Epidermal Growth Factor Receptor (EGFR) tyrosine kinase [1]
IC50 (EGFR inhibitory activity) = 0.15 μM [1]

Khelline inhibits the proliferation of HeLa and MCF-7 cells [1]. The Ca2+ influx route that is triggered by elevated K+ and norepinephrine is inhibited by khellin [2].

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EGFR Inhibitory Activity: Using a solid-phase ELISA assay, khellin (compound 1) was evaluated for its ability to inhibit the autophosphorylation of EGFR kinases. It exhibited moderate EGFR inhibitory activity with an IC50 of 0.15 μM. [1]
Anti-proliferative Activity: The anti-proliferative activity of khellin was determined using a standard MTT-based colorimetric assay on two human tumor cell lines. Against MCF-7 (human breast adenocarcinoma cell line), the IC50 was 0.30 μM. Against HeLa (human cervical carcinoma cell line), the IC50 was 0.38 μM. [1]

The study references previous work (Ruckstuhl & Landry, 1981) showing that khellin inhibits cAMP and cGMP phosphodiesterase activities from bovine lung, with higher selectivity for cAMP phosphodiesterase. This mechanism may contribute to its relaxant effects through increased cyclic nucleotide levels. [2]

EGFR Inhibitory Assay (ELISA): The level of EGFR was quantified by sandwich ELISA in MCF-7 and HeLa cells. Cells were treated for 24 hours with 0-50 μg/mL of khellin and then homogenized by three freeze/thaw cycles. The cell homogenate was coated onto 96-well microtiter plates and incubated. After washing and blocking, plates were incubated with primary antibody, then biotin-labeled secondary antibody, followed by peroxidase-conjugated streptavidin. The enzyme reaction was carried out by adding substrate solution (TMB and H₂O₂). Color development was stopped with 1M HCl, and optical absorbance was measured at 450 nm using a microplate reader. [1]
Anti-proliferative Assay (MTT Assay): MCF-7 and HeLa cells were seeded at 7×10³ cells/well in 96-well plates. After 24 hours, exponentially growing cells were exposed to khellin at final concentrations ranging from 0.10 to 100 μg/mL. After 48 hours, cell survival was determined by adding MTT solution (5 mg/mL in PBS). After 4 hours, 10% SDS in 0.01N HCl was added, and plates were incubated at 37°C for 18 hours. Optical absorbance was measured at 570 nm. IC50 values were determined from replicates of six wells from at least two independent experiments. [1]
Cell Culture: Human breast adenocarcinoma (MCF-7) and human cervical carcinoma (HeLa) cell lines were obtained from ATCC. Cells were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 units/mL penicillin G sodium, 100 units/mL streptomycin sulfate, and 250 mg/mL amphotericin B. Cells were maintained at sub-confluence at 37°C in humidified air containing 5% CO₂. For sub-culturing, monolayer cells were harvested after trypsin/EDTA treatment at 37°C. Cells were used when confluence reached 75%. Khellin was dissolved in DMSO (99.9%) and diluted 1000-fold in the assays. [1]

The document notes that khellin is an active principle from Ammi visnaga that has been used in folk medicine and has demonstrated antispasmodic and coronary vasodilator effects. The concentrations used in this study (10⁻⁵ to 6.4×10⁻⁴ M) produced relaxant effects without indication of toxicity in the isolated tissue preparations. [2]

[1]. Molecular modeling study bioactive natural product of khellin analogues as a novel potential pharmacophore of EGFR inhibitors. J Enzyme Inhib Med Chem. 2013 Dec;28(6):1171-81.

[2]. Relaxant actions of khellin on vascular smooth muscle. J Pharm Pharmacol . 1989 Apr;41(4):236-41.

Khellin is a furanochrome ketone compound with methoxy groups at positions 4 and 9 and a methyl group at position 7 of its basic tricyclic skeleton. It is the main component of the plant Ammi visnaga, an herbal remedy used to treat various ailments, primarily acting as a vasodilator. It possesses vasodilatory, bronchodilatory, anti-asthmatic, and cardiovascular properties. It is an organic heterocyclic tricyclic compound belonging to the oxoheterocyclic and furanochrome ketone class. Its function is associated with 5H-furano[3,2-g]chromene-5-one. Khellin has been reported in Dioscorea deltoidea, Dioscorea parviflora, and other organisms with relevant data. It is a vasodilator and also has bronchodilatory effects. It has been used to treat angina, asthma, and in combination with ultraviolet A to treat vitiligo. (Excerpt from Martindale Pharmacopoeia, 30th edition, p. 1024) See also: Visnaga daucoides fruit (partial).

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Background and Source: Khellin is a naturally occurring furochromone that can be isolated from the fruits and seeds of Ammi visnaga L., a perennial herbaceous plant that grows wild in many Eastern Mediterranean countries. The powdered fruit has been used in Egyptian folk medicine as a diuretic for treatment of kidney and bladder stones. Clinical and therapeutic effectiveness of khellin has been demonstrated for coronary, respiratory and urologic indications. [1]
Role in Drug Discovery: Khellin served as the parent natural product compound for the development of novel furochromone derivatives with potential EGFR inhibitory activity. The study investigated the structure-activity relationships (SAR) of khellin derivatives, with compound 5 showing the most potent EGFR inhibitory activity (IC50 = 0.04 μM), comparable to the positive control erlotinib (IC50 = 0.03 μM). [1]
Molecular Docking: Molecular docking simulations were performed to position khellin and its derivatives into the EGFR active site (PDB: 1M17). The docking score (binding free energy) for khellin was -11.38 kcal/mol. The study revealed that the benzofuran ring system of khellin derivatives was responsible for fitting into the position occupied by the quinazoline nucleus of erlotinib. [1]

C14H12O5

260.24

260.068

82-02-0

3828

White to light yellow solid powder

1.3±0.1 g/cm3

482.1±0.0 °C at 760 mmHg

150-154ºC

218.8±28.7 °C

0.0±1.2 mmHg at 25°C

1.595

1.77

0

5

2

19

405

0

HSMPDPBYAYSOBC-UHFFFAOYSA-N

InChI=1S/C14H12O5/c1-7-6-9(15)10-11(16-2)8-4-5-18-12(8)14(17-3)13(10)19-7/h4-6H,1-3H3

4,9-dimethoxy-7-methylfuro[3,2-g]chromen-5-one

Khellin Viscardan LynamineKhellin Amiptan Kelicor KeloidBenecardin Chellin Coronin Eskel RykellinMefurina

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)

DMSO : ~16.67 mg/mL (~64.06 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.)