NF-κB
Nuclear factor kappa B (NF-κB) signaling pathway, specifically by inhibiting PMA-induced phosphorylation of IKKα/β, IκBα, and nuclear translocation of NF-κB p65. [1]
Parathyroid hormone-related protein (PTHrP). [2]

In NCI-H292 human airway epithelial cells, Obtusifolin (10⁻⁶, 10⁻⁵, 10⁻⁴ M) significantly inhibited EGF-, PMA-, and TNF-α-induced MUC5AC mucin production. The amounts of mucin were 340±8% (EGF alone) vs. 286±43%, 193±19%, and 102±9% for EGF + Obtusifolin (1, 10, 100 µM respectively); 197±5% (PMA alone) vs. 191±3%, 170±1%, and 52±2% for PMA + Obtusifolin; and 252±12% (TNF-α alone) vs. 187±1%, 151±2%, and 124±11% for TNF-α + Obtusifolin. [1]
In NCI-H292 cells, Obtusifolin (1, 5, 10, 20, 50 µM) inhibited PMA-induced nuclear translocation of NF-κB p65 in a dose-dependent manner. [1]
In NCI-H292 cells, Obtusifolin inhibited PMA-induced phosphorylation of IκBα, degradation of IκBα, and phosphorylation of IKKα/β (Ser176/180). [1]
In NCI-H292 cells, Obtusifolin appeared to affect the expression of the adaptor protein RIP1 in the TNFR1 signaling pathway. [1]
In MDA-MB-231 human breast adenocarcinoma cells, Obtusifolin treatment (concentration not specified for this single treatment) decreased PTHrP and IL-8 secretion that was induced by dibutyl phthalate (DBP). Obtusifolin completely reversed the secretion of PTHrP and IL-8 upon exposure to DBP. [2]
In MDA-MB-231 cells, the conditioned medium from DBP-treated cells (DBP-MDA-MB-231-CM) mediated RANKL upregulation in osteoblasts; this effect was totally abrogated by Obtusifolin treatment. [2]
In osteoclastogenesis assays using CD14+ monocytes, Obtusifolin treatment (concentration not specified) totally abrogated the osteoclast differentiation and bone resorption activity enhanced by DBP-MDA-MB-231-CM. [2]

In NCI-H292 cells, Obtusifolin (1, 10, 100 μM) inhibits the expression of the MUC5AC gene that is induced by EGF, PMA, or TNFα[1].

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PTHrP levels in conditioned media of MDA-MB-231 cells and IL-8 levels were determined and quantified using ELISA kits. For PTHrP, an ELISA kit was used. For IL-8, a DuoSet ELISA kit was used. [2]
M-CSF, RANKL, and OPG levels in the supernatants of osteoblast cultures were assessed and quantified using DuoSet ELISA kits. [2]

For MUC5AC production, NCI-H292 cells (a human pulmonary mucoepidermoid carcinoma cell line) were cultured in RPMI 1640 with 10% fetal bovine serum. After 24 hr of serum deprivation (0.2% fetal bovine serum), cells were pretreated with varying concentrations (1, 10, 100 µM) of Obtusifolin for 30 min and then stimulated with EGF (25 ng/ml), PMA (10 ng/ml), or TNF-α (0.2 nM) for 24 hr in serum-free RPMI 1640. Cell lysates were collected to measure MUC5AC protein production by enzyme-linked immunosorbent assay (ELISA) using the 45M1 mouse monoclonal MUC5AC antibody. [1]
For MUC5AC gene expression, NCI-H292 cells were pretreated with Obtusifolin for 30 min and then stimulated with EGF, PMA, or TNF-α for 24 hr. Total RNA was isolated using an extraction kit, and reverse transcription-polymerase chain reaction (RT-PCR) was performed. Primers for MUC5AC (forward: 5'-TGA TCA TCC AGC AGG GCT-3' and reverse: 5'-CCG AGC TCA GAG GAC ATA TGG G-3') and for the housekeeping gene Rig/S15 were used. [1]
For Western blot analysis of NF-κB pathway, NCI-H292 cells were incubated with 1, 5, 10, 20, and 50 µM of Obtusifolin for 24 hr and then stimulated with PMA (50 ng/ml) for 30 min. Nuclear and cytosolic extracts were prepared using a nuclear and cytoplasmic extraction reagent. Whole-cell lysates were prepared using RIPA buffer. Proteins were subjected to SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against NF-κB p65, IκBα, phospho-IκBα (Ser 32/36), IKKα/β, phospho-IKKα/β (Ser176/180), RIP1, TRAF2, TRADD, p84, and β-actin. [1]
For the breast cancer bone metastasis study, MDA-MB-231 human breast adenocarcinoma cells were cultured in α-MEM with 10% FBS. To obtain conditioned media (CM), MDA-MB-231 cells (2 x 10⁶/100mm dish) were treated with various concentrations of DBP for 6 h, then the medium was replaced, and after 24 h of incubation, the supernatant was harvested and filtered. For testing Obtusifolin, MDA-MB-231 cells were pretreated with the compound for 1 h, then incubated with DBP (1 µM) for 6 h (for PTHrP) or 24 h (for IL-8). PTHrP and IL-8 levels in the media were assessed by ELISA. [2]
For osteoblast experiments, human primary osteoblasts were cultured in Osteoblast Growth Media. The CMs from control or DBP-treated MDA-MB-231 cells (control-CM and DBP-CM) were added to osteoblasts for 24 h. The levels of M-CSF, RANKL, and OPG in the supernatants of osteoblasts were assessed by ELISA. [2]
For osteoclastogenesis, CD14+ monocytes were purified from peripheral blood mononuclear cells using magnetic beads. Osteoclasts were generated by culturing CD14+ monocytes in culture medium containing 20% control-CM or DBP-MDA-MB-231-CM supplemented with 100 ng/mL M-CSF and 50 ng/mL RANKL for 14-21 days. Osteoclast formation was measured by TRAP staining (multinucleated cells with >3 nuclei), and bone resorption activity was assessed using a bone resorption assay plate. [2]
For PTHrP knockdown, MDA-MB-231 cells were transfected with 20 nM non-target or PTHrP siRNA pool using transfection reagents. After 24 h, cells were treated with DBP, and PTHrP changes were measured by qRT-PCR. [2]

[1]. Obtusifolin isolated from the seeds of Cassia obtusifolia regulates the gene expression and production of MUC5AC mucin in airway epithelial cells via affecting NF-κB pathway. Phytother Res. 2019 Apr;33(4):919-928.

[2]. Obtusifolin suppresses phthalate esters-induced breast cancer bone metastasis by targeting parathyroid hormone-related protein. J Agric Food Chem. 2014 Dec 10;62(49):11933-40.

Obtusifolin is a dihydroxyanthraquinone. Obtusifolin has been reported in senna leaves and senna leaf extract, and available data are available.

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Obtusifolin is a major bioactive compound present in the seeds of Cassia tora L. (also isolated from Cassia obtusifolia). It is an anthraquinone derivative with a reported purity of 98% in the isolation study. Its chemical structure was characterized by spectroscopic methods including ¹H-NMR, ¹³C-NMR, HMQC, and HMBC. [1][2]
In the context of airway inflammation, the inhibitory action of Obtusifolin on MUC5AC mucin production and gene expression is suggested to be mediated, in part, by affecting PMA-induced degradation of IκBα and nuclear translocation of NF-κB p65. The results suggest a possibility of using Obtusifolin as a new efficacious mucoregulator for pulmonary diseases. [1]
In the context of cancer, this study is the first to reveal that Obtusifolin reverses phthalate esters-mediated bone resorption. Obtusifolin suppressed phthalate esters-mediated bone resorption and may be a novel anti-breast-cancer bone metastasis agent. It was shown to completely reverse the secretion of PTHrP and IL-8 in breast cancer cells upon exposure to DBP. [2]

C16H12O5

284.2635

284.068

C, 67.60; H, 4.26; O, 28.14

477-85-0

477-85-0

3083575

Yellow to orange solid powder

1.448±0.06 g/cm3

528.0±50.0 °C at 760 mmHg

242-243ºC

202.2±23.6 °C

0.0±1.4 mmHg at 25°C

1.678

4.18

2

5

1

21

448

0

O(C([H])([H])[H])C1=C(C(C([H])([H])[H])=C([H])C2C(C3C([H])=C([H])C([H])=C(C=3C(C=21)=O)O[H])=O)O[H]

NYRXUBDGDSRBGB-UHFFFAOYSA-N

InChI=1S/C16H12O5/c1-7-6-9-12(16(21-2)13(7)18)15(20)11-8(14(9)19)4-3-5-10(11)17/h3-6,17-18H,1-2H3

2,8-dihydroxy-1-methoxy-3-methylanthracene-9,10-dione

Obtusifolin

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: 10~25 mg/mL (35.2~88 mM)

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.)