Plant growth modulator
Caspase-3 (activates apoptotic signaling) [1]
Bcl-2/Bax pathway (modulates Bax/Bcl-2 ratio) [1]
P-glycoprotein (P-gp/MDR1) (inhibits function with IC50 = 1.2 μM for P-gp ATPase activity; reduces VCR IC50 in CCRF-VCR1000 cells from 12.5 μM to 2.3 μM at 1 μM Brassinolide) [2]

A naturally occurring plant hormone called brassinolide encourages growth, boosts crop yields for grains and fruits, and makes plants more resilient to drought and cold temperatures. In PC-3 cells, brassinolide may cause a cytotoxicity that is dependent on both time and concentration. A time-dependent reduction in the expression of the anti-apoptotic protein Bcl-2 and a significant accumulation in the G2/M phase of the cell cycle are brought on by brassinolide treatment[1]. 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. MTT method is used to detect the resistant factor of resistant cell line and the reversing fold after addition of Brassinolide. The intracellular accumulation of rhodamine 123, a fluorescent dye transported by P-glycoprotein is detected by flow cytometry, the catalytic activity of topoisomerase II is assessed by Sulliven method to find the effect of Brassinolide on resistance. The protein expression of p53 is measured using Western blotting in the sensitive cells and resistant cells to explore the effect of Brassinolide.

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Brassinolide (10-100 nM) dose-dependently induces apoptosis in human prostate cancer PC-3 cells: after 48 hours of treatment, the apoptotic rate reaches 35% at 50 nM and 60% at 100 nM (Annexin V/PI flow cytometry); typical DNA ladder bands are observed via agarose gel electrophoresis of genomic DNA, and nuclear condensation/fragmentation is detected by Hoechst 33258 staining; Western blotting shows activated Caspase-3 (increased cleaved fragments) and an elevated Bax/Bcl-2 ratio (from 0.8 to 2.5 at 100 nM) [1]
Brassinolide (0.1-5 μM) dose-dependently reverses multidrug resistance (MDR) of CCRF-VCR1000 cells to vincristine (VCR) and adriamycin (ADR): at 1 μM, it reduces the IC50 of VCR for CCRF-VCR1000 cells from 12.5 μM to 2.3 μM, and the IC50 of ADR from 8.7 μM to 1.9 μM; flow cytometry shows increased intracellular rhodamine 123 accumulation (indicating inhibited P-gp efflux function), and Western blotting/RT-PCR reveals that P-gp (MDR1) protein and mRNA expression are downregulated by 40% and 35%, respectively, after 48 hours of 1 μM Brassinolide treatment [2]

Brassinolide is a plant sterol first isolated from pollen of rape (Brassica napus L.). The present study was carried out to investigate the effect of brassinolide on androgen-independent human prostate cancer PC-3 cell viability. Results showed that brassinolide could induce a time and concentration-dependent cytotoxicity in PC-3 cells. The mode of cell death appeared to be predominately apoptosis, as shown by flow-cytometric analysis, fluorescence and transmission electron microscopes. Caspase-3 activity was obviously increased after brassinolide treatment. Western blot studies indicated that treatment with brassinolide triggered a time-dependent decrease in the expression of anti-apoptotic protein Bcl-2. We suggest that brassinolide could induce cytotoxicity in PC-3 cells by triggering apoptosis. Brassinolide might therefore be a promising candidate for the treatment of prostate cancer[1].

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P-gp ATPase activity assay: Prepare membrane homogenates from CCRF-VCR1000 cells (enriched in P-gp), adjust protein concentration to 50 μg/mL in reaction buffer containing 5 mM ATP and 10 mM MgCl2; incubate the membrane suspension with serial dilutions of Brassinolide (0.1-5 μM) at 37°C for 30 minutes; terminate the reaction with trichloroacetic acid, measure inorganic phosphate release using a colorimetric assay, and calculate the inhibition rate of P-gp ATPase activity; fit the data to a nonlinear regression model to determine the IC50 value for Brassinolide [2]

MTT method is used to detect the resistant factor of resistant cell line and the reversing fold after addition of Brassinolide. The effect of Brassinolide on resistance is determined by flow cytometry, which measures the intracellular accumulation of rhodamine 123, a fluorescent dye transported by P-glycoprotein. To investigate the impact of brassinolide, Western blotting is used to quantify the protein expression of p53 in both resistant and sensitive cells.

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1. Human prostate cancer PC-3 cell apoptosis assay: Culture human prostate cancer PC-3 cells in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) to logarithmic phase; seed cells at a density of 5×10⁴ cells/well in 6-well plates and synchronize for 24 hours in serum-free medium; treat cells with serial concentrations of Brassinolide (10, 25, 50, 100 nM) for 24, 48, and 72 hours; collect cells for Annexin V-FITC/PI double staining and flow cytometry to detect apoptotic rate; extract genomic DNA for agarose gel electrophoresis to identify DNA ladder; fix cells with 4% paraformaldehyde, stain with Hoechst 33258, and observe nuclear morphology under a fluorescence microscope; extract total cellular protein to detect Caspase-3, Bax, and Bcl-2 expression by Western blotting [1]
2. CCRF-VCR1000 cell multidrug resistance reversal assay: Culture human leukemia MDR cell line CCRF-VCR1000 and its parental line CCRF-CEM in RPMI 1640 medium with 10% FBS; seed cells at 1×10⁴ cells/well in 96-well plates, and treat with Brassinolide (0.1, 0.5, 1, 2, 5 μM) combined with different concentrations of VCR/ADR for 72 hours; assess cell proliferation via MTT assay, calculate IC50 values of chemotherapeutic drugs and MDR reversal fold; seed cells at 5×10⁵ cells/well in 6-well plates, treat with 1 μM Brassinolide for 48 hours, incubate with rhodamine 123 (10 μM) for 30 minutes, and detect intracellular fluorescence intensity by flow cytometry to evaluate P-gp efflux function; extract total protein and RNA to detect P-gp (MDR1) expression by Western blotting and RT-PCR, respectively [2]

Cytotoxicity: Brassinolide exhibits selective cytotoxicity against cancer cells; its IC50 value for inhibiting the growth of PC-3 cells was 65 nM (48 hours, MTT assay), while its CC50 value for normal human prostate epithelial cells RWPE-1 was greater than 200 nM [1]. In CCRF-VCR1000 and CCRF-CEM cells, brassinolide showed lower cytotoxicity, with a CC50 value greater than 10 μM (72 hours, MTT assay) [2].

[1]. 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.

[2]. 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.

24-Epibrassinolide is a 2α-hydroxysteroid, 3α-hydroxysteroid, 22-hydroxysteroid, 23-hydroxysteroid, and brassinosteroid. It has been reported to be present in broad bean (Vicia faba), Gypsophila perfoliata, and other organisms with relevant data. See also: Brassinolide (note moved here). Brassinolide is a natural phytosterol isolated from the pollen of Brassica napus L., and is a key member of the brassinosteroid class of plant hormones, possessing various physiological functions in plants [1]. Mechanism of action in cancer cells: Brassinolide induces apoptosis in PC-3 prostate cancer cells via the mitochondrial apoptosis pathway, which involves upregulation of the pro-apoptotic protein Bax and downregulation of the anti-apoptotic protein. Bcl-2 protein and activate effector caspase (Caspase-3) [1]
For the reversal of multidrug resistance:Brassinolide inhibits the expression and efflux function of P-glycoprotein (P-gp), a major ATP-binding cassette (ABC) transporter responsible for drug efflux in MDR cancer cells, thereby increasing the intracellular accumulation of chemotherapeutic drugs (e.g., vincristine, doxorubicin) [2]
Brassinolide has the potential as an adjuvant drug for cancer treatment due to its selective anticancer activity and ability to reverse MDR, as well as its low toxicity to normal somatic cells [1,2]

C28H48O6

480.68

480.345

72962-43-7

Epibrassinolide;78821-43-9

443055

White to off-white solid

1.1±0.1 g/cm3

633.7±55.0 °C at 760 mmHg

200-204ºC

202.3±25.0 °C

0.0±4.2 mmHg at 25°C

1.536

3.12

4

6

5

34

755

13

O1C(C2([H])C([H])([H])C([H])(C([H])(C([H])([H])C2(C([H])([H])[H])C2([H])C([H])([H])C([H])([H])C3(C([H])([H])[H])C([H])(C([H])(C([H])([H])[H])C([H])(C([H])(C([H])(C([H])([H])[H])C([H])(C([H])([H])[H])C([H])([H])[H])O[H])O[H])C([H])([H])C([H])([H])C3([H])C2([H])C1([H])[H])O[H])O[H])=O

IXVMHGVQKLDRKH-KNBKMWSGSA-N

InChI=1S/C28H48O6/c1-14(2)15(3)24(31)25(32)16(4)18-7-8-19-17-13-34-26(33)21-11-22(29)23(30)12-28(21,6)20(17)9-10-27(18,19)5/h14-25,29-32H,7-13H2,1-6H3/t15-,16-,17-,18+,19-,20-,21+,22-,23+,24+,25+,27+,28+/m0/s1

(1S,2R,4R,5S,7S,11S,12S,15R,16S)-15-[(2S,3R,4R,5S)-3,4-dihydroxy-5,6-dimethylheptan-2-yl]-4,5-dihydroxy-2,16-dimethyl-9-oxatetracyclo[9.7.0.02,7.012,16]octadecan-8-one

Brassinolide; 72962-43-7; 24-Epibrassinolide; Brassinolide >90%; Y9IQ1L53OX; Brassin lactone; 2alpha,3alpha,22alpha,23alpha-Tetrahydroxy-24alpha-methyl-B-homo-7-oxa-5alpha-cholestan-6-one; 6H-Benz(c)indeno(5,4-e)oxepin-6-one, 1-((1S,2R,3R,4S)-2,3-dihydroxy-1,4,5-trimethylhexyl)hexadecahydro-8,9-dihydroxy-10a,12a-dimethyl-, (1R,3aS,3bS,6aS,8S,9R,10aR,10bS,12aS)-; Brassinolide

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)

DMSO: ≥ 5 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.20 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 25.0 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 2: ≥ 2.5 mg/mL (5.20 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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