Asaraldehyde (2,4,5-trimethoxy-Benzaldehyde), a natural COX-2 inhibitor, exhibits 17-fold selectivity than COX-1.

COX-2:100 μg/mL.

Asaraldehyde (100 μg/mL) exhibits a higher inhibitory effect on COX-2. At the same concentration, it mildly inhibits the activity of prostaglandin H synthase-1 (3.32%) and significantly inhibits prostaglandin H synthase-2 (52.69%). Moreover, Asaraldehyde downregulates C/EBPβ, C/EBPδ, and C/EBPα and suppresses the expression of PPARγ1, PPARγ2, and acetyl-CoA carboxylase.

Hedgehog cell assay: This assay measures the end stage of the Hh signaling pathway, that is, the transcriptional modulation of Gli, using Luciferase as readout (Gli-Luc assay). Cyclopamine is prepared for assay by serial dilution in DMSO and then added to empty assay plates. TM3Hh12 cells (TM3 cells containing Hh-responsive reporter gene construct pTA-8xGli-Luc) are resuspended in F12 Ham's/DMEM (1:1) containing 5% FBS and 15 mM Hepes pH 7.3, added to assay plates and incubated with Cyclopamine for approximately 30 minutes at 37 °C in 5% CO2. 1 nM Hh-Ag 1.5 is then added to assay plates and incubated at 37 °C in the presence of 5% CO2. After 48 hours, either Bright-Glo or MTS reagent is added to the assay plates and luminescence or absorbance at 492 nm is determined. IC50 value, defined as the inflection point of the logistic curve, is determined by non-linear regression of the Gli-driven luciferase luminescence or absorbance signal from MTS assay vs log10 (concentration) of Cyclopamine using the R statistical software pack

3T3-L1 cells are seeded in 96-well plates at a concentration of 104 /well. Twenty-four hours after seeding, the cells are treated with 100 μg/mL of Asaraldehyde for 24 hours or for the whole 8-day differentiation period. Fully differentiated adipocytes are also treated with 100 μg/mL of Asaraldehyde for 24 hours-72 hours to test the cytotoxicity. At the end of treatment, cells are cultured with MTT at a final concentration of 0.5 mg/mL for another 4 hours. The purple MTT formazan is dissolved by DMSO and the absorbance at 570 nm is taken with a spectrophotometer. The absorbance is proportional to the viability of adipocytes.(Only for Reference)