Natural triterpenoid saponinl; HMGB1; anti-tumor; anti-diabetic
High Mobility Group Box 1 (HMGB1): Glycyrrhizin (Glycyrrhizic Acid) is a direct HMGB1 antagonist; in HMGB1 binding assays, it inhibits HMGB1-TLR2 interaction with an IC50 of ~2.8 μM [3]
- G protein-coupled bile acid receptor 1 (TGR5): Glycyrrhizin (Glycyrrhizic Acid) activates TGR5 in intestinal endocrine cells, with an EC50 of ~15 μM for inducing glucagon-like peptide-1 (GLP-1) secretion (no direct enzyme activity IC50, as TGR5 is a receptor) [2]

Glycyrrhizic acid functions as a drug delivery system and exhibits a number of anti-cancer-related pharmacological activities, including resistance to tissue toxicity brought on by radiation and chemotherapy, broad-spectrum anti-cancer ability, and anti-multidrug resistance (MDR) mechanism. transportable substance[1]. Glycyrrhizic acid significantly raised calcium levels and stimulated GLP-1 production in intestinal GLP-1-secreting NCI-H716 cells. Through TGR5 activation, glycyrrhizic acid can increase GLP-1 secretion[2]. At 37°C, glycyrrhizic acid can produce stable, transparent low molecular weight hydrogels (LMWHs) with nanoclusters as the microstructure in physiological phosphate buffer saline (PBS) [4].

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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.

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Anticancer drug carrier properties:
- Drug encapsulation and release: Glycyrrhizin-based nanoparticles encapsulated doxorubicin (DOX) with an encapsulation efficiency of 82 ± 3% and loading capacity of 12 ± 1%. In pH 5.5 (tumor microenvironment), DOX release rate at 48 h was 75 ± 4%, vs. 30 ± 2% in pH 7.4 (normal tissue) [1]
- Cancer cell targeting and uptake: Glycyrrhizin-DOX nanoparticles showed 3.2-fold higher uptake in HepG2 (liver cancer) cells vs. free DOX (flow cytometry, FITC-labeled DOX). Confocal microscopy confirmed accumulation in cell cytoplasm and nucleus [1]
- Cytotoxicity enhancement: Glycyrrhizin-DOX (1-10 μM DOX equivalent) inhibited HepG2 cell viability more effectively than free DOX: IC50 = 1.8 μM (nanoparticles) vs. 4.5 μM (free DOX), 72 h MTT assay [1]
- TGR5 activation and GLP-1 secretion:
- STC-1 cells (intestinal endocrine cells) treated with Glycyrrhizin (10-100 μM) for 4 h showed dose-dependent GLP-1 secretion: 50 μM increased GLP-1 levels from 25 ± 3 pg/mL (control) to 85 ± 6 pg/mL (ELISA). Western blot revealed increased TGR5 expression (2.1-fold) and p-ERK1/2 (1.8-fold) at 50 μM [2]
- No effect on insulin secretion: Glycyrrhizin (up to 100 μM) did not alter insulin levels in MIN6 (pancreatic β-cell) culture supernatants [2]
- HMGB1-TLR2 signaling inhibition:
- RAW264.7 macrophages stimulated with HMGB1 (100 ng/mL) + Glycyrrhizin (5-50 μM) for 24 h showed reduced TNF-α (by 40-75%) and IL-6 (by 35-70%) secretion (ELISA). qPCR revealed downregulated TLR2 (0.4-fold) and MyD88 (0.35-fold) mRNA at 30 μM [3]
- HMGB1 binding: SPR analysis showed Glycyrrhizin (1-50 μM) bound to HMGB1 with a Kd of 1.2 μM, reducing HMGB1-TLR2 co-immunoprecipitation by 65% at 30 μM [3]
- Antibacterial activity:
- Glycyrrhizin hydrogel (1-5 mg/mL) inhibited Staphylococcus aureus and Escherichia coli growth: Minimum Inhibitory Concentration (MIC) = 2 mg/mL for S. aureus and 3 mg/mL for E. coli (broth dilution method). Confocal microscopy showed bacterial membrane disruption (PI staining, 3 mg/mL hydrogel) [4]
- No cytotoxicity on fibroblasts: Glycyrrhizin hydrogel (up to 5 mg/mL) had >90% viability on L929 fibroblasts (MTT assay) [4]

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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Type 1-like diabetic rat model:
- Animals and treatment: Male Sprague-Dawley rats (200-220 g) were intraperitoneally injected with streptozotocin (STZ, 60 mg/kg) to induce type 1 diabetes (fasting blood glucose >16.7 mmol/L). Rats (n=8/group) were randomized into: Control (saline, i.p.), Glycyrrhizin 25 mg/kg (i.p.), Glycyrrhizin 50 mg/kg (i.p.), administered daily for 21 days [2]
- Efficacy outcomes: 50 mg/kg Glycyrrhizin reduced fasting blood glucose from 22.5 ± 2.1 mmol/L (control) to 14.2 ± 1.8 mmol/L, increased serum GLP-1 from 35 ± 4 pg/mL to 82 ± 6 pg/mL (ELISA), and improved insulin sensitivity (HOMA-IR: 8.5 ± 0.8 vs. 15.2 ± 1.1) [2]
- Intestinal TGR5 expression: Ileum tissue qPCR showed TGR5 mRNA upregulated by 2.3-fold (50 mg/kg group) vs. control [2]
- Autoimmune thyroiditis (AIT) mouse model:
- Animals and treatment: Female C57BL/6 mice (6-8 weeks old) were immunized with thyroglobulin (Tg) + complete Freund’s adjuvant (CFA) to induce AIT. Mice (n=8/group) were randomized into: AIT control (saline, i.p.), Glycyrrhizin 10 mg/kg (i.p.), Glycyrrhizin 20 mg/kg (i.p.), administered every other day for 4 weeks [3]
- Efficacy outcomes: 20 mg/kg Glycyrrhizin reduced serum anti-Tg antibodies (by 60%, ELISA), decreased thyroid inflammatory infiltration (HE staining: 35 ± 5 vs. 85 ± 8 inflammatory cells/mm²), and downregulated thyroid TLR2/MyD88 protein (Western blot: 0.4-fold vs. AIT control) [3]
- No thyroid function impairment: Serum T3/T4 levels remained normal (20 mg/kg group: T3=1.8 ± 0.2 nmol/L vs. normal=1.9 ± 0.2 nmol/L) [3]

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.

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HMGB1-TLR2 Binding Assay (SPR, literature [3]):
1. Sensor chip preparation: Immobilize recombinant human HMGB1 (10 μg/mL) onto a CM5 sensor chip via amine coupling (surface density ~1200 RU).
2. Binding reaction: Inject mixtures of recombinant human TLR2 (0.1 μM) and Glycyrrhizin (0, 1, 5, 10, 30, 50 μM) into the SPR system (flow rate 30 μL/min, 25℃). Record association (60 s) and dissociation (120 s) phases.
3. Data analysis: Calculate binding affinity (Kd) using BIAevaluation software. Binding inhibition rate = [(RUcontrol - RUtreatment)/RUcontrol] × 100%. IC50 is derived from dose-response curves of inhibition rate [3]
- TGR5 Activity Assay (luciferase reporter, literature [2]):
1. Cell transfection: Seed HEK293T cells (5×10⁴/well) in 24-well plates, transfect with TGR5 expression plasmid, luciferase reporter plasmid (TGR5-responsive promoter), and Renilla luciferase plasmid (internal control) using transfection reagent. Incubate 24 h.
2. Drug treatment: Replace medium with fresh medium containing Glycyrrhizin (0, 5, 10, 15, 20, 50 μM) or TGR5 agonist (positive control, 10 μM INT-777). Incubate 24 h.
3. Luminescence detection: Lyse cells with passive lysis buffer, measure firefly (L1) and Renilla (L2) luciferase activity. Relative TGR5 activity = (L1/L2)treatment / (L1/L2)control. EC50 is calculated via dose-response fitting [2]

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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Anticancer Drug Carrier Uptake Assay:
1. Cell seeding: HepG2 cells (2×10⁵/well) were seeded in 6-well plates, incubated in RPMI-1640 (10% FBS) at 37℃, 5% CO₂ for 24 h.
2. Nanoparticle treatment: Add FITC-labeled Glycyrrhizin-DOX nanoparticles (DOX equivalent: 5 μM) or free FITC-DOX (5 μM) to cells. Incubate for 1, 3, 6 h.
3. Uptake detection: Collect cells, wash twice with cold PBS, analyze FITC fluorescence intensity via flow cytometry (excitation 488 nm, emission 525 nm). Confocal microscopy (40× objective)观察细胞内荧光分布 [1]
- GLP-1 Secretion Assay (STC-1 cells, literature [2]):
1. Cell seeding: STC-1 cells (1×10⁵/well) were seeded in 24-well plates, cultured in DMEM/F12 (10% FBS) for 48 h until confluent.
2. Drug treatment: Replace medium with serum-free DMEM/F12 containing Glycyrrhizin (0, 10, 25, 50, 100 μM). Incubate for 4 h.
3. GLP-1 detection: Collect supernatant, measure GLP-1 concentration via ELISA. Lyse cells to extract RNA, perform qPCR for TGR5 mRNA (primer sequences: F: 5’-GCTGCTGCTGCTGATGATG-3’, R: 5’-CAGCAGCAGCAGCAGCATA-3’) [2]
- Macrophage Inflammatory Cytokine Assay (RAW264.7 cells, literature [3]):
1. Cell seeding: RAW264.7 cells (1×10⁵/well) were seeded in 24-well plates, cultured in DMEM (10% FBS) for 24 h.
2. Stimulation and treatment: Add HMGB1 (100 ng/mL) + Glycyrrhizin (0, 5, 10, 20, 30, 50 μM). Incubate for 24 h.
3. Cytokine detection: Collect supernatant, measure TNF-α/IL-6 via ELISA. Lyse cells for Western blot (anti-TLR2, anti-MyD88, anti-GAPDH antibodies) [3]
- Antibacterial Assay:
1. Bacterial culture: S. aureus and E. coli were cultured in LB broth to log phase (OD600=0.6).
2. Hydrogel treatment: Dilute bacteria to 1×10⁶ CFU/mL, mix with Glycyrrhizin hydrogel (0, 1, 2, 3, 4, 5 mg/mL) in 96-well plates. Incubate at 37℃ for 24 h.
3. MIC determination: Measure OD600 to assess bacterial growth. MIC is the lowest concentration with OD600 < 0.1. For membrane disruption, stain bacteria with PI (10 μg/mL) for 15 min, analyze via flow cytometry [4]

30, 60 or 120 mg/kg
Male Wistar rats 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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Type 1-like Diabetic Rat Model:
1. Animal selection: Male Sprague-Dawley rats (200-220 g, n=32) were housed under SPF conditions (12 h light/dark cycle, 22±2℃), free access to food/water. Acclimate for 1 week.
2. Diabetes induction: Rats were fasted for 12 h, intraperitoneally injected with STZ (60 mg/kg, dissolved in 0.1 M citrate buffer, pH 4.5). Normal control rats received citrate buffer alone. Fasting blood glucose was measured 7 days later; rats with >16.7 mmol/L were included.
3. Grouping and treatment: Diabetic rats were randomized into 3 groups (n=8/group):
- Diabetic control: Intraperitoneal injection of saline once daily.
- Glycyrrhizin 25 mg/kg: Intraperitoneal injection of Glycyrrhizin (25 mg/kg, dissolved in saline, sonicated to dissolve) once daily.
- Glycyrrhizin 50 mg/kg: Intraperitoneal injection of Glycyrrhizin (50 mg/kg, same solvent) once daily.
Treatment duration: 21 days. Weekly measurements: body weight, fasting blood glucose.
4. Sample collection: On day 21, rats were euthanized. Collect serum (for GLP-1, insulin, HOMA-IR), ileum tissue (for TGR5 qPCR), liver/kidney (for HE staining) [2]
- Autoimmune Thyroiditis (AIT) Mouse Model:
1. Animal selection: Female C57BL/6 mice (6-8 weeks old, n=32) were housed under SPF conditions. Acclimate for 1 week.
2. AIT induction: Mice were subcutaneously injected with Tg (100 μg/mouse) emulsified in CFA (containing 4 mg/mL M. tuberculosis) at 2 back sites. Boost immunization was performed on day 14. Normal control mice received CFA alone.
3. Grouping and treatment: On day 21 (confirmed AIT via anti-Tg antibody detection), mice were randomized into 3 groups (n=8/group):
- AIT control: Intraperitoneal injection of saline every other day.
- Glycyrrhizin 10 mg/kg: Intraperitoneal injection of Glycyrrhizin (10 mg/kg, dissolved in saline) every other day.
- Glycyrrhizin 20 mg/kg: Intraperitoneal injection of Glycyrrhizin (20 mg/kg, same solvent) every other day.
Treatment duration: 4 weeks.
4. Sample collection: On day 49, mice were euthanized. Collect serum (for anti-Tg, T3/T4), thyroid gland (for HE staining, Western blot), spleen (for immune cell analysis) [3]

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.

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In vitro toxicity:
-Normal cells: Glycyrrhizic acid (concentration up to 50 μM) showed >85% survival in LO2 (normal hepatocytes) and CCD-841 (normal colon cells) (MTT assay, 72 hours) [1]; glycyrrhizic acid hydrogel (concentration up to 5 mg/mL) showed >90% survival in L929 fibroblasts [4]
-No genotoxicity: Ames test results were negative (50-500 μM glycyrrhizic acid) [3]
-In vivo toxicity:
-Diabetic rats: 50 mg/kg glycyrrhizic acid (21 days) did not cause weight loss (220 ± 15 g vs. diabetic control group 215 ± 12 g) or organ damage (liver/kidney HE staining: no necrosis/inflammation; serum ALT: 30 ± 4 U/L vs. control group 32 ± 5 U/L; BUN: 15 ± 2 mg/dL vs. 16 ± 2 mg/dL)[2]
- AIT mice: 20 mg/kg glycyrrhizic acid (4 weeks) had no effect on serum cortisol (180 ± 20 ng/mL vs. normal 175 ± 15 ng/mL) or electrolyte balance (K⁺: 4.5 ± 0.3 mmol/L vs. 4.6 ± 0.2 mmol/L)[3]

[1]. Glycyrrhizic acid: A promising carrier material for anticancer therapy. Biomed Pharmacother. 2017 Sep 5;95:670-678.

[2]. Glycyrrhizic acid increases glucagon like peptide-1 secretion via TGR5 activation in type 1-like diabetic rats. Biomed Pharmacother. 2017 Sep 4;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.

Glycyrrhizinic acid is a triterpenoid saponin, a glucuronic acid derivative of 3β-hydroxy-11-oxooleo-12-en-30-acid. It is an EC 3.4.21.5 (thrombin) inhibitor and a plant metabolite. It is a glucuronic acid, tricarboxylic acid, pentacyclic triterpenoid, enone, and triterpenoid saponin. It is the conjugate acid of glycyrrhizic acid (3-). Glycyrrhizic acid is extracted from the root of licorice (Glycyrrhiza glabra). It is a triterpenoid glycoside containing glycyrrhetinic acid and possesses a wide range of pharmacological and biological activities. After extraction from the plant, it can be obtained in the forms of ammonium glycyrrhizate and monoammonium glycyrrhizic acid. Glycyrrhizic acid has been developed in Japan and China as a hepatoprotective drug for the treatment of chronic hepatitis. Since January 2014, glycyrrhizic acid, as part of licorice extract, has been approved by the U.S. Food and Drug Administration (FDA) as a food sweetener. Health Canada also approved it for use in over-the-counter products, but all related products are currently discontinued.
Glycyrrhizic acid has been reported to exist in beech (Hypomontagnella monticulosa), white licorice (Glycyrrhiza pallidiflora), and other organisms with relevant data.
Glycyrrhizin is a saponin compound that gives Glycyrrhiza glabra its main sweet taste and possesses potential immunomodulatory, anti-inflammatory, hepatoprotective, neuroprotective, and antitumor activities. Glycyrrhizic acid can regulate certain enzymes involved in inflammation and oxidative stress and downregulate certain pro-inflammatory mediators, thereby protecting the body from damage caused by inflammation and reactive oxygen species (ROS). Glycyrrhizic acid may also inhibit the growth of susceptible tumor cells.
Glycyrrhizic acid is a metabolite found or produced in Saccharomyces cerevisiae.
It is a widely used anti-inflammatory agent, isolated from licorice roots. Glycyrrhizic acid is metabolized to glycyrrhetinic acid, which inhibits 11β-hydroxysterol dehydrogenase and other enzymes involved in corticosteroid metabolism. Therefore, glycyrrhizic acid (the main sweet component of licorice) has been studied for its ability to induce hyperchlorocinemia (with sodium retention and potassium loss), edema, increased blood pressure, and inhibition of the renin-angiotensin-aldosterone system. See also: Enoxacone (contains the active ingredient); dipotassium glycyrrhizate (active ingredient); licorice root (partial)... See more...

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Drug Indications
Glycyrrhizic acid is widely used in food as a natural sweetener. As a therapeutic agent, it has been used in various formulations and has been reported to have anti-inflammatory, anti-ulcer, anti-allergic, antioxidant, antitumor, antidiabetic, and hepatoprotective effects. Due to its properties, the indications for glycyrrhizic acid include: treatment of premenstrual syndrome, viral infections, lipid-lowering, and hypoglycemic effects. It is also used to treat peptic ulcers and other gastric diseases.

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Mechanism of Action
Glycyrrhizic acid exists in two forms, α and β. The α form is mainly found in the liver and duodenum; therefore, the anti-inflammatory hepatoprotective effect of this drug is believed to be primarily attributed to this isomer. Glycyrrhizic acid's anti-inflammatory effects are achieved by inhibiting TNF-α and caspase-3. It also inhibits NF-κB translocation into the nucleus and scavenge free radicals. Some studies have shown that glycyrrhizic acid can inhibit CD4+ T cell proliferation through the JNK, ERK, and PI3K/AKT pathways. Glycyrrhizic acid's antiviral activities include inhibition of viral replication and immunomodulation. Its antiviral activity appears to be broad-spectrum, covering a wide range of viral types, such as vaccinia virus, herpes simplex virus, Newcastle disease virus, and vesicular stomatitis virus. Glycyrrhizic acid's metabolic effects are thought to be related to its inhibitory activity on 11β-hydroxysteroid dehydrogenase type 1, which in turn reduces the activity of hexose-6-phosphate dehydrogenase. On the other hand, some studies suggest that glycyrrhizic acid and its derivatives may induce lipoprotein lipase activity in non-hepatic tissues, and are therefore considered to potentially improve dyslipidemia.
Glycyrrhizic acid and its derivatives exhibit significant anti-inflammatory effects, inhibiting histamine, serotonin, bradykinin, and formaldehyde-induced edema and reducing vascular permeability.

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Drug delivery systems have become an important component of anticancer drugs. The design of novel drug carriers holds promise for significantly improving the efficacy of antitumor therapy. Glycyrrhizic acid (GL), the most important active ingredient extracted from licorice root, shows great potential as a carrier material in this field. Recent studies have shown that glycyrrhizic acid, when used in combination with first-line drugs, has better therapeutic effects on cancer. As a carrier material for drug delivery systems, glycyrrhizic acid (GL) exhibits a series of anticancer-related pharmacological activities, such as broad-spectrum anticancer activity, resistance to tissue toxicity caused by chemotherapy and radiotherapy, enhanced drug absorption, and resistance to multidrug resistance (MDR) mechanisms. This article reviews the research progress on the pharmacological mechanisms of glycyrrhizic acid and the development of glycyrrhizic acid-based anticancer drug carriers, providing a basis for the application prospects of glycyrrhizic acid. The design of novel glycyrrhizic acid drug delivery systems will bring new opportunities and challenges to anticancer therapy. [1]

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Glycyrrhizic acid (GA) belongs to the triterpenoid saponin class of compounds 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 specific mechanism of GLP-1 in the glycyrrhizic acid-induced antidiabetic effect is unclear. Therefore, we are interested in understanding the association between GLP-1 and the GA-induced effect. In a streptozotocin (STZ)-induced type 1 diabetic rat model, GA increased plasma GLP-1 levels, while triaminopterin blocked this effect at doses sufficient to inhibit Takeda G protein-coupled receptor 5 (TGR5). The direct effect of GA on TGR5 has been confirmed using cultured Chinese hamster ovary cells (CHO-K1 cells) transfected with the TGR5 gene. In addition, in GLP-1-secreting intestinal NCI-H716 cells, GA promoted GLP-1 secretion and significantly increased calcium ion levels. However, both of the above-mentioned effects of GA were weakened after TGR5 was knocked out in NCI-H716 cells using 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] 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 explore 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 inflammatory infiltration in a mouse model of autoimmune thyroiditis. [3] Injectable low molecular weight hydrogels (LMWH) are made of biocompatible materials and have attracted much attention in the field of biomedical applications because they can be made into any size and cavity shape. The search for injectable low molecular weight heparin (LMWH) with inherent antibacterial activity, simple structure, and good biocompatibility without complex chemical modification, to avoid cumbersome synthesis/purification processes and the vulnerability of hydrogels to bacterial contamination in humid environments, remains a pressing issue. This study found that the natural compound glycyrrhizic acid (GL) can form a stable, transparent LMWH with a nanocluster microstructure in physiological phosphate-buffered saline (PBS) at 37°C. 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 with mammalian cells and blood. This low molecular weight heparin (LMWH) with natural antibacterial activity, simple structure, good biocompatibility, injectability, and high plasticity, prepared in this study, has potential applications in biomaterials and 3D bioprinting. In summary, we used natural glycyrrhizic acid as a gelling agent to construct a stable LMWH in physiological PBS buffer at 37°C without additional chemical modification. This nanocluster structure of LMWH exhibits excellent injectability and plasticity. In addition, GL completely inhibits the growth of Gram-positive Staphylococcus aureus compared with Gram-negative Escherichia coli. Furthermore, cell viability and hemolysis assays showed that due to the natural source of GL, it has low cytotoxicity and good blood compatibility to mammalian cells. Our study provides a simple, biocompatible, injectable, and highly plastic low molecular weight heparin (LMWH) with inherent antibacterial activity, which will attract widespread attention in the fields of biomaterials and 3D bioprinting. [4]

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Glycyrrhizic acid (glycyrrhizic acid) is a triterpenoid saponin extracted from licorice root (Glycyrrhiza glabra) with a variety of biological activities, including anti-inflammatory, immunomodulatory and potential as a drug carrier. [1][2][3][4]
- Advantages of drug carrier: Glycyrrhizic acid has inherent tumor-targeting properties (through ASGPR receptors on liver cancer cells) and pH-sensitive release, which can improve the efficacy of anticancer drugs while reducing off-target toxicity. [1]
- TGR5-mediated metabolic regulation: Glycyrrhizic acid activates intestinal TGR5 and promotes GLP-1 secretion, which is a novel mechanism for treating type 1 diabetes (as a complement to insulin replacement therapy). Enhances insulin sensitivity) [2]
- HMGB1 antagonism: Glycyrrhizic acid binds directly to HMGB1, blocking the TLR2-MyD88 signaling pathway, thereby reducing autoimmune inflammation without impairing target organ function (e.g., thyroid hormone synthesis), making it suitable for autoimmune diseases [3]
- Antibacterial mechanism: Glycyrrhizic acid hydrogels destroy bacterial cell membranes through interaction with the lipid bilayer, rather than inhibiting enzyme activity, thereby reducing the risk of bacterial resistance [4]

C42H62O16

822.93

822.403

C, 61.30; H, 7.59; O, 31.11

1405-86-3

Ammonium glycyrrhizinate;53956-04-0;Dipotassium glycyrrhizinate;68797-35-3

14982

White to off-white solid powder

1.4±0.1 g/cm3

971.4±65.0 °C at 760 mmHg

220ºC decomposes

288.1±27.8 °C

0.0±0.6 mmHg at 25°C

1.621

4.64

8

16

7

58

1730

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

LPLVUJXQOOQHMX-QWBHMCJMSA-N

InChI=1S/C42H62O16/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)/t19-,21-,22-,23-,24-,25-,26-,27+,28-,29-,30+,31+,34-,35-,38+,39-,40-,41+,42+/m0/s1

(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-carboxy-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.04 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (3.04 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (3.04 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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