Discovery of β₂- adrenoceptor agonists in Curcuma zedoaria Rosc using label-free cell phenotypic assay combined with...

Traditional Chinese Medicines (TCMs) have been widely used in clinical practice, and provided a rich source for discovering new drug leads. However, efficient identification of active molecules responsible for the therapeutic effects of complex TCMs is still highly challenging. Here, we combined label-free cell phenotypic assay with two dimensional liquid chromatography (2DLC) to identify potential β₂-adrenoceptor (β₂-AR) agonists related to anti-asthmatic effect of Curcuma zedoaria Rosc (C.zedoaria), a commonly used TCM. The ethyl acetate extract of C.zedoaria was first fractionated into 26 fractions. Label-free cell phenotypic profiling was then used to locate the active sites. Orthogonal second-dimensional (D2) separation was performed on two fractions displaying agonistic effect at the β₂-AR, combined with screening of the D2 fractions to track the activity. Finally, this approach led to the isolation of three known diarylheptanoids, among which diarylheptanoid b exhibited the most potent agonistic activity with an EC₅₀ value of 5.93 μM. This result was further demonstrated through the chemical synthesis of diarylheptanoid b. It is the first time to discover that diarylheptanoids could activate the β₂-AR, which may be responsible for the anti-asthmatic effect of C.zedoaria observed traditionally and in clinical application. This study also demonstrates the potential of this integrated strategy for identifying active ingredients and determining the basis of therapeutic materials in complex TCMs.