Global Natural Products Social (GNPS)-Based Molecular-Networking-Guided Isolation of Phenolic Compounds from Ginkgo biloba Fruits and the Identification of Estrogenic Phenolic Glycosides

Ginkgo biloba L. stands as one of the oldest living tree species, exhibiting a diverse range of biological activities, including antioxidant, neuroprotective, anti-inflammatory, and cardiovascular activities. As part of our ongoing discovery of novel bioactive components from natural sources, we directed our focus toward the investigation of potential bioactive compounds from G. biloba fruit. The profiles of its chemical compounds were examined using a Global Natural Products Social (GNPS)-based molecular networking analysis. Guided by this, we successfully isolated and characterized 11 compounds from G. biloba fruit, including (E)-coniferin (1), syringin (2), 4-hydroxybenzoic acid 4-O-beta-D-glucopyranoside (3), vanillic acid 4-O-beta-D-glucopyranoside (4), syringic acid 4-O-beta-D-glucopyranoside (5), (E)-ferulic acid 4-O-beta-D-glucoside (6), (E)-sinapic acid 4-O-beta-D-glucopyranoside (7), (1 ' R,2 ' S,5 ' R,8 ' S,2 ' Z,4 ' E)-dihydrophaseic acid 3 '-O-beta-D-glucopyranoside (8), eucomic acid (9), rutin (10), and laricitrin 3-rutinoside (11). The structural identification was validated through a comprehensive analysis involving nuclear magnetic resonance (NMR) spectroscopic data and LC/MS analyses. All isolated compounds were evaluated using an E-screen assay for their estrogen-like effects in MCF-7 cells. As a result, compounds 2, 3, 4, 8, and 9 promoted cell proliferation in MCF-7 cells, and these effects were mitigated by the ER antagonist, ICI 182,780. In particular, cell proliferation increased most significantly to 140.9 +/- 6.5% after treatment with 100 mu M of compound 2. The mechanism underlying the estrogen-like effect of syringin (2) was evaluated using a Western blot analysis to determine the expression of estrogen receptor alpha (ER alpha). We found that syringin (2) induced an increase in the phosphorylation of ER alpha. Overall, these experimental results suggest that syringin (2) can potentially aid the control of estrogenic activity during menopause.