Discovery and Characterization of the Key Constituents in Ginkgo biloba Leaf Extract With Potent Inhibitory Effects on Human UDP-Glucuronosyltransferase 1A1.

Human UDP-glucuronosyltransferase 1A1 (hUGT1A1) is one of the most essential phase II enzymes in humans. Dysfunction or strong inhibition of hUGT1A1 may result in hyperbilirubinaemia and clinically relevant drug/herb-drug interactions (DDIs/HDIs). Recently, a high-throughput fluorescence-based assay was constructed by us to find the compounds/herbal extracts with strong inhibition against intracellular hUGT1A1. Following screening of over one hundred of herbal products, the extract of Ginkgo biloba leaves (GBL) displayed the most potent hUGT1A1 inhibition in HeLa-UGT1A1 cells (Hela cells overexpressed hUGT1A1). Further investigations demonstrated that four biflavones including bilobetin, isoginkgetin, sciadopitysin and ginkgetin, are key constituents responsible for hUGT1A1 inhibition in living cells. These biflavones potently inhibit hUGT1A1 in both human liver microsomes (HLM) and living cells, with the IC50 values ranging from 0.075 to 0.41 µM in living cells. Inhibition kinetic analyses and docking simulations suggested that four tested biflavones potently inhibit hUGT1A1-catalyzed NHPN-O-glucuronidation in HLM via a mixed inhibition manner, showing the K i values ranging from 0.07 to 0.74 µM. Collectively, our findings uncover the key constituents in GBL responsible for hUGT1A1 inhibition and decipher their inhibitory mechanisms against hUGT1A1, which will be very helpful for guiding the rational use of GBL-related herbal products in clinical settings.

Inhibition of human carboxylesterases by magnolol: Kinetic analyses and mechanism.

Magnolol, the most abundant bioactive constituent of the Chinese herb Magnolia officinalis, has been found with multiple biological activities, including anti-oxidative, anti-inflammatory and enzyme-regulatory activities. In this study, the inhibitory effects and inhibition mechanism of magnolol on human carboxylesterases (hCEs), the key enzymes responsible for the hydrolytic metabolism of a variety of endogenous esters as well as ester-bearing drugs, have been well-investigated. The results demonstrate that magnolol strongly inhibits hCE1-mediated hydrolysis of various substrates, whereas the inhibition of hCE2 by magnolol is substrate-dependent, ranging from strong to moderate. Inhibition of intracellular hCE1 and hCE2 by magnolol was also investigated in living HepG2 cells, and the results showed that magnolol could strongly inhibit intracellular hCE1, while the inhibition of intracellular hCE2 was weak. Inhibition kinetic analyses and docking simulations revealed that magnolol inhibited both hCE1 and hCE2 in a mixed manner, which could be partially attributed to its binding at two distinct ligand-binding sites in each carboxylesterase, including the catalytic cavity and the regulatory domain. In addition, the potential risk of the metabolic interactions of magnolol via hCE1 inhibition was predicted on the basis of a series of available pharmacokinetic data and the inhibition constants. All these findings are very helpful in deciphering the metabolic interactions between magnolol and hCEs, and also very useful for avoiding deleterious interactions via inhibition of hCEs.

Natural constituents of St. John's Wort inhibit the proteolytic activity of human thrombin.

Thrombin, a multifunctional serine protease responsible for the proteolytic hydrolysis of soluble fibrinogen, plays a pivotal role in the blood coagulation cascade. Currently, thrombin inhibitor therapy has been recognized as an effective therapeutic strategy for the prevention and treatment of thrombotic diseases. In this study, the inhibitory effects of natural constituents in St. John's Wort against human thrombin are carefully investigated by a fluorescence-based biochemical assay. The results clearly demonstrate that most of naphthodianthrones, flavonoids and biflavones exhibit strong to moderate inhibition on human thrombin. Among all tested compounds, hypericin shows the most potent inhibitory capability against thrombin, with the IC50 value of 3.00 µM. Further investigation on inhibition kinetics demonstrates that hypericin is a potent and reversible inhibitor against thrombin-mediated Z-GGRAMC acetate hydrolysis, with the Ki value of 2.58 µM. Inhibition kinetic analyses demonstrate that hypericin inhibits thrombin-mediated Z-GGRAMC acetate hydrolysis in a mixed manner, which agrees well with the results from docking simulations that hypericin can bind on both catalytic cavity and anion binding exosites. All these findings suggest that hypericin is a natural thrombin inhibitor with a unique dianthrone skeleton, which can be used as a good candidate to develop novel thrombin inhibitors with improved properties.