Natural triterpenoid saponinl; HMGB1; anti-tumor; anti-diabetic

Dipotassium glycyrrhizinate (0-400 μM, 4 days) suppresses the mRNA levels of atopic dermatitis-related genes (NELL2, CA2, AQP3 and HAS3 gene levels) in cells produced by IL-4 and IL-13 [1]. Dipotassium glycyrrhizinate (0-400 μM, 4 days) partially recovered the atopic dermatitis-like phenotype (spongy cell spaces) in an IL-4/IL-13-induced AD-like skin equivalent mouse [1]. 0-100 μM, 24 hours) reduces sunitinib-induced autophagy and cell death in CCC-HEH-2 cells [5].

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Atopic dermatitis (AD) is a chronic inflammatory skin disease that is not fully understood. Defects in skin barrier function and dysregulation of the Th2 immune response are thought to be pivotal in AD pathogenesis. In this study, we used keratinocytes and AD-like skin equivalent models using Th2 cytokines IL-4 and IL-13. The keratinocytes and AD-like skin model were used to investigate the effect of dipotassium glycyrrhizinate (KG), which is widely used as an anti-inflammatory agent for AD treatment. KG decreased AD-related gene expression in keratinocytes stimulated with Th2 cytokines. KG alleviated AD-like phenotypes and gene expression patterns and inhibited release of AD-related cytokines in the AD-like skin equivalent models. These findings indicate KG has potential effectiveness in AD treatment and AD-like skin equivalent models may be useful for understanding AD pathogenesis.[1]

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Direct effects of Glycyrrhizic acid (GA) on TGR5 in cells transfected TGR5 [2] As in our previous report, we used CHO-K1 cells to transfect the exogenous TGR5 receptor gene. Then, Western blot analysis was applied to confirm the success of experiments. The expressed TGR5 in these CHO-K1 cells was characterized as functional. [2] Then, these cells were applied to estimate the direct effect of GA on the TGR5 receptor. The glucose uptake using intracellular 2-NBDG content as indicator was significantly enhanced by GA treatment in cells that expressed TGR5 (Fig. 3A). Triamterene dose-dependently blocked the GA-induced glucose uptake in these cells (Fig. 3B). Moreover, the cyclic AMP (cAMP) levels were also dose-dependently increased by GA in TGR5-CHO-K1 cells (Fig. 3C) and reduced by triamterene in the same manner (Fig. 3D). However, GA did not affect CHO-K1 cells that lacked TGR5 expression in terms of the increase in 2-NBDG uptake (Fig. 3A) or elevation of cAMP levels (Fig. 3B).

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Glycyrrhizic acid (GA) enhances GLP-1 secretion via TGR5 in intestinal cells [2] Cultured intestinal NCI-H716 cells that have TGR5 receptors are often applied to investigate the GLP-1 release. A marked elevation in GLP-1 secretion (Fig. 4A) or calcium influx (Fig. 4B) was also observed in NCI-H716 cells incubated with GA. However, both effects of GA were deleted after silencing TGR5 gene that was expressed in NCI-H716 cells. It is totally similar to our previous report showing the promotion of GLP-1 release by butelinic acid via TGR5 activation.

Dipotassium glycyrrhizinate (150 mg/kg, i.p.) raises cardiovascular GLP-1 levels and lessens type 1 diabetes symptoms brought on by streptozotocin (65 mg/kg, i.p.) [2]. (50 mg/kg, intraperitoneal injection, once daily for 4 weeks) prevents the development of prostatitis in a model of autoimmune inflammation caused by potassium iodide in rats [3]. Intraperitoneal injection of 50–200 mg/kg dipotassium glycyrrhizinate.

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Glycyrrhizic acid elevated plasma GLP-1 levels in streptozotocin-induced type 1 diabetic rats (STZ-treated rats), while triamterene was sufficient to inhibit Takeda G protein-coupled receptor 5 (TGR5) was blocked [1]. In mice, glycyrrhizic acid (50 mg/kg, ip) dramatically lowers TgAb, HMGB1, TNF-α, IL-6, and IL-1β levels [3].

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Effects of Glycyrrhizic acid (GA) on the changes of plasma glucose in type 1-like diabetic rats [2]
In STZ-induced diabetic rats, injection of Glycyrrhizic acid (GA) attenuated the hyperglycemia in a dose-dependent manner (Fig. 1A). Moreover, plasma insulin levels in STZ-induced diabetic rats (5.35 ± 2.11 pmol/l, n = 8) have been markedly reduced compared to those in normal rats (141.4 ± 10.2 pmol/l, n = 8). However, GA at the highest dose failed to modify the plasma insulin levels in these diabetic rats (5.66 ± 1.39 pmol/l, n = 8). It means that endogenous insulin did not involve in the effect of GA in this animal model. Hyperglycemia was also attenuated in these diabetic rats by sitagliptin at a dose that effectively inhibited the dipeptidyl peptidase-4 (DPP-4). The decrease of hyperglycemia by GA injection was significantly enhanced by sitagliptin in diabetic rats (Fig. 1A). Moreover, triamterene dose-dependently inhibited the GA-induced changes in these diabetic rats regardless of sitagliptin-pretreatment (Fig. 1B).

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Effects of Glycyrrhizic acid (GA) on changes of plasma GLP-1 level in diabetic rats [2]
In the type 1-like diabetic rats, Glycyrrhizic acid (GA) induced a dose-dependent elevation of the plasma GLP-1 levels (Fig. 2A). This effect of Glycyrrhizic acid (GA) was significantly enhanced in diabetic rats by pretreatment with sitagliptin at a dose effective to inhibit DPP-4, a GLP-1 inactivating enzyme (Fig. 2A). Moreover, similar to the changes in blood sugar, the GA-induced increase in plasma GLP-1 level was also reversed by triamterene dose-dependently in these diabetic rats regardless of the pretreatment with sitagliptin (Fig. 2B)

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. The mRNA expression of HMGB1 was significantly higher at 8 and 16 weeks in the NaI group than it was in the control group. Serum levels of thyroglobulin antibodies, HMGB1, tumor necrosis factor alpha, IL-6, and IL-1β were significantly increased in the NaI group, but they were dramatically attenuated with Glycyrrhizic acid (GL) injection. The prevalence of thyroiditis and the infiltration of lymphocytes were significantly decreased in the NaI + Glycyrrhizic acid (GL) group. Glycyrrhizic acid (GL) administration also significantly reduced the protein expression of TLR2, MyD88, HMGB1 and nuclear transcription factor κB in the thyroid gland and attenuated the severity of thyroiditis.[3]
Conclusion: HMGB1 may play a crucial role in autoimmune thyroiditis by causing inflammatory infiltration, thus increasing the severity of autoimmune thyroiditis. Glycyrrhizic acid (GL) effectively attenuated thyroiditis in the iodine-induced NOD.H-2h4 mice via a molecular mechanism related to the inhibition of TLR2-HMGB1 signaling[3].

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Idiopathic pulmonary fibrosis is a progressive and lethal form of interstitial lung disease that lacks effective therapies at present. Glycyrrhizic acid (GA), a natural compound extracted from a traditional Chinese herbal medicine Glycyrrhiza glabra, was recently reported to benefit lung injury and liver fibrosis in animal models, yet whether GA has a therapeutic effect on pulmonary fibrosis is unknown. In this study, we investigated the potential therapeutic effect of GA on pulmonary fibrosis in a rat model with bleomycin (BLM)-induced pulmonary fibrosis. The results indicated that GA treatment remarkably ameliorated BLM-induced pulmonary fibrosis and attenuated BLM-induced inflammation, oxidative stress, epithelial-mesenchymal transition, and activation of transforming growth factor-beta signaling pathway in the lungs. Further, we demonstrated that GA treatment inhibited proliferation of 3T6 fibroblast cells, induced cell cycle arrest and promoted apoptosis in vitro, implying that GA-mediated suppression of fibroproliferation may contribute to the anti-fibrotic effect against BLM-induced pulmonary fibrosis. In summary, our study suggests a therapeutic potential of GA in the treatment of pulmonary fibrosis.[6]

Antibacterial assay [4]
The antibacterial activity of GL gel was evaluated by growth inhibition efficiency and minimum inhibitory concentration (MIC). The Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were used as the representative gram-positive and gram-negative bacteria, respectively. In brief, a single colony was inoculated into 5 mL Luria-Bertani (LB) broth medium and incubated at 37 ◦C overnight with shaking at 200 rpm. After overnight pre-culture, bacterial suspensions were diluted to 106 cfu mL-1 in LB medium, and then supplemented with PBS or GL gel at final concentrations ranging from 0 to 2 mM. The cultures were then incubated at 37 ◦C overnight with gentle shaking. The optical density of the culture was measured using a Synergy H1 microplate reader at a wavelength of 600 nm. MIC was determined as the lowest concentration of antibacterial materials when no visible growth of bacterium was observed.1 The same procedure was followed for realtime monitoring the growth kinetics of bacteria treated with PBS or GL gel at different concentrations, except that the OD600 of each culture was recorded at different time points during the incubation process. Especially, 106 cfu mL-1 of S. aureus and E. coli were incubated with PBS or GL gel at a final concentration of MIC, respectively. After 2 h of incubation, the bacterial suspension was diluted 100 times with PBS and 100 μL of the diluted bacteria were spread on the agar culture plate and incubated at 37 ◦C overnight.

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Live/dead fluorescent staining [4]
After treatment with PBS or GL gel at MIC for 2 h, both S. aureus and E. coli were harvested by centrifugation and washed with 0.9 % NaCl 3 times. The bacteria were then stained with SYTO 9 and propidium iodide (PI) from live/dead staining kit (L7012) for 30 min in the dark. The bacteria were then visualized with a LSM 700 confocal laser scanning microscope imaging system.

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Morphological characterization of bacteria [4]
SEM was used to study the morphologies of bacteria after treatment with GL gel. 106 cfu mL-1 of S. aureus and E. coli were incubated with PBS or GL gel at a final concentration of MIC for 2 h, and were collected by centrifugation and fixed with 2.5 % (w/w) glutaraldehyde for 2 h. After washing with PBS 3 times, the bacteria were dropped onto silicon wafer and dried overnight. The bacteria on the surface of silicon wafer were sequentially dehydrated with graded ethanol aqueous solutions (30 %, 50 %, 70 %, 80 %, 90 % and 100 %) for 10 min. After drying overnight, the samples were coated with gold and imaged using a Phenom World-Pro X scanning electron microscopy.

Cell viability assay [4]
Cell viability assay in L929 fibroblasts was carried out to determine the in vitro cytotoxicity of GL gel. Briefly, the L929 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) in an atmosphere of 5 % CO2 at 37 ◦C. The cells were harvested and seeded in 96-well plate at a seeding density of 1 × 104 cells per well. After overnight incubation, the DMEM media were replaced by fresh media containing as-prepared GL gel at certain concentrations. After incubating for 24 h, the culture media was replaced by 100 μL media containing 10 % CellTiter-BlueTM reagent and incubated for 2 h at 37 ◦C. The fluorescence intensity was measured at 550/590 nm (Ex/Em) using Synergy H1 microplate reader. The relative cell viability was calculated by comparison to the negative control which is L929 cells treated with PBS.

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Cell imaging [4]
The L929 cells were seeded in 24-well plate with a seeding density of 2 × 104 cells per well. After overnight culture, the cells were treated with DMEM containing 2 mM of GL gel and co-cultured at 37 ◦C for 24 h. The treated cells were washed with PBS and fixed with 4% (w/v) paraformaldehyde for 15 min at room temperature. Then, the cells were stained with FITCphalloidin for 1 h, followed by DAPI staining of the cell nucleic acid for 15 min. After washing, the cells were imaged by a LSM 700 confocal laser scanning microscope imaging system.

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Hemolysis assay [4]
The red blood cells (RBCs) were isolated from rat blood by centrifugation at 2000 rpm for 5 min. The precipitated RBCs were washed with PBS for 3 times before use. The RBCs were suspended in PBS at the volume concentration of 2 %, and supplemented with GL gel at final concentrations ranging from 1 to 2 mM. The RBCs treated with PBS and water were set as the negative control and positive control, respectively. After 2 h of incubation at 37 ◦C, the treated RBCs were centrifuged at 2000 rpm for 5 min. The absorbance of the supernatant at 545 nm was measured using a microplate reader. The hemolysis ratio was calculated using the following equation.

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CHO-K1 cells used to transfect TGR5 [2]
CHO-K1 cells were prepared from CHO cell line using the ovary from adult Chinese hamster as described previously. In the current experiment, the expression vector of human TGR5 cDNA was used to transfect into CHO-K1 cells according to our previous report. The next day, successful transfection was confirmed by Western blot. Then, cells expressing TGR5 were used for Glycyrrhizic acid (GA) treatment.

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Measurement of glucose uptake in cells [2]
The 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) was used to determine the glucose uptake using this fluorescent glucose analog as described in our previous report. The fluorescence intensity in each cell sample was determined using a fluorescence spectrofluorometer. Protein was assayed by BCA assay kit. Then, uptake of 2-NBDG was quantified in the cells that received Glycyrrhizic acid (GA) treatment. The effectiveness of triamterene was also compared with vehicle-treated group after pretreatment for 30 min.

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Assay of cAMP in cells [2]
Following our previous method, the cells were treated with Glycyrrhizic acid (GA) for 72 h. Intracellular cAMP level was then determined by the ELISA kit, similar to our previous method. Each measurement was performed in duplicate.

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Assay of extracellular GLP-1 [2]
We used NCI-H716 cells (5 × 105 cells per well) to treat with Glycyrrhizic acid (GA) at the indicated concentration under 37 °C for 1 h. Then, supernatants were employed for analysis of extracellular GLP-1 level using ELISA kit, according to our previous method. Each duplicate measurement performed on the indicated samples.

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Cell Culture and Treatment [6]
Murine fibroblast cell line 3T6 was purchased from the Cell Bank of China Academy of Sciences. The cells were cultured in DMEM supplemented with 10% FBS at 37°C in a humidified atmosphere of 95% air and 5% CO2. Glycyrrhizic acid (GA) was dissolved in DMSO to make 180 mM concentrated stocks, which were diluted with PBS to make the 9 mM working solution. 3T6 cells were treated with 5, 10, 25, 50, 100, and 200 μM GA for 24 h, and the cytotoxicity was then analyzed by measuring the activity of lactate dehydrogenase (LDH) in the conditioned culture medium using the LDH Activity Assay Kit. Low (25 μM), medium (50 μM), and high (100 μM) doses of GA were selected to treat the cells in later experiments.

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Proliferation Assay [6]
Cell proliferation was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. 3T6 cells were seeded in 96-well microplates at a density of 3,000 cells per well and cultured at 37°C for 24 h. Glycyrrhizic acid (GA) was added into each well to the indicated final concentration (0, 25, 50, or 100 μM). After 24 or 48 h GA treatment, MTT was added into the culture medium to a final concentration of 0.2 mg/ml for 4 h incubation at 37°C. Thereafter, the medium was aspirated and the formazan crystals were dissolved completely in 200 μl DMSO per well. The optical density (OD) at 490 nm was recorded by an ELX-800 microplate reader. Each assay point was done in five replicates.

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Flow Cytometric Analysis of Cell Cycle and Apoptosis [6]
Following Glycyrrhizic acid (GA) treatment for 24 h, the cells were analyzed for cell cycle and apoptosis by flow cytometry. For cell cycle analysis, the cells were harvested, fixed in 70% ethanol at 4°C for 2 h, and incubated with the propidium iodide (PI) solution for 30 min at 37°C in the dark, followed by analysis in FACSCalibur flow cytometer. Cell apoptosis was assayed with the Annexin V-FITC/PI Apoptosis Detection Kit according to the manufacturer’s instructions. Following staining, the apoptotic status of the cells was analyzed by flow cytometry.

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Scratch Wound Assay [6]
Cell migration was assessed by the well-established in vitro scratch wound assay (Liang et al., 2007). The confluent monolayer of 3T6 cells was incubated with 5 μM mitomycin-C for 2 h to inhibit cell proliferation. A scratch was evenly created by horizontally crossing the surface of the cell monolayer with a 200 μl pipette tip. The detached cells were washed off with serum-free medium, and the cells were cultured with serum-free medium containing the indicated concentration of Glycyrrhizic acid (GA) for 24 h at 37°C in a 5% CO2 incubator. The cells were photographed under an inverted microscope at 0, 6, 12, and 24 h post-scratching, and the rate of wound closure was calculated as (original gap distance - gap distance at the indicated time point)/original gap distance × 100%.

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Transwell Assay [6]
3T6 cells were pre-treated with 5 μM mitomycin-C for 2 h, and resuspended in culture medium containing the indicated concentration of Glycyrrhizic acid (GA). 2 × 104 cells in 200 μl suspension were plated in one Transwell chamber pre-coated with Matrigel. The Transwell chamber was then placed into a 24-well plate with each well-containing 800 μl culture medium supplemented with 20% FBS. The cells were cultured for 24 h at 37°C in an atmosphere of 5% CO2. Thereafter, the cells and the Matrigel on the top surface of the Transwell membrane were wiped off, and the cells on the bottom surface of the membrane were fixed with paraformaldehyde and stained with crystal violet. The cells were observed under a 200 × inverted microscope, and the numbers of the invading cells were counted in five fields on each membrane.

Animal/Disease Models: Iodine-induced autoimmune thyroiditis mouse model [3]
Doses: 50 mg/kg
Route of Administration: intraperitoneal (ip) injection, one time/day, Results lasting for 4 weeks: diminished serum TgAb, HMGB1, TNF-α, IL-6, IL-1β levels. Reduce the prevalence and lymphocyte infiltration of thyroiditis and reduce the severity of thyroiditis.

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Assay of plasma glucose and GLP-1 levels in diabetic rats [2]
Diabetic rats were orally treated with 5 mg/kg/day sitagliptin to inhibit DPP-4 or vehicle for two week before the administration of Glycyrrhizic acid (GA). Rats were fasted overnight without water restriction before the experiment. Then, Glycyrrhizic acid (GA) at desired dose was administrated to rats by intraperitoneal injection with or without pretreatment with sitagliptin for 30 min. The blood samples were collected 1 h after administration of Glycyrrhizic acid (GA) from the femoral vein of rats under pentobarbital anaesthesia (30 mg/kg, i.p.). The plasma glucose levels were measured in an automatic analyzer as described previously. The plasma GLP-1 levels were estimated with a commercial ELISA kit, as described in our previous report.

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A total of 80 male NOD.H-2h4 mice were randomly divided into a control or iodine supplement (NaI) group at four weeks of age, and the control group was fed with regular water, whereas the NaI group was supplied with 0.005% sodium iodine water. Another 24 male NOD.H-2h4 mice were also randomized into three groups (eight mice per group) as follows: control, NaI, and GL treatment after iodine supplementation (NaI + GL). The NOD.H-2h4 mice were fed with 0.005% sodium iodide water for eight weeks to enhance autoimmune thyroiditis. After iodine treatment, the mice received intraperitoneal injections of GL for four weeks. The severity of lymphocytic infiltration in the thyroid gland was measured by histopathological studies. The serum levels of HMGB1, tumor necrosis factor alpha, interleukin (IL)-6, IL-1β, and thyroglobulin antibody titers were measured using an enzyme-linked immunosorbent assay. HMGB1 expression was measured by immunohistochemical staining and real-time polymerase chain reaction. TLR2, HMGB1, MyD88, and nuclear transcription factor κB were measured by Western blot.[3]

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Establishment of BLM-induced Pulmonary Fibrosis in Rats and Glycyrrhizic acid (GA) Treatment [6]
Forty male 8-weeks-old Sprague-Dawley rats, weighing around 250 g, were used in this study. The rats were randomly divided into five groups with eight rats in each group: control, BLM, BLM+GA50, BLM+GA100, and BLM+GA200. Induction of pulmonary fibrosis with BLM was conducted according to a previously described method (Thrall et al., 1979). All rats were anesthetized with 10% hydrate chloride at 3.5 ml/kg body weight (bw). Following anesthesia, a midline cut of the neck skin was made, and the trachea was exposed by blunt dissection. The needle of 1 ml syringe was inserted into the trachea, and bleomycin, dissolved in 100 μl sterile saline, was injected into the rat’s lungs at a dose of 5 mg/kg bw, while an equal volume of saline was injected into the rats from the Control group. The rats were rotated immediately after injection to ensure an even distribution of BLM in the lungs, and then the neck skin incision was sewn. Thereafter, the rats from the BLM+GA50, BLM+GA100, and BLM+GA200 groups received an intraperitoneal injection of GA at a dose of 50, 100, and 200 mg/kg bw respectively every day for a total of 28 days, and the rest animals received saline. The rats were sacrificed 28 days after BLM induction, the bronchoalveolar lavage fluids (BALFs) were collected by intratracheal instillation and draining of 1.5 ml saline for three times, and then the lungs were excised for further analysis.

Absorption, Distribution and Excretion
Glycyrrhizic acid is mainly absorbed after presystemic hydrolysis and formation of glycyrrhetinic acid. Therefore, after oral administration of a dose of 100 mg of glycyrrhizic acid, this major metabolite appears in plasma in a concentration of 200 ng/ml while glycyrrhizic acid cannot be found. The finding of a minimal amount of glycyrrhizic acid in urine suggests the existence of a partial absorption in the gastrointestinal tract.
Glycyrrhizic acid presents a biphasic elimination from the central compartment with a dose-dependent second elimination phase. The majority of the administered dose is eliminated by the bile in which glycyrrhizic acid can be eliminated unchanged and undergoes enterohepatic cycling. On the other hand, the major metabolite, glycyrrhetinic acid, forms glucuronide and sulfate conjugates. These conjugates are efficiently transported into the bile and duodenum where commensal bacteria hydrolizes the conjugate for the formation of glycyrrhetinic acid and further reabsorption. This reabsorption behavior seems to be related to the activity of 3-alpha-hydroxysteroid dehydrogenase which transports very efficiently the metabolite from the plasma to the bile. About 1.1-2.5% of the administered dose of glycyrrhizic acid can be found in urine which corresponds to the minimal cycling and reabsorption of this compound.
The apparent volume of distribution of glycyrrhizic acid either in the central compartment and in steady-state are in the range of 37-64 ml/kg and 59-98 ml/kg, respectively.
The constant reabsorption of glycyrrhetic acid in the duodenum causes a delay in the terminal plasma clearance. The reported total body clearance of glycyrrhizic acid is reported to be in the range of 16-25 ml.kg/h.
GLYCYRRHIZIN WAS ABSORBED IN RAT SMALL INTESTINE; THERE WAS NO DETECTABLE AMT OF GLYCYRRHETINIC ACID IN BLOOD AFTER BOLUS INJECTION OF GLYCYRRHIZIN INTO PORTAL VEIN; GLYCYRRHETINIC ACID WAS PRESENT IN DETECTABLE AMT IN BLOOD AFTER ORAL ADMIN.
Glycyrrhizic acid (GZA) and glycyrrhetinic acid (GRA) can be determined rapidly and precisely by high-performance liquid chromatography (HPLC) in biological fluids and tissues from experimental animals and humans. From plasma and tissues, glycyrrhizic acid and glycyrrhetinic acid are extracted by organic solvents and the extracts can directly be used for HPLC. From bile or urine, extraction and determination of glycyrrhizic acid and glycyrrhetinic acid are more difficult due to interfering endogenous compounds and conjugation of glycyrrhetinic acid with glucuronides or sulfates. Extraction of glycyrrhizic acid and glycyrrhetinic acid from urine or bile can be performed by ion-pairing followed by extraction with organic solvents or by solid phase extraction. Glycyrrhetinic acid conjugates can be determined by chromatographic separation or by pretreatment with beta-glucuronidase. The pharmacokinetics of glycyrrhetinic acid and glycyrrhizic acid can be described by a biphasic elimination from the central compartment with a dose-dependent second elimination phase. Depending on the dose, the second elimination phase in humans has a half-life of 3.5 hours for glycyrrhizic acid and between 10-30 hours for glycyrrhetinic acid. The major part of both glycyrrhetinic acid or glycyrrhizic acid is eliminated by the bile. While glycyrrhizic acid can be eliminated unmetabolized and undergoes enterohepatic cycling, Glycyrrhetinic acid is conjugated to glycyrrhetinic acid glucuronide or sulfate prior to biliary excretion. Orally administered glycyrrhizic acid is almost completely hydrolyzed by intestinal bacteria and reaches the systemic circulation as glycyrrhetinic acid.
Glycyrrhizic acid is currently of clinical interest for treatment of chronic hepatitis. It is also applied as a sweetener in food products and chewing tobacco. In some highly exposed subgroups of the population, serious side effects such as hypertension and electrolyte disturbances have been reported. In order to analyze the health risks of exposure to this compound, the kinetics of glycyrrhizic acid and its active metabolites were evaluated quantitatively. Glycyrrhizic acid and its metabolites are subject to complex kinetic processes, including enterohepatic cycling and presystemic metabolism. In humans, detailed information on these processes is often difficult to obtain. Therefore, a model was developed that describes the systemic and gastrointestinal tract kinetics of glycyrrhizic acid and its active metabolite glycyrrhetic acid in rats. Due to the physiologically based structure of the model, data from earlier in vitro and in vivo studies on absorption, enterohepatic cycling, and presystemic metabolism could be incorporated directly. The model demonstrates that glycyrrhizic acid and metabolites are transported efficiently from plasma to the bile, possibly by the hepatic transfer protein 3-alpha-hydroxysteroid dehydrogenase. Bacterial hydrolysis of the biliary excreted metabolites following reuptake of glycyrrhetic acid causes the observed delay in the terminal plasma clearance of glycyrrhetic acid. These mechanistic findings, derived from analysis of experimental data through physiologically based pharmacokinetic modeling, can eventually be used for a quantitative health risk assessment of human exposure to glycyrrhizic acid containing products. Copyright 2000 Academic Press.
To assess the multiplicity for the biliary excretion of xenobiotic conjugates, glycyrrhizic acid (glycyrrhizin) was studied in rats after intravenous (IV) injection of 10 mg/kg glycyrrhizic acid and IV infusion of inhibitors, dibromosulfophthalein and indocyanine green. Indocyanine green did not affect the biliary excretion of glycyrrhizic acid, whereas dibromosulfophthalein reduced it significantly. The plasma level of glycyrrhizic acid was increased by dibromosulfophthalein, but not by indocyanine green. In Eisai hyperbilirubinemic rats, the biliary excretion of glycyrrhizic acid was severely impaired, resulting in an increased plasma level. The findings suggested that the biliary excretion of glycyrrhizic acid is mediated by the system shared by liquiritigenin glucuronides and dibromosulfophthalein, but not by indocyanine green, and that the system is hereditarily defective in Eisai hyperbilirubinemic rats.

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Metabolism / Metabolites
When orally administered, glycyrrhizic acid is almost completely hydrolyzed by intestinal bacteria for the formation of glycyrrhetinic acid, which is an active metabolite and can enter systemic circulation, and two molecules of glucuronic acid. This metabolite is transported and taken in the liver for its metabolization to form glucuronide and sulfate conjugates.
BOLUS INJECTION OF GLYCYRRHIZIN GIVEN RATS IN PORTAL VEIN, GAVE RISE IN BLOOD LEVEL OF SUBSTANCE WHICH APPEARS TO BE GLUCURONIC ACID CONJUGATE FORMED AS METABOLITE OF GLYCYRRHETINIC ACID.

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Biological Half-Life
Depending on the dose, the second elimination phase in humans has a half-life of 3.5 hours.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Licorice (Glycyrrhiza glabra) root contains glycyrrhizin (also called glycyrrhizic acid or glycyrrhizinic acid) and a mixture of the potassium and calcium salts of glycyrrhizic acid. Glycyrrhizin is metabolized to the active glycyrrhetinic acid in the intestine. Deglycyrrhizinated licorice (DGL) has had glycyrrhizin removed. Licorice is a purported galactogogue, and is included in some Asian proprietary mixtures to increase milk supply; however, no scientifically valid clinical trials support this use. In fact, licorice usually reduces serum prolactin, which might decrease milk production in the early stages of lactation. Women taking licorice have experienced elevated blood pressure. Galactogogues should never replace evaluation and counseling on modifiable factors that affect milk production. Some mothers in Türkiye reportedly use licorice to improve the taste and quality of their milk.
Glycyrrhizin is detectable in the breastmilk of some women taking licorice, but studies measuring glycyrrhetinic acid have not been performed. Licorice has been used safely and effectively in combination with other herbs given to infants as a tea for the short-term treatment of colic. However, two infants whose mothers had an excessive intake of an herbal tea that contained licorice had signs of anethole toxicity. Because both of these papers reported on herbal mixtures, the effect(s) of licorice alone cannot be determined. Licorice and licorice extract are "generally recognized as safe" (GRAS) as foods by the U.S. Food and Drug Administration. Long-term, excessive use of licorice can cause hypertension, hypokalemia, and disturbances of adrenal hormones, and therefore should probably be avoided during nursing.
Dietary supplements do not require extensive pre-marketing approval from the U.S. Food and Drug Administration. Manufacturers are responsible to ensure the safety, but do not need to prove the safety and effectiveness of dietary supplements before they are marketed. Dietary supplements may contain multiple ingredients, and differences are often found between labeled and actual ingredients or their amounts. A manufacturer may contract with an independent organization to verify the quality of a product or its ingredients, but that does not certify the safety or effectiveness of a product. Because of the above issues, clinical testing results on one product may not be applicable to other products. More detailed information about dietary supplements is available elsewhere on the LactMed Web site.
◉ Effects in Breastfed Infants
Two breastfed infants, aged 15 and 20 days, were admitted to the hospital for a reported lack of weight gain in the previous 7 to 10 days, caused by "difficult feeding". The parents reported restlessness and vomiting during the past day. One of the mothers also reported feeling drowsy and weak. On examination, the infants were afebrile but had hypotonia, lethargy, emesis, weak cry, poor sucking and weak responses to painful stimuli. Infant laboratory values, electrocardiograms and blood pressures were normal, and septic work-ups were negative. Both mothers had both been drinking more than 2 liters daily of an herbal tea mixture reportedly containing licorice, fennel, anise, and goat's rue to stimulate lactation. After the mothers discontinued breastfeeding and the herbal tea, the infants improved within 24 to 36 hours. Symptoms of the affected mother also resolved rapidly after discontinuing the herbal tea. After 2 days, breastfeeding was reinstituted with no further symptoms in the infants. Both infants were doing well at 6 months of age. The authors attributed the maternal and infant symptoms to anethole, which is found in both anise and fennel; however, the anethole levels were not measured in breastmilk, nor were the teas tested for their content.
◉ Effects on Lactation and Breastmilk
A woman with a history of excessive licorice intake had amenorrhea, severe headaches, hypertension, hypokalemia. She had elevated serum prolactin levels that remained abnormal for one month after licorice discontinuation and normalized by 6 months after discontinuation.
In a study of 25 men and 25 women, the baseline and thyrotropin-stimulated serum prolactin levels were measured to determine normal serum prolactin values. Subjects who regularly ingested licorice had lower basal and lower stimulated serum prolactin concentrations.
A traditional, nonstandardized decoction of peony and licorice roots called Shaoyao-Gancao-Tang in Chinese and Shakuyaku-Kanzo-To in Japanese was studied in women with elevated serum prolactin caused by long-term (>6 months) ingestion of risperidone. Patients received either bromocriptine 5 mg daily for 4 weeks followed by 4 weeks of 22.5 grams daily of the peony-licorice decoction (equivalent to 25 mg of glycyrrhetinic acid), or the same drugs in the reverse order. Evaluation of serum prolactin found that both treatments reduced serum prolactin by 21 to 28% from baseline at 4 and 8 weeks.
Forty women who complained of an insufficient milk supply at 5 days postpartum were given a combination herbal supplement as 2 capsules of Lactare (Pharma Private Ltd., Madras, India; currently available from TTK Pharma, Chennai, India) 3 times daily. Each capsule contained wild asparagus 200 mg, ashwagandha (Withania somnifera) 100 mg, fenugreek 50 mg, licorice 50 mg, and garlic 20 mg. By day 4 of therapy, no infants required supplementary feeding. Infants were weighed before and after each feeding on the fifth day of maternal therapy to determine the amount of milk ingested. On the day of the test weighing, infants' milk intake averaged 388 mL, and the fluid and caloric intake was considered adequate. This study cannot be considered as valid evidence of a galactogogue effect of these herbs because it lacks randomization, blinding, a placebo control, and maternal instruction in breastfeeding technique. Additionally, infants were breastfed only 6 to 8 times daily, which is insufficient to maximize milk supply at this stage of lactation.
Women who were between 14 and 90 days postpartum and reported lactation failure were given instructions on breastfeeding technique and encouraged to exclusively breastfeed. If their infant had gained less than 15 grams in 1 week, they were randomized to receive either two tablespoonfuls of a mixture containing wild asparagus or an identical placebo for 4 weeks. In each 100 grams, the mixture contained Asparagus racemosus 15 grams, Anethum soiva 1 gram, Ipomea digitata 1 gram, Glycyrrhiza glabra 1 gram, Spinacia oleracea 2.5 grams, Cuminum cyminum 0.5 gram, and panchatrinamol 1 gram. Of the 64 women randomized, 11 did not complete the trial. Serum prolactin measurements were made before a morning nursing before treatment and after 4 weeks of treatment. Infant weight gains and the number of supplemental feedings were recorded initially and after 4 weeks of therapy. No differences were found in the changes in serum prolactin, infant weight gain or amount of supplementation between the treatment and placebo groups after 4 weeks of therapy. No side effects or changes in liver function tests occurred during the study.
A study in Japan compared the use of a mixture of 13 herbs, including licorice, to ergonovine for their effects on lactation and serum prolactin in postpartum women. The herbal mixture, called Xiong-gui-tiao-xue-yin, was given in a randomized fashion to 41 women in a dose of 2 grams of a dried aqueous extract 3 times daily. A comparable group of 41 women were randomized to receive methylergonovine 0.375 mg daily. Therapy was started on the day of delivery, but the duration of therapy was not specified. Plasma oxytocin and prolactin were measured on days 1 and 6; milk volumes were measured daily, although the method of measuring milk volume was not specified. Serum prolactin was higher on days 1 and 6 in the women who received the herbals; plasma oxytocin was lower on day 1 in the women who received the herbal, but not different on day 6. Milk volumes were greater on days 4, 5, and 6 in women who received the herbal mixture. This study has serious flaws that make its interpretation impossible. First, milk volume measurement is subject to considerable variability depending on the measurement method used, but the method was not specified. Second, methylergonovine has caused decreases in serum prolactin and milk production in some studies. Because of the lack of a placebo group, the differences found could be a negative effect of methylergonovine rather than a positive effect of the herbal preparation. Because this study used a multi-ingredient combination product in which licorice was only one component, the results might be different from studies in which licorice was used alone.
In an uncontrolled, non-blinded multicenter study in India, 1132 patients who reported inadequate milk supply were give a mixture (Lactancia, Corona Remedies Pvt. Ltd.) To take in a dose of 30 grams twice daily. The product contains Asparagus racemosus (wild asparagus, shatavari), Cuminum cyminum (cumin), Glycyrrhiza glabra (licorice), Spinacia oleracea (spinach) as well as amino acids, vitamins, minerals and DHA. Most of the mothers (1049) had improved lactation and increased infant weight. However, with no placebo control group, results cannot be attributed to the product.

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Protein Binding
Glycyrrhizic acid does not bind to any plasma proteins as it is not absorbed systemically. On the other hand, its main active metabolite, glycyrrhetinic acid presents a very large binding to serum proteins such as albumin.

[1]. Ameliorating effect of dipotassium glycyrrhizinate on an IL-4- and IL-13-induced atopic dermatitis-like skin-equivalent model. Arch Dermatol Res. 2019 Mar;311(2):131-140.

[2]. Glycyrrhizic acid increases glucagon like peptide-1 secretion via TGR5 activation in type 1-like diabetic rats. Biomed Pharmacother. 2017 Nov;95:599-604.

[3]. Glycyrrhizin, a Direct HMGB1 Antagonist, Ameliorates Inflammatory Infiltration in a Model of Autoimmune Thyroiditis via Inhibition of TLR2-HMGB1 Signaling. Thyroid. 2017 May;27(5):722-731.

[4]. A Simple Injectable Moldable Hydrogel Assembled from Natural Glycyrrhizic Acid with Inherent Antibacterial Activity. ACS Appl. Bio Mater. 2020, 3, 1, 648–653.

[5]. Autophagic degradation of CCN2 (cellular communication network factor 2) causes cardiotoxicity of sunitinib. Autophagy. 2022 May;18(5):1152-1173.

[6]. Glycyrrhizic acid alleviates bleomycin-induced pulmonary fibrosis in rats. Front Pharmacol. 2015 Oct 1;6:215.

Glycyrrhizinate dipotassium is an organic molecular entity.
A widely used anti-inflammatory agent isolated from the licorice root. It is metabolized to GLYCYRRHETINIC ACID, which inhibits 11-BETA-HYDROXYSTEROID DEHYDROGENASES and other enzymes involved in the metabolism of CORTICOSTEROIDS. Therefore, glycyrrhizic acid, which is the main and sweet component of licorice, has been investigated for its ability to cause hypermineralocorticoidism with sodium retention and potassium loss, edema, increased blood pressure, as well as depression of the renin-angiotensin-aldosterone system.
See also: Glycyrrhizin (has active moiety); Glycyrrhiza Glabra (part of); Glycyrrhizinate dipotassium; xylitol (component of) ..

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Glycyrrhizic acid (GA) is belonged to triterpenoid saponin that is contained in the root of licorice and is known to affect metabolic regulation. Recently, glucagon like peptide-1 (GLP-1) has widely been applied in diabetes therapeutics. However, the role of GLP-1 in GA-induced anti-diabetic effects is still unknown. Therefore, we are interested in understanding the association of GLP-1 with GA-induced effects. In type 1-like diabetic rats induced by streptozotocin (STZ-treated rats), GA increased the level of plasma GLP-1, which was blocked by triamterene at a dose sufficient to inhibit Takeda G-protein-coupled receptor 5 (TGR5). The direct effect of GA on TGR5 has been identified using the cultured Chinese hamster ovary cells (CHO-K1 cells) transfected TGR5 gene. Moreover, in intestinal NCI-H716 cells that secreted GLP-1, GA promoted GLP-1 secretion with a marked elevation of calcium levels. However, both effects of GA were reduced by ablation of TGR5 with siRNA in NCI-H716 cells. Therefore, we demonstrated that GA can enhance GLP-1 secretion through TGR5 activation.[2]
Taken together, we demonstrated for the first time that GA could increase plasma GLP-1 level via TGR5 activation in tyep 1-like diabetic rats. Therefore, GA could have future applications in the clinic. [2]

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igh mobility group box-1 (HMGB1), a non-histone protein, plays an important role in autoimmune diseases. However, the significance of HMGB1 in the pathogenesis of autoimmune thyroiditis has not been reported. The purpose of this study was to explore whether HMGB1 participates in the pathogenesis of autoimmune thyroiditis, and whether glycyrrhizin (GL), a direct inhibitor of HMGB1, attenuates the severity of thyroid inflammatory infiltration in a murine model of autoimmune thyroiditis.[3]

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Injectable low-molecular-weight hydrogels (LMWHs) from biocompatible materials have attracted much attention in biomedical applications because they can adapt any desired sizes and cavity shapes. Searching for simple, biocompatible injectable LMWHs owning inherent antibacterial activity without complicated chemical modification remains an open question to avoid the tedious synthesis/purification process and the easy bacterial infection of hydrogels in a moist environment. In this work, glycyrrhizic acid (GL), a naturally occurring compound, was found to form a stable transparent LMWH at 37 °C in physiological phosphate buffered saline (PBS) with nanoclusters as the microstructures. Moreover, this hydrogel exhibited great injectable and moldable properties. The antibacterial study showed that the growth of Gram-positive Staphylococcus aureus (S. aureus) could be completely inhibited by GL, whereas noneffect on Gram-negative Escherichia coli (E. coli) was observed. In addition, cell viability and hemolysis assay revealed that GL had good biocompatibility and hemocompatibility to mammalian cells because of its natural origin. Our simple biocompatible injectable moldable LMWH with inherent antibacterial ability has potential in the area of biomaterials and 3D bioprinting.
In summary, we used natural glycyrrhizic acid as a gelator to construct a stable LMWH at 37 °C in physiological PBS without additional chemical modification. This nanocluster-structured LMWH exhibited great injectable and moldable properties. Moreover, the growth of Gram-positive S. aureus could be completely inhibited by GL compared with Gram-negative E. coli. In addition, cell viability and hemolysis assays revealed that GL has low cytotoxicity and good hemocompatibility to mammalian cells because of its natural origin. Our work provides a simple, biocompatible injectable moldable LMWH with inherent antibacterial ability, which will be of great interest in the area of biomaterials and 3D bioprinting.[4]

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Excessive macroautophagy/autophagy is one of the causes of cardiomyocyte death induced by cardiovascular diseases or cancer therapy, yet the underlying mechanism remains unknown. We and other groups previously reported that autophagy might contribute to cardiomyocyte death caused by sunitinib, a tumor angiogenesis inhibitor that is widely used in clinic, which may help to understand the mechanism of autophagy-induced cardiomyocyte death. Here, we found that sunitinib-induced autophagy leads to apoptosis of cardiomyocyte and cardiac dysfunction as the cardiomyocyte-specific Atg7-/+ heterozygous mice are resistant to sunitinib. Sunitinib-induced maladaptive autophagy selectively degrades the cardiomyocyte survival mediator CCN2 (cellular communication network factor 2) through the TOLLIP (toll interacting protein)-mediated endosome-related pathway and cardiomyocyte-specific knockdown of Ccn2 through adeno-associated virus serotype 9 (AAV9) mimics sunitinib-induced cardiac dysfunction in vivo, suggesting that the autophagic degradation of CCN2 is one of the causes of sunitinib-induced cardiotoxicity and death of cardiomyocytes. Remarkably, deletion of Hmgb1 (high mobility group box 1) inhibited sunitinib-induced cardiomyocyte autophagy and apoptosis, and the HMGB1-specific inhibitor glycyrrhizic acid (GA) significantly mitigated sunitinib-induced autophagy, cardiomyocyte death and cardiotoxicity. Our study reveals a novel target protein of autophagic degradation in the regulation of cardiomyocyte death and highlights the pharmacological inhibitor of HMGB1 as an attractive approach for improving the safety of sunitinib-based cancer therapy. [5]

C42H60K2O16

899.1128

898.315

68797-35-3

Ammonium glycyrrhizinate;53956-04-0;Glycyrrhizic acid;1405-86-3

656852

White to off-white solid powder

6

16

5

60

1720

19

C[C@]12CC[C@](C[C@H]1C3=CC(=O)[C@@H]4[C@]5(CC[C@@H](C([C@@H]5CC[C@]4([C@@]3(CC2)C)C)(C)C)O[C@@H]6[C@@H]([C@H]([C@@H]([C@H](O6)C(=O)[O-])O)O)O[C@H]7[C@@H]([C@H]([C@@H]([C@H](O7)C(=O)[O-])O)O)O)C)(C)C(=O)O.[K+].[K+]

BIVBRWYINDPWKA-VLQRKCJKSA-L

InChI=1S/C42H62O16.2K/c1-37(2)21-8-11-42(7)31(20(43)16-18-19-17-39(4,36(53)54)13-12-38(19,3)14-15-41(18,42)6)40(21,5)10-9-22(37)55-35-30(26(47)25(46)29(57-35)33(51)52)58-34-27(48)23(44)24(45)28(56-34)32(49)50;;/h16,19,21-31,34-35,44-48H,8-15,17H2,1-7H3,(H,49,50)(H,51,52)(H,53,54);;/q;2*+1/p-2/t19-,21-,22-,23-,24-,25-,26-,27+,28-,29-,30+,31+,34-,35-,38+,39-,40-,41+,42+;;/m0../s1

dipotassium;(2S,3S,4S,5R,6R)-6-[(2S,3R,4S,5S,6S)-2-[[(3S,4aR,6aR,6bS,8aS,11S,12aR,14aR,14bS)-11-carboxy-4,4,6a,6b,8a,11,14b-heptamethyl-14-oxo-2,3,4a,5,6,7,8,9,10,12,12a,14a-dodecahydro-1H-picen-3-yl]oxy]-6-carboxylato-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylate

Dipotassium glycyrrhizinate; Glycyrrhizinate dipotassium; 68797-35-3; Dipotassium glycyrrhizate; CHEBI:79402; CA2Y0FE3FX; Neubormitin; Glycyrrhizic acid dipotassium;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~50 mg/mL (~55.61 mM)
DMSO : ~20.83 mg/mL (~23.17 mM)

Solubility in Formulation 1: 100 mg/mL (111.22 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)