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.

Accurate and sensitive detection of Catechol-O-methyltransferase activity by liquid chromatography with fluorescence detection.

Catechol-O-methyltransferase (COMT) is a major drug metabolizing enzyme in humans. COMT expression is directedly associated with various mental diseases and cancers due to its essential role in catalyzing metabolic inactivation of endogenous catecholamines and catechol estrogens. However, a practical method to precisely measure COMT activities in biological samples is lacking. In the current study, we established a liquid chromatography-fluorescence detection (LC-FD) method based on fluorometric detection of the methylated product of 3-BTD, a fluorescent probe for COMT, to sensitively quantify COMT activities in human erythrocytes and cell homogenates. Assay validation of the established LC-FD based method was conducted for selectivity and sensitivity, range of linearity, precision and accuracy, recovery, biological matrices effect and stability. The limit of quantification for 3-BTMD (the methylated product of 3-BTD by COMT) of this method was 0.0083 nM, which is nearly 10-fold lower than that for previously published methods. The method was precise with intra- and inter-day relative standard deviation (RSD) lower than 5%. In addition, this method showed an excellent anti-interference ability with no effects of the endogenous substances on the fluorometric detection of 3-BTMD. The practical use of this method was established by its successful application for the measurement of COMT activities in individual human erythrocytes (n = 13), and in cell homogenates generated from four different human cell lines. Our results suggest that this method will be of great value in accurately determining the native activity of COMT in biological samples, which is beneficial for a complete understand of the role of COMT both in physiological and pathological conditions.

An ultra-sensitive and easy-to-use assay for sensing human UGT1A1 activities in biological systems.

The human UDP-glucuronosyltransferase 1A1 (UGT1A1), one of the most essential conjugative enzymes, is responsible for the metabolism and detoxification of bilirubin and other endogenous substances, as well as many different xenobiotic compounds. Deciphering UGT1A1 relevance to human diseases and characterizing the effects of small molecules on the activities of UGT1A1 requires reliable tools for probing the function of this key enzyme in complex biological matrices. Herein, an easy-to-use assay for highly-selective and sensitive monitoring of UGT1A1 activities in various biological matrices, using liquid chromatography with fluorescence detection (LC-FD), has been developed and validated. The newly developed LC-FD based assay has been confirmed in terms of sensitivity, specificity, precision, quantitative linear range and stability. One of its main advantages is lowering the limits of detection and quantification by about 100-fold in comparison to the previous assay that used the same probe substrate, enabling reliable quantification of lower amounts of active enzyme than any other method. The precision test demonstrated that both intra- and inter-day variations for this assay were less than 5.5%. Furthermore, the newly developed assay has also been successfully used to screen and characterize the regulatory effects of small molecules on the expression level of UGT1A1 in living cells. Overall, an easy-to-use LC-FD based assay has been developed for ultra-sensitive UGT1A1 activities measurements in various biological systems, providing an inexpensive and practical approach for exploring the role of UGT1A1 in human diseases, interactions with xenobiotics, and characterization modulatory effects of small molecules on this conjugative enzyme.

Study on the Role of Cytc in Response to BmNPV Infection in Silkworm, Bombyx mori (Lepidoptera).

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the primary pathogens of the silkworm. Cytochrome c (cytc) showed a significant response to BmNPV infection in our previous transcriptome study. However, little is known about the role of Bombyx mori cytc (Bmcytc) in resistance to BmNPV infection. In this study, the expression levels analysis of Bmcytc showed stable expression levels in selected tissues of the resistant strain AN following BmNPV infection, while there was downregulation in the susceptible strain p50, except in the malpighian tubule. To further study the role of Bmcytc in viral infection, Bmcytc was knocked down with siRNA in vitro, resulting in significant downregulation of selected downstream genes of the mitochondrial pathway, including Bmapaf, Bmcaspase-Nc, and Bmcaspase-1; this was also confirmed by overexpression of Bmcytc using the pIZT/V5-His-mCherry insect vector, except Bmcaspase-1. Moreover, knockdown of Bmcytc significantly promoted the infection process of BmNPV in vitro, while the infection was inhibited by overexpression of Bmcytc at the early stage and subsequently increased rapidly. Based on these results, we concluded that Bmcytc plays a vital role in BmNPV infection by regulating the mitochondrial apoptosis pathway. Our work provides valuable data for the clarification of the mechanism of silkworm resistance to BmNPV infection.