Screening, Identification, and Potential Interaction of Active Compounds from Eucommia ulmodies Leaves Binding with Bovine Serum Albumin

Abstract

The aqueous extract of Eucommia ulmoides leaves has been commonly known as Du-zhong tea as a functional health food for the treatment of hypertension, hypercholesterolemia, and fatty liver. This study developed a centrifugal ultrafiltration-high-performance liquid chromatography (HPLC) method for screening and identification of bioactive compounds in E. ulmoides leaves binding with bovine serum albumin (BSA). Six active compounds were screened, isolated, and elucidated by their ultraviolet (UV), electrospray ionization-mass spectrometry (ESI-MS), and nuclear magnetic resonance (NMR) data as geniposidic acid (1), caffeic acid (2), chlorogenic acid (3), quercetin-3-O-sambubioside (4), rutin (5), and isoquercitrin (6). The interaction between active compounds and BSA was investigated in the absence and presence of other compounds by quenching the intrinsic BSA fluorescence. The results indicated that the structures significantly affected the binding process. The values of binding constants for compounds 2-6 were in the range of 10(5)-10(6) mol L(-1), while geniposidic acid (1) hardly quenching the BSA intrinsic fluorescence. However, the quenching process of geniposidic acid was easily affected in the presence of other active compounds. The formation of the geniposidic acid-phenylpropanoid (flavonoid) complex could increase the binding affinity of geniposidic acid with BSA; however, the increased steric hindrance of the complex may make phenylpropanoid or flavonoid dissociate from BSA and then decrease their affinities.

Simultaneous ligand fishing and identification of human serum albumin binders from Eucommia ulmoides bark using surface plasmon resonance-high performance liquid chromatography–tandem mass spectrometry

“…Geniposidic acid, chlorogenic acid, quercetin (+)-pinoresinol-4-O-␤-d-glucopyranoside (PG) were isolated and characterized from E. ulmoides in our laboratory and their structures were identified by UV, MS/MS and NMR [5,6]. The purities of them were determined to be over 97% by HPLC area normalization method.…”

“…To our best knowledge, E. ulmoides bark is rich in iridoids, lignans, flavonoids and phenolic acids [4]. Acid is known to achieve better separation for compounds with hydroxyl groups by reducing peak tailing [5,6], and acid as additive could provide high MS signal intensity [7]. Therefore, mobile phase (methanol-water, acetonitrile-water) with different concentrations of acetic acid or formic acid, gradient program (gradient time, gradient shape and initial composition of the mobile phase), column temperature and detection wavelength (relatively higher absorption) were investigated in the course of optimizing separation conditions.…”

“…The conventional approach is generally based on the bioactive-guided fractionation, which is difficult to achieve the biochemical fingerprint for the process of modernization and globalization of herb medicines. We have previously developed DPPH-HPLC/ultrafiltration-HPLC methods to analyze radical scavengers and bovine serum albumin (BSA) binders in E. ulmoides leaf and phosphodiesterase inhibitory lignans in E. ulmoides bark [5][6][7].…”

“…Up to now, many ligand fishing methods combined with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) have been employed, which include, but are not limited to, centrifugation [8], ultrafiltration [6,9,10], equilibrium dialysis [11], microdialysis [12], magnetic beads [13], affinity chromatography [14]. All of the methods combined bioactivity assay and analysis power to accommodate simultaneous readouts of biochemical fingerprint and unequivocal structural information of bioactive compounds [15].…”

Analysis of Xanthine Oxidase Inhibitors fromPuerariae flosUsing Centrifugal Ultrafiltration Coupled with HPLC-MS

“…4 Based on these features, active compounds could be retained by membrane together with enzyme due to the binding with enzyme. Thus, CU became a useful technique for screening active compounds bound to biomacromolecules such as bovine serum albumin, 5 α-glucosidase, 6 quinone reductase-2, 7 deoxyribonucleic acid (DNA) and liposomes. 8,9 High performance liquid chromatography-mass spectrometry (HPLC-MS or LC-MS) has been widely applied for the simultaneous separation and identification of active compounds in complex mixtures.…”