Glycyrrhizin Content Research Articles

Effects of glucose, fructose and 5-hydroxymethyl-2-furaldehyde on the presystemic metabolism and absorption of glycyrrhizin in rabbits

Our previous study reported that co-administration of honey significantly increased the serum levels of glycyrrhetic acid (GA) after oral administration of glycyrrhizin (GZ) in rabbits. The components of honey are sucrose, glucose, fructose and 5-hydroxymethyl-furaldehyde (HMF). To clarify the causative component(s) in honey that altered the metabolic pharmacokinetics of GZ, rabbits were given GZ (150 mg kg(-1)) with and without glucose (5 g/rabbit), fructose (5 g/rabbit) and HMF (1 mg kg(-1)), respectively, in crossover designs. An HPLC method was used to determine concentrations of GZ and GA in serum as well as GA and 3-dehydroglycyrrhetic acid (3-dehydroGA) in faeces suspension. A noncompartment model was used to calculate the pharmacokinetic parameters and analysis of variance was used for statistical comparison. Our results indicated that the area under curve (AUC) of GA was significantly increased by 29% when HMF was coadministered, whereas the pharmacokinetics of GZ and GA were not significantly altered by coadministration of glucose or fructose. An in-vitro study, using faeces to incubate GZ and GA individually, indicated that HMF significantly inhibited the oxidation of GA to 3-dehydroGA and this may explain the enhanced GA absorption in-vivo. It was concluded that HMF is the causative component in honey that affects the presystemic metabolism and pharmacokinetics of GZ in-vivo.

Application of pressure-controlled colon delivery capsule to oral administration of glycyrrhizin in dogs.

A colon delivery system has been used to improve the bioavailability of glycyrrhizin, a glycoside of glycyrrhetic acid. The bioavailability of glycyrrhizin is low when administered in conventional oral galenic dosage forms because glycyrrhizin is enzymatically hydrolysed both in the stomach and in the intestine. It was reasoned that if large amounts of glycyrrhizin were directly delivered to the colon, enzymatic activity should be reduced due to saturation so that intact glycyrrhizin could be absorbed into the systemic circulation. Based on this assumption, pressure-controlled colon delivery capsules (PCDCs) were used as a colon delivery system. Eight types of glycyrrhizin solution were prepared and were introduced into PCDCs. After oral administration of the test PCDCs to beagle dogs, blood samples were obtained over 24 h and plasma glycyrrhizin concentrations were measured by an HPLC method. With PCDCs containing aqueous glycyrrhizin and propylene glycol solutions, plasma glycyrrhizin levels were extremely low and the bioavailabilities of glycyrrhizin were 0.6% and 0.4%, respectively. When Labrasol was added to both types of glycyrrhizin solution, the bioavailability was improved to 4.6% for aqueous solution and 3.8% for propylene glycol solution. When a surfactant, Polysorbate 80, was added in combination with Labrasol, synergistic effects were not obtained. Furthermore, dose-dependent effects of Polysorbate 80 were not obtained. Labrasol, which is a component of self-emulsifying drug delivery systems (SEDDS), has been shown to strongly improve the bioavailability of glycyrrhizin from the colon.

Preparation of PLGA Microspheres Encapsulating Highly Concentrated Glycyrrhizin and the Bile Elimination of Glycyrrhizin after Subcutaneous Administration in Rats

The aim of this study was to prepare poly (lactide-co-glycolide) (PLGA) microspheres encapsulating highly concentrated glycyrrhizin (GZ), a hydrophilic drug, and to compare the release characteristics of GZ in in vitro experiments and GZ elimination into bile after subcutaneous administration in rats. The preparation was carried out based on water drying using a (w/o)/w emulsion. The encapsulation rate of GZ in microspheres was 76% when the GZ concentration in the outer water phase was equal to that in the inner water phase for the preparation of (w/o)/w emulsion. The release of GZ from the microspheres showed a biphasic zero-order profile, that is, the behavior boundary was approximately 12 h. The release of GZ from the microspheres at the periods of 0.5-8 h and 12-672 h was 0.18 mg/h and 0.0012 mg/h, respectively. On the other hand, 0.25% of GZ administered (5.0 mg) was eliminated into bile by 12 h, and the bile clearance rate (1.13 ml/h) of GZ after the subcutaneous administration of the microspheres was the same as that (1.13 ml/h) after the administration of GZ solution. From the results, it is suggested that the initial controlled release (0.18 mg/h) of GZ from microspheres may be beneficial for the hepatic bioavailability of GZ.

Determination of Flavonoids in Licorice Using Acid Hydrolysis and Reversed-Phase HPLC and Evaluation of the Chemical Quality of Cultivated Licorice

Licorice contains flavonoids and triterpenoids as the major bioactive components. Most of the flavonoids are the glycosidic form of liquiritigenin (LIQ), isoliquiritigenin (ISO) and formononetin (FOR). A reversed-phase HPLC method for the quantification of LIQ, ISO and FOR in licorice was developed. This method does not measure each glycoside but measures the aglycones using acid hydrolysis. All calibration curves showed good linear regression (r > 0.9998). The method showed good precision for intraday (RSD < 2.14 %) and interday (RSD < 0.51 %) assays. The limit of detection was 0.031 microg for LIQ, 0.011 microg for ISO and 0.006 microg for FOR. The limit of quantification was 0.31 microg for LIQ, 0.11 microg for ISO and 0.06 microg for FOR. The flavonoid contents along with the glycyrrhizin content of cultivated licorice from seedling plants in Japan and commercial wild licorice were investigated. This new method could be extremely useful for evaluating the quality of licorice.

Si-Ni-San, a traditional Chinese prescription, and its active ingredient glycyrrhizin ameliorate experimental colitis through regulating cytokine balance

Si-Ni-San, a traditional Chinese medicinal formula, exerts an important function in the treatment of inflammatory bowel diseases based upon thousands of years of clinical practice, but the underlying mechanism is still unclear. In this study, we investigated the therapeutic potential of Si-Ni-San and its ingredient glycyrrhizin in trinitrobenzene sulfonic acid (TNBS)-induced experimental colitis in mice, a well-characterized murine model for Crohn's disease. Si-Ni-San and glycyrrhizin significantly ameliorated TNBS-induced colitis with reduced mortality and recovery of body weights. In addition, Si-Ni-San and glycyrrhizin dose-dependently decreased macroscopic inflammation scores, microscopic histoligical scores, and myeloperoxidase activity. Furthermore, Si-Ni-San and glycyrrhizin caused a decrease in pro-inflammatory cytokines including IFN-γ, IL-12, TNF-α and IL-17 and an increase in regulatory cytokine IL-10 in colon of the mice. It should be noticed the therapeutic effect of Si-Ni-San at 450 mg/kg was much better than that of its contained content of glycyrrhizin at 10 mg/kg. In conclusion, Si-Ni-San and glycyrrhizin significantly ameliorated TNBS-induced colitis in mice through regulating pro- and anti-inflammtory cytokine production.

Short Communication: Quantitative Estimation of Glycyrrhizin in Marketed Formulation by HPTLC

A simple, precise, accurate and rapid high performance thin layer chromatographic (HPTLC) method has been developed and validated for the quantitative estimation of glycyrrhizin in marketed pharmaceutical dosage forms. The stationary phase used was precoated silica gel 60F254. A mobile phase containing 2- propanol, ethyl acetate, water and ammonia (50:35:25:2 v/v) was used to resolve the spots of glycyrrhizin. Detection of spots was carried out by UV at 257 nm. The Rf value was found to be 0.38 ± 0.01. The method was validated in terms of linearity, accuracy and precision. The proposed method can be used to determine the concentration of glycyrrhizin in marketed formulations.

Acute kidney injury following hypokalemic rhabdomyolysis: complication of chronic heavy cola consumption in an adolescent boy

A 16-year-old boy presented with acute kidney injury (AKI) which was attributed to chronic heavy cola consumption. Habitual heavy cola ingestion might lead to hypokalemic rhabdomyolysis by its glycyrrhizin content. AKI has been described rarely in association with this clinical picture. It is important for physicians to keep heavy cola and other soft drink consumption in mind as a cause for hypokalemic rhabdomyolysis and AKI.

Simultaneous Determination of Paeoniflorin and Glycyrrhizin in Sayuk-san by HPLC/DAD

A high performance liquid chromatographic (HPLC) method for the simultaneous determination of marker constituents, paeoniflorin and glycyrrhizin was established for the quality control of traditional herbal medicinal preparation, Sayuk-san (SYS). Separation and quantification were successfully achieved with a Waters XTerra RP18 column (, ) by gradient elution of a mixture of acetonitrile and water containing 0.03% phosphoric acid (pH 2.03) at a flow rate of 1.0 mL/min. The diode-array UV/vis detector (DAD) was used for the detection and the wavelength for quantification was set at 230 nm. The presence of paeoniflorin and glycyrrhizin in this decoction was ascertained by retention time, spiking with each authentic standard and UV spectrum. All two compounds showed good linearity (>0.996) in a relatively wide concentration ranges. The R.S.D. for intra-day and inter-day precision was less than 7.3% and the limits of detection (LOD) were less than 55.7 ng. The mean recovery of each compound was with R.S.D. values less than 4.6%. This method was successfully applied to the determination of contents of paeoniflorin and glycyrrhizin in three commercial products of SYS. These results suggest that the developed HPLC method is simple, effective and could be readily utilized as a quality control method for commercial SYS products.

Glycyrrhizin and Licorice Significantly Affect the Pharmacokinetics of Methotrexate in Rats

Glycyrrhizin (GZ) and licorice (root of Glycyrrhiza uralensis) are worldwide food additives and important oriental phytomedicines. This study investigated the biological fate of GZ by orally giving GZ and licorice decoction (LD) to rats. The serum concentrations of GZ and glycyrrhetic acid (GA) were determined by high performance liquid chromatography. The results showed that GZ was not detected and GA was present in serum until 3 days postdosing of GZ and LD. To evaluate the effects of GZ and licorice on the pharmacokinetics of methotrexate (MTX), an important immunosuppressant with a narrow therapeutic window, rats were orally given MTX with and without GZ and LD in different dosage regimens. The serum MTX concentration was determined by fluorescence polarization immunoassay. The results revealed that the AUC and MRT of MTX were significantly increased by GZ and LD. In conclusion, the concurrent use of GZ or licorice with MTX should be with caution.

Expressed sequence tags from rhizomes of Glycyrrhiza uralensis

Glycyrrhiza uralensis (Leguminosae, Fabaceae) is the most important medicinal plant used as a natural sweetener, in Chinese herbal medicines, and as a tobacco flavoring agent. Here, we report the generation of 56,857 expressed sequence tags (ESTs) derived from two full-length cDNA libraries produced from rhizomes (stolons) of two strains of G. uralensis, together with differential expression data obtained by MegasortTM analysis. The MegasortTM analysis was performed using two plant organs, namely, thickened roots and a rhizome-like organ, which differed in their glycyrrhizin content. After clustering of the 5′-end of ESTs, 5,542 contigs and 4,932 singletons were generated. A total of 10,474 nonredundant sequences were annotated on the basis of the TAIR database, of which 7,905 (75.5%) sequences exhibited homology to the sequences of registered genes. The gene functions of these 7,905 sequences were classified into Biological Process and Molecular Function on the basis of Gene Ontology (GO).

Survey of Glycyrrhizae Radix resources in Mongolia: chemical assessment of the underground part of Glycyrrhiza uralensis and comparison with Chinese Glycyrrhizea Radix

In order to reveal the chemical characteristics of Glycyrrhiza uralensis growing in Mongolia and to clarify whether it can be the source of Glycyrrhizae Radix used in Japan, eight major bioactive constituents in the underground parts of G. uralensis collected in Mongolia were quantitatively analyzed and compared with Glycyrrhizae Radix produced in China. Most of the 15 samples from eastern, southern and western parts of Mongolia contained 26.95-58.55 mg/g of glycyrrhizin, exceeding the criterion (25 mg/g) assigned in the Japanese Pharmacopoeia. The sample collected in Tamsagiyn hooly, Dornod province, in eastern Mongolia was of the highest content 58.55 mg/g. The contents of three flavanone constituents (liquiritin apioside, liquiritin and liquiritigenin) and three chalcones (isoliquiritin apioside, isoliquiritin and isoliquiritigenin) varied significantly according to collection places; the subtotal of the three flavanones ranged from 3.00 to 26.35 mg/g, and the subtotal of the three chalcones ranged from 1.13 to 10.50 mg/g. The content of glycyrrhizin and subtotal contents of flavanones and chalcones in the underground parts of G. uralensis from Mongolia were obviously lower than wild samples, but higher than cultivated samples derived from the same species produced in China. Glycycoumarin, a species-specific constituent of G. uralensis, was detected in all Mongolian samples. Its contents in samples from eastern Mongolia, Sergelen and Tamsagiyn hooly of Dornod province were very high and were compatible with Tohoku-kanzo derived from wild Chinese G. uralensis. The present study suggested that Mongolian G. uralensis could be a source of Glycyrrhizae Radix, mostly of Japanese Pharmacopoeia grade. However, the producing area should be taken into consideration to ensure relatively high quality. In addition, planned use and promotion of cultivation must be advocated to avoid confronting Mongolian Glycyrrhiza with the same threat as its congener in China. Our study sheds some light on selecting cultivation areas and superior strains, which are important tasks to promote cultivation.

Pharmacokinetics of glycyrrhizin in normal and albumin‐deficient rats

The pharmacokinetics of glycyrrhizin (GZ) was compared in albumin-deficient rats (NAR) and normal rats (SDR) after intravenous administration. The study sought to clarify the relationship between GZ concentration and its elimination rate in serum, liver and bile when the serum protein binding of GZ decreased. Serum protein binding in SDR and NAR, respectively, was 99.7% and 68.2% for a GZ concentration of 2.5 microg/ml. At steady-state conditions after i.v. infusion of GZ (0.5-2.0 mg/h), the relationship between the GZ concentration in serum and liver was linear in the SDR but nonlinear in the NAR. For both NAR and SDR, the GZ liver level and the elimination rate was nonlinear, indicating that the elimination of GZ from liver into bile was the rate-limiting step regardless of serum protein binding, and that the liver GZ level was extremely high when serum protein binding was decreased. It is concluded that a typical dose of GZ in chronic hepatitis patients whose serum albumin level is low will not cause a decrease of therapeutic effect compared with patients with a normal serum albumin level.

Production of Glycyrrhizin in Callus Cultures of Licorice

Licorice plants, Glycyrrhiza glabra, G. uralensis, and G. inflata, were investigated for callus induction using Murashige and Skoog (MS) medium combined with auxins and cytokinins. After 4 weeks of culture, 33-100% of leaf or stem explants formed calli. Maximum of shoot induction from callus cultures was achieved by G. inflata stem explants cultured on MS medium supplemented with 1 mg/l alpha-naphthaleneacetic acid (NAA) and 0.5 mg/l 6-benzyladenine (BA) (67%) which also gave maximum shoot formation per explant (two shoots per explant). These results indicated that all three Glycyrrhiza species regenerated shoots from callus cultures on MS medium combined with NAA and BA or only thidiazuron (TDZ; 0.1 and 0.5 mg/l). Glycyrrhizin contents of G. uralensis calli induced using MS medium in combination with NAA and BA [(27.60 +/- 8.47) microg/g DW] or TDZ alone [(36.52 +/- 2.45) microg/ g DW] were higher than those found in other combinations.

Ri-mediated Transformation of Glycyrrhiza uralensis with a Squalene Synthase Gene (GuSQS1) for Production of Glycyrrhizin

Root of Glycyrrhiza uralensis, one of the most important medicinal plants, containing bioactive triterpene saponins (glycyrrhizin). Squalene synthase (SQS) plays a regulatory role in the biosynthesis of triterpene saponins. In the present investigation, SQS coding sequence from G. uralensis was cloned by polymerase chain reaction (PCR) and a transgenic system was developed for G. uralensis through Agrobacterium rhizogenes-mediated transformation. The SQS gene placed under a CaMV 35S promoter was transferred into G. uralensis using A. rhizogenes strain ACCC10060. The transformed hairy roots were selected on Murashige and Skoog (1962)-containing phosphinothricin (PPT) and root lines were established. The integration of SQS gene was confirmed by PCR and Southern blot. Three transgenic root lines UP1, UP24, UP31 were obtained and their growth rates were detected. The result showed that transgenic root lines but UP1 line grew faster than control hairy roots; high-performance liquid chromatography (HPLC) analysis demonstrated the highest glycyrrhizin content of transgenic roots was 2.5 mg/g dry weight and was about 2.6 times higher than control hairy roots.

Approach to enhancing the distribution of glycyrrhizin in the liver: intravenous infusion studies in a rat model of bile duct ligation

We adopted a pharmaceutical approach to enhancing the distribution of glycyrrhizin (GZ) in the liver based on continuous administration of GZ for chronic hepatitis therapy. The present study characterizes the changes in GZ concentration in serum, bile, and liver under steady-state conditions after drip GZ infusion (0.5-2.0 mg/ml/h) in rats with bile duct fistulas. Eight hours after infusion, approximately 90% of GZ was excreted into bile as an intact compound regardless of the GZ dose tested. We observed a non-linear relationship between steady-state serum GZ concentrations and GZ dose (i.e. serum GZ concentrations abruptly increased with increasing GZ dose). GZ concentrations in the liver 8 h after infusion paralleled those in serum under steady-state conditions, but did not parallel those in bile. These results led us to conclude that under our experimental conditions transport activity responsible for the excretion of GZ to bile was in a rate-limiting step compared to that responsible for the uptake of GZ by hepatocytes. We therefore predicted that GZ concentration in the liver will increase with obstructing an efflux transport system such as canalicular multi-specific organic anion transporters. Probenecid among inhibitors or substrates for efflux transporters significantly enhanced the liver GZ concentration in the infusion studies. In conclusion, the concomitant administration of probenecid would be useful for liver protection therapy under continuous injection of the limited amount of GZ.

グリチルリチン注射剤市販品の物性および成分含量の比較検討

We evaluated the quality of parenteral injections (22 products) containing monoammonium glycyrrhizinate,the main extract from the Glycyrrhizae radix.The pH and osmolarity ratio of each product were within the range stated in the package insert.The glycyrrhizic acid,glycine,and L-cysteine concentrations in the injection products were 2.07-2.51 mg/mL,16.0-21.4 mg/mL,and 1.04-1.30 mg/mL,respectively.The concentrations of glycyrrhetinic acid and glycyrrhetic acid-3-Oglucuronide,impurities that cause pseudo-aldosteronism,were 0.173-2.24μg/mL and 0.777-8.29μg/mL,respectively,showing that there was a wide variation in the concentrations of these 2 substances among the products.Further,the results of discriminant analysis on impurity proportions suggested that it is possible to distinguish bulk powders from each other based on impurity contents.In conclusion,it is important to determine the proportions of impurities in glycyrrhizic acid products and their bulk powders in order to assess their quality.

Glycyrrhiza glabraculturein vitro— Saponin production

Our experiment has demonstrated changes in the concentration of glycyrrhetin and glycyrrhizin depending on the concentration of Pb(NO3)2 and CrCl3 and the elicitation time. Both abiotic elicitors caused an increase in saponin content. Compared to a callus culture, the amount of glycyrrhetin in Glycyrrhiza glabra intact plant was lower, about 0.0058 % of dry weight (DW) measured by HPLC. Unfortunately, glycyrrhizin concentration in callus culture was still very low — comparative intact plant samples contained about 1.6545 % DW of glycyrrhizin.

Effects of arbuscular mycorrhizal fungi on the growth, nutrient uptake and glycyrrhizin production of licorice (Glycyrrhiza uralensis Fisch)

The growth of licorice in arid areas faces nutritional and environmental stresses. Arbuscular mycorrhizal (AM) fungi have been shown to increase the abilities of plants to develop. However, little is known regarding the role of AM fungi in licorice (Glycyrrhiza uralensis) growth. In the present study, by inoculation with two AM fungi, Glomus mosseae (Nicolson & Gerdemann) Gerd. & Trappe and Glomus veriforme (P. Karst.), the effects on licorice growth in sand were examined by measuring plant height, number of leaves, shoot and root fresh weight, and by analyzing morphological parameters of the root system in sand. The influence of the two microorganisms on the accumulation of mineral nutritions and bioactive components in licorice were also investigated. The results showed that mycorrhyzae were of the Arum-type and their colonization frequency (F %), colonization intensity (M %) and colonization intensity (m %) of AM fungi inoculation were found to be 80.0–84.6%, 49.4–60.0% and 58.4–71.9%, respectively. The inoculation significantly improved plant growth during early and late growth stages in comparison with the control. Moreover, inoculation of G. mosseae and G. versiforme, alone or in combination, improved plant phosphorus acquisition in the leaf over non-inoculation plants. In addition, mycorrhiza formation enhanced the glycyrrhizin concentration in roots, but resulted in a considerable reduction of the root oxidase activity. The results indicate that the inoculation with AM fungi could be a useful approach to increase the licorice pharmic quality.

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

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.

Effects of Long Term Administration of Shakuyaku-kanzo-To and Shosaiko-To on Serum Potassium Levels

Typical adverse effects of Shakuyaku-kanzo-To (SKT), an herbal medicine containing licorice, is a licorice-induced pseudoaldosteronism with hypokalemia and hypertension. The risk factors for this side effect are still unclear. To identify the risk factors, we surveyed 37 cases of licorice-induced pseudoaldosteronism in the literature and serum potassium levels in our patients receiving SKT and Shosaiko-To (SST), which contain 6 g and 1.5 g of licorice in the daily dose, respectively. In the case report survey, pseudoaldosteronism developed a median 35 (range 2-231) days after the administration of SKT, which is shorter than after SST (450, range 150-2190 days) and other licorice products including glycyrrhizin (210, range 14-730 days). A significant correlation was observed between the glycyrrhizin contents in the licorice preparations and the dosing periods for developing pseudoaldosteronism (r=-0.700, p<0.01). A negative correlation was also observed between serum potassium level and dosing period for SKT, but not for SST. The difference in age (65.3+/-11.6 vs. 57.2+/-17.3 y) and dosing period (57.3+/-66.3 vs. 19.0+/-24.3 days) between the patients with and without hypokalemia after the administration of SKT was statistically significant (p<0.05). The occurrence rate of hypokalemia including pseudoaldosteronism was around 80% with SKT administration for more than 30 days for patients exceeding 60 years old. It was suggested that patient age (>60 y) and dosing period of SKT (>30 days) might be risk factors for developing pseudoaldosteronism or hypokalemia as well as coadministration of drugs inducing hypokalemia.