Cell experiment:

The lymphocytic mouse leukemia L1210 cells (ATCC, CCL 219) are plated sparsely at 104 cells per well in 24-well cluster plates in Dulbecco’s modified Eagle medium containing 10% fetal calf serum, 4 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin sulfate, and Verbascoside (solubilized in DMSO). After a 2-day incubation period at 37℃ in a humidified atmosphere (5% CO2 in air), growth is monitored by counting cell numbers in a Coulter-counter. IC50 values are calculated on the basis of the linear regression lines established for each compound tested[1].

Animal experiment:

Rats[2]To induce atopic dermatitis (AD)-like symptoms, 2,4-dinitrochlorobenzene (DNCB) is used. Briefly, the dorsal hair of the mice is removed using an electronic clipper 2 days before DNCB treatment. An application of 200 μL of 1% DNCB (in acetone:olive oil = 4:1) is made to the shaved dorsal skin for sensitization. The repeated challenge is performed on the same site with 0.2% DNCB once every 3 days for about 2 weeks. The mice are divided into 4 groups (n = 6 per group): (1) vehicle-treated controls, (2) DNCB-treated only, (3) 1% Verbascoside (in acetone:olive oil 4:1)-treated only, and (4) DNCB + 1% Verbascoside-treated group[2].

Verbascoside (Acteoside) is a hydroxytyrosol and phenylpropanoid sugar ester and an inhibitor of PKC and NF-κB [1].

Verbascoside has been found to decrease tyrosinase activity and melanin biosynthesis by regulating ERK signaling, and inhibit mycotoxin-mediated cell damage. In addition, Verbascoside has been reported to decrease the RANKL-stimulated differentiation of RAW264.7 cells and osteoclast formation. Moreover, Verbascoside has shown the inhibition of osteoclast formation in bone marrow macrophages （BMMs）with IC50 value of 4.8μM. Apart from these, Verbascoside has also been revealed to prevent these RANKL-induced increases in p-p38, p-ERK, p-JNK and the levels of phosphorylated MAPKs by immunometric analysis in macrophages. Furthermore, Verbascoside has been reported to inhibit RANKL-induced activation of NF-κB-DNA binding [1].

References:
[1] Lee SY1, Lee KS, Yi SH, Kook SH, Lee JC. Acteoside suppresses RANKL-mediated osteoclastogenesis by inhibiting c-Fos induction and NF-κB pathway and attenuating ROS production. PLoS One. 2013 Dec 4;8(12):e80873. doi: 10.1371/journal.pone.0080873. eCollection 2013.