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Osthol is a prenylated coumarin which is abundant in the fruits of Cnidium monnieri Cusson (Umbelliferae). We previously isolated osthol from C. monnieri fruits and reported antifibrotic activity. Diverse biological activity of osthol including antitumor, anti-inflammatory, and neuroprotective activities have been also reported. In the present study, we investigated the melanogenesis inhibitory activity of osthol in B16F10 melanoma cells. In addition, due to the importance of extraction yield for further development as cosmetic products, the extraction conditions for the maximum yield from the isolation of osthol from C. monnieri fruits were optimized using response surface methodology.
The fruits of C. monnieri were purchased from a local herbal market in Chungbuk, Korea, in November 2013. They were identified by the herbarium of College of Pharmacy at Chungbuk National University, where a voucher specimen was deposited (CBNU201311-CM). Osthol was isolated from the fruits of C. monnieri as previously reported (Shin et al., 2011 Shin E, Lee C, Sung SH, Kim YC,
Hwang BY, Lee MK. 2011.Antifibrotic activity of coumarins from Cnidium monnieri fruits extract in HSC-T6 hepatic stellate cells. J Nat Med 65:370–374. The purity of osthol is >98%, as measured by HPLC analysis.
B16F10 mouse melanoma cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 μg/ml streptomycin. Cells were maintained at 37°C in a humidified atmosphere of 95% air–5% CO2.
B16F10 melanomas were treated with osthol at concentrations of 1, 3, 10, and 30 μM for 72 h. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in an ELISA plate reader.
B16F10 melanomas were treated with osthol at concentration of 1, 3, and 10 μM for 72 h. After washing with phosphate buffered saline (PBS), the cells were harvested and the melanin was solubilized via vortexing in 1N NaOH–10% DMSO at 80°C. The melanin contents were measured by an absorbance value at 405 nm with synthetic melanin as a standard.
Tyrosinase inhibitory activity was evaluated using mushroom tyrosinase.
Osthol, a coumarin of Cnidium monnieri fruits, inhibits melanin content in B16F10 melanoma cells by inhibition of the expression of melanogenesis genes, such as tyrosinase, TRP-1, and TRP-2. In addition, optimization of extraction factors such as extraction solvent, extraction time, and sample/solvent ratio investigated and the osthol yield under optimal conditions was found to be 15.0 mg/g dried samples, which gives a strong support for economic efficiency. Therefore, osthol is a promising natural compound in the field of pigmentation disorders and the cosmetics industry.
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