plant growth modulator[1]
Brassinolide-d5 functions as a plant hormone through binding to its specific receptor, BRASSINOSTEROID INSENSITIVE 1 (BRI1), a leucine-rich repeat receptor-like kinase (LRR-RLK) located on the plant cell surface. Upon binding, BRI1 dimerizes with its co-receptor BAK1 (BRI1-ASSOCIATED RECEPTOR KINASE 1), triggering a phosphorylation cascade that activates the transcription factors BES1 and BZR1 (via dephosphorylation by protein phosphatase 2A). These transcription factors then translocate to the nucleus and regulate the expression of genes involved in cell elongation, division, differentiation, vascular development, stress responses, and photomorphogenesis.

Brassinolide is a plant sterol first isolated from pollen of rape (Brassica napus L.). Brassinolide can induce a time and concentration-dependent cytotoxicity in PC-3 cells. The mode of cell death appears to be predominately apoptosis, as shown by flow- cytometric analysis, fluorescence and transmission electron microscopes. Caspase-3 activity is obviously increased after Brassinolide treatment. Western blot studies indicate that treatment with Brassinolide triggered a time-dependent decrease in the expression of anti-apoptotic protein Bcl-2, which suggests that Brassinolide can induce cytotoxicity in PC-3 cells by triggering apoptosis. Brassinolide might therefore be a promising candidate for the treatment of prostate cancer[1]. Brassinolide is a plant growth modulator, on multidrug resistance (MDR) of human T lymphoblastoid cell line CCRF- VCR 1000 which is obtained by progressive addition of vincristine (VCR) to sensitive CCRF-CEM cells, and to explore preliminarily the mechanism of reversing action. After treatment of Brassinolide under the concentration of 0.001-10 μg/mL, the resistance of CCRF-VCR is reversed partly with the reversing folds respectively as 4.4-11.6. The intracellular accumulation of rhodamine 123 is significantly reduced in the resistant cells. After treatment of Brassinolide, the accumulation increased, the level of fluorescent dye is situated between resistant cells and sensitive cells. No alteration of the catalytic activity of topoisomerase II is found among three groups . The level of protein expression of p53 in resistant cells is higher than that of sensitive cells. After Brassinolide treatment, the expression of p53 in CCRF-VCR cells restored to the level of sensitive cells. Brassinolide can effectively reverse the resistance of CCRF-VCR cells by inhibiting the effusion of drug transported by P-glucoprotein. To down regulate the abnormal expression of p53 maybe one of the mechanisms of reversing MDR for Brassinolide[2]. Stable heavy isotopes of hydrogen, carbon, and other elements have been incorporated into drug molecules, largely as tracers for quantitation during the drug development process. Deuteration has gained attention because of its potential to affect the pharmacokinetic and metabolic profiles of drugs[3].

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In vitro, unlabeled Brassinolide exhibits high biological activity in plant bioassays at nanomolar to picomolar concentrations. The rice lamina inclination bioassay (EC50 ~0.1-1 nM) and bean second internode elongation assay are standard tests for brassinosteroid activity. Brassinolide-d5 is not typically used in these bioassays but is used to study brassinosteroid metabolism and signaling. In plant cell cultures, Brassinolide (1-100 nM) induces cell elongation, promotes cell division, and activates expression of brassinosteroid-responsive genes such as SAUR-AC1, TCH4, and EXPANSIN. The deuterated analog has identical activity to unlabeled Brassinolide at the receptor level due to minimal kinetic isotope effect.

In vivo (in planta), unlabeled Brassinolide regulates numerous growth and developmental processes: stem and root elongation, vascular differentiation, leaf expansion, senescence, flowering, stress tolerance, and photomorphogenesis. In Arabidopsis thaliana, brassinosteroid-deficient mutants (e.g., det2, dwf4, cpd, bri1) exhibit dwarfism, dark green curled leaves, delayed flowering, and reduced fertility, which can be rescued by exogenous Brassinolide application (0.1-10 nM, sprayed or applied to growth medium). Brassinolide-d5 is used as a tracer to study the biosynthesis, transport, metabolism, and signaling of brassinosteroids rather than as a growth regulator itself. In animal systems, high concentrations (≥1 microM) of Brassinolide exhibit concentration- and time-dependent cytotoxicity in PC-3 human prostate cancer cells.

No standardized receptor-binding protocols for Brassinolide-d5 exist. For BRI1 receptor binding assays: Express recombinant BRI1 ectodomain (BRI1-ECD) in insect cells or E. coli. Incubate BRI1-ECD with [3H]-Brassinolide (1-10 nM) in binding buffer (25 mM HEPES, pH 7.5, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 1 mM DTT) for 1-2 hours at 4degC. Add increasing concentrations of unlabeled Brassinolide (0.1 nM to 10 microM) to compete. Separate bound and free radioligand by gel filtration or polyethylene glycol (PEG) precipitation. Measure radioactivity by scintillation counting. For binding studies with Brassinolide-d5, it would be used as a tracer at low concentrations or as internal standard for quantitation. For BRI1 kinase activity assay: Incubate BRI1 recombinant protein with ATP (10 microM) and Brassinolide (1-100 nM). Measure autophosphorylation by anti-phosphothreonine/tyrosine Western blot or by measuring ADP production using coupled enzyme assay.

No plant cell-based protocols specifically using Brassinolide-d5 exist. For standard brassinosteroid bioassays: Rice (Oryza sativa L. cv. “Kasalath”) lamina inclination bioassay: Excise the second leaf sheath from 8-10 day old etiolated rice seedlings, cut into 5 mm segments, and float on water in 24-well plates containing Brassinolide (0.001-10 nM in 0.01% ethanol). Incubate in dark at 30degC for 2-3 days. Measure the angle between the lamina and the sheath. For gene expression studies: Treat 7-10 day old Arabidopsis seedlings with 1-100 nM Brassinolide for 1-6 hours. Extract total RNA, reverse transcribe to cDNA, and perform qPCR for brassinosteroid-responsive marker genes (SAUR-AC1, TCH4, EXP8, CPD, DWF4). Brassinolide-d5 is not used as the test compound in these assays; it is used as internal standard for quantifying endogenous brassinosteroid levels or for tracer studies.

For brassinosteroid metabolism/tracer studies using Brassinolide-d5: Treat 2-4 week old Arabidopsis thaliana plants or 8-10 day old rice seedlings with Brassinolide-d5 (10-100 nM in 0.01% ethanol/Tween 20 solution) via foliar spray or root application. Alternatively, apply to leaf axils or directly to the apical meristem using a microsyringe (0.5-2 microL of 1-10 microM solution). Harvest plant tissues (leaf blades, sheaths, roots, inflorescences) at 0.5, 1, 2, 6, 12, 24, 48 hours. Homogenize in cold methanol:chloroform:water (4:2:1). Add additional Brassinolide-d5 (if needed) as internal standard. Purify extracts by solid-phase extraction (C18 cartridge) and/or immunoaffinity column. Analyze by LC-MS/MS using multiple reaction monitoring (MRM). Quantify parent Brassinolide-d5 and detect deuterated metabolites (e.g., 6-deoxobrassinolide, castasterone, 3-epibrassinolide) based on characteristic mass shifts.

No dedicated pharmacokinetic data for Brassinolide-d5 exist. Unlabeled Brassinolide in plants: Absorption through leaves and roots is rapid (minutes to hours). Translocation occurs through both xylem and phloem. In Arabidopsis, the half-life of Brassinolide is approximately 2-8 hours due to rapid metabolism via hydroxylation, glycosylation, sulfonation, and side chain cleavage. Major catabolic pathways include C-6 oxidation, C-23 hydroxylation, and C-2/C-3 epimerization. The deuterium label is stable and does not exchange with the environment. In animals (where Brassinolide is not a hormone but may have anticancer activity), no PK studies have been published.

No dedicated toxicity data for Brassinolide-d5 exist. Unlabeled Brassinolide has very low toxicity in animals. Acute oral LD50 in rats >2000 mg/kg. No significant toxicity in subchronic studies at doses up to 100 mg/kg/day. In humans, brassinosteroids have not been evaluated in clinical trials. Phytotoxicity can occur at high concentrations (≥1 microM) in some plant species, causing leaf epinasty, growth inhibition, and chlorosis. In animal cell lines (PC-3 human prostate cancer cells), Brassinolide induces apoptosis and cell cycle arrest at G2/M phase at concentrations ≥10 microM, but such concentrations are unlikely to be achieved systemically. Brassinolide-d5 is for research use only, not for human consumption.

[1]. Reversing effect of brassinolide on multidrug resistance of-CCRF-VCR1000 cells and a preliminary investigation on its mechanisms. Yao Xue Xue Bao. 2005 Feb;40(2):117-21.

[2]. Brassinolide, a plant sterol from pollen of Brassica napus L., induces apoptosis in human prostate cancer PC-3 cells. Pharmazie. 2007 May;62(5):392-5.

Brassinolide-d5 is a research-use only stable isotope-labeled compound, not approved for diagnostic, therapeutic, or agricultural use as a growth regulator. It has not been evaluated in clinical trials. Its primary application is as a tracer/internal standard for studying brassinosteroid biosynthesis, metabolism, signaling, and transport by LC-MS/MS in: (1) Metabolic flux analysis of brassinosteroid pathways; (2) Quantitation of endogenous brassinosteroid levels in plants (Arabidopsis, rice, tomato, etc.); (3) Identification and characterization of new brassinosteroid metabolites; (4) Enzyme assays for brassinosteroid biosynthetic enzymes (e.g., DWF4, CPD, ROT3); (5) Mechanism studies of brassinosteroid signaling (BRI1-BAK1 interaction). Brassinosteroids are the sixth class of plant hormones.

C28H43D5O6

485.71

Typically exists as solid at room temperature

Brassin lactone-d5

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)

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.)