Cell experiment:

B16 melanoma cells are cultured in 24-well plates at a density of 1×104 cells/well. After 24 h, the cells are incubated for 72 h in the presence of Isosakuranetin at the indicated concentrations. The culture medium is removed and replaced with 500 μL of fresh culture medium containing 10% sterile filtered MTT. After 3 h, the formazan crystals are dissolved in 500 μL/well isopropanol and absorbance is measured at 570 nm against 630 nm. Inhibition of proliferation (%) is expressed as the percentage of viable treated cells in comparison with control cells[1].

Animal experiment:

Male Sprague-Dawley rats weighing 250 to 300 g are habituated to the temperature, humidity, and lighting (12 h light/dark cycle, lights on at 7: 00 a.m.) controlled vivarium and singly housed for at least 1 week before the studies begin. The rotarod test is used to evaluate the potential motor-impairing effect of Isosakuranetin. For the time course studies, baseline measurement is immediately followed by an injection of Isosakuranetin, and then the paw-withdrawal threshold is measured every 15 min until the drug effect dissipates to a point that the paw withdrawal threshold is not significantly different from the predrug data or until 75 min passes[2].

Isosakuranetin is a flavanone flavonoid which can be found in the fruit of Citrus bergamia.

Isosakuranetin stimulates melanin accumulation in a dose-dependent manner. In addition, Isosakuranetin at 30 μM gradually increases the expression of Tyr, TRP1, and TRP2 after 48 and 72 h. The MTT assay shows that Isosakuranetin decreases the number of cells at concentrations greater than 45 μM. Further analysis of trypan blue staining demonstrates that Isosakuranetin does not affect the viability of B16 melanocytes, but shows that Isosakuranetin decreases their proliferation rate. The tyrosinase activity of B16 melanocytes treated with 15 and 30 μM Isosakuranetin for 72 h is significantly increased by 2- and 3.2-fold, respectively, in comparison with that of the control cells[1].

Isosakuranetin dose-dependently increases the paw-withdrawal threshold within the dose range of 1.5 to 6 mg/kg. Post hoc analysis reveals that Isosakuranetin increases the paw-withdrawal threshold and statistical significance is seen at times 30 and 40 min for the dose of 3 mg/kg and 30 to 60 min for the dose of 6 mg/kg[2].

[1]. Drira R, et al. Isosakuranetin, a 4'-O-methylated flavonoid, stimulates melanogenesis in B16BL6 murine melanoma cells. Life Sci. 2015 Dec 15;143:43-9. [2]. Jia S, et al. Antinociceptive Effects of Isosakuranetin in a Rat Model of Peripheral Neuropathy. Pharmacology. 2017;100(3-4):201-207.