralensis,indicating a successful transfer of genetic information by ion implantation.

Molecular study on the role of vacuolar transporters in glycyrrhetinic acid production in engineered Saccharomyces cerevisiae

Glycyrrhetinic acid (GA) and glycyrrhizin (GL), active ingredients derived from Glycyrrhiza, Glycyrrhizae Radix (The Japanese Pharmacopia), exhibit various pharmacological effects, such as anti-inflammatory and anti-allergic activities, and a side effect of licorice-induced pseudoaldosteronism. GA is also produced as a metabolite of GL in gastrointestinal bacterial flora when Glycyrrhiza-containing Kampo medicines are orally administered. The present study aimed to confirm that a galacturonic-glycyrrhizin (gala-GL), an analog of GL contained in Glycyrrhiza, contributes to gastrointestinal GA production as well as GL. Gala-GL was hydrolyzed to GA by the intestinal flora, although the hydrolysis rate was slower than that of GL. This is the first report to reveal hydrolyzation of gala-GL into GA in the intestinal flora. The gala-GL content in the 16 Kampo extracts containing 1.0-4.0 g/daily dose of Glycyrrhiza and 8 commercially available Shakuyakukanzoto (SKT) products containing 2.0-4.8 g/daily dose of Glycyrrhiza were 2.2-9.5 and 2.9-7.2 mg/daily dose, respectively. The gala-GL contents corresponded to 7.3-10.9% and 8.0-9.4% of the total GL contents (sum of GL and gala-GL) for the Kampo extracts and SKT products, respectively. These results suggest that gala-GL also can be as sources of gastrointestinal GA, although the contents in Glycyrrhiza-containing Kampo medicines and the hydrolytic rate of conversion to GA in intestinal bacterial flora were much smaller than those of GL.

We hypothesized that roasted Glycyrrhizae Radix (Glycyrrhizin Radix Praeparata, GRP) might modify anti-diabetic action due to compositional changes. Then we examined the anti-diabetic effect and mechanism of raw Glycyrrhizae Radix (GR) and GRP extracts and their major respective components, glycyrrhizin and glycyrrhetinic acid. In partial pancreatectomized (Px) diabetic mice, both GR and GRP improved glucose tolerance, but only GRP enhanced glucose-stimulated insulin secretion as much as exendin-4. Both GR and GRP extracts enhanced insulin-stimulated glucose uptake through peroxisome proliferation-activated receptor (PPAR)-gamma activation in 3T3-L1 adipocytes. Consistently with the results of the mice study, only GRP and glycyrrhetinic acid enhanced glucose-stimulated insulin secretion in isolated islets. In addition, they induced mRNA levels of insulin receptor substrate-2, pancreas duodenum homeobox-1, and glucokinase in the islets, which contributed to improving beta-cell viability. In conclusion, GRP extract containing glycyrrhetinic acid improved glucose tolerance better than GR extract by enhancing insulinotropic action. Thus, GRP had better anti-diabetic action than GR.

ABSTRACTGlycyrrhetinic acid is a major metabolite of glycyrrhizin, which is one of the main components of licorice roots and is considered to be one of the pharmacologically active substances in licorice. A new hybridoma cell line, named G-2A6, was generated by fusing mouse myeloma cells and splenocytes, which were immunized using glycyrrhetinic-acid–keyhole limpet hemocyanin to produce a monoclonal antibody (mAb) against glycyrrhetinic acid. Using the anti-glycyrrhetinic acid mAb, we attempted to develop a simple, rapid, and highly sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA). The developed icELISA had a range from 3.91 to 125 ng/mL with low coefficients of variation (less than 5%) and demonstrated a high recovery rate of glycyrrhetinic acid spiked into licorice powder (average = 101.76%). In addition, the icELISA could determine the glycyrrhetinic acid concentration in glycyrrhetinic-acid-spiked human serum with simple pretreatment, which suggests that the developed ELISA system using anti-glycyrrhetinic acid mAb would prove to be an effective and useful tool for determining glycyrrhetinic acid in various fields such as the analysis of Glycyrrhiza plants and pharmacokinetic studies of glycyrrhetinic acid during the administration of glycyrrhetinic acid, glycyrrhizin, and/or licorice-based medical agents.

We studied the effects of shakuyaku-kanzo-toh (a Chinese herbal medicine) and its components on testosterone production by rat gonads. We used paeoniflorin as a main component of shakuyaku (paeoniae radix), glycyrrhizin as a main component of kanzo (glycyrrhizae radix) and glycyrrhetinic acid as a main metabolite of glycyrrhizin. Oral administration of shakuyaku-kanzo-toh, glycyrrhizin, and glycyrrhetinic acid decreased in vitro basal testosterone production in Leydig cells by LH stimulation. Glycyrrhizin and glycyrrhetinic acid caused a significant decrease in testosterone production with an accumulation of 17 alpha-hydroxyprogesterone when incubated with isolated Leydig cells, while paeoniflorin showed no such effect. The inhibitory effect of glycyrrhetinic acid was far more potent than that of glycyrrhizin, causing about 90% inhibition at 10 micrograms/ml. Glycyrrhizin and glycyrrhetinic acid did not change the cyclic AMP or progesterone level in the Leydig cells. When 14C-labeled androstenedione was incubated with microsomal fraction of testicular or ovarian tissue, glycyrrhizin and glycyrrhetinic acid inhibited the conversion of androstenedione to testosterone, indicating that these compounds inhibit the activity of 17 beta-hydroxysteroid dehydrogenase (EC. 1.1.1.64). The ED50 of glycyrrhetinic acid was about 4 microM.

Analysis and pharmacokinetics of glycyrrhizic acid and glycyrrhetinic acid in humans and experimental animals

Introduction: Radix Glycyrrhiza has been used in China for thousand years to treat cancer. However, focus on its tumor-suppressing mechanism has been concentrated on its effect on tumor cell growth and apoptosis.Objectives: With the aid of a panel of human breast cancer cell lines, we reveal that glycyrrhetinic acid (GA), a major component of Radix Glycyrrhiza, is actually a significantly more potent agent to suppress invasion than cell survival.Results: GA effectively inhibits breast cancer cell MMP-2/MMP-9 expression; GA-induced reduction in the MMP-2/9 expression is apparently mediated by GA’s ability to specifically inhibit the p38 MAPK activity and its downstream AP1 activation. Moreover, we show that GA down regulates the levels of Fra-1 and c-Jun, two main components of AP1 transcription complex in invasive breast cancer cells and that AP1-specific inhibitor abrogates breast cancer cell invasion. These results suggest that GA impairs the p38 MAPK-AP1 signaling axis, leading to the repression of breast cancer cell invasion. Finally, we demonstrate that GA effectively suppresses breast tumor outgrowth and pulmonary metastasis without causing animal weight loss or eliciting liver/kidney toxicity to the recipient animals.Conclusion: This study indicates that GA represents a good candidate compound for the potential development of therapeutic drug.

Pharmacokinetic comparisons of two different combinations of Shaoyao-Gancao Decoction in rats: Competing mechanisms between paeoniflorin and glycyrrhetinic acid