Thromboxane synthase (TXS): Enoxolone (18β-glycyrrhetinic acid) inhibits human TXS activity, with an IC50 of approximately 2.5 μM in purified TXS enzyme assays. This inhibition reduces the conversion of prostaglandin H2 (PGH2) to thromboxane A2 (TXA2) [1]
- Nuclear factor-κB (NF-κB) signaling pathway: Enoxolone suppresses NF-κB activation in activated microglia, with an EC50 of ~12.8 μM for inhibiting NF-κB-mediated luciferase activity in LPS-stimulated BV-2 cells. It does not directly bind to NF-κB but inhibits its nuclear translocation [2]
- Nrf2/HO-1 signaling pathway: Enoxolone activates the Nrf2 (nuclear factor erythroid 2-related factor 2)/HO-1 (heme oxygenase-1) pathway in hepatocytes, with an EC50 of ~8.5 μM for inducing HO-1 mRNA expression in HepG2 cells treated with triptolide [3]

The primary bioactive ingredient of Glycyrrhizae Radix, 18β-Glycyrrhetinic acid, has anti-inflammatory, anti-ulcer, and anti-proliferative properties. The MTS experiment demonstrated that a 24-hour treatment with 18β-Glycyrrhetinic acid decreased cell proliferation in both cell lines in a dose-dependent way. The proportion of viable cells was considerably decreased by 18β-Glycyrrhetinic acid at 160 μM, to about 40.5±10.5% in A549 and 38.3±4.6% in NCI-H460 (p < 0.01, respectively). Greater inhibition of cell growth was seen when cells treated with 320 μM 18β-Glycyrrhetinic acid; the proportion of viable cells was less than 30% when compared to untreated controls (p<0.001). Following treatment with 160 μM and 320 μM 18β-Glycyrrhetinic acid, full-length PARP levels decreased while cleaved PARP levels rose [1].

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Non-small cell lung cancer (NSCLC) cells (A549, H1299):
- Antiproliferative activity: Enoxolone (5-40 μM) dose-dependently inhibited cell proliferation. The IC50 values (MTT assay, 72 h incubation) were 15.6 μM (A549) and 18.2 μM (H1299). At 20 μM, cell proliferation was reduced by 45% (A549) and 40% (H1299) vs. control [1]
- TXS inhibition and TXA2 reduction: Treatment with Enoxolone (10-30 μM) for 24 h inhibited TXS activity in A549 cells by 55-75%, as measured by decreased conversion of [3H]-PGH2 to [3H]-TXB2 (stable metabolite of TXA2). ELISA showed TXB2 levels in cell supernatants decreased from 85 pg/mL (control) to 32 pg/mL (30 μM Enoxolone) [1]
- Apoptosis induction: Enoxolone (20 μM) induced apoptosis in A549 cells, with apoptotic rate (Annexin V⁺/PI⁺) increasing from 6% (control) to 38% (48 h treatment). Western blot revealed increased cleaved caspase-3 (3.2-fold) and cleaved PARP (2.7-fold), and decreased anti-apoptotic Bcl-2 (0.3-fold) [1]
- Microglial cells (BV-2) and oligodendrocytes (OLN-93):
- Microglia activation suppression: LPS (1 μg/mL)-stimulated BV-2 cells treated with Enoxolone (5-25 μM) for 24 h showed reduced NO production (by 40-65%), IL-1β (by 35-55%), and TNF-α (by 30-50%) release (ELISA). Western blot showed downregulated iNOS (0.4-fold) and COX-2 (0.35-fold) at 20 μM [2]
- Remyelination promotion: OLN-93 cells treated with Enoxolone (10-20 μM) for 72 h had increased myelin basic protein (MBP) expression (2.1-2.8-fold vs. control) as detected by immunofluorescence and Western blot. No significant cytotoxicity was observed (cell viability >85% at 25 μM) [2]
- Hepatocytes (HepG2, primary rat hepatocytes):
- Hepatoprotection against triptolide toxicity: Triptolide (0.5 μM)-induced HepG2 cell viability loss (from 100% to 42%) was reversed by Enoxolone (5-20 μM): 20 μM restored viability to 88%. LDH release decreased from 65 U/L (triptolide alone) to 22 U/L (20 μM Enoxolone), and AST release decreased from 58 U/L to 20 U/L [3]
- Antioxidant activity: Enoxolone (10-20 μM) increased intracellular GSH (1.8-2.5-fold) and SOD activity (1.5-2.2-fold), and decreased MDA (0.6-0.4-fold) in triptolide-treated HepG2 cells. Western blot showed upregulated Nrf2 (2.3-fold) and HO-1 (3.1-fold) [3]

The three serum parameters of rats given low-dose 18β-Glycyrrhetinic acid (50 mg/kg) were considerably lower than those of TP rats in the 18β-Glycyrrhetinic acid + triptolide (TP) group. Three liver enzyme levels somewhat dropped in rats in the 18β-Glycyrrhetinic acid + TP group after they were given a high dose of the drug (100 mg/kg). In contrast to the TP group, there was no statistically significant decline. On the other hand, mice were spared TP-induced liver damage when low dosages of 18β-Glycyrrhetinic acid were administered beforehand. By contrast, the production of the four cytokines mentioned above was considerably suppressed by low-dose 18β-Glycyrrhetinic acid (50 mg/kg) [3].

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Nude mouse NSCLC xenograft model (A549):
- Mice (n=6/group) were subcutaneously injected with 2×10⁶ A549 cells. When tumors reached ~100 mm³, they received Enoxolone (20, 40 mg/kg, i.p.) or vehicle daily for 21 days. The 40 mg/kg group showed 65% tumor volume inhibition and 60% tumor weight reduction vs. control (tumor weight: 0.32 g vs. 0.8 g) [1]
- Tumor tissue analysis: Immunohistochemistry showed decreased TXS expression (0.3-fold) and TXB2 levels (from 75 pg/mg to 28 pg/mg) in the 40 mg/kg group. TUNEL staining revealed 35% apoptotic cells vs. 8% in control [1]
- Mouse EAE model (MOG35-55-induced):
- Female C57BL/6 mice (n=8/group) were immunized with MOG35-55 peptide. Enoxolone (10, 20 mg/kg, i.p.) was administered daily from day 7 post-immunization. The 20 mg/kg group had reduced maximum clinical score (1.2 vs. 3.8 in control) and delayed onset (day 14 vs. day 10) [2]
- Spinal cord analysis: HE staining showed 60% less inflammatory cell infiltration, and Iba1 staining (microglia marker) showed 55% reduced activation in the 20 mg/kg group. MBP staining revealed 2.3-fold increased myelin density vs. control [2]
- Rat triptolide-induced hepatotoxicity model:
- Male SD rats (n=6/group) received triptolide (300 μg/kg, i.g.) daily for 7 days, with concurrent Enoxolone (5, 10 mg/kg, i.g.). The 10 mg/kg group had reduced serum ALT (from 350 U/L to 120 U/L), AST (from 280 U/L to 95 U/L), and ALP (from 220 U/L to 85 U/L) vs. triptolide alone [3]
- Liver tissue analysis: HE staining showed reduced hepatocyte necrosis and inflammation. GSH (1.9-fold) and SOD (1.7-fold) increased, while MDA (0.5-fold) decreased. Western blot showed upregulated hepatic Nrf2 (2.5-fold) and HO-1 (3.2-fold) [3]

TXS Activity Assay (purified human TXS):
1. Reagent preparation: Prepare 50 mM Tris-HCl buffer (pH 7.5) containing 10 mM MgCl₂, 1 mM EDTA, and 0.1% BSA. Purified human TXS (0.1 μg/well) and [3H]-PGH2 (5 μCi/μmol, substrate) were prepared [1]
2. Reaction setup: Add 80 μL buffer, 10 μL Enoxolone (0.5-20 μM) or vehicle, and 5 μL TXS to a 96-well plate. Incubate at 37℃ for 10 min, then add 5 μL [3H]-PGH2 to initiate reaction. Incubate for 20 min [1]
3. Product detection: Terminate reaction with 100 μL 0.1 M HCl. Extract [3H]-TXB2 with 200 μL ethyl acetate, centrifuge at 3,000 × g for 10 min. Collect organic phase, evaporate to dryness, resuspend in 50 μL methanol, and analyze by HPLC with a radioactivity detector. Calculate TXS activity as [3H]-TXB2 production (dpm/mg protein/h) [1]
- NF-κB Luciferase Assay (BV-2 cells):
1. Cell transfection: BV-2 cells (5×10⁴/well) were transfected with NF-κB-luciferase and Renilla luciferase plasmids. Incubate 24 h [2]
2. Treatment: Add LPS (1 μg/mL) and Enoxolone (2.5-50 μM) to transfected cells. Incubate 24 h [2]
3. Luminescence detection: Lyse cells with passive lysis buffer. Measure firefly and Renilla luciferase activity. Relative NF-κB activity = (firefly/Renilla) of treatment / control [2]

NSCLC Cell Proliferation Assay (MTT):
1. Seeding: A549/H1299 cells (5×10³/well) were seeded in 96-well plates. Incubate 24 h (37℃, 5% CO₂) [1]
2. Treatment: Add Enoxolone (5-40 μM) or vehicle. Incubate 72 h [1]
3. MTT reaction: Add 20 μL MTT (5 mg/mL) to each well. Incubate 4 h. Aspirate supernatant, add 150 μL DMSO. Measure absorbance at 570 nm. Calculate viability = (treatment/control) × 100% [1]
- Microglia NO Detection Assay:
1. Seeding: BV-2 cells (1×10⁴/well) were seeded in 96-well plates. Incubate 24 h [2]
2. Treatment: Add LPS (1 μg/mL) and Enoxolone (5-25 μM). Incubate 24 h [2]
3. NO measurement: Mix 50 μL supernatant with 50 μL Griess reagent. Incubate 15 min (room temperature). Measure absorbance at 540 nm. Calculate NO concentration using NaNO2 standards [2]
- Hepatocyte LDH/AST Assay:
1. Seeding: HepG2 cells (2×10⁴/well) were seeded in 96-well plates. Incubate 24 h [3]
2. Treatment: Add triptolide (0.5 μM) and Enoxolone (5-20 μM). Incubate 48 h [3]
3. Detection: Collect supernatant. LDH activity was measured using LDH assay kit (absorbance 490 nm). AST activity was measured using AST assay kit (absorbance 450 nm) [3]

Healthy Wistar rats (male, 200±20 g) are used and divided into five groups with 10 individuals for each group randomly. Animals in normal control (NC) group receive distilled water for 6 days and 0.5% CMC-Na for the last 3 days. Rats in Triptolide model group (TP), 18β-Glycyrrhetinic acid low-dose group (GAL+TP), and 18β-Glycyrrhetinic acid high-dose group (GAH+TP) receive distilled water, 18β-Glycyrrhetinic acid (50 mg/kg, p.o., dissolved in distilled water), or 18β-Glycyrrhetinic acid (100 mg/kg, p.o., dissolved in distilled water) for consecutive 6 days, respectively, and liver injury is induced by TP (2.4 mg/kg, p.o., suspended in 0.5% CMC-Na) for the last 3 days. Animals in the above three groups receive TP 6 hours after distilled water or 18β-Glycyrrhetinic acid treatment on the last 3 day
Rats

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NSCLC Xenograft Model (Nude Mice):
1. Animals: 6-8 week-old male nude mice (n=18). Acclimate 1 week [1]
2. Tumor induction: Subcutaneously inject 2×10⁶ A549 cells (0.2 mL, 1:1 Matrigel/PBS) into right flank [1]
3. Grouping/Treatment: When tumors reach ~100 mm³, randomize into 3 groups: Vehicle (10% DMSO/PBS, i.p.), Enoxolone 20 mg/kg (i.p.), 40 mg/kg (i.p.). Administer daily for 21 days [1]
4. Sample collection: Measure tumor volume (length×width²/2) and weight weekly. Euthanize mice on day 21. Collect tumors for IHC, Western blot, and TXB2 assay [1]
- EAE Model (C57BL/6 Mice):
1. Animals: 6-8 week-old female C57BL/6 mice (n=24). Acclimate 1 week [2]
2. EAE induction: Subcutaneously inject 200 μg MOG35-55 (emulsified in CFA) at 4 back sites. IP inject 200 ng pertussis toxin on day 0/2 [2]
3. Grouping/Treatment: Randomize into 3 groups on day 7: Vehicle (10% DMSO/PBS, i.p.), Enoxolone 10 mg/kg, 20 mg/kg (i.p.). Administer daily for 14 days. Score clinical signs daily (0=normal to 5=death) [2]
4. Sample collection: Euthanize on day 21. Collect spinal cord for HE, Iba1, and MBP staining [2]
- Hepatotoxicity Model (SD Rats):
1. Animals: 8-week-old male SD rats (n=24). Acclimate 1 week [3]
2. Hepatotoxicity induction: Administer triptolide (300 μg/kg, i.g.) daily for 7 days [3]
3. Grouping/Treatment: Randomize into 4 groups: Normal (saline, i.g.), Triptolide alone, Triptolide + Enoxolone 5 mg/kg, 10 mg/kg (i.g.). Administer Enoxolone concurrently with triptolide [3]
4. Sample collection: Euthanize on day 8. Collect blood for ALT/AST/ALP assay. Collect liver for HE staining, antioxidant assay, and Western blot [3]

In vitro toxicity: - Non-small cell lung cancer cells: Enoxone (concentration up to 40 μM) showed no cytotoxicity to normal bronchial epithelial cells (BEAS-2B), with cell viability >85% [1] - Microglia: Enoxone (concentration up to 25 μM) showed no cytotoxicity to normal astrocytes, with cell viability >90% [2] - Hepatocytes: Enoxone (concentration up to 20 μM) showed no cytotoxicity to normal primary rat hepatocytes, with cell viability >88% [3] - In vivo toxicity: - Nude mice: Enoxone (40 mg/kg, 21 days) did not cause weight loss (22.5±1.2 g vs. control group 23.1±1.0 g) or organ damage (liver/kidney). HE staining normal)[1] - EAE mice: Enoxone (20 mg/kg, 14 days) did not cause leukopenia (white blood cell count: 6.8±0.7×10⁹/L vs. control group 7.0±0.6×10⁹/L)[2] - Rats: Enoxone (10 mg/kg, 7 days) had no effect on serum urea nitrogen (15.2±1.8 mg/dL vs. normal value 14.8±1.5 mg/dL) or creatinine (0.8±0.1 mg/dL vs. normal value 0.7±0.1 mg/dL)[3]

[1]. 18β-Glycyrrhetinic acid suppresses cell proliferation through inhibiting thromboxane synthase in non-small cell lung cancer. PLoS One. 2014 Apr 2;9(4):e93690.

[2]. 18β-glycyrrhetinic acid suppresses experimental autoimmune encephalomyelitis through inhibition of microglia activation and promotion of remyelination. Sci Rep. 2015 Sep 2;5:13713.

[3]. Protective Effect of 18β-Glycyrrhetinic Acid against Triptolide-Induced Hepatotoxicity in Rats. Evid Based Complement Alternat Med. 2017;2017:3470320.

Glycyrrhetinic acid is a pentacyclic triterpenoid compound formed by the substitution of oleanolic-12-ene at positions 3, 11, and 30 with hydroxyl, carbonyl, and carboxyl groups, respectively. It possesses immunomodulatory effects and is also a plant metabolite. It is a pentacyclic triterpenoid compound, a cyclic terpene ketone, and a hydroxy monocarboxylic acid. It is the conjugate acid of glycyrrhetinic acid. It is derived from the hydride of oleanolic acid. Enoxetine (glycyrrhizic acid) has been used in basic scientific research on epigenetic mineralocorticoid hyperparathyroidism (AME). Enoxetine has been reported to exist in plants of the genus Glycyrrhiza (such as Glycyrrhiza glabra) and other organisms with relevant data. Enoxetine is a pentacyclic triterpenoid aglycone metabolite of glycyrrhizic acid, a product of the plant Glycyrrhiza glabra, which has potential expectorant and prokinetic effects. After administration, enoxetine can inhibit prostaglandin metabolism by inhibiting 15-hydroxyprostaglandin dehydrogenase [NAD(+)] and prostaglandin reductase 2. Therefore, this drug can enhance the activity of prostaglandins E2 and F2α, thereby inhibiting gastric acid secretion, while stimulating pancreatic secretion and the secretion of intestinal and respiratory mucus, ultimately leading to enhanced intestinal motility and antitussive effects. In addition, this drug can also inhibit 11β-hydroxysteroid dehydrogenase and other enzymes in the kidneys involved in the conversion of cortisol to cortisone.
Glycyrrhizic acid is an oleanolic acid derived from licorice and has certain anti-allergic, antibacterial, and antiviral properties. It can be used topically to treat allergic or infectious skin inflammations, or orally to exert its aldosterone effect in electrolyte regulation.
See also: glycyrrhizic acid (the active ingredient of the following substances); ammonium glycyrrhizate (active ingredient); licorice root (partial). Enoxetine (18β-glycyrrhetinic acid) is a natural triterpenoid compound derived from licorice root (Glycyrrhiza glabra) that has antitumor, anti-inflammatory and hepatoprotective activities [1][2][3] - In non-small cell lung cancer, enoxetine exerts antiproliferative effects by inhibiting TXS, reducing TXA2 (a pro-tumor mediator) and inducing apoptosis. TXS overexpression can reverse its antitumor effect, confirming that TXS is a key target [1]
- In experimental autoimmune encephalomyelitis (EAE), enoxazone treats autoimmune neuroinflammation through a dual mechanism: inhibiting microglia activation (by inhibiting NF-κB) and promoting oligodendrocyte myelin regeneration (by upregulating MBP), thereby simultaneously addressing inflammation and myelin loss [2]
- In terms of hepatotoxicity, enoxazone protects hepatocytes by activating Nrf2/HO-1, enhancing antioxidant capacity (GSH/SOD), and reducing oxidative stress (MDA) without interfering with the therapeutic effect of triptolide [3]
- Enoxone has a good safety profile in preclinical models, supporting its potential for clinical development in non-small cell lung cancer (NSCLC), autoimmune neurological diseases, and drug-induced liver injury [1][2][3]

C30H46O4

470.68

470.339

471-53-4

10114

White to off-white solid powder

1.1±0.1 g/cm3

588.3±50.0 °C at 760 mmHg

292 - 295ºC

323.7±26.6 °C

0.0±3.7 mmHg at 25°C

1.563

6.57

2

4

1

34

965

9

CC1(C)[C@@H](O)CC[C@]([C@@]1([H])CC[C@@]([C@@]2(CC[C@]3(CC[C@](C(O)=O)(C[C@]3(C2=C4)[H])C)C)C)5C)(C)[C@@]5([H])C4=O

MPDGHEJMBKOTSU-YKLVYJNSSA-N

InChI=1S/C30H46O4/c1-25(2)21-8-11-30(7)23(28(21,5)10-9-22(25)32)20(31)16-18-19-17-27(4,24(33)34)13-12-26(19,3)14-15-29(18,30)6/h16,19,21-23,32H,8-15,17H2,1-7H3,(H,33,34)/t19-,21-,22-,23+,26+,27-,28-,29+,30+/m0/s1

(2S,4aS,6aS,6bR,8aR,10S,12aS,12bR,14bR)-10-hydroxy-2,4a,6a,6b,9,9,12a-heptamethyl-13-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-2-carboxylic acid

Glycyrrhetin; Enoxolone; Glycyrrhetic acid; BRN 2229654; NSC 35347; NSC-35347; BRN-2229654; BRN2229654; NSC35347

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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: ≥ 2.17 mg/mL (4.61 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 21.7 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: ≥ 2.17 mg/mL (4.61 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 21.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 2% DMSO+30% PEG 300+5% Tween 80+ddH2O: 10mg/mL

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