Cell experiment:

Diosmetin is dissolved in DMSO which is maintained at a constant concentration in control samples (2%). HepG2 cells are maintained in a humidified atmosphere of 5% CO2 at 37℃, and cultured in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin. HepG2 cell density is adjusted to 2×104 cells/100 μL, and the cells are seeded into 96-well plates and placed in an incubator overnight (37℃ in 5% CO2) to allow for attachment and recovery. MTT analyses are performed. Briefly, cells are pretreated with 5, 10, 15 and 20 μg/mL diosmetin for 24 h. A total of 20 μL MTT solution (5 mg/mL in PBS) solution is transferred to each well to yield a final 120 μL/well and to separate wells a total of 10 μL CCK8 (5 mg/mL in PBS) is transferred. The plates are incubated for 4 h at 37℃ in 5% CO2 and the absorbance is recorded at wavelengths of 595 nm and 450 nm, respectively. The half maximal inhibitory concentration (IC50) of diosmetin is calculated[1].

Animal experiment:

Experimental acute pancreatitis is induced in mice by seven intraperitoneal injection of cerulein (50 μg/kg) at hourly intervals. Diosmetin (100 mg/kg) or vehicle is pretreated 2 h before the first cerulein injection. After 6 h, 9 h, 12 h of the first cerulein injection, the severity of acute pancreatitis is evaluated biochemically and morphologically[2].

Diosmetin (DIO) is an agonist of the aryl hydrocarbon receptor (AHR). It potently inhibited the enzyme activity of cytochrome P450 1A1 (CYP1A1) in a dose-dependent manner with an IC50 value of approximately 30 nM, in microsomes from MCF-7 cells [1].

AHR belongs to the Per, ARNT, Sim/basic-helix-loop-helix superfamily of ligand-activated transcription factors. AHR mediates the toxic effects of polycyclic aromatic hydrocarbons, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyls. These chemicals all bind to AHR, and result in the activation of a battery of genes, including the cytochromes P450 CYP1A1, CYP1A2, and CYP1B1 [2].

In MCF-7 cells, at 24 h after the incubation of diosmetin, CYP1A1 mRNA was increased in a dose-dependent manner. In MCF-7 cells, diosmetin at 2.5 μM modestly increased CYP1A1 enzyme activity, with an activity increase in cells, while diosmetin at 5 μM did not increase the enzyme activity compared to controls in cells. Compared with controls, diosmetin dose-dependently increased the capacity of nuclear extracts to bind an oligonucleotide containing the AhR-binding sequence of CYP1A1 [1].

In the presence of CYP1A inhibitor, the concentration of diosmetin ranged from 25 μM at 0 h to 22 μM. In the absence of CYP1A inhibitor, the concentration of diosmetin ranged from 25 μM at 0 h to 15 μM [3].

No in vivo result from the administration of diosmetin had been found.

References:
[1].  Ciolino HP, Wang TT and Yeh GC. Diosmin and diosmetin are agonists of the aryl hydrocarbon receptor that differentially affect cytochrome P450 1A1 activity. Cancer Res, 1998, 58(13):2754-60.
[2].  Gonzalez FJ and Fernandez-Salguero P. The Aryl Hydrocarbon Rreceptor Studies Using the AHR-Null Mice. Drug Metabolism and Disposition, 1998, 26(12): 1194-1198.
[3].  Androutsopoulos VP and Spandidos DA. The flavonoids diosmetin and luteolin exert synergistic cytostatic effects in human hepatoma HepG2 cells via CYP1A-catalyzed metabolism, activation of JNK and ERK and P53/P21 up-regulation. J Nutr Biochem, 2013, 24(2):496-504.