Mechanistic Study of Icaritin-Induced Inactivation of Cytochrome P450 2C9.

Icaritin (ICT) is a prenylflavonoid derivative that has been approved by National Medical Products Administration for the treatment of hepatocellular carcinoma. This study aims to evaluate the potential inhibitory effect of ICT against cytochrome P450 (CYP) enzymes and to elucidate the inactivation mechanisms. Results showed that ICT inactivated CYP2C9 in a time-, concentration-, and NADPH-dependent manner with Ki = 1.896 µM, Kinact = 0.02298 minutes-1, and Kinact/Ki = 12 minutes-1 mM-1, whereas the activities of other CYP isozymes was minimally affected. Additionally, the presence of CYP2C9 competitive inhibitor, sulfaphenazole, superoxide dismutase/catalase system, and GSH all protected CYP2C9 from ICT-induced activity loss. Moreover, the activity loss was neither recovered by washing the ICT-CYP2C9 preincubation mixture nor the addition of potassium ferricyanide. These results, collectively, implied the underlying inactivation mechanism involved the covalent binding of ICT to the apoprotein and/or the prosthetic heme of CYP2C9. Furthermore, an ICT-quinone methide (QM)-derived GSH adduct was identified, and human glutathione S-transferases (GST) isozymes GSTA1-1, GSTM1-1, and GSTP1-1 were shown to be substantially involved in the detoxification of ICT-QM. Interestingly, our systematic molecular modeling work predicted that ICT-QM was covalently bound to C216, a cysteine residue located in the F-G loop downstream of substrate recognition site (SRS) 2 in CYP2C9. The sequential molecular dynamics simulation confirmed the binding to C216 induced a conformational change in the active catalytic center of CYP2C9. Lastly, the potential risks of clinical drug-drug interactions triggered by ICT as a perpetrator were extrapolated. In summary, this work confirmed that ICT was an inactivator of CYP2C9. SIGNIFICANCE STATEMENT: This study is the first to report the time-dependent inhibition of CYP2C9 by icaritin (ICT) and the intrinsic molecular mechanism behind it. Experimental data indicated that the inactivation was via irreversible covalent binding of ICT-quinone methide to CYP2C9, while molecular modeling analysis provided additional evidence by predicting C216 as the key binding site which influenced the structural confirmation of CYP2C9's catalytic center. These findings suggest the potential of drug-drug interactions when ICT is co-administered with CYP2C9 substrates clinically.

Comparison of Bioactive Phenolic Compounds and Antioxidant Activities of Different Parts of Taraxacum mongolicum.

Herbs derived from Taraxacum genus have been used as traditional medicines and food supplements in China for hundreds of years. Taraxacum mongolicum is a famous traditional Chinese medicine derived from Taraxacum genus for the treatment of inflammatory disorders and viral infectious diseases. In the present study, the bioactive phenolic chemical profiles and antioxidant activities of flowers, leaves, and roots of Taraxacum mongolicum were investigated. Firstly, a high performance liquid chromatography method combined with segmental monitoring strategy was employed to simultaneously determine six bioactive phenolic compounds in Taraxacum mongolicum samples. Moreover, multivariate statistical analysis, including hierarchical clustering analysis, principal component analysis, and partial least squares discriminant analysis were performed to compare and discriminate different parts of Taraxacum mongolicum based on the quantitative data. The results showed that three phenolic compounds, caftaric acid, caffeic acid, and luteolin, could be regarded as chemical markers for the differences of flowers, leaves, and roots of Taraxacum mongolicum. In parallel, total phenolic contents, total flavonoid contents and antioxidant activities of different parts of Taraxacum mongolicum were also evaluated and compared. It is clear that Taraxacum mongolicum had antioxidant properties, and the antioxidant capacities of different parts of Taraxacum mongolicum in three antioxidant assays showed a similar tendency: Flowers ≈ leaves > roots, which revealed a positive relationship with their total phenolic and flavonoid contents. Furthermore, to find the potential antioxidant components of Taraxacum mongolicum, the latent relationships of the six bioactive phenolic compounds and antioxidant activities of Taraxacum mongolicum were investigated by Pearson correlation analysis. The results indicated caftaric acid and caffeic acid could be the potential antioxidant ingredients of Taraxacum mongolicum. The present work may facilitate better understanding of differences of bioactive phenolic constituents and antioxidant activities of different parts of Taraxacum mongolicum and provide useful information for utilization of this herbal medicine.

Green Extraction of Phenolic Acids from Artemisia argyi Leaves by Tailor-Made Ternary Deep Eutectic Solvents.

The Artemisia argyi leaf (AL) has been used as a traditional medicine and food supplement in China and other Asian countries for hundreds of years. Phytochemical studies disclosed that AL contains various bioactive constituents. Among bioactive constituents, phenolic acids have been recognized as the main active compounds in AL. To the best of our knowledge, no research has been focused on extraction method for the bioactive phenolic acids from AL. Nowadays, deep eutectic solvents (DESs) are emerging as a new type of green and sustainable solvent for efficient extraction of bioactive compounds from natural products. In the present study, an environmentally friendly extraction method based on DESs was established to extract bioactive phenolic acids from ALs. Diverse tailor-made solvents, including binary and ternary DESs, were explored for simultaneous extraction of four phenolic acids (3-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, and 4,5-di-O-caffeoylquinic acid) from AL. The results indicated that the ternary DES composed of a 2:1:2 molar ratio of choline chloride, malic acid, and urea showed enhanced extraction yields for phenolic acids compared with conventional organic solvents and other DESs. Subsequently, the extraction parameters for the four phenolic acids by selected tailor-made DESs, including liquid-solid ratios, water content (%) in the DESs, and extraction time, were optimized using response surface methodology and the optimal extraction conditions were: extraction time, 23.5 min; liquid-solid ratio, 57.5 mL/g (mL of DES/g dry weight of plant material); water content, 54%. The research indicated that DESs were efficient and sustainable green extraction solvents for extraction of bioactive phenolic acids from natural products. Compared to the conventional organic solvents, the DESs have a great potential as possible alternatives to those organic solvents in health-related areas such as food and pharmaceuticals.

Extraction, Purification, and Biological Activities of Polysaccharides from Branches and Leaves of Taxus cuspidata S. et Z.

Taxus cuspidata S. et Z. is an excellent natural source of bioactive polysaccharides and has various biological activities. The objective of this study was to evaluate the effect of antidiabetic and antitumor activities of polysaccharides from Taxus cuspidata branches and leaves (TCBL) and to determine the optimum extraction technology of TCBL using a low-temperature and high-efficiency enzyme and ultrasound-assisted coupled extraction (EUCE) method. Optimal technology parameters were determined as follows: an extraction temperature of 51 °C, an extraction time of 33 min, a ratio of material to liquid of 1:19 (g:mL), and an enzyme concentration of 0.10 mg·mL-1. Under the optimized conditions, the polysaccharide yield from TCBL obtained by EUCE was 4.78% ± 0.18%. The four purified polysaccharides (Pe1, Pe2, Pe3, Pe4) from TCBL are mainly composed of arabinose, galactose, glucose, a small amount of xylose, and mannose. This composition was assessed by HPIC analysis. The antidiabetic activity and antitumor activity of polysaccharides from TCBL were assayed in vitro. Among the four purified polysaccharides from TCBL, purified Pe4 had the highest inhibitory capacity against α-glucosidase, and its IC50 value was 123.0 µg·mL-1. Pe1 had the highest antitumor capacity against MCF7 cells and HepG2 cells, with IC50 values of 169.0 and 132.0 µg·mL-1. Pe4 had the highest antitumor effect on human cervical cancer cells (Hela), and its IC50 value was 89.9 µg·mL-1. Pe4 polysaccharide demonstrated a good α-glucosidase inhibitory activity and antitumor capacity against Hela cells. Therefore, Pe4 polysaccharide from TCBL is a beneficial source of potential inhibitors of type II diabetes and human cervical cancer activity.

Direct determination of the tumor marker AFP via silver nanoparticle enhanced SERS and AFP-modified gold nanoparticles as capturing substrate.

The authors describe a rapid and direct SERS-based immunoassay for the determination of AFP, an important marker for diagnosis of hepatocellular carcinoma. Silver nanoparticles (AgNPs; 36 nm i.d.) serve as a support to immobilize antibody and as a SERS intensifier, and AFP-modified gold nanoparticles are employed as capturing substrate. Direct and quantitative detection of AFP is accomplished with a limit detection as low as 5 ng·mL-1. Compared to assays based on the use of metal nanoparticles, the use of gold-silver nanoparticle heterodimers as an active SERS substrate can save costs because only a single antibody is required. Moreover, the high selectivity and good linear relationship of detecting AFP in fetal bovine serum indicates its potential applicability for the direct analysis of clinical samples. Graphical abstract Direct and quantitative determination of AFP antigen utilizing SERS has been was successfully presented and applied to detect alpha fetoprotein antigen in fetal bovine serum with detection limit of 2 ng•mL-1.

Colorimetric Signal Amplification Assay for Mercury Ions Based on the Catalysis of Gold Amalgam.

Mercury is a major threat to the environment and to human health. It is highly desirable to develop a user-friendly kit for on-site mercury detection. Such a method must be able to detect mercury below the threshold levels (10 nM) for drinking water defined by the U.S. Environmental Protection Agency. Herein, we for the first time reported catalytically active gold amalgam-based reaction between 4-nitrophenol and NaBH4 with colorimetric sensing function. We take advantage of the correlation between the catalytic properties and the surface area of gold amalgam, which is proportional to the amount of the gold nanoparticle (AuNP)-bound Hg(2+). As the concentration of Hg(2+) increases until the saturation of Hg onto the AuNPs, the catalytic performance of the gold amalgam is much stronger due to the formation of gold amalgam and the increase of the nanoparticle surface area, leading to the decrease of the reduction time of 4-nitrophenol for the color change. This sensing system exhibits excellent selectivity and ultrahigh sensitivity up to the 1.45 nM detection limit. The practical use of this system for Hg(2+) determination in tap water samples is also demonstrated successfully.

Saccharomyces cerevisiae cell surface display technology: Strategies for improvement and applications.

Microbial cell surface display technology provides a powerful platform for engineering proteins/peptides with enhanced properties. Compared to the classical intracellular and extracellular expression (secretion) systems, this technology avoids enzyme purification, substrate transport processes, and is an effective solution to enzyme instability. Saccharomyces cerevisiae is well suited to cell surface display as a common cell factory for the production of various fuels and chemicals, with the advantages of large cell size, being a Generally Regarded As Safe (GRAS) organism, and post-translational processing of secreted proteins. In this review, we describe various strategies for constructing modified S. cerevisiae using cell surface display technology and outline various applications of this technology in industrial processes, such as biofuels and chemical products, environmental pollution treatment, and immunization processes. The approaches for enhancing the efficiency of cell surface display are also discussed.

Hydrophobic paper-based SERS platform for direct-droplet quantitative determination of melamine.

A novel hydrophobic-SERS substrate platform based on silver nanoparticles (AgNPs) functionalized commercially available filter paper was reported. Compared with conventional substrates, not only could this novel SERS substrate meet the requirements of simple and large-scale preparation, but also realized direct droplet-detection with reusable property. In this work, the fabrication, physical characterization, and SERS sensitivity of the substrates to spot-on food determination were studied. The experimental results show the method exhibited high reproducibility with less than 9% spot-to-spot variation in Raman intensity. Furthermore, the method was successfully applied to detect melamine in diluted milk with the instrument detection limit (IDL) down to 1 ppm in a linear correlation (1 ppm-1000 ppm). In addition, this SERS substrate could also be applied successfully in other fields, such as the determination of pesticide (thiram) and dye (malachite green) in real environment.

Rapid fabrication of three-dimensional flower-like gold microstructures on flexible substrate for SERS applications.

In this work, hierarchical flower-like gold microstructures (HFGMs) are synthesized on a flexible PET film substrate by electrochemical deposition method as a SERS sensor without surfactant or directing agent. The effects of deposition current and electrolyte concentration on morphology and SERS performance are discussed to find the optimal preparation conditions. SERS activity of samples with different morphologies was investigated by comparing experiments and the mechanism was discussed. It shows the HFGMs with 3D nanoflakes and nanosheet tips present outstanding SERS activity with ultrasensitive detection limits. The lowest concentration of R6G that can be detected is 10-10 M, and the characteristic signals of thiram can be detected as low as 0.1 ppm. The HFGMs substrate show great potential of application in trace detection by SERS.

Application of metabolomics in toxicity evaluation of traditional Chinese medicines.

Traditional Chinese medicines (TCM) have a long history of use because of its potential complementary therapy and fewer adverse effects. However, the toxicity and safety issues of TCM have drawn considerable attention in the past two decades. Metabolomics is an "omics" approach that aims to comprehensively analyze all metabolites in biological samples. In agreement with the holistic concept of TCM, metabolomics has shown great potential in efficacy and toxicity evaluation of TCM. Recently, a large amount of metabolomic researches have been devoted to exploring the mechanism of toxicity induced by TCM, such as hepatotoxicity, nephrotoxicity, and cardiotoxicity. In this paper, the application of metabolomics in toxicity evaluation of bioactive compounds, TCM extracts and TCM prescriptions are reviewed, and the potential problems and further perspectives for application of metabolomics in toxicological studies are also discussed.

Fabrication of a Flexible Gold Nanorod Polymer Metafilm via a Phase Transfer Method as a SERS Substrate for Detecting Food Contaminants.

Surface enhanced Raman scattering (SERS) has been widely used in detection of food safety due to the nondestructive examination property. Here, we reported a flexible SERS film based on a polymer-immobilized gold nanorod polymer metafilm. Polystyrene-polyisoprene-polystyrene (SIS), a transparent and flexible, along with having excellent elasticity, polymer, was chosen as the main support of gold nanorods. A simple phase transfer progress was adopted to mix the gold nanorods with the polymer, which can further be used in most water-insoluble polymers. The SERS film performed satisfactorily while being tested in a series of standard Raman probes, like crystal violet (CV) and malachite green (MG). Moreover, the excellent reproducibility and elastic properties make the film a promising substrate in practical detection. Hence, the MG detection on the fish surface and trace thiram detection on orange pericarp were inspected with detection results of 1 × 10-10 and 1 × 10-6 M, which were below the demand of the National standard of China, exactly matching the realistic application requirements.

Rapid fabrication of flexible and transparent gold nanorods/poly (methyl methacrylate) membrane substrate for SERS nanosensor application.

Flexible substrates have been proposed for daily-life applications in SERS detection due to the prominent sample collection properties such as they can be wrapped around non-planar object surface. Combining the noble metals with polymers, flexible SERS substrates could be fabricated with advantages of light weight, transparency and high SERS sensitivity. Herein, we prepare a gold nanorods (AuNRs)/poly(methyl methacrylate) (PMMA) film as flexible SERS substrate by self-assembling a uniformly AuNRs array layer on PMMA template. This AuNRs/PMMA film performs excellently on thiram trace detection with the lowest detection concentration of 0.5 ppb. The fabricated substrates were applied for practical detection with cucumber by directly covering the AuNRs/PMMA flexible film on the target surface. Furthermore, the high SERS sensitivity as well as great reproducibility present a wide range of prospections for the further application of non-plane surface.

Aptasensor for electrochemical sensing of angiogenin based on electrode modified by cationic polyelectrolyte-functionalized graphene/gold nanoparticles composites.

Herein, a label-free and highly sensitive electrochemical aptasensor for the detection of angiogenin was proposed based on a conformational change of aptamer and amplification by poly(diallyldimethyl ammonium chloride) (PDDA)-functionalized graphene/gold nanoparticles (AuNPs) composites-modified electrode. PDDA-functionalized graphene (P-GR) nanosheets as the building block in the self-assembly of GR nanosheets/AuNPs heterostructure enhanced the electrochemical detection performance. The electrochemical aptasensor has an extraordinarily sensitive response to angiogenin in a linear range from 0.1pM to 5nM with a detection limit of 0.064pM. The developed sensor provides a promising strategy for the cancer diagnosis in medical application in the future.

A non-aggregation spectrometric determination for mercury ions based on gold nanoparticles and thiocyanuric acid.

We provide a highly sensitive and selective colorimetric assay to detect mercury ions (Hg(2+)) in aqueous environment using thiocyanuric acid (TCA) molecule-functionalized gold nanoparticles (AuNPs). This method is based on the thiophilicity of Hg(2+) and AuNPs as well as the unique optical properties of TCA-functionalized AuNPs. In the presence of TCA, AuNPs aggregate due to the strong attraction between thiol groups of TCA and surface-bounded AuCl4(-)/AuCl2(-) ions, which induces the visible color change from red to blue. With the addition of Hg(2+), Hg(2+) is more apt to interact with thiols than AuNPs. Thus, Hg(2+) can remove the AuNPs of the TCA-functionalized AuNPs and trigger AuNP aggregation redisperse again. This assay can selectively detect Hg(2+) with the detection limits as low as 0.5 nM in aqueous solution.

A colorimetric aptamer biosensor based on cationic polymer and gold nanoparticles for the ultrasensitive detection of thrombin.

A colorimetric assay for the ultrasensitive determination of thrombin based on cationic polymer and gold nanoparticles was presented, in which unmodified gold nanoparticles (AuNPs) was used as probes and 21-mer thrombin-binding aptamer (TBA) as sensing elements. Upon the addition of thrombin, TBA interacted specifically with thrombin to form a G-quadruplex structure. As a result, the conformation change facilitated the cationic polymer, poly(diallyldimethylammonium chloride) (PDDA)-induced AuNP aggregation. Thus, the visible change in color from wine-red to blue-purple was readily seen by the naked eye. The colorimetric sensor could detect thrombin down to 1 pM with high selectivity in the presence of other interferring proteins. Furthermore, the assay was successfully employed to determine thrombin in human serum sample, which suggested its great potential for diagnostic purposes.