Epocholeone targets plant growth and stress response pathways. As a brassinosteroid analog, it binds to the plant brassinosteroid receptor BRI1 (Brassinosteroid Insensitive 1), a plasma membrane-localized leucine-rich repeat receptor-like kinase (LRR-RLK). Binding of the ligand activates a signaling cascade involving the transcription factors BZR1 and BZR2/BES1, leading to the expression of genes involved in cell elongation, division, differentiation, photosynthesis, and stress tolerance. The propionyl ester groups on Epocholeone may facilitate its uptake by plant cells and enhance its stability compared to natural brassinosteroids, which have hydroxyl groups that are more prone to modification and degradation. The compound's bioactivity is distinct from brassinolide and 24-epibrassinolide, ranking comparably to 14-OH-brassinolide in Arabidopsis root elongation assays. In addition to its plant targets, Epocholeone is being studied for potential effects on mammalian targets, such as inflammatory pathways (e.g., modulating key immune system receptors), though these remain to be fully characterized.

In vitro studies on plant systems demonstrate that Epocholeone exhibits bioactivity distinct from that of brassinolide and 24-epibrassinolide. In Arabidopsis root elongation assays, Epocholeone ranks similarly to 14-OH-brassinolide. The compound promotes root elongation, cell expansion, and shoot growth. In vitro, Epocholeone can be used to study brassinosteroid signaling pathways in plant cell cultures. For example, treatment of Arabidopsis seedlings (grown on agar plates) with Epocholeone (0.001-10 uM) results in increased hypocotyl length and root growth. The activity can be quantified by measuring the growth parameters and the expression of brassinosteroid-responsive genes (e.g., SAUR-AC1, CPD, DWF4) by qPCR. The EC50 for growth promotion is typically in the nanomolar to low micromolar range. In mammalian systems, Epocholeone has been reported to have potential therapeutic applications in treating inflammatory diseases and immune system disorders, as it may function by modulating key receptors. However, detailed in vitro data on its effects on mammalian cells (e.g., immune cells, endothelial cells) is not available in the public domain. It is being investigated for its ability to modulate inflammatory pathways and immune responses, but no specific EC50 or IC50 values are provided.

In vivo studies of Epocholeone are primarily in plant models. In agricultural field trials, Epocholeone has been shown to increase crop yield. Specifically, a 0.003% solution (3000× dilution) applied to wheat results in a validated 10% yield increase. The compound can control fungal or physiological diseases of crops. In greenhouse or field studies, Epocholeone is typically applied as a foliar spray or as a seed treatment. The optimal concentration varies by crop and target disease. For disease control, Epocholeone likely acts by enhancing the plant's innate immune system (systemic acquired resistance or induced systemic resistance) rather than directly killing the pathogen. In a plant disease model, Epocholeone treatment reduces the severity of fungal infections (e.g., powdery mildew, rust, Fusarium) and physiological disorders (e.g., fruit cracking, sunburn). The growth-promoting effects are observed in various crops, including wheat, rice, corn, soybeans, and vegetables, leading to increased biomass, grain yield, and improved quality parameters (e.g., protein content, grain weight). In mammalian models, Epocholeone is being explored for its potential to treat inflammatory and immune disorders. It is a novel compound under investigation for its therapeutic applications in these areas, but no specific in vivo efficacy data (e.g., in rodent models of arthritis, asthma, or inflammatory bowel disease) is available in the public domain. The compound is not yet registered under EU Regulation 1107/2009 for agricultural use in Europe but is available for research and field trials in regions with active registrations.

Non-cell-based assays for Epocholeone in plant systems include brassinosteroid-binding assays using the BRI1 receptor. A radioligand binding assay can be performed using microsomal membranes from Arabidopsis plants expressing BRI1 or from insect cells expressing recombinant BRI1. Membranes (50-100 ug protein) are incubated with 0.5 nM 3H-brassinolide or 3H-24-epibrassinolide and increasing concentrations of unlabeled Epocholeone (0.1-1000 nM) in binding buffer (25 mM MES pH 5.5, 10 mM MgCl2, 0.1% BSA) for 60-90 minutes at 4degC. Non-specific binding is determined in the presence of 10 uM unlabeled brassinolide. Bound and free are separated by centrifugation or filtration. The IC50 for displacement is determined, and the Ki is calculated using the Cheng-Prusoff equation. The relative binding affinity of Epocholeone to BRI1 compared to natural brassinolide can be calculated. For functional assays, a BRI1-dependent phosphorylation assay can be performed. Recombinant BRI1 kinase domain is incubated with Epocholeone (1-1000 nM) and ATP (0.1 mM) for 30 minutes at 30degC, and the autophosphorylation of BRI1 is detected by Western blotting using an anti-phospho-tyrosine or anti-phospho-BRI1 antibody. The EC50 for activation is determined. For a cell-free reporter assay, a BRI1-dependent BZR1 phosphorylation assay can be set up using the Arabidopsis BZR1 protein as a substrate. For plant pathogen assays, Epocholeone can be tested in vitro for direct antifungal activity using a mycelial growth inhibition assay. The compound is added to potato dextrose agar (PDA) at various concentrations (10-1000 ug/mL), and a fungal disc is placed in the center. The diameter of the fungal colony is measured after 3-7 days. The percentage of inhibition is calculated. Epocholeone is not expected to have strong direct antifungal activity; rather, it induces plant defense responses. For mammalian systems, receptor binding assays (e.g., with glucocorticoid or other nuclear receptors) could be performed, but no such data is available.

For plant-based studies, Arabidopsis thaliana seeds are surface-sterilized and germinated on MS (Murashige and Skoog) agar plates containing various concentrations of Epocholeone (0, 0.01, 0.1, 1, 10 uM). The plates are placed vertically to allow root growth. After 7-10 days, the primary root length and hypocotyl length are measured. The number of lateral roots is counted. The percentage of growth stimulation is calculated relative to control. For gene expression analysis, seedlings are treated with Epocholeone (1 uM) for 0.5, 1, 2, 4, 8, 12 hours, then harvested for RNA extraction. qPCR is performed using primers for brassinosteroid-responsive marker genes (e.g., BR6ox2, CPD, DWF4, SAUR-AC1). For crop plants (e.g., wheat, rice), plants are grown in pots or in the field. Epocholeone is formulated as a wettable powder or suspension concentrate and sprayed onto the leaves at the appropriate growth stage (e.g., tillering stage, booting stage). The application rate is typically expressed as grams of active ingredient per hectare. For yield trials, multiple plots are used with replication (e.g., 4 replicates per treatment). At harvest, the yield per plant or per unit area (e.g., kg/ha) is measured. For disease control, plants are inoculated with a fungal pathogen (e.g., powdery mildew) before or after Epocholeone treatment. Disease severity is assessed by visual scoring of lesion coverage or by measuring fungal biomass (e.g., by qPCR). For mammalian studies, if Epocholeone is being investigated for anti-inflammatory activity, a standard protocol using mice would involve a model of inflammation. For example, in a carrageenan-induced paw edema model, mice (BALB/c, 6-8 weeks) are administered Epocholeone (orally or i.p.) at doses of 1-50 mg/kg, 1 hour before subplantar injection of 1% carrageenan (50 uL). Paw volume is measured by plethysmometry at 0, 1, 2, 3, 4, 6 hours. The percentage inhibition of edema is calculated. In an LPS-induced sepsis model, mice are injected i.p. with LPS (10 mg/kg), and Epocholeone is administered at the same time or 30 minutes before. Survival is monitored for 24-48 hours, and serum levels of pro-inflammatory cytokines (TNF-alpha, IL-6, IL-1beta) are measured by ELISA. In an autoimmune disease model (e.g., collagen-induced arthritis), Epocholeone would be administered daily for 2-3 weeks, and clinical scores (swelling, redness of paws) and histopathology of joints would be assessed. However, no such data is publicly available for Epocholeone; these are hypothetical protocols based on its described anti-inflammatory potential.

Pharmacokinetic data for Epocholeone is not available. As a plant growth regulator, it is not intended for human use, and its PK in mammals is likely not a priority. For agricultural applications, it is applied as a foliar spray, and its uptake, translocation, and metabolism in plants can be studied. A patented crystalline form enables formulation of stable suspension concentrates or wettable powders. The compound is likely to be absorbed through the leaf cuticle and translocated via the phloem to other parts of the plant. Its metabolism in plants would involve ester hydrolysis and glycosylation. In mammals, if administered systemically, the ester groups would likely be hydrolyzed by esterases, releasing the active metabolite(s). The LogP (estimated 4-5) suggests good passive permeability. However, detailed PK parameters (t1/2, Vd, CL, AUC, F) are not reported. For in vitro assays, Epocholeone is soluble in DMSO and organic solvents, and should be stored as a powder at -20degC.

No toxicity data is available for Epocholeone in mammals. It is considered safe for use as a plant growth regulator when applied according to label instructions. In acute toxicity studies in rats, it would likely have an LD50 > 2000 mg/kg (oral), indicating low acute toxicity. It is not expected to be genotoxic or carcinogenic. Standard safety precautions for handling (gloves, lab coat, safety goggles) should be followed. The compound is for research use only and is not for human use. In Europe, it is not approved under EU 1107/2009, so it is not commercially available for agricultural use there; it is available for research and development (R&D) and field trials in regions with active registrations.

[1]. Watanabe T, et, al. Synthesis and biological activity of 2,3-diol stereoisomers of 28-homobrassinolide and brassinolide. J. CHEM. RESEARCH (S), 1998; 744-5.

Epocholeone is a semi-synthetic brassinosteroid analog and plant growth regulator. It is also known by the trade name (Aizengmei) in China. It is used to control fungal or physiological diseases of crops and to increase yield (e.g., wheat yield increase of 10% at 0.003% solution). The compound is not a drug and is not FDA-approved. It is supplied as a solid powder, soluble in DMSO and ethanol. It should be stored at -20degC, protected from light and moisture. The compound is a valuable tool for studying brassinosteroid signaling in plants and for agricultural research aimed at improving crop yield and stress tolerance. It is also a subject of preclinical research for potential anti-inflammatory and immune-modulating effects in mammals, though this is still in early stages. This compound is not for human or veterinary use.

C35H56O7

588.81

588.403

162922-31-8

87556584

White to off-white solid powder

1.11g/cm3

642.9ºC at 760mmHg

262.8ºC

0mmHg at 25°C

1.521

6.747

0

7

11

42

1020

13

CCC(O[C@H]1C[C@H]2[C@]([C@H]3CC[C@@]4([C@@H]([C@@H]([C@H]5O[C@@H]5C(C(C)C)CC)C)CC[C@H]4[C@@H]3COC2=O)C)(C)C[C@H]1OC(CC)=O)=O

FHOXQLTVOMNIOR-QZPAGEHASA-N

InChI=1S/C35H56O7/c1-9-21(19(4)5)32-31(42-32)20(6)23-12-13-24-22-18-39-33(38)26-16-27(40-29(36)10-2)28(41-30(37)11-3)17-35(26,8)25(22)14-15-34(23,24)7/h19-28,31-32H,9-18H2,1-8H3/t20-,21-,22-,23+,24-,25-,26+,27-,28+,31+,32+,34+,35+/m0/s1

[(1S,2R,4R,5S,7S,11S,12S,15R,16S)-2,16-dimethyl-15-[(1S)-1-[(2R,3R)-3-[(3S)-2-methylpentan-3-yl]oxiran-2-yl]ethyl]-8-oxo-4-propanoyloxy-9-oxatetracyclo[9.7.0.02,7.012,16]octadecan-5-yl] propanoate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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