Na⁺/K⁺-ATPase complexes: The document mentions that hellebrigenin, like other bufadienolides, possesses higher binding affinity and inhibition for α1β1 than for α2β1 and α3β1 Na⁺/K⁺-ATPase complexes. [1]
Akt: Hellebrigenin inhibits Akt expression and phosphorylation (at Thr308 and Ser473). This is not a direct binding target but a downstream effect. [1]

Cytotoxicity in HepG2 Cells: Hellebrigenin potently reduced the viability of HepG2 cells in a dose- and time-dependent manner. The IC50 values were 0.40 ± 0.05 μmol/L, 0.13 ± 0.01 μmol/L, and 0.10 ± 0.01 μmol/L after 24 h, 48 h, and 72 h of treatment, respectively. [1]
Colony Formation Inhibition: Hellebrigenin treatment (62.5, 125, and 400 nmol/L for 12 h) led to a dose-dependent decrease in the number of colonies formed by HepG2 cells over 10 days. [1]
Cell Cycle Analysis: Flow cytometry analysis with PI staining showed that hellebrigenin induced a concentration- and time-dependent accumulation of HepG2 cells in the G2/M phase. [1]
DNA Damage (Comet Assay): Treatment with hellebrigenin (62.5 and 125 nmol/L for 48 h) significantly damaged DNA, as evidenced by increased Tail Length, Tail DNA%, and Olive Tail Moment in the comet assay (P < 0.001). [1]
Immunofluorescence for DNA Damage: Confocal microscopy revealed an accumulation of p-H2AX (Ser¹³⁹), a sensitive marker for DNA double-stranded breaks, in hellebrigenin-treated cells. [1]
Apoptosis Detection (Hoechst 33258): After treatment with hellebrigenin (62.5, 125, and 250 nmol/L for 48 h), HepG2 cells exhibited morphological features of early apoptosis, indicated by bright blue fluorescence from nuclear condensation. [1]
Apoptosis Quantification (Annexin V-FITC/PI): Flow cytometry showed that the percentage of apoptotic cells increased significantly from 2.4% to 17.9% in a concentration-dependent manner and from 1.9% to 15.3% in a time-dependent manner after hellebrigenin treatment. [1]
Mitochondrial Membrane Potential (ΔΨm): JC-1 staining assay demonstrated that hellebrigenin treatment resulted in a marked collapse of ΔΨm, decreasing from 99.8% to 63.8% in a concentration-dependent manner and from 99.5% to 62.4% in a time-dependent manner. [1]
Western Blot Analysis: Hellebrigenin treatment modulated the expression of various proteins involved in cell cycle, DNA damage response, and apoptosis. This includes up-regulation of p-ATM (Ser¹⁹⁸¹), p-Chk2 (Tyr⁶⁸), p-CDK1 (Tyr¹⁵), Cyclin B1, Bax, cleaved caspase-9, cleaved caspase-3, and cleaved PARP. It also caused down-regulation of p-CDC25C (Ser²¹⁶), translocation of Bax to mitochondria, and release of cytochrome c from mitochondria to the cytosol. Furthermore, hellebrigenin markedly down-regulated total Akt and its phosphorylated forms, p-Akt (Thr³⁰⁸) and p-Akt (Ser⁴⁷³). [1]
Akt Silencing (siRNA) Experiment: Transfection with Akt siRNA (100 nmol/L) significantly blocked the G2/M cell cycle arrest but enhanced apoptosis induced by hellebrigenin, as shown by an increased subG1 population and PARP cleavage. [1]
Akt Activation with hIGF-I: Pretreatment with hIGF-I (100 ng/mL), an activator of Akt, remarkably attenuated the hellebrigenin-induced cytotoxicity. [1]

Cell Viability Assay (MTT): HepG2 cells (5000/well) were seeded in 96-well plates and exposed to various concentrations of hellebrigenin (8 nmol/L to 1000 nmol/L) for 24, 48, and 72 h. After treatment, MTT solution (5 mg/mL) was added for 4 h. The formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm. Cell viability was expressed as a percentage relative to the control (0.2% DMSO) group. [1]
Colony Formation Assay: HepG2 cells (300/well) were incubated with hellebrigenin (62.5, 125, and 400 nmol/L) for 12 h. The medium was then replaced with fresh medium, and cells were cultured for 10 days to allow colony formation. Colonies were fixed with methanol at -20°C for 30 min and stained with 1% crystal violet. [1]
Cell Cycle Analysis: HepG2 cells (300,000/well) were treated with hellebrigenin (62.5, 125, and 250 nmol/L for 48 h; or 125 nmol/L for 24, 36, and 48 h). Cells were fixed with 75% ethanol at 4°C overnight, then incubated with PI (0.2 mg/mL) in the dark at 37°C for 15 min. PI fluorescence was analyzed by flow cytometry, and cell cycle phase distribution was determined using software. [1]
Comet Assay: HepG2 cells (300,000/well) were treated with hellebrigenin (62.5 and 125 nmol/L) for 48 h. Cells were mixed with comet agarose and transferred to comet slides. Slides were incubated in lysis buffer at 4°C overnight, then subjected to alkaline electrophoresis (40 V) for 30 min. After fixation and air-drying, cells were stained with Vista green DNA dye. Comets were photographed, and parameters like tail length, tail DNA%, and olive tail moment were evaluated for at least 20 cells per sample using image analysis software. [1]
Immunofluorescence for p-H2AX: HepG2 cells treated with hellebrigenin (62.5 and 125 nmol/L for 48 h) were fixed with 4% formaldehyde and permeabilized with 0.1% Triton X-100. Cells were blocked and incubated with a primary antibody against p-H2AX (Ser¹³⁹), followed by an Alexa Fluor 647-conjugated secondary antibody and DAPI staining. Images were acquired using a laser scanning confocal microscope. [1]
Apoptosis Detection (Hoechst 33258): HepG2 cells (5000/well) were treated with hellebrigenin (62.5, 125, and 250 nmol/L) for 48 h. Cells were then incubated with Hoechst 33258 (10 μg/mL) in the dark for 30 min. After washing with PBS, cells were observed under a fluorescence microscope with excitation at 350 nm and emission at 460 nm. [1]
Apoptosis Quantification (Annexin V-FITC/PI): HepG2 cells treated with hellebrigenin were harvested and stained using an Annexin V-FITC/PI staining assay kit according to the manufacturer's protocol. The stained cells were then analyzed by flow cytometry to quantify the percentage of early and late apoptotic cells. [1]
Mitochondrial Membrane Potential Assay (JC-1): HepG2 cells (300,000/well) were treated with hellebrigenin (62.5, 125, and 250 nmol/L for 24 h). Cells were then stained with JC-1 (10 μmol/L) for 30 min in the dark. JC-1 fluorescence (red for aggregates, green for monomers) was detected using a flow cytometer. [1]
Western Blot Analysis: Cells (2,000,000/dish) were treated with hellebrigenin (62.5 and 125 nmol/L for 48 h; or 125 nmol/L for 24 and 48 h). Total protein lysates, as well as cytosolic and mitochondrial fractions, were prepared. Nuclear extracts were also prepared. Protein concentration was determined using a BCA protein assay kit. Equal amounts of protein (50 μg) were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked and immunoblotted with specific primary antibodies. Immunoreactive bands were visualized using enhanced chemiluminescent substrates. [1]
Akt siRNA Interference: HepG2 cells (2,000,000/dish) at ~60% confluency were transfected with siRNA duplexes against human Akt (100 nmol/L) using a lipid-based transfection reagent. After 36 h, cells were harvested to confirm Akt knockdown by Western blot. Transfected cells were then exposed to hellebrigenin for 48 h, followed by analysis of cell viability, cell cycle profile, and apoptosis induction. [1]

The document notes that bufadienolides, including hellebrigenin, are known to have cardiotonic effects and cardiotoxicity, which is mainly attributed to the inhibition of Na⁺/K⁺-ATPase and the subsequent increase in intracellular calcium levels. This is a general class effect, and no specific toxicity data for hellebrigenin was generated in this study. [1]

[1]. Hellebrigenin induces cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells through inhibition of Akt. Chem Biol Interact. 2014 Aug 5;219:184-94.

Hellebrigenin has been reported to have been found in toads (Bufo gargarizans), toads (Bufo bufo), and other organisms with available data.

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Background: Hellebrigenin is a bufadienolide, a type of cardioactive steroid, found in the skin secretions of toads and in plants of the Helleborus and Kalanchoe genera. It was isolated from the traditional Chinese medicine Venenum Bufonis. [1]
Known Anti-cancer Activity: Previous studies have shown hellebrigenin to be active against several cancer cell lines, including HL-60, HCT-8, A549, Hsa83, MCF-7, PC-3, KB, and HeLa. It has also been reported to overcome ABCB1- or ABCC1-mediated multidrug resistance and apoptosis resistance in cancer cells. [1]
Mechanism of Action: This study is the first to report that hellebrigenin induces DNA double-stranded breaks, leading to activation of the ATM-Chk2 pathway, which in turn causes G2/M cell cycle arrest. It also triggers the mitochondrial apoptotic pathway, characterized by Bax translocation, loss of mitochondrial membrane potential, cytochrome c release, and caspase activation. The study further demonstrates that inhibition of Akt is involved in the regulation of this hellebrigenin-induced cell cycle arrest and apoptosis. [1]
Therapeutic Potential: The findings support the potential of hellebrigenin as a chemotherapeutic agent for the treatment of liver cancer. However, the authors note that attention should be paid to its potential cardiotoxicity, and strategies like structural modification or controlled-release formulations may be needed to improve its safety and efficacy. [1]

C24H32O6

416.5073

416.22

465-90-7

259577

White to off-white solid powder

1.419g/cm3

626.3ºC at 760 mmHg

175 °C

215ºC

1.675

2.535

3

6

2

30

834

8

C[C@]12CC[C@H]3[C@H]([C@]1(CC[C@@H]2C4=COC(=O)C=C4)O)CC[C@]5([C@@]3(CC[C@@H](C5)O)C=O)O

TVKPTWJPKVSGJB-XHCIOXAKSA-N

InChI=1S/C24H32O6/c1-21-8-5-18-19(6-10-23(28)12-16(26)4-9-22(18,23)14-25)24(21,29)11-7-17(21)15-2-3-20(27)30-13-15/h2-3,13-14,16-19,26,28-29H,4-12H2,1H3/t16-,17+,18-,19+,21+,22-,23-,24-/m0/s1

(3S,5S,8R,9S,10S,13R,14S,17R)-3,5,14-trihydroxy-13-methyl-17-(6-oxopyran-3-yl)-2,3,4,6,7,8,9,11,12,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthrene-10-carbaldehyde

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 and light.

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.)