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 primarily absorbed after first-pass hydrolysis to glycyrrhetinic acid. Therefore, after oral administration of 100 mg glycyrrhizic acid, the plasma concentration of the major metabolite, glycyrrhetinic acid, is 200 ng/ml, while glycyrrhizic acid is undetectable. Only trace amounts of glycyrrhizic acid are detected in urine, suggesting partial absorption in the gastrointestinal tract. Glycyrrhizic acid undergoes biphasic elimination in the central nervous system, with the second elimination phase being dose-dependent. The majority of the administered dose is excreted via bile, with glycyrrhizic acid being excreted unchanged and undergoing enterohepatic circulation. On the other hand, the major metabolite, glycyrrhetinic acid, forms glucuronide and sulfate conjugates. These conjugates are efficiently transported to the bile and duodenum, where symbiotic bacteria hydrolyze them to generate glycyrrhetinic acid, which is further reabsorbed. This reabsorption behavior appears to be related to the activity of 3α-hydroxysteroid dehydrogenase, an enzyme that very efficiently transports metabolites from plasma to bile. Approximately 1.1–2.5% of the administered dose of glycyrrhizic acid is detectable in urine, indicating extremely low circulating and reabsorption rates. The apparent volumes of distribution of glycyrrhizic acid in the central compartment and at steady state are 37–64 ml/kg and 59–98 ml/kg, respectively. Persistent reabsorption of glycyrrhetinic acid in the duodenum leads to a delayed terminal plasma clearance. Systemic clearance of glycyrrhizic acid has been reported to be in the range of 16–25 ml·kg/h. Glycyrrhizic acid is absorbed in the rat small intestine; after rapid intravenous injection of glycyrrhizic acid, glycyrrhetinic acid is undetectable in the blood; after oral administration, glycyrrhetinic acid is detectable in the blood. High-performance liquid chromatography (HPLC) can rapidly and accurately determine glycyrrhizic acid (GZA) and glycyrrhetinic acid (GRA) in the body fluids and tissues of laboratory animals and humans. Glycyrrhizic acid and glycyrrhetinic acid are extracted from plasma and tissues with organic solvents, and the extracts can be directly used for HPLC analysis. Extraction and determination of glycyrrhizic acid and glycyrrhetinic acid from bile or urine are challenging due to interference from endogenous compounds and the binding of glycyrrhetinic acid with glucuronide or sulfate. Methods for extracting glycyrrhizic acid and glycyrrhetinic acid from urine or bile include ion-pair conjugation followed by organic solvent extraction or solid-phase extraction. Glycyrrhetinic acid conjugates can be determined by chromatographic separation or β-glucuronidase pretreatment. The pharmacokinetics of glycyrrhetinic acid and glycyrrhetinic acid can be described using a biphasic elimination model, with elimination starting in the central compartment and exhibiting a dose-dependent second elimination phase. Depending on the dose, the second elimination phase half-life of glycyrrhizic acid in humans is 3.5 hours, while that of glycyrrhetinic acid is 10–30 hours. Both glycyrrhetinic acid and glycyrrhetinic acid are largely excreted via bile. Glycyrrhizic acid can be excreted directly without metabolism and undergoes enterohepatic circulation; however, glycyrrhetinic acid binds to glycyrrhetinic acid glucuronide or sulfate before bile excretion. Orally administered glycyrrhizic acid is almost completely hydrolyzed by intestinal bacteria and enters the systemic circulation as glycyrrhetinic acid. Glycyrrhizic acid currently has clinical applications in the treatment of chronic hepatitis. It is also used as a sweetener in food and chewing tobacco. Serious side effects such as hypertension and electrolyte disturbances have been reported in some high-exposure populations. To analyze the health risks of exposure to this compound, this study quantitatively assessed the kinetics of glycyrrhizic acid and its active metabolites. Glycyrrhizic acid and its metabolites are affected by complex kinetic processes, including enterohepatic circulation and first-pass metabolism. Detailed information on these processes is often difficult to obtain in humans. Therefore, we developed a model to describe the systemic and gastrointestinal kinetics of glycyrrhizic acid and its active metabolite, glycyrrhetinic acid, in rats. Because this model is based on physiological structures, it can directly integrate previous in vitro and in vivo data on absorption, enterohepatic circulation, and first-pass metabolism. The model suggests that glycyrrhizic acid and its metabolites can be efficiently transported from plasma to bile, which may be related to the hepatic transporter 3α-hydroxysteroid dehydrogenase. Following reabsorption, glycyrrhetinic acid's metabolites, excreted via bile, undergo bacterial hydrolysis, leading to the observed delayed terminal clearance of glycyrrhetinic acid in plasma. These mechanistic findings stem from the analysis of experimental data and were modeled using a physiologically based pharmacokinetic model, ultimately enabling the quantitative assessment of health risks in humans exposed to products containing glycyrrhetinic acid. Copyright 2000 Academic Press.
To assess the diversity of bile excretion of xenobiotic conjugates, this study investigated glycyrrhetinic acid (glycyrrhizin) in rats. Rats were intravenously injected with 10 mg/kg glycyrrhetinic acid, along with the inhibitors dibromosulfonphthalein and indocyanine green. Indocyanine green did not affect the bile excretion of glycyrrhetinic acid, while dibromosulfonphthalein significantly reduced its excretion. Dibromosulfonphthalein increased plasma concentrations of glycyrrhetinic acid, while indocyanine green had no such effect. In Eisai hyperbilirubinemia rats, bile excretion of glycyrrhetinic acid was severely impaired, resulting in elevated plasma concentrations. The results indicate that the bile excretion of glycyrrhizic acid is mediated by a system shared by glycyrrhizin glucuronide and sodium dibromosulfonamide, rather than indocyanine green, and this system is genetically defective in Eisai hyperbilirubinemia rats. Metabolism/Metabolites After oral administration, glycyrrhizic acid is almost completely hydrolyzed by intestinal bacteria, producing the active metabolite glycyrrhetinic acid (which enters systemic circulation) and two molecules of glucuronide. This metabolite is transported to the liver and absorbed, where it is metabolized to form glucuronide and sulfate conjugates. Rapid intravenous injection of glycyrrhizic acid into rats resulted in an increase in the level of a substance in the blood, which appears to be the glucuronide conjugate formed from the metabolism of glycyrrhetinic acid. Biological Half-Life Depending on the dose, the half-life of the second elimination phase in humans is 3.5 hours.

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
Licorice (Glycyrrhiza glabra) root contains glycyrrhizic acid (also known as glycyrrhizic acid or glycyrrhizic acid root glycoside) as well as a mixture of potassium and calcium glycyrrhizate salts. Glycyrrhizic acid is metabolized in the intestine to the active ingredient, glycyrrhetinic acid. Deglycyrrhizinated licorice (DGL) has had glycyrrhizic acid removed. Licorice is considered a galactagogue, and some proprietary Asian formulations also contain licorice to increase milk production; however, there are currently no scientifically valid clinical trials to support this use. In fact, licorice typically lowers serum prolactin levels, which may reduce milk production in the early stages of lactation. Women taking licorice may experience elevated blood pressure. Lactagogues should never replace assessment and consultation regarding controllable factors affecting milk production. Some mothers in Turkey have reported using licorice to improve the taste and quality of their breast milk.
Glycyrrhizic acid has been detected in the breast milk of some women who have taken licorice, but studies measuring glycyrrhetinic acid have not been conducted. Licorice, when blended with other herbs to make a tea, has been used safely and effectively for short-term treatment of infant colic. However, two infants developed anise brain poisoning symptoms after their mothers consumed excessive amounts of herbal tea containing licorice. Because both papers reported herbal blends, the effectiveness of licorice alone cannot be determined. The U.S. Food and Drug Administration (FDA) classifies licorice and licorice extract as "Generally Recognized As Safe" (GRAS). Long-term excessive consumption of licorice can lead to high blood pressure, hypokalemia, and adrenal hormone disorders; therefore, breastfeeding women should avoid its use. Dietary supplements do not require extensive premarket approval from the FDA. Manufacturers are responsible for ensuring product safety but are not required to prove the safety and effectiveness of dietary supplements before they are marketed. Dietary supplements may contain multiple ingredients, and the ingredients listed on the label often differ from the actual ingredients or amounts. Manufacturers may commission independent agencies to verify the quality of their products or their ingredients, but this does not guarantee the product's safety and effectiveness. Given the above issues, clinical trial results for one product may not be applicable to other products. For more detailed information on dietary supplements, please visit other pages on the LactMed website.
◉ Impact on Breastfed Infants
Two breastfed infants, aged 15 and 20 days respectively, were admitted to the hospital due to insufficient weight gain over the past 7 to 10 days, reportedly due to "feeding difficulties." The parents reported that the infants had been irritable and vomiting in the past day. One mother also reported feeling drowsy and weak. Examination revealed that the infants had no fever but exhibited hypotonia, lethargy, vomiting, weak crying, poor sucking ability, and sluggish response to painful stimuli. Laboratory tests, electrocardiograms, and blood pressure were normal, and tests for sepsis were negative. Both mothers had been drinking more than 2 liters daily of a herbal tea mixture reportedly containing licorice, fennel, star anise, and goat's bean to stimulate lactation. After the mothers stopped breastfeeding and drinking the herbal tea, the infants' symptoms improved within 24 to 36 hours. The affected mothers also experienced rapid symptom relief after stopping the herbal tea. Two days later, breastfeeding was resumed, and the infants did not experience any further symptoms. Both infants were in good health at 6 months of age. The authors attributed the symptoms in both mother and infant to anethole, which is found in fennel and cumin; however, they did not test for anethole content in breast milk or herbal teas.
◉ Effects on Lactation and Breast Milk
A woman with a history of long-term excessive licorice intake experienced amenorrhea, severe headaches, hypertension, and hypokalemia. Her serum prolactin levels were elevated and remained abnormal for one month after discontinuing licorice, returning to normal after six months.
In a study involving 25 men and 25 women, researchers measured basal and post-thyroid-stimulating prolactin levels to determine normal serum prolactin values. Subjects who regularly consumed licorice had lower basal and post-stimulation serum prolactin concentrations.
A study investigated women with elevated serum prolactin levels due to long-term (>6 months) risperidone use using a traditional, non-standardized peony and licorice decoction called Shaoyao Gancao Tang (Chinese) and Shaoyao Gancao Tang (Japanese). Patients received two treatment regimens: one was 5 mg of bromocriptine daily for 4 weeks; the other was 22.5 g of peony and licorice decoction daily (equivalent to 25 mg of glycyrrhetinic acid) for 4 weeks; and a third regimen involved taking both medications first, followed by the reverse order. Serum prolactin level assessments showed that both treatment regimens reduced serum prolactin levels by 21% to 28% from baseline at weeks 4 and 8 of treatment. Forty women complaining of insufficient breast milk 5 days postpartum received a compound herbal supplement, two Lactare capsules (manufactured by Madras Pharma Private Ltd., India; currently sold by TTK Pharma, Chennai, India) three times daily. Each capsule contained 200 mg of wild asparagus, 100 mg of Withania somnifera, 50 mg of fenugreek, 50 mg of licorice, and 20 mg of garlic. By day 4 of treatment, none of the infants required supplemental feeding. On day 5 of the mother's treatment, the infant was weighed before and after each feeding to determine the amount of milk consumed. On the day of the weighing test, the average milk intake of the infant was 388 ml, and fluid and calorie intake was considered adequate. Because this study lacked randomization, double-blinding, placebo control, and guidance on breastfeeding techniques for mothers, it cannot be considered valid evidence that these herbs have a galactagogue effect. Furthermore, the infant was only breastfed 6 to 8 times per day, which is insufficient to maximize milk supply at this stage of lactation. Women who reported lactation failure 14 to 90 days postpartum received guidance on breastfeeding techniques and were encouraged to exclusively breastfeed. If their infants gained less than 15 grams in one week, they were randomly assigned to receive either two tablespoons of a mixture containing wild asparagus or the same placebo for 4 weeks. Each 100g mixture contained: 15g asparagus, 1g dill, 1g sweet potato, 1g licorice, 2.5g spinach, 0.5g cumin, and 1g panthenol. Of the 64 randomly assigned women, 11 did not complete the trial. Serum prolactin levels were measured before morning breastfeeding and 4 weeks after treatment. Infant weight gain and the number of supplemental feedings were recorded and again at the beginning of treatment and 4 weeks after treatment. After 4 weeks of treatment, there were no differences between the treatment group and the placebo group in changes in serum prolactin, infant weight gain, or the number of supplemental feedings. No side effects or abnormal liver function tests occurred during the study period. A Japanese study compared the effects of a mixture of 13 herbs (including licorice) and ergonovine on lactation and serum prolactin levels in postpartum women. The herbal mixture, named "Xiong Gui Tiao Xue Yin," was randomly assigned to 41 women at a dose of 2 grams of the dried aqueous extract three times daily. Another 41 women were randomly assigned to receive 0.375 mg of ergonovine daily. Treatment began on the day of delivery, but the duration was not specified. Plasma oxytocin and prolactin levels were measured on days 1 and 6; milk volume was measured daily, but the method of measurement was not specified. Women taking the herbal remedy had higher serum prolactin levels on days 1 and 6 than those taking ergonovine; women in the herbal remedy group had lower plasma oxytocin levels on day 1, but there was no difference between the two groups on day 6. Women in the herbal remedy group had higher milk production on days 4, 5, and 6 than the control group. However, this study has serious flaws that make its results uninterpretable. First, milk production measurements can vary considerably depending on the measurement method, but this study did not specify the method used. Second, some studies have shown that ergonovine can cause a decrease in serum prolactin levels and milk production. Due to the lack of a placebo group, the observed differences may be a negative effect of ergonovine rather than a positive effect of the herbal preparation. Because this study used a multi-ingredient compound preparation, with licorice being only one ingredient, the results may differ from those of studies using licorice alone. In a non-controlled, non-blinded, multicenter study in India, 1132 patients who reported insufficient breast milk took a compound preparation (Lactancia, Corona Remedies Pvt. Ltd.) at 30 grams twice daily. This product contains asparagus (wild asparagus, satagwari), cumin, licorice, spinach, as well as amino acids, vitamins, minerals, and DHA. Most mothers (1049 participants) experienced improved lactation, and their infants also gained weight. However, since there was no placebo control group, the results cannot be attributed to the product.

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Protein Binding
Glycyrrhizic acid does not bind to any plasma proteins because it is not absorbed systemically. On the other hand, its main active metabolite, glycyrrhetinic acid, binds very strongly 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. It is a widely used anti-inflammatory agent extracted from licorice root. Glycyrrhizic acid is metabolized to glycyrrhetinic acid, which inhibits 11β-hydroxysteroid dehydrogenase and other enzymes involved in corticosteroid metabolism. Therefore, glycyrrhizic acid (the main sweet component of licorice) has attracted attention due to its ability to induce hyperchloremicity (with sodium retention and potassium loss), edema, increased blood pressure, and inhibition of the renin-angiotensin-aldosterone system. See also: glycyrrhizic acid (with the active fraction); licorice root (its fraction); Glycyrrhizinate dipotassium; xylitol (component)... Glycyrrhizic acid (GA) belongs to the triterpenoid saponins found in licorice root and is known to affect metabolic regulation. In recent years, glucagon-like peptide-1 (GLP-1) has been widely used in the treatment of diabetes. However, the mechanism of action of GLP-1 in the GA-induced antidiabetic effect is unclear. Therefore, we are committed to investigating the association between GLP-1 and GA-induced effects. In a streptozotocin (STZ)-induced type 1 diabetic rat model, GA increased plasma GLP-1 levels, while triamterene blocked this effect at a dose sufficient to inhibit Takeda G protein-coupled receptor 5 (TGR5). We confirmed the direct effect of GA on TGR5 using cultured Chinese hamster ovary cells (CHO-K1 cells) transfected with the TGR5 gene. In addition, GA promoted GLP-1 secretion and significantly increased calcium levels in GLP-1-secreting intestinal NCI-H716 cells. However, both of these effects of GA were attenuated in NCI-H716 cells after TGR5 knockout with siRNA. Therefore, we confirmed that GA can enhance GLP-1 secretion by activating TGR5. [2] In summary, we have for the first time confirmed that GA can increase plasma GLP-1 levels in type 1 diabetic rats by activating TGR5. Therefore, GA may have clinical application value in the future. [2]

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High-mobility group box 1 (HMGB1) is a non-histone protein that plays an important role in autoimmune diseases. However, the significance of HMGB1 in the pathogenesis of autoimmune thyroiditis has not been reported. This study aims to investigate whether HMGB1 is involved in the pathogenesis of autoimmune thyroiditis and whether glycyrrhizic acid (GL), a direct inhibitor of HMGB1, can reduce the severity of thyroid inflammation in a mouse model of autoimmune thyroiditis. [3]

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Injectable low molecular weight hydrogels (LMWHs) made of biocompatible materials have attracted much attention in the field of biomedical applications because they can be made into any desired size and cavity shape. Finding injectable LMWHs with inherent antibacterial activity without complex chemical modification, simple structure, and good biocompatibility, in order to avoid the cumbersome synthesis/purification process and the problem of hydrogels being susceptible to bacterial infection in humid environments, remains an urgent problem to be solved. This study found that the natural compound glycyrrhizic acid (GL) can form stable transparent LMWHs in physiological phosphate buffer (PBS) at 37°C, and its microstructure is a nanocluster. Furthermore, this hydrogel exhibits excellent injectability and plasticity. Antibacterial studies showed that GL completely inhibited the growth of Gram-positive Staphylococcus aureus (S. aureus) but had no inhibitory effect on Gram-negative Escherichia coli (E. coli). In addition, cell viability and hemolysis assays indicated that GL, due to its natural origin, has good biocompatibility and blood compatibility with mammalian cells. This low molecular weight heparin (LMWH) with natural antibacterial capabilities, simple structure, good biocompatibility, injectability, and high plasticity, prepared by us, has potential applications in biomaterials and 3D bioprinting. In summary, we constructed a stable LMWH in physiological PBS buffer at 37°C using natural glycyrrhizic acid as a gelling agent without additional chemical modification. This nanocluster structure of LMWH exhibits excellent injectability and plasticity. Furthermore, compared to Gram-negative Escherichia coli, GL completely inhibited the growth of Gram-positive Staphylococcus aureus. In addition, cell viability and hemolysis assays showed that GL has low cytotoxicity and good blood compatibility to mammalian cells due to its natural origin. Our study provides a simple, biocompatible, injectable, malleable, and inherently antibacterial low molecular weight heparin, which will be of great significance in the fields of biomaterials and 3D bioprinting. [4]

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Excessive macroautophagy/autophagy is one of the causes of cardiomyocyte death induced by cardiovascular disease or cancer treatment, but its underlying mechanism is unclear. We and other research teams have previously reported that autophagy may be involved in cardiomyocyte death induced by sunitinib (a widely used clinical tumor angiogenesis inhibitor), which helps to understand the mechanism of autophagy-induced cardiomyocyte death. In this study, we found that sunitinib-induced autophagy leads to cardiomyocyte apoptosis and cardiac dysfunction because cardiomyocyte-specific Atg7-/+ heterozygous mice are resistant to sunitinib. Sunitinib-induced maladaptive autophagy selectively degrades cardiomyocyte survival mediator CCN2 (cell communication network factor 2) through the TOLLIP (Toll interacting protein) mediated endosomal-associated pathway. Knockdown of Ccn2 in cardiomyocytes using adeno-associated virus type 9 (AAV9) mimics sunitinib-induced cardiac dysfunction in vivo, suggesting that autophagic degradation of CCN2 is one of the causes of sunitinib-induced cardiotoxicity and cardiomyocyte death. Notably, the loss of HMGB1 (high-mobility group box 1) inhibited sunitinib-induced cardiomyocyte autophagy and apoptosis, while the HMGB1-specific inhibitor glycyrrhizic acid (GA) significantly reduced 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 that pharmacological inhibitors of HMGB1 are a promising approach to improve the safety of sunitinib-based cancer treatments. [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.)