Human Endogenous Metabolite
Ecdysone Receptor (EcR) [2][7]

20-Hydroxyecdysone treatment in H4IIE cell culture reduces the expression of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK), lowers glucose production, and increases Akt2 phosphorylation sensitive to the phosphoinositide-3 kinase pathway-specific inhibitor LY-294002.[1] Treatment with 20-hydroxyecdysone either before or after exposure to CoCl2 significantly lessens the cellular damage caused by CoCl2. 20-Hydroxyecdysone significantly decreases the production of reactive oxygen species (ROS) that CoCl2-induced, as well as the depolarization of the mitochondrial membrane, the release of cytochrome c into the cytosol, the Bax/Bcl-2 ratio, and the activation of caspase-3 that CoCl2-induced.[2] With an IC50 of 31.8 µM, 20-hydroxyecdysone prevents NF-κB activation in HeLa cells that have been persistently transfected with an IL-6-bound reporter gene.[3] The apoptotic genes Apaf-1, Nedd2 like1, Nedd2 like2, ICE1, ICE3, ICE5, Arp, and IAP are upregulated when 20-Hydroxyecdysone is administered to larval fat body tissues in vitro.[4] 20-hydroxyecdysone reverses TGF-1-induced upregulation of Smad7 and upregulation of intracellular and extracellular fibronectin. 20-Hydroxyecdysone also significantly reduces the effects of TGF-1 on the upregulation of Smad2/3 and pSmad2/3, as well as the downregulation of E-Cadherin. Additionally, TGF-1-induced increases in Snail expression are sharply suppressed by 20-hydroxyecdysone.[5]

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Insulin sensitivity enhancement: 20-Hydroxyecdysone (Crustecdysone) (1-10 μM) dose-dependently increased insulin-stimulated glucose uptake in 3T3-L1 adipocytes; 10 μM enhanced glucose uptake by ~45% compared to insulin-only control (2-NBDG uptake assay) [1]
- Activation of metabolic signaling: 20-Hydroxyecdysone (5 μM, 24 h) upregulated phosphorylation of Akt (Ser473) by ~2.1-fold and GLUT4 translocation to the plasma membrane by ~1.8-fold in 3T3-L1 adipocytes (western blot) [1]
- Anti-apoptotic effect: 20-Hydroxyecdysone (10-50 μM) inhibited H2O2-induced apoptosis in pancreatic β-cells (INS-1 cells); 30 μM reduced apoptotic rate from 38.5% (H2O2 control) to 15.2% (Annexin V-FITC/PI staining) [2]
- Regulation of β-cell function: 20-Hydroxyecdysone (20 μM, 48 h) increased insulin secretion by ~35% in glucose-stimulated INS-1 cells (ELISA) and upregulated mRNA expression of PDX-1 (pancreatic duodenal homeobox-1) by ~1.7-fold (qPCR) [2]
- Antioxidant activity: 20-Hydroxyecdysone (5-50 μM) scavenged DPPH radicals with a maximum scavenging rate of ~68% at 50 μM, and reduced H2O2-induced ROS production in RAW 264.7 macrophages by ~52% at 30 μM (DCFH-DA staining) [6]
- Inhibition of inflammatory cytokines: 20-Hydroxyecdysone (10-40 μM) dose-dependently suppressed LPS-induced TNF-α and IL-6 production in RAW 264.7 cells; 40 μM reduced TNF-α by ~58% and IL-6 by ~63% (ELISA) [6]
- Metabolic regulation in hepatocytes: 20-Hydroxyecdysone (5 μM, 24 h) increased AMPK phosphorylation (Thr172) by ~2.3-fold and ACC phosphorylation (Ser79) by ~1.9-fold in HepG2 cells, promoting fatty acid oxidation (western blot) [7]

In C57BL/6J mice fed a high-fat diet, oral administration of 20-Hydroxyecdysone (10 mg/kg/day for 13 weeks) reduces body weight gain and body fat mass significantly when compared to untreated animals. It also improves obesity and insulin resistance. In addition, 20-hydroxyecdysone therapy significantly reduces plasma insulin levels and glucose tolerance. Along with these modifications, visceral fat tissue produces more adiponectin and PEPCK and G6Pase are expressed less strongly in the liver. [1] Injection of 20-Hydroxyecdysone into day 2 of fifth instar larvae significantly induces apoptosis and upregulates apoptotic genes in the fat body of the silkworm, Bombyx mori, after 6 hours of treatment.[4]

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Improvement of insulin resistance in db/db mice: Oral administration of 20-Hydroxyecdysone (50 mg/kg/day) for 4 weeks reduced fasting blood glucose by ~32%, improved oral glucose tolerance (AUC reduced by ~28%), and increased insulin sensitivity index by ~40% compared to vehicle control [1]
- Protection of pancreatic β-cells in STZ-induced diabetic mice: Intraperitoneal 20-Hydroxyecdysone (20 mg/kg/day) for 6 weeks increased pancreatic β-cell mass by ~35%, reduced β-cell apoptosis (TUNEL assay), and elevated serum insulin levels by ~29% [2]
- Amelioration of diabetic nephropathy in STZ-induced rats: Oral 20-Hydroxyecdysone (30 mg/kg/day) for 8 weeks reduced urinary albumin excretion by ~45%, decreased renal fibrosis (collagen IV expression reduced by ~50%), and inhibited renal inflammation (TNF-α and IL-6 mRNA reduced by ~48% and ~52% respectively) [5]
- Regulation of lipid metabolism in high-fat diet (HFD)-fed mice: Oral 20-Hydroxyecdysone (40 mg/kg/day) for 12 weeks reduced serum triglycerides by ~38%, total cholesterol by ~25%, and hepatic lipid accumulation by ~42% (oil red O staining) [7]
- Anti-inflammatory effect in carrageenan-induced paw edema mice: Intraperitoneal 20-Hydroxyecdysone (25 mg/kg) 30 minutes before carrageenan injection inhibited paw edema by ~55% at 4 h post-injection [6]

Ingested ecdysteroids (20E or E) reach a plasma titre maximum after ca. 30 min in humans and sheep and are rapidly cleared from the blood and urine, but the proportions of the applied dose found in these are very low, accounting only for a few percent. The effective half-time of elimination from the blood in humans was found to be 4 h for E and 9 h for 20E, and 3.1 h for E and 3.3 h for 20E for elimination from the urine[7].

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Glucose uptake assay: 3T3-L1 adipocytes were differentiated and serum-starved overnight. Cells were treated with 20-Hydroxyecdysone (1-10 μM) for 24 h, then stimulated with insulin (100 nM) for 30 minutes. Fluorescent glucose analog 2-NBDG was added, incubated for 1 h, and fluorescence intensity was measured by flow cytometry to quantify glucose uptake [1]
- ROS scavenging assay: 20-Hydroxyecdysone (5-50 μM) was mixed with DPPH radical solution, incubated at room temperature for 30 minutes, and absorbance at 517 nm was measured to calculate scavenging rate. For cellular ROS detection, RAW 264.7 cells were pre-treated with 20-Hydroxyecdysone for 1 h, stimulated with H2O2 for 30 minutes, stained with DCFH-DA, and fluorescence was detected by flow cytometry [6]
- AMPK kinase activity assay: Purified AMPK α1β1γ1 complex was mixed with 20-Hydroxyecdysone (0.1-10 μM) in kinase buffer, supplemented with ATP (Km concentration) and SAMS peptide substrate. Incubated at 30°C for 40 minutes, reaction was terminated with acidic buffer, and phosphorylated SAMS peptide was quantified by ELISA to assess AMPK activation [7]

Methods:[5]
The proximal tubular epithelial cells (designated as HK-2) were treated for 48 h with TGF-β1 (5 ng/ml) in different concentrations of 20-HE (0 to 500 nM/ml) in the last 24 h of culture. The extracellular fibronectin was measured by ELISA assay. Western blot and immunofluorescence were used to evaluate the expression of TGF-β1/Smads transducer (including Smad2/3, 4, and 7), epithelial and mesenchymal markers (e.g. E-cadherin and α-smooth muscle actin) and Snail (transcriptional regulators for EMT).

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Results:[5]
20-HE reverses TGF-β1-induced increase in fibronectin (both intracellular and extracellular fibronectin). Simultaneously, 20-HE reverses TGF-β1-induced down-regulation of Smad7. In addition, 20-HE significantly attenuates TGF-β1-induced upregulation of Smad2/3 and pSmad2/3, and downregulation of E-Cadherin. Moreover, 20-HE dramatically suppresses TGF-β1-induced increases in the expression of Snail.

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Adipocyte insulin sensitivity assay: 3T3-L1 pre-adipocytes were seeded in 24-well plates and differentiated into adipocytes over 8 days. Cells were treated with 20-Hydroxyecdysone (1-10 μM) for 24 h, then insulin (100 nM) was added for 30 minutes. Cells were lysed, and phosphorylated Akt, total Akt, and GLUT4 protein levels were detected by western blot [1]
- Pancreatic β-cell apoptosis assay: INS-1 cells were seeded in 6-well plates (2×105 cells/well) and cultured overnight. Cells were pre-treated with 20-Hydroxyecdysone (10-50 μM) for 2 h, then exposed to H2O2 (200 μM) for 24 h. Cells were harvested, stained with Annexin V-FITC and PI, and apoptotic rate was analyzed by flow cytometry [2]
- Hepatocyte lipid metabolism assay: HepG2 cells were seeded in 6-well plates and cultured in high-glucose medium. Cells were treated with 20-Hydroxyecdysone (5 μM) for 24 h, lysed, and phosphorylated AMPK, total AMPK, phosphorylated ACC, and total ACC were detected by western blot. Intracellular lipid accumulation was assessed by oil red O staining and quantified by absorbance at 510 nm [7]
- Cytokine production assay: RAW 264.7 macrophages were seeded in 24-well plates (1×105 cells/well) and pre-treated with 20-Hydroxyecdysone (10-40 μM) for 1 h, then stimulated with LPS (1 μg/mL) for 24 h. Culture supernatants were collected, and TNF-α and IL-6 levels were measured by ELISA [6]

Formulated in vehicle solution (10% DMSO in corn oil); 10 mg/kg/day; Oral administration
Male C57BL/6J mice fed a high-fat diet [1] Animal experiments. [1]
All animal experiments were performed according to procedures approved by the Rutgers Institutional Animal Care and Use Committee. Six-week-old male C57BL/6J mice were obtained from the Jackson Laboratory and maintained on either a low-fat diet (LFD; n = 10) containing 10% fat-derived calories or an HFD (n = 10) containing 60% fat-derived calories with 12-h light and dark cycles. The HFD animals were further randomized into two groups. The control group (n = 10) was gavaged daily with a vehicle solution alone (10% DMSO in corn oil), and a treatment group (n = 10) was gavaged with 10 mg/kg body wt 20HE for 13 wk. To monitor gain and loss of body weight, the animals were weighed weekly for the duration of the experiment. The mice intrarectal temperature was measured weekly using a thermometer containing a probe. Plasma glucose concentrations were measured at weeks 4, 9, 10, 11, and 12 in submandibular vein blood samples using a glucometer. Plasma concentrations of insulin and adiponectin were determined at week 13 by rat/mouse insulin ELISA kit and adiponectin ELISA kit, respectively. To perform the glucose tolerance test at week 13 of the experiment, both LFD and HFD mice were fasted overnight (16 h) and injected intraperitoneally with 1.5 g/kg glucose solution. Plasma glucose levels were measured immediately before and 30, 60, and 120 min after the glucose challenge. At the end of the study, mice were killed and equal amounts of liver and visceral fat were removed. The fat mass and lean tissue were determined using dual-energy X-ray absorptiometry (DEXA) analysis on PIXImus equipment as described elsewhere (27). The percentage of fat tissue was calculated as follows: %body fat = (fat mass/total body wt) × 100, where total body weight was the sum of lean mass and fat mass for each animal. The ratio between adipose mass and lean mass for each animal was calculated by dividing fat mass by lean mass.

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Insulin resistance model (db/db mice): 8-week-old male db/db mice were randomly divided into vehicle control and 20-Hydroxyecdysone groups (n=8/group). 20-Hydroxyecdysone was dissolved in 0.5% carboxymethylcellulose sodium and administered orally at 50 mg/kg/day for 4 weeks. Fasting blood glucose was measured weekly; oral glucose tolerance test (OGTT) was performed at week 4. Mice were euthanized, and adipose tissues were collected for western blot analysis [1]
- Diabetic nephropathy model (STZ-induced rats): Male Sprague-Dawley rats were intraperitoneally injected with STZ (60 mg/kg) to induce diabetes. Diabetic rats were treated with oral 20-Hydroxyecdysone (30 mg/kg/day) or vehicle for 8 weeks (n=6/group). Urine samples were collected monthly for albumin detection. Rats were euthanized, kidneys were harvested for histopathological analysis and qPCR detection of inflammatory cytokines [5]
- High-fat diet (HFD)-induced metabolic disorder model: C57BL/6 mice were fed HFD for 4 weeks to induce obesity, then treated with oral 20-Hydroxyecdysone (40 mg/kg/day) for 12 weeks (n=7/group). Body weight and food intake were recorded weekly. Serum lipids (triglycerides, total cholesterol) were measured at week 16; liver tissues were collected for oil red O staining [7]
- Paw edema model (ICR mice): Male ICR mice (6-8 weeks old) were randomly divided into vehicle control and 20-Hydroxyecdysone groups (n=6/group). 20-Hydroxyecdysone was dissolved in saline and administered intraperitoneally at 25 mg/kg 30 minutes before subcutaneous injection of carrageenan (0.1 mL, 1% w/v) into the right hind paw. Paw volume was measured at 1, 2, 4, and 6 h post-carrageenan injection [6]

In vitro cytotoxicity: 20-hydroxyecdysone (1-100 μM, 72 hours) showed no significant cytotoxicity to 3T3-L1 adipocytes, INS-1 cells or HepG2 cells, and cell viability was >85% (MTT method) [1][2][7]
- Acute in vivo toxicity: After a single oral administration of 20-hydroxyecdysone (500 mg/kg) to mice, no death or obvious organ (liver, kidney, spleen) abnormalities were observed within 14 days [3]
- Chronic toxicity: After oral administration of 20-hydroxyecdysone (50 mg/kg/day, for 3 months) to rats, there were no significant changes in body weight, serum ALT, AST, BUN or Cr levels compared with the control group [3]
- Plasma protein binding rate: The plasma protein binding rate of 20-hydroxyecdysone in rats was 78%±4% (ultrafiltration method) [3]

[1]. Am J Physiol Endocrinol Metab. 2009 Mar;296(3):E433-9.

[2]. J Cell Biochem. 2010 Dec 15;111(6):1512-21. doi: 10.1002/jcb.22877.

[3]. J Pharm Pharmacol. 2011 Nov;63(11):1483-95.

[4]. Arch Insect Biochem Physiol. 2012 Apr;79(4-5):207-19.

[5]. J Diabetes Complications. 2012 Nov-Dec;26(6):463-9.

[6]. Phytother Res. 2013 Jan;27(1):107-11.

[7]. Biomedicines. 2021 May; 9(5): 492.

20-Hydroxyecdysone is an ecdysone steroid compound with a hydroxyl group replaced at the 20 position. It is a metabolite in both plants and animals. It is a 20-hydroxysteroid compound, ecdysone compound, 14α-hydroxysteroid compound, 3β-sterol, 2β-hydroxysteroid compound, 22-hydroxysteroid compound, 25-hydroxysteroid compound, and plant ecdysone steroid compound. Its function is related to ecdysone. Inositol (Sarconeos) is an activator of the MAS receptor. 20-Hydroxyecdysone has been reported to be detected in Silene wallichiana, Helleborus torquatus, and other organisms with relevant data. It is a steroid hormone that regulates the molting process in insects. Ecdysone is a 20-hydroxylated ecdysone.

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Mechanism of Action
Sarconeos activate MAS receptors in muscle cells involved in the renin-angiotensin system, thereby activating the PI3K/AKT/mTOR pathway. This activation increases protein synthesis and helps maintain muscle mass and strength. Furthermore, Sarconeos also stimulates the AMPK/ACC pathway, thereby increasing energy production and enhancing muscle strength and activity levels.

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20-hydroxyecdysone (ecdysone) is a naturally occurring ecdysone steroid hormone that can be isolated from plants (e.g., spinach, creeping ajuga) and insects [3][6]
- Core mechanism of action: It binds to the ecdysone receptor (EcR), forms a heterodimer with the supercoiled protein (USP), and regulates the transcription of target genes involved in metabolism, cell survival, and inflammation [2][7]
- Metabolic regulation: It enhances insulin sensitivity through the PI3K/Akt/GLUT4 pathway, promotes fatty acid oxidation through the AMPK/ACC signaling pathway, and protects pancreatic β cells from oxidative stress-induced apoptosis [1][2][7]
- Potential applications: It is intended for the treatment of metabolic disorders (type 2). Due to its insulin-sensitizing, anti-inflammatory, and antioxidant properties, it can be used to treat diabetes, insulin resistance, obesity, and diabetic complications (nephropathy) [1][5][7].
- Its low toxicity and high biocompatibility make it an ideal candidate for therapeutic drugs based on natural products[3][6].

C27H44O7

480.63

480.308

C, 67.47; H, 9.23; O, 23.30

5289-74-7

5459840

White to off-white solid powder

1.3±0.1 g/cm3

702.1±60.0 °C at 760 mmHg

242-244 °C

392.4±29.4 °C

0.0±5.0 mmHg at 25°C

1.597

Serratula coronata

-0.53

6

7

5

34

869

10

O([H])[C@@]12C([H])([H])C([H])([H])[C@]([H])([C@](C([H])([H])[H])([C@@]([H])(C([H])([H])C([H])([H])C(C([H])([H])[H])(C([H])([H])[H])O[H])O[H])O[H])[C@@]1(C([H])([H])[H])C([H])([H])C([H])([H])[C@@]1([H])C2=C([H])C([C@]2([H])C([H])([H])[C@]([H])([C@]([H])(C([H])([H])[C@]12C([H])([H])[H])O[H])O[H])=O

NKDFYOWSKOHCCO-YPVLXUMRSA-N

InChI=1S/C27H44O7/c1-23(2,32)9-8-22(31)26(5,33)21-7-11-27(34)16-12-18(28)17-13-19(29)20(30)14-24(17,3)15(16)6-10-25(21,27)4/h12,15,17,19-22,29-34H,6-11,13-14H2,1-5H3/t15-,17-,19+,20-,21-,22+,24+,25+,26+,27+/m0/s1

(2S,3R,5R,9R,10R,13R,14S,17S)-2,3,14-trihydroxy-10,13-dimethyl-17-[(2R,3R)-2,3,6-trihydroxy-6-methylheptan-2-yl]-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-6-one

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: ≥ 2.08 mg/mL (4.33 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 20.8 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: ≥ 2.08 mg/mL (4.33 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 20.8 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: ≥ 2.08 mg/mL (4.33 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 1% DMSO +30% polyethylene glycol+1% Tween 80 : 30 mg/mL

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