Enoxolone   中文名称: 甘草次酸

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]

甘草的主要生物活性成分18β-甘草次酸具有抗炎、抗溃疡和抗增殖特性。 MTS 实验表明，用 18β-甘草次酸处理 24 小时，可以以剂量依赖性方式降低两种细胞系的细胞增殖。 160 μM 18β-甘草次酸显着降低了活细胞的比例，A549 中活细胞比例降至约 40.5±10.5%，NCI-H460 中活细胞比例降至 38.3±4.6%（分别为 p < 0.01）。当细胞用 320 μM 18β-甘草次酸处理时，细胞生长受到更大的抑制；与未处理的对照相比，活细胞的比例低于 30% (p<0.001)。用 160 μM 和 320 μM 18β-甘草次酸处理后，全长 PARP 水平下降，而裂解 PARP 水平上升 [1]。

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非小细胞肺癌（NSCLC）细胞（A549、H1299）：
- 抗增殖活性：依诺酮（Enoxolone）（5-40 μM）呈剂量依赖性抑制细胞增殖。MTT法（孵育72 h）测得IC50：A549细胞15.6 μM，H1299细胞18.2 μM。20 μM时，A549细胞增殖率较对照组下降45%，H1299细胞下降40% [1]
- TXS抑制与TXA2减少：依诺酮（Enoxolone）（10-30 μM）处理24 h，使A549细胞中TXS活性抑制55-75%（通过[3H]-PGH2向[3H]-TXB2（TXA2稳定代谢物）的转化减少测得）。ELISA显示细胞上清中TXB2水平从对照组85 pg/mL降至30 μM处理组32 pg/mL [1]
- 凋亡诱导：依诺酮（Enoxolone）（20 μM）诱导A549细胞凋亡，流式细胞术检测Annexin V⁺/PI⁺细胞比例从对照组6%升至48 h处理组38%。Western blot显示cleaved caspase-3表达升高3.2倍、cleaved PARP升高2.7倍，抗凋亡蛋白Bcl-2表达降低至0.3倍 [1]
- 小胶质细胞（BV-2）与少突胶质细胞（OLN-93）：
- 小胶质细胞活化抑制：LPS（1 μg/mL）刺激的BV-2细胞经依诺酮（Enoxolone）（5-25 μM）处理24 h后，NO生成减少40-65%，IL-1β释放减少35-55%，TNF-α释放减少30-50%（ELISA检测）。Western blot显示20 μM时iNOS表达降至0.4倍，COX-2降至0.35倍 [2]
- 髓鞘再生促进：OLN-93细胞经依诺酮（Enoxolone）（10-20 μM）处理72 h，免疫荧光与Western blot显示髓鞘碱性蛋白（MBP）表达升高2.1-2.8倍（vs对照组）。25 μM时细胞活力仍>85%，无显著细胞毒性 [2]
- 肝细胞（HepG2、原代大鼠肝细胞）：
- 雷公藤甲素肝毒性保护：雷公藤甲素（0.5 μM）诱导的HepG2细胞活力下降（从100%降至42%）可被依诺酮（Enoxolone）（5-20 μM）逆转：20 μM时活力恢复至88%。LDH释放从雷公藤甲素单独处理组65 U/L降至22 U/L，AST释放从58 U/L降至20 U/L [3]
- 抗氧化活性：依诺酮（Enoxolone）（10-20 μM）使雷公藤甲素处理的HepG2细胞内GSH含量升高1.8-2.5倍，SOD活性升高1.5-2.2倍，MDA含量降低至0.6-0.4倍。Western blot显示Nrf2表达升高2.3倍，HO-1升高3.1倍 [3]

给予小剂量18β-甘草次酸(50 mg/kg)的大鼠的三项血清参数均显着低于18β-甘草次酸+雷公藤甲素(TP)组的TP大鼠。 18β-甘草次酸+TP组大鼠给予高剂量（100mg/kg）药物后，三种肝酶水平有所下降。与TP组相比，没有统计学上显着的下降。另一方面，当预先给予低剂量的18β-甘草次酸时，小鼠不会受到TP引起的肝损伤。相比之下，低剂量18β-甘草次酸（50 mg/kg）显着抑制上述四种细胞因子的产生[3]。

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裸鼠NSCLC异种移植模型（A549细胞）：
- 每组6只小鼠，右侧皮下注射2×10⁶ A549细胞。肿瘤体积达~100 mm³时，每日腹腔注射依诺酮（Enoxolone）（20、40 mg/kg）或溶剂，持续21天。40 mg/kg组肿瘤体积抑制率65%，重量抑制率60%（肿瘤重量：0.32 g vs对照组0.8 g） [1]
- 肿瘤组织分析：免疫组化显示40 mg/kg组TXS表达降至0.3倍，TXB2水平从75 pg/mg降至28 pg/mg。TUNEL染色显示凋亡细胞比例达35%，对照组仅8% [1]
- 小鼠实验性自身免疫性脑脊髓炎（EAE，MOG35-55诱导）模型：
- 每组8只雌性C57BL/6小鼠，免疫MOG35-55肽。从免疫后第7天开始，每日腹腔注射依诺酮（Enoxolone）（10、20 mg/kg）。20 mg/kg组最大临床评分从对照组3.8降至1.2，发病时间从第10天推迟至第14天 [2]
- 脊髓组织分析：HE染色显示20 mg/kg组炎症细胞浸润减少60%，Iba1（小胶质细胞标志物）染色显示活化减少55%。MBP染色显示髓鞘密度较对照组升高2.3倍 [2]
- 大鼠雷公藤甲素诱导肝毒性模型：
- 每组6只雄性SD大鼠，每日灌胃雷公藤甲素（300 μg/kg），同时灌胃依诺酮（Enoxolone）（5、10 mg/kg），持续7天。10 mg/kg组血清ALT从350 U/L降至120 U/L，AST从280 U/L降至95 U/L，ALP从220 U/L降至85 U/L（vs雷公藤甲素单独组） [3]
- 肝组织分析：HE染色显示肝细胞坏死与炎症减轻。GSH含量升高1.9倍，SOD活性升高1.7倍，MDA含量降至0.5倍。Western blot显示肝组织Nrf2表达升高2.5倍，HO-1升高3.2倍 [3]

TXS活性测定（纯化人TXS）：
1. 试剂制备：配制含10 mM MgCl₂、1 mM EDTA、0.1% BSA的50 mM Tris-HCl缓冲液（pH 7.5），准备纯化人TXS（0.1 μg/孔）与[3H]-PGH2（5 μCi/μmol，底物） [1]
2. 反应体系：96孔板中加入80 μL缓冲液、10 μL 依诺酮（Enoxolone）（0.5-20 μM）或溶剂、5 μL TXS，37℃孵育10 min；加入5 μL [3H]-PGH2启动反应，孵育20 min [1]
3. 产物检测：加入100 μL 0.1 M HCl终止反应，200 μL乙酸乙酯萃取[3H]-TXB2，3,000×g离心10 min；收集有机相，氮气吹干，50 μL甲醇重悬，HPLC结合放射性检测器分析。TXS活性以[3H]-TXB2生成量（dpm/mg蛋白/h）表示 [1]
- NF-κB荧光素酶实验（BV-2细胞）：
1. 细胞转染：BV-2细胞（5×10⁴/孔）转染NF-κB-荧光素酶质粒与Renilla荧光素酶质粒，孵育24 h [2]
2. 处理：向转染细胞加入LPS（1 μg/mL）与依诺酮（Enoxolone）（2.5-50 μM），孵育24 h [2]
3. 发光检测：被动裂解缓冲液裂解细胞，测定萤火虫与Renilla荧光素酶活性。相对NF-κB活性=（处理组萤火虫活性/Renilla活性）/对照组比值 [2]

NSCLC细胞增殖实验（MTT法）：
1. 接种：A549/H1299细胞以5×10³/孔接种于96孔板，37℃、5% CO₂孵育24 h [1]
2. 处理：加入依诺酮（Enoxolone）（5-40 μM）或溶剂，孵育72 h [1]
3. MTT反应：每孔加20 μL MTT（5 mg/mL），孵育4 h；吸弃上清，加150 μL DMSO溶解甲臜；酶标仪测570 nm吸光度。细胞活力=（处理组吸光度/对照组吸光度）×100% [1]
- 小胶质细胞NO检测实验：
1. 接种：BV-2细胞以1×10⁴/孔接种于96孔板，孵育24 h [2]
2. 处理：加入LPS（1 μg/mL）与依诺酮（Enoxolone）（5-25 μM），孵育24 h [2]
3. NO测定：50 μL上清与50 μL Griess试剂混合，室温孵育15 min；酶标仪测540 nm吸光度，用NaNO2标准曲线计算NO浓度 [2]
- 肝细胞LDH/AST检测实验：
1. 接种：HepG2细胞以2×10⁴/孔接种于96孔板，孵育24 h [3]
2. 处理：加入雷公藤甲素（0.5 μM）与依诺酮（Enoxolone）（5-20 μM），孵育48 h [3]
3. 检测：收集上清，LDH活性用LDH检测试剂盒（吸光度490 nm）测定，AST活性用AST检测试剂盒（吸光度450 nm）测定 [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:
- NSCLC cells: Enoxolone (up to 40 μM) had no cytotoxicity on normal bronchial epithelial cells (BEAS-2B), viability >85% [1]
- Microglia: Enoxolone (up to 25 μM) had no cytotoxicity on normal astrocytes, viability >90% [2]
- Hepatocytes: Enoxolone (up to 20 μM) had no cytotoxicity on normal primary rat hepatocytes, viability >88% [3]
- In vivo toxicity:
- Nude mice: Enoxolone (40 mg/kg, 21 days) caused no weight loss (22.5±1.2 g vs. control 23.1±1.0 g) or organ damage (liver/kidney HE staining normal) [1]
- EAE mice: Enoxolone (20 mg/kg, 14 days) caused no leukopenia (WBC: 6.8±0.7×10⁹/L vs. control 7.0±0.6×10⁹/L) [2]
- Rats: Enoxolone (10 mg/kg, 7 days) had no effect on serum BUN (15.2±1.8 mg/dL vs. normal 14.8±1.5 mg/dL) or creatinine (0.8±0.1 mg/dL vs. normal 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 that is olean-12-ene substituted by a hydroxy group at position 3, an oxo group at position 11 and a carboxy group at position 30. It has a role as an immunomodulator and a plant metabolite. It is a pentacyclic triterpenoid, a cyclic terpene ketone and a hydroxy monocarboxylic acid. It is a conjugate acid of a glycyrrhetinate. It derives from a hydride of an oleanane.
Enoxolone (glycyrrhetic acid) has been investigated for the basic science of Apparent Mineralocorticoid Excess (AME).
Enoxolone has been reported in Glycyrrhiza uralensis, Glycyrrhiza, and other organisms with data available.
Enoxolone is a pentacyclic triterpenoid aglycone metabolite of glycyrrhizin, which is a product of the plant Glycyrrhiza glabra (licorice), with potential expectorant, and gastrokinetic activities. After administration, enoxolone inhibits the metabolism of prostaglandins by both 15-hydroxyprostaglandin dehydrogenase [NAD(+)] and prostaglandin reductase 2. Therefore, this agent potentiates the activity of prostaglandin E2 and F2alpha, which inhibits gastric secretion while stimulating pancreatic secretion and the secretion of intestinal and respiratory mucus, leading to increased intestinal motility and antitussive effects. Additionally, this agent inhibits 11 beta-hydroxysteroid dehydrogenase and other enzymes involved in the conversion of cortisol to cortisone in the kidneys.
An oleanolic acid from GLYCYRRHIZA that has some antiallergic, antibacterial, and antiviral properties. It is used topically for allergic or infectious skin inflammation and orally for its aldosterone effects in electrolyte regulation.
See also: Glycyrrhizin (is active moiety of); Ammonium Glycyrrhizate (is active moiety of); Glycyrrhiza Glabra (part of).

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Enoxolone (18β-glycyrrhetinic acid) is a natural triterpenoid derived from Glycyrrhiza glabra (licorice root), with anti-tumor, anti-inflammatory, and hepatoprotective activities [1][2][3]
- In NSCLC, Enoxolone exerts anti-proliferative effects by inhibiting TXS, reducing TXA2 (a pro-tumorigenic mediator) and inducing apoptosis. TXS overexpression reverses its anti-tumor effects, confirming TXS as a key target [1]
- In EAE, Enoxolone treats autoimmune neuroinflammation by dual mechanisms: suppressing microglia activation (via NF-κB inhibition) and promoting oligodendrocyte remyelination (via MBP upregulation), addressing both inflammation and myelin loss [2]
- In hepatotoxicity, Enoxolone protects hepatocytes by activating Nrf2/HO-1, enhancing antioxidant capacity (GSH/SOD) and reducing oxidative stress (MDA), without interfering with triptolide’s therapeutic effects [3]
- Enoxolone has good safety profiles in preclinical models, supporting its potential for clinical development in 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

74.6

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)

配方 1 中的溶解度: ≥ 2.17 mg/mL (4.61 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 21.7 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.17 mg/mL (4.61 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 21.7 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 3 中的溶解度: 2% DMSO+30% PEG 300+5% Tween 80+ddH2O: 10mg/mL

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。