A systematic study on the influence of the main ingredients of an ivy leaves dry extract on the β2-adrenergic responsiveness of human airway smooth muscle cells

The bronchospasmolytic and secretolytic effects of ivy leaves dry extracts can be explained by an increased beta(2)-adrenergic responsiveness of the bronchi. Recently, it was shown that alpha-hederin inhibits the internalization of beta(2)-adrenergic receptors (beta(2)AR) under stimulating conditions. alpha-Hederin pretreated alveolar type II cells and human airway smooth muscle cells revealed an increased beta(2)AR binding and an elevated intracellular cAMP level, respectively. In order to identify whether additional compounds also mediate an increased beta(2)-adrenergic responsiveness, we examined the ingredients of an ivy leaves dry extract (EA 575) protocatechuic acid, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, kaempferol-3-O-rutinoside, 3,4-, 3,5- and 4,5-dicaffeoylquinic acid, hederacoside B, and beta-hederin. Within all the tested substances, only B-hederin inhibited the internalization of GFP-tagged beta(2)AR in stably transfected HEK293 cells. Using fluorescence correlation spectroscopy beta-hederin (1 mu M, 24 h) pretreated HASM cells showed a statistically significant increase in the beta(2)AR binding from 33.0 +/- 8.9% to 44.1 +/- 11.5% which was distributed with 36.0 +/- 9.5% for tau(bound1) and 8.1 +/- 2.6% for tau(bound2), respectively (n = 8, p < 0.05). The increased binding was selectively found for the receptor ligand complex with unrestricted lateral mobility (tau(bound1) of 0.9 +/- 0.1 ms, D-1 = 9.1 +/- 0.2 mu m(2)/s, n = 8), whereas the binding of beta(2)AR with hindered lateral mobility (tau(bound2) of 64.2 +/- 47.6 ms, D-2 = 0.15 +/- 0.02 mu m(2)/s, n = 8) was not affected. Compared to control cells, a statistically significant increase of 17.5 +/- 6.4% (n = 4, p < 0.05) and 24.2 +/- 5.8% (n = 4, p < 0.001) in the cAMP formation was found for beta-hederin pretreated HASM cells after stimulation with 10 mu M of terbutaline and simultaneous stimulation with 10 mu M terbutaline and 10 mu M forskolin, respectively. Within this systematic study focusing on the influence of the ingredients of an ivy leaves dry extract on HASM cells it was possible to identify H-hederin as further component presumably responsible for the beta(2)-mimetic effects. (C) 2014 The Authors. Published by Elsevier Ltd.