Psoralidin inhibits NOTCH1 signaling, leading to downregulation of NOTCH1 and its downstream target HES1 [2].

It also inhibits NF-κB (p65) expression [2].

Psoralen treatment (10, 15, 20, and 25 μM; 24 hours) was sensitive to clear breast cancer cell (BCC) populations (ALDH- cells, ALDH+ cells, and commercial BSCS), with an IC50 ranging from 18 to 21 μM. On the other hand, MCF-12A cells treated with psoralidin (30 μM; 24 hours) were able to significantly induce ALDH- cells, ALDH+ cells, and commercial BCSC[2]. Psoralidin's effects on treating ALDH- and ALDH+ cells in AIDS [2]

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Psoralidin inhibits cell viability in ALDH⁻ breast cancer cells, ALDH⁺ breast cancer stem cells, and commercial breast cancer stem cells, with IC₅₀ values ranging from 18 to 21 μM after 24 h treatment. Normal mammary epithelial cells (MCF-12A) are resistant to psoralidin [2].

It significantly inhibits colony formation in both ALDH⁻ and ALDH⁺ cells when treated at their respective IC₅₀ concentrations [2].

It inhibits mammosphere formation in ALDH⁻ and ALDH⁺ cells, reducing both the number and size of mammospheres [2].

It induces apoptosis in ALDH⁻ cells, ALDH⁺ cells, and commercial breast cancer stem cells at 30 μM, with apoptotic rates of 53.60%, 44.1%, and 45.9%, respectively [2].

It downregulates NOTCH1 and HES1 protein expression in ALDH⁻ and ALDH⁺ cells [2].

It increases E-cadherin expression and decreases β-catenin and vimentin expression in ALDH⁻ and ALDH⁺ cells [2].

It inhibits migration in wound healing assays: ALDH⁻ cells show 29.4% migration, ALDH⁺ cells show 19.77% migration, and commercial breast cancer stem cells show 82% migration compared to vehicle-treated controls [2].

It inhibits invasion in transwell assays: ALDH⁻ cells show 0.65-fold, ALDH⁺ cells show 0.30-fold, and commercial breast cancer stem cells show 0.46-fold invasion compared to vehicle-treated controls [2].

It inhibits NF-κB (p65) and BCL-2 expression, and induces BAX expression in ALDH⁻ and ALDH⁺ cells [2].

It induces cleaved caspase-3, cleaved caspase-9, and cleaved PARP in ALDH⁻ and ALDH⁺ cells [2].

Psoralen (5 mg/kg) reduces inflammation in BALB/c infrared-irradiated lungs by modifying the expression of pro-inflammatory cytokines that are crucial in inflammation [1].

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Psoralidin (5 mg/kg, intraperitoneal) administered 30 min before and 1 h after thoracic irradiation (20 Gy) in BALB/c mice inhibits IR-induced mRNA expression of TNF-α, TGF-β, IL-6, IL-1α, IL-1β, and ICAM-1 in lung tissue at 12 h and 1 week post-irradiation [1].

In vivo pull-down assay confirms direct binding of psoralidin to FLAP in lung extracts of IR-irradiated mice [1].

Recombinant human FLAP was expressed in E. coli with a C-terminal 6His-tag and purified via Ni-NTA affinity chromatography, ion exchange, and gel filtration. The direct interaction between FLAP and psoralidin was measured using isothermal titration calorimetry (ITC) at 25°C. FLAP (0.01 mM) was titrated with psoralidin (0.1 mM) in Tris buffer (20 mM, pH 7.5) containing 150 mM NaCl. Each titration consisted of 29 injections of 10 μL with 300-second intervals. Data were fitted to a single-site binding model. The binding affinity (Kd) was determined to be 21.0 μM, with ΔH = -18.5 ± 1.5 kcal/mol, ΔS = -26.5 cal/mol/degree, and a 1:1 stoichiometry. Psoralidin did not bind to 5-LOX or COX-2 under the same ITC conditions [1].

For pull-down assays, psoralidin-sepharose 4B beads were prepared by coupling psoralidin to sepharose 4B. Cellular lysates (500 μg) from irradiated cells or purified FLAP (10 μg) were incubated with psoralidin-sepharose beads overnight at 4°C. After washing, bound proteins were analyzed by immunoblotting [1].

Cell viability assay [2]
Cell Types: ALDH- cells, ALDH+ cells, commercial breast cancer stem cells (BSCS) and normal mammary epithelial cells (MCF-12A)
Tested Concentrations: 10, 15 , 50 and 25. μM
Incubation Duration: 24 hrs (hours)
Experimental Results: IC50 for ALDH- cells, ALDH+ cells, commercial BCSC was 18 to 21 μM.

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Apoptosis analysis [2]
Cell Types: ALDH- cells, ALDH+ cells and commercial BCSC
Tested Concentrations: 20 and 30 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: No obvious apoptosis was observed in all three cell types after 20 μM treatment death induction. However, at 30 μMin, 53.60%, 44.1%, and 45.9% of ALDH- cells, ALDH+ cells, and commercial BCSCs were apoptotic, respectively.

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Cell viability was measured using trypan blue exclusion or MTT assay. Cells were treated with various concentrations of psoralidin (10, 15, 20, 25 μM) or DMSO for 24 h [2].

Apoptosis was quantified using Annexin V-FITC and propidium iodide staining. Cells were treated with psoralidin at IC₅₀ values or 30 μM for 24 h, then analyzed by flow cytometry [2].

Mammosphere formation assay: Single-cell suspensions of ALDH⁻ and ALDH⁺ cells were cultured at 4,000 cells per 2 mL in DMEM or MammoCult medium with supplements in ultra-low attachment plates. Cells were treated with psoralidin at IC₅₀ concentrations and cultured for 2–3 weeks, after which mammospheres were counted [2].

Soft agar colony formation assay: ALDH⁻ and ALDH⁺ cells (5 × 10³) were treated with psoralidin at IC₅₀ concentrations and cultured for 10 days. Colonies were stained with crystal violet and counted [2].

Invasion assay: Cells (1 × 10⁵) were seeded in Matrigel-coated transwell chambers with 8-μm pores and treated with psoralidin at IC₅₀ concentrations. After 24 h, invasive cells on the lower membrane were stained with crystal violet and counted [2].

Wound healing migration assay: Cells were grown to confluence, a linear wound was created, and the medium was replaced with fresh medium containing psoralidin at IC₅₀ concentrations. Wound closure was monitored every 2 h for up to 36 h using a Biostation CT, and the wound distance was measured [2].

Western blot analysis: Cells were treated with psoralidin at IC₅₀ concentrations for 12 or 24 h. Whole-cell lysates were prepared, and proteins were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against NOTCH1, HES1, E-cadherin, β-catenin, vimentin, p65, BCL-2, BAX, caspase-3, caspase-9, and PARP [2].

Immunofluorescence staining: Cells grown on coverslips were treated with psoralidin at IC₅₀ concentrations for 24 h, fixed, and incubated with antibodies against E-cadherin or β-catenin, followed by secondary antibodies. Nuclei were stained with DAPI, and images were captured by confocal microscopy [2].

siRNA transfection: ALDH⁻ and ALDH⁺ cells were transfected with NOTCH1 siRNA (20 nM) or control siRNA using transfection reagent. After 36 h, cell proliferation was measured by trypan blue exclusion, and protein expression was analyzed by western blot [2].

Animal/Disease Models: balb/c (Bagg ALBino) mouse[1]
Doses: 5 mg/kg
Route of Administration: intraperitoneal (ip) injection; 30 minutes before and 1 hour after IR irradiation (20 Gy).
Experimental Results: Anti-inflammatory effects on mice irradiated with infrared rays.

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Male BALB/c mice (8 weeks, 22–25 g) were divided into groups (n=5 per group). Mice were anesthetized with ketamine (80 mg/kg) and xylazine (16 mg/kg) intraperitoneally. The thorax was irradiated with 20 Gy using a Gamma Cell 40 Exactor (0.81 Gy/min). Psoralidin (5 mg/kg) was administered intraperitoneally in DMSO (500 μL) 30 min before and 1 h after irradiation. Control mice received sham irradiation. Lungs were collected at 12 h and 1 week post-irradiation. Lung lysates were prepared for RNA isolation and real-time RT-PCR [1].

For in vivo pull-down, lung lysates (500 μg) were incubated with psoralidin-sepharose 4B beads, and bound FLAP was detected by immunoblotting [1].

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Male BALB/c mice (8 weeks, 22–25 g) were divided into groups (n=5 per group). Mice were anesthetized with ketamine (80 mg/kg) and xylazine (16 mg/kg) intraperitoneally. The thorax was irradiated with 20 Gy using a Gamma Cell 40 Exactor (0.81 Gy/min). Psoralidin (5 mg/kg) was administered intraperitoneally in DMSO (500 μL) 30 min before and 1 h after irradiation. Control mice received sham irradiation. Lungs were collected at 12 h and 1 week post-irradiation. Lung lysates were prepared for RNA isolation and real-time RT-PCR [1].

For in vivo pull-down, lung lysates (500 μg) were incubated with psoralidin-sepharose 4B beads, and bound FLAP was detected by immunoblotting [1].

Psoralidin did not affect the viability of normal mammary epithelial cells (MCF-12A) at concentrations up to 25 μM [2].

[1]. Psoralidin, a dual inhibitor of COX-2 and 5-LOX, regulates ionizing radiation (IR)-induced pulmonary inflammation.Biochem Pharmacol. 2011 Sep 1;82(5):524-34.

[2]. Silencing NOTCH signaling causes growth arrest in both breast cancer stem cells and breast cancer cells.Br J Cancer. 2013 Nov 12;109(10):2587-96.

Psoralidin is a coumarin compound with a structure in which coumarin is substituted at positions 3 and 9 with hydroxyl groups, and at position 2 with an isopentenyl group. It is a plant metabolite and an estrogen receptor agonist. Psoralidin belongs to the coumarin, polyphenol, and δ-lactone classes, and its function is related to that of other coumarin compounds. It has been reported to be found in common bean (Phaseolus lunatus), three-lobed lentil (Dolichos trilobus), and other organisms with relevant data. See also: Cullen corylifolium fruit (partial).

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Psoralidin is a naturally occurring furanocoumarin derived from the seeds of the Asian medicinal plant Psoralea corylifolia. It exhibits a variety of biological activities, including antioxidant, antibacterial, antidepressant, anticoagulant, anti-inflammatory, antiallergic, ROS modulatory, and anticancer activities [2].

This study demonstrates that psoralidin targets both breast cancer cells and breast cancer stem cells by inhibiting NOTCH1 signaling, leading to growth arrest, inhibition of epithelial-mesenchymal transition (EMT), and induction of apoptosis. It downregulates pro-survival signaling (NF-κB, BCL-2) and activates pro-apoptotic pathways (BAX, caspase cascade). The compound also inhibits migration and invasion, which are associated with EMT [2].

C20H16O5

336.34

336.099

18642-23-4

5281806

White to off-white solid powder

1.4±0.1 g/cm3

458.8±34.0 °C at 760 mmHg

290-292°

231.3±25.7 °C

0.0±1.2 mmHg at 25°C

1.689

5.03

2

5

2

25

554

0

O1C2C([H])=C(C([H])=C([H])C=2C2C(=O)OC3C([H])=C(C(C([H])([H])/C(/[H])=C(\C([H])([H])[H])/C([H])([H])[H])=C([H])C=3C1=2)O[H])O[H]

YABIJLLNNFURIJ-UHFFFAOYSA-N

InChI=1S/C20H16O5/c1-10(2)3-4-11-7-14-17(9-15(11)22)25-20(23)18-13-6-5-12(21)8-16(13)24-19(14)18/h3,5-9,21-22H,4H2,1-2H3

3,9-dihydroxy-2-(3-methylbut-2-enyl)-[1]benzofuro[3,2-c]chromen-6-one

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 : ~50 mg/mL (~148.66 mM)

Solubility in Formulation 1: 2.08 mg/mL (6.18 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.18 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 20.8 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.)