NF-κB p65 (binding energy: -6.67 kcal/mol, Ki: 12.86 μM; 3 hydrogen bonds) [1]
NF-κB p50 (binding energy: -6.23 kcal/mol, Ki: 27.05 μM) [1]
IκBα (inhibitor of kappa-B alpha) (binding energy: -5.19 kcal/mol, Ki: 156.00 μM) [1]
TAK-1 (transforming growth factor-β-activating kinase-1) (binding energy: -6.25 kcal/mol, Ki: 26.22 μM) [1]
IKKα (IκB kinase alpha) (binding energy: -6.15 kcal/mol, Ki: 30.89 μM) [1]

Through the Nrf2-p62-keap1 feedback loop, isodeoxyelephant causes protective autophagy in lung cancer cells.

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Isodeoxylephantopin suppressed the proliferation of both invasive (MDA-MB-231) and non-invasive (MCF-7, T47D, MDA-MB-468, MDA-MB-453) breast cancer cell lines in a dose- and time-dependent manner. For MDA-MB-231 cells, 10 μM Isodeoxylephantopin for 72 hrs suppressed cell proliferation by 50%; 25 μM for 12 hrs suppressed proliferation by 27% (compared to 10% for DET). In multiple cell lines (T47D, MCF-7, MDA-MB-468, MDA-MB-453), treatment with 1-100 μM Isodeoxylephantopin for 72 hrs decreased cell viability in a dose-dependent manner. Treatment with 25 μM for 24-72 hrs suppressed viability time-dependently. [1]
Isodeoxylephantopin suppressed colony formation of breast cancer cells. MCF-7 and MDA-MB-231 cells exposed to 1-25 μM Isodeoxylephantopin for 24 hrs showed a drastic decrease in colony formation after 7 days, even at 2.5 μM. [1]
Isodeoxylephantopin sensitized breast cancer cells to doxorubicin. Pretreatment of MCF-7 cells with 1 μM and 10 μM Isodeoxylephantopin before 0.25 μM doxorubicin reduced viability by 17% and 58%, respectively (compared to 7% reduction with doxorubicin alone). [1]
Isodeoxylephantopin induced apoptosis as shown by AO/PI dual staining (increased dead cells with red fluorescence, membrane blebbing and nuclear condensation), cell cycle analysis (3.5-fold increase in sub-G1 population at 25 μM; 2-fold increase in G2/M phase), annexin V/PI staining (13.1% annexin V positive cells at 10 μM vs 1.7% in control), and DNA laddering (concentration-dependent). [1]
Isodeoxylephantopin suppressed expression of anti-apoptotic proteins Bcl-xL and Bcl-2, invasive protein MMP-9, and induced cleavage of caspase 7, caspase 9 and PARP. It also suppressed mRNA transcripts of survivin and cyclin D1, and induced mRNA expression of proapoptotic Bax. [1]
Isodeoxylephantopin disrupted mitochondrial membrane potential. JC-1 staining showed reduction in red fluorescence (intact mitochondria) and increase in green fluorescence (depolarized mitochondria) after treatment. [1]
Isodeoxylephantopin induced ROS generation in MDA-MB-231 cells: 10 μM for 1 hr produced 2.8-fold increase in ROS (H2DCFDA staining). NAC pre-treatment almost completely suppressed ROS generation and also suppressed PS externalization induced by Isodeoxylephantopin. [1]
Isodeoxylephantopin reduced migration of MDA-MB-231 cells in wound healing assay. After 48 hrs, wound area was 14% in control, 28% at 1 μM, and 40% at 2.5 μM; healed area was 85% in control, 71% at 1 μM, and 59% at 2.5 μM. [1]
Isodeoxylephantopin inhibited NF-κB activation. Pretreatment with 10 μM Isodeoxylephantopin for 6 hrs suppressed okadaic acid (100 nM, 4 hrs)-induced p65 nuclear translocation as shown by immunocytochemistry. [1]
Isodeoxylephantopin modulated lncRNA expression in MDA-MB-231 cells: up-regulated GAS5 (1.3, 2.3, 3.2-fold at 1, 2.5, 5 μM respectively), NKILA (4.7, 5.1, 7.5-fold), ANRIL, lincRNA-Tnfaip3 and HOTAIR in a concentration-dependent manner; conversely, down-regulated oncogenic H19 expression. [1]

Molecular docking analysis was performed to examine the interaction of Isodeoxylephantopin and DET with NF-κB associated proteins. SMILE IDs of the compounds were obtained from PubChem database and converted to .pdb files using CORINA 3D server. The 3D structures of p65 (PDB ID 1NFI, chain A), p50 (chain B), IκBα (chain E), TAK-1 (PDB ID 2EVA) and IKKα (PDB ID 5TQY) were procured. AutoDock Tool 4 was used for the identification of binding affinities and poses of ligands and proteins. Binding energies (kcal/mol) and Ki (dissociation constant, μM) values were calculated. For Isodeoxylephantopin with p65: binding energy -6.67 kcal/mol, Ki 12.86 μM, with 3 hydrogen bonds (residues Cys120, Phe298, Asn229). For p50: -6.23 kcal/mol, Ki 27.05 μM. For IκBα: -5.19 kcal/mol, Ki 156.00 μM. For TAK-1: -6.25 kcal/mol, Ki 26.22 μM. For IKKα: -6.15 kcal/mol, Ki 30.89 μM. [1]

Cell viability was assessed by MTT assay measuring mitochondrial reductase activity. Cells (5000 per well in 96-well plates) were treated with different concentrations of Isodeoxylephantopin (1-100 μM) for 12-72 hrs, then purple formazan formation was measured. [1]
Clonogenic assay: Cells were treated with Isodeoxylephantopin (1-25 μM) for 24 hrs, then the agent was washed off and cells were allowed to form colonies for 7 days. Colonies were stained with crystal violet (0.25%) and counted manually. [1]
Live/dead cell discrimination assay using acridine orange (AO) and propidium iodide (PI) dual staining. AO permeates live and dead cells (green fluorescence); PI enters only dead cells with compromised membrane (red fluorescence). Cells were treated with 5-25 μM Isodeoxylephantopin for 24 hrs, washed, stained with AO/PI, and examined under fluorescence microscope. [1]
Phosphatidylserine externalization assay: Annexin V/PI staining was used. Cells were exposed to 10 μM Isodeoxylephantopin for 24 hrs, then stained with Alexa Fluor 488 conjugated annexin V antibody and analyzed by flow cytometry. [1]
DNA laddering assay: Cells were treated with Isodeoxylephantopin, then lysed in buffer containing EDTA, Tris (pH 8.0), RNase A, and SDS at 37°C for 30 min. Lysate was deproteinized with proteinase K at 55°C for 2 hrs. DNA was precipitated with chloroform and isopropanol, washed with 70% ethanol, air-dried, dissolved in Tris-EDTA buffer, and electrophoresed on 1.5% agarose gel containing ethidium bromide. DNA bands were visualized using gel documentation system. [1]
Cell cycle analysis: After treatment with various concentrations of Isodeoxylephantopin, cells were washed with PBS, fixed with 70% chilled methanol, treated with RNase A, stained with PI, and analyzed by flow cytometry using Cell Quest software. [1]
Mitochondrial membrane potential (Δψ) measurement: Cells were exposed to 10-50 μM Isodeoxylephantopin, washed, incubated in the dark with JC-1 (10 μg/mL) at 37°C for 20 minutes, washed again, and imaged under fluorescence microscopy. Green fluorescence indicates depolarized mitochondria; red fluorescence indicates intact mitochondria. [1]
Western blot analysis: Whole cell lysate from untreated and Isodeoxylephantopin-treated cells was separated by SDS-PAGE, transferred onto nitrocellulose membrane, probed with primary antibodies (for Bcl-xL, Bcl-2, p65, MMP-9, PARP, cleaved caspase 7, cleaved caspase 9) and secondary antibodies, and detected using ECL reagent. [1]
Immunocytochemistry for NF-κB p65 cellular localization: Cells were fixed with paraformaldehyde, permeabilized with PBST, probed with primary and secondary antibodies, counterstained with DAPI, and imaged under fluorescence microscope. Cells were treated with 10 μM Isodeoxylephantopin for 6 hrs, then cultured with 100 nM okadaic acid for 4 hrs. [1]
Cell migration assay (scratch/wound healing): At 70% confluency, monolayer cells were wounded with a sterile culture tip, debris washed off, then Isodeoxylephantopin applied. Wounded area was examined at 0, 9, 24 and 48 hrs by phase contrast microscope. Healed area and wound size were calculated using ImageJ software. [1]
ROS generation estimation: Control and treated cells were stained with 10 μM H2DCFDA for 1 hr in the dark, then analyzed by flow cytometry using Cell Quest software. [1]
Semi-quantitative and quantitative RT-PCR: Total RNA was isolated using trizol reagent, reverse transcribed using high capacity cDNA synthesis kit. Semi-quantitative RT-PCR was performed to examine mRNA transcripts of cyclin D1, survivin, and Bax, with PCR products electrophoresed on 1.5% agarose gel, quantified by densitometry using ImageJ, and normalized to GAPDH. Quantitative real-time PCR was performed using SYBR Green/ROX qPCR Master Mix and real-time system to examine lncRNA expression (GAS5, NKILA, ANRIL, lincRNA-Tnfaip3, HOTAIR, H19), normalized to ACTB and 5SrRNA. [1]

In silico ADMET analysis predicted the following properties for Isodeoxylephantopin: lipophilicity (log P) 2.25, topological polar surface area 78.92, molecular weight 344.36, hydrogen bond acceptors 6, hydrogen bond donors 0, rotatable bonds 3. Isodeoxylephantopin was predicted to be permeable to blood-brain barrier and intestine, with no evidence of carcinogenic or genotoxic effects. [1]

In silico ADMET analysis showed no evidence of carcinogenic and genotoxic effects for Isodeoxylephantopin. [1]

[1]. Isodeoxyelephantopin, a Sesquiterpene Lactone Induces ROS Generation, Suppresses NF-κB Activation, Modulates LncRNA Expression and Exhibit Activities Against Breast Cancer. Sci Rep. 2019 Nov 29;9(1):17980.

[2]. Treatment of Hominis placenta pharmacopuncture for a patient with mild neurocognitive disorder: Case report. J Pharmacopuncture. 2019 Dec; 22(4): 279–283.

Deoxyelephantopin is a sesquiterpene compound. It has been reported to have been found in rough elephant skin grass (Elephantopus scaber), and relevant data are available for reference.

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Isodeoxylephantopin (IDET) and deoxylephantopin (DET) are major sesquiterpene lactone constituents from Elephantopus scaber Linn. Both contain an α-methylene-γ-lactone ring which may contribute to cytotoxic activities. The γ-lactone ring oxygen atom at C-2 is in β-orientation in DET and α-orientation in IDET. The presence of C11-C13 exocyclic methylene in conjugation with γ-lactone contributes to cytotoxicity. Isodeoxylephantopin was more effective in reducing breast cancer cell proliferation compared to DET. [1]
Isodeoxylephantopin obeys Lipinski's rule of five (molecular weight ≤500, logP ≤5, H-bond donors ≤5, H-bond acceptors ≤10). [1]
Isodeoxylephantopin modulates multiple cell signaling molecules including ROS generation, NF-κB activation, lncRNA expression (up-regulates tumor suppressors GAS5, NKILA, and down-regulates oncogenic H19), and induces apoptosis through mitochondrial pathway and caspase activation. The compound suppresses NF-κB-regulated tumorigenic proteins and induces proapoptotic Bax. [1]
A previous study (not this one) demonstrated that Isodeoxylephantopin exhibits anti-inflammatory activities only in cancer cells but not in normal lymphocytes. [1]

C19H20O6

344.3585

344.125

38927-54-7

6325056

White to off-white solid

1.3±0.1 g/cm3

584.3±50.0 °C at 760 mmHg

150-153℃

258.1±30.2 °C

0.0±1.6 mmHg at 25°C

1.556

1.51

0

6

3

25

741

4

O1C(C(=C([H])[H])[C@@]2([H])[C@@]1([H])C([H])=C(C([H])([H])[H])C([H])([H])[C@]1([H])C([H])=C(C(=O)O1)C([H])([H])[C@]2([H])OC(C(=C([H])[H])C([H])([H])[H])=O)=O |t:12|

JMUOPRSXUVOHFE-MCYOVBASSA-N

InChI=1S/C19H20O6/c1-9(2)17(20)24-15-8-12-7-13(23-19(12)22)5-10(3)6-14-16(15)11(4)18(21)25-14/h6-7,13-16H,1,4-5,8H2,2-3H3/b10-6+/t13-,14+,15-,16-/m0/s1

[(3S,4R,8R,9E,12S)-10-methyl-5-methylidene-6,14-dioxo-7,13-dioxatricyclo[10.2.1.04,8]pentadeca-1(15),9-dien-3-yl] 2-methylprop-2-enoate

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)

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.)