Adenine phosphate-Cu nanozyme with multienzyme mimicking activity for efficient degrading phenolic compounds and detection of hydrogen peroxide, epinephrine and glutathione.

BACKGROUND: With the development of nanotechnology, various nanomaterials with enzyme-like activity (nanozymes) have been reported. Due to their superior properties, nanozymes have shown important application potential in the fields of bioanalysis, disease detection, and environmental remediation. However, only a few nanomaterials with multi-enzyme mimicry activity have been reported. In this study, a novel multienzyme mimic was synthesized through a simple and rapid preparation protocol by coordinating copper ions with N3, N6 (amino), N7, and N9 on adenine phosphate. RESULTS: The prepared adenine phosphate-Cu complex exhibits significant peroxidase, laccase, and oxidase mimicking activities. The Michaelis-Menten constant (Km) and the maximal velocity (Vmax) values of the peroxidase, laccase, and oxidase mimicking activities of AP-Cu nanozyme are 0.052 mM, 0.14 mM, and 2.49 mM; and 0.552 µM min-1, 6.70 µM min-1, and 2.24 µM min-1, respectively. Then, based on its laccase mimicking activity, the nanozyme was applied in the degradation of phenolic compounds. The calculated kinetic constant for the degradation of 2,4-dichlorophenol is 0.468 min-1 and the degradation efficiency of 2,4-dichlorophenol (0.1 mM) reaches 96.14% at 7 min. Finally, based on the multienzyme mimicking activity of adenine phosphate-Cu nanozyme, simple colorimetric sensing methods with high sensitivity and good selectivity were developed for the detection of hydrogen peroxide, epinephrine, and glutathione in the ranges of 20.0-200.0 µM (R2 = 0.9951), 5.0-100.0 µM (R2 = 0.9970), and 5.0-200.0 µM (R2 = 0.9924) with the limits of quantitation of 20.0 µM, 5.0 µM, and 5.0 µM, respectively. SIGNIFICANCE: In short, the synthesis of nanozymes with multi-enzyme mimicry activity through coordination between copper ions and small molecule mimicry enzymes provides new ideas for the design and research of multi-enzyme mimics.

Zeolitic imidazolate frameworks as light-responsive oxidase-like mimics for the determination of adenosine triphosphate and discrimination of phenolic pollutants.

In this study, 3,3',5,5'-tetramethylbenzidine (TMB) was selected as a chromogenic substrate to evaluate the light-responsive oxidase-like activity of different zeolitic imidazolate frameworks (ZIFs). The synthesized ZIFs were systematically characterized by scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and X-ray diffraction analysis. Several main operational parameters, including ZIFs and TMB concentrations, pH value, radiation time, and working current, in the reaction process were optimized. The kinetic measurement results show that ZIF-90 exhibits higher affinity to the substrate than horseradish peroxidase. Furthermore, given that adenosine triphosphate (ATP) can specifically combine with Zn2+ binding site and destroy the structure of ZIF-90, a specific and sensitive colorimetric method was established for the quantitative detection of ATP within the range 10 - 240 µM. In addition, on the basis that phenolic pollutants can impact the reaction kinetics diversely on different ZIFs, a sensor array was constructed and successfully applied to differentiate five phenolic pollutants in lake water samples. This work is expected to shed light on the establishment of ZIF-based light-responsive oxidase-like nanozymes for the highly selective colorimetric detection and sensor array.

[Effects of short-segment pedicle screw internal fixation surgery combined with hyperbaric oxygen treatment for acute spinal injury on the morphology and function of the spine].

OBJECTIVE: To explore effect of short-segment pedicle screw internal fixation combined with hyperbaric oxygen in treating acute spinal fractures and its influence on recovery of spinal nerve function. METHODS: A total of 96 patients with acute spinal fracture admitted from February 2017 to March 2020 were divided into combined group and control group, with 48 cases in each group. Both groups were treated with short-segment pedicle screw internal fixation. The combined group was given hyperbaric oxygen after surgery. The operation time, surgical blood loss, incision length and other general operation conditions between two groups were recorded. The differences in spinal morphology and function, Ameraican Spinal Injury Assiciation(ASIA) neurological function grade, serum inflammatory factors and ability of daily living activities were observed before and after surgery. RESULTS: There was no significant difference in operation time, surgical blood loss, and incision length between combined group and control group(P>0.05). There were no significant differences in anterior height ratio and Cobb angle between two groups before surgery, 1 week and 6 months after surgery(P>0.05). The height ratio of anterior margin of the injured spine was significantly improved in both groups at 1 week and 6 months after surgery compared with preoperative period (P<0.05), and Cobb angle was significantly reduced in both groups compared with preoperative period (P<0.05). There was no statistically significant difference in serum interleukin-6(IL-6), interleukin-8(IL-8), and tumor necrosis factor-α(TNF-α) levels between two groups at 1 d after surgery(P>0.05);the serum IL-6, IL-8, and TNF-α levels of combined group were lower than those of control group at 1 week after surgery (P<0.05). At 6 months after surgery, ASIA neurological function grade of combined group was C grade in 2 cases, D grade in 23 cases, E grade in 22 cases. In control group, 7 cases was grade C, 26 cases was grade D, 13 cases was grade E, and the difference between two groups was statistically significant(P<0.05). The Barthel score of combined group was higher than that of control group at 1 month and 3 months after surgery, and the difference was statistically significant (P<0.05);at 6 months after surgery, there was no significant difference in Barthel score between two groups(P>0.05). CONCLUSION: Short-segment pedicle screw internal fixation combined with hyperbaric oxygen for the treatment of acute spinal fractures is beneficial to the recovery of spinal nerve function after surgery, and has a certain effect on the early improvement of the patients' activities of daily living.

Preparation of yolk-shell structure NH2-MIL-125 magnetic nanoparticles for the selective extraction of nucleotides.

Yolk-shell structure magnetic metal-organic framework nanoparticles were prepared via post solvothermal method and employed as a magnetic solid-phase extraction adsorbent for selective pre-concentration of 5'-ribonucleotides by π stacking interaction, hydrogen bonding, and the strong interaction between titanium ions (Ti4+) and phosphate group. The properties of the materials were confirmed by scanning electron microscopy, transmission electron microscopy, X-ray diffraction, X-ray photoelectron spectrometry, vibrating sample magnetometer, infrared spectroscopy, thermogravimetric analysis, and Brunauer-Emmett-Teller analysis. The main parameters affecting the adsorption-desorption process, including adsorbent amount, incubation time, incubation temperature, sample pH, shaking speed, elution solution, and elution time, were systematically optimized. Finally, 1.0 mg of adsorbent mixed with 1.0 mL sample solution (10.0 mmol⋅L-1 NaCl, pH 3.0) and shaken at 135 rpm for 5 min at 40 °C, washed with 1.0 mL Na3PO4-NH3∙H2O under vortex for 5 min were selected as optimized adsorption-desorption conditions. The binding performance of adsorbent towards five nucleotides was evaluated by static adsorption experiments. The data are well-fitted to the Langmuir isotherm model and the maximum adsorption capacity is 27.8 mg g-1 for adenosine 5'-monophosphate. The limit of detection of the method is 19.44-38.41 ng mL-1. Under the optimal conditions, the adsorbent was successfully applied to magnetic solid-phase extraction and high performance liquid chromatography determination of five nucleotides in octopus, chicken, fish, and pork samples.

A portable personal glucose meter method for enzyme activity detection and inhibitory activity evaluation based on alkaline phosphatase-mediated reaction.

In this study, an effective and portable method for enzyme activity detection and inhibitory activity evaluation was developed based on the alkaline phosphatase (ALP)-mediated reaction in a personal glucose meter (PGM). In this method, ALP catalyzes the hydrolysis of substrate amifostine (WR-2721) to produce ethanethiol (WR-1065), which can trigger the reduction of ferricyanide (K3[Fe(CN)6]), an electron transfer mediator in glucose test strips, to ferrocyanide ([K4Fe(CN)6]) and generate a PGM-detectable signal. Thus, WR-1065 can be directly quantified by a PGM as simply as detecting glucose in blood. After being systematically optimized, the method was applied to evaluate the inhibitory activity of ten small-molecule compounds and six Cordyceps sinensis (CS) extracts on ALP. The results showed that adenosine-5-monophosphate and theophylline had high inhibitory activity, but two CS extracts have promotion potency on ALP with the values of -20.7 ± 1.3% and -46.6 ± 2.1%, respectively. Moreover, the binding sites and modes of small-molecule compounds to ALP were investigated by molecular docking, while a new substrate competitor with theoretically good inhibitory activity against ALP was designed by scaffold hopping. Finally, the accuracy of the PGM method for enzyme activity detection was assessed by detecting ALP from milk samples, and the recovery ranged from 87.7% to 116.9%. These results indicate that it is feasible to evaluate enzyme activity and the inhibitory activity of small-molecule compounds and CS extracts on ALP using a PGM based on ALP-mediated reaction. Graphical abstract.

A simple and portable method for ß-Glucosidase activity assay and its inhibitor screening based on a personal glucose meter.

In this study, a simple and portable enzyme activity assay and inhibitor screening method was developed based on ß-Glucosidase-mediated cascade reaction in a personal glucose meter (PGM). The inhibition of castanospermine (ß-Glucosidase inhibitor) on ß-Glucosidase leads to reducing the yields of glucose and saligenin produced by the catalysis hydrolysis of D (-)-Salicin. The ferricyanide (K3 [Fe(CN)6]) can be reduced by the products of glucose and saligenin to form ferrocyanide ([K4[Fe(CN)6]) in the glucose strips, and thereby get the electron to generate PGM detectable signals. This strategy can realize the direct determination of glucose and saligenin using PGM as simple as measuring the glucose in blood. Under the optimum experimental conditions, quantitative detection of ß-Glucosidase in crude almond sample was achieved within the ranges of 1.0-9.0 U/mL with the limit of detection of 0.45 U/mL. The recoveries of ß-Glucosidase spiked with two different concentrations (3.0 and 6.0 U/mL) in the crude bitter almond extracts were determined as 96.2% and 84.3%, respectively. Furthermore, gallic acid, protocatechualdehyde, cryptochlorogenic acid, epigallocatechin, epicatechin and vanillic acid exhibited good inhibitory effect (all higher than 40%) on ß-Glucosidase. In addition, tea polyphenol extracts of raw Pu-erh and Fuding white tea had good inhibition potency and the % of inhibition were (29.0 ± 3.5)% and (21.1 ± 2.2)% on ß-Glucosidase, respectively. Finally, molecular docking study indicated that hydrogen bonding plays an important role in the interaction between the compounds and ß-Glucosidase. The enzyme activity assay and inhibitor screening method developed in present study using PGM based on ß-Glucosidase-mediated cascade reaction would be of value for expanding the application of PGM in non-glucose target analysis.

Evaluation inhibitory activity of catechins on trypsin by capillary electrophoresis-based immobilized enzyme microreactor with chromogenic substrate.

In this study, a capillary electrophoresis-based online immobilized enzyme microreactor was developed for evaluating the inhibitory activity of green tea catechins and tea polyphenol extracts on trypsin. The immobilized trypsin activity and other kinetic parameters were evaluated by measuring the peak area of the hydrolyzate of chromogenic substrate S-2765. The results indicated that the activity of the immobilized trypsin remained approximately 90.0% of the initial immobilized enzyme activity after 30 runs. The value of Michaelis-Menten constant (Km ) was (0.47 ± 0.08) mM, and the half-maximal inhibitory concentration (IC50 ) and inhibition constant (Ki ) of benzamidine were measured as 3.34 and 3.00 mM, respectively. Then, the inhibitory activity of four main catechins (epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate) and three tea polyphenol extracts (green tea, white tea, and black tea) on trypsin were investigated. The results showed that four catechins and three tea polyphenol extracts had potential trypsin inhibitory activity. In addition, molecular docking results illustrated that epigallocatechin gallate, epicatechin gallate, epicatechin, and epigallocatechin were all located not only in the catalytic cavity, but also in the substrate-binding pocket of trypsin. These results indicated that the developed method is an effective tool for evaluating inhibitory activity of catechins on trypsin.

Tyrosinase-mediated dopamine polymerization modified magnetic alginate beads for dual-enzymes encapsulation: Preparation, performance and application.

In this study, a simple and efficient method to obtain entrapment of mixtures of double enzymes is developed. As a proof of principle, double enzymes (tyrosinase (TYR) and ß-glucosidase (ß-Glu)) were co-immobilized in magnetic alginate-polydopamine (PDA) beads using in situ TYR-mediated dopamine polymerization and internal setting strategy-mediated magnetic alginate-PDA gelation. The leakage of enzymes from the magnetic alginate beads was significantly reduced by exploiting the double network cross-linking of alginate and PDA, which was induced by the d-(+)-Gluconic acid δ-lactone (GDL) and TYR, respectively. The physicochemical properties of the prepared magnetic alginate beads were characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and scanning electron microscopy (SEM) analysis. After that, the enzymatic reaction conditions and the performance of the entrapped TYR and ß-Glu, such as enzyme kinetics and inhibition kinetics, were investigated. The Michaelis-Menten constants (Km) of the entrapped TYR and ß-Glu were determined as 2.72 and 3.45 mM, respectively. The half-maximal inhibitory concentrations (IC50) of kojic acid and castanospermine for the entrapped TYR and ß-Glu were determined as 13.04 and 56.23 µM, respectively. Finally, the entrapped double enzymes magnetic alginate beads were successfully applied to evaluate the inhibitory potency of six kinds of tea polyphenols extracts. Black tea and white tea showed high inhibition activity against TYR were (36.14 ± 1.43)% and (36.76 ± 2.35)%, respectively, while the black tea and dark tea showed high inhibition activity against ß-Glu were (37.89 ± 6.70)% and (21.28 ± 4.68)%, respectively.

Modulation of electroosmotic flow in capillary electrophoresis by plant polyphenol-inspired gallic acid/polyethyleneimine coatings: Analysis of small molecules.

Plant polyphenols can form functional coatings on various materials through self-polymerization. In this paper, a series of modified capillary columns, which possess diversity of charge characteristics for modulating electroosmotic flow (EOF), were prepared by one-step co-deposition of gallic acid (GA), a plant-derived polyphenol monomer, and branched polyethyleneimine (PEI). The physicochemical properties of the prepared columns were characterized by Fourier transform infrared spectroscopy (FT-IR), UV-Vis spectroscopy and scanning electron microscopy (SEM). The magnitude and direction of EOF of GA/PEI co-deposited columns were modulated by changing a series of coating parameters, such as post-incubation of FeCl3, co-deposition time, and deposited amounts of GA and PEI with different relative molecular mass (PEI-600, PEI-1800, PEI-10000, and PEI-70000). Furthermore, the separation efficiencies of the prepared GA/PEI co-deposited columns were evaluated by separations of small molecules, including organic acids, polar nucleotides, phenols, nucleic acid bases and nucleosides. Results indicated that modulating of EOF plays an important role in enhancing the separation performance and reversing the elution order of the analytes. Finally, the developed method was successfully applied to quantitative analysis of acidic compounds in four real samples. The recoveries were in the range of 73.5%-85.8% for citric acid, benzoic acid, sorbic acid, salicylic acid and ascorbic acid in beverage and fruit samples, 101.6%-104.9% for cinnamic acid, vanillic acid, and ferulic acid in Angelica sinensis sample, while 84.6%-97.8% for guanosine-5'-monophosphate, uridine-5'-monophosphate, cytosine-5'- monophosphate and adenosine-5'-monophosphate in Cordyceps samples. These results indicated that the co-deposition of plant polyphenol-inspired GA/PEI coatings can provide new opportunities for EOF modulation of capillary electrophoresis.

Extraction of nucleobases, nucleosides and nucleotides by employing a magnetized graphene oxide functionalized with hydrophilic phytic acid and titanium(IV) ions.

A magnetite@graphene oxide nanocomposite was first coated with polyethylenimine and then modified with phytic acid and titanium(IV) ions. The high loading with Ti(IV) and the good hydrophilicity of PEI and PA result in a material that can be applied to the efficient extraction of highly polar nucleobases, nucleosides and nucleotides. The physicochemical properties of the composite were investigated by scanning electron microscopy, transmission electron microscopy, energy dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, water contact angle measurements, thermogravimetric analysis, and vibrating sample magnetometry. A series of parameters that affect extraction and elution under the conditions of immobilized metal affinity chromatography (IMAC) and hydrophilic interaction liquid chromatography (HILIC) were examined. The analytes were eluted from the nanocomposites using 10 mM trisodium phosphate as the elution solution in the IMAC mode, and 50% methanol-water as elution solution in the HILIC mode. Figures of merit include (a) an intra-day precision of 0.1-1.0% in the IMAC mode; (b) an intra-day precision of 0.4%-0.8% in the HILIC mode; (c) detection limits between 1.8-2.8 ng mL-1 in the IMAC mode; and (d) detection limits of 4.0-10.5 ng mL-1 in the HILIC mode. The method was applied to the extraction of the nucleotides cytidine-5'-monophosphate (CMP), uridine-5'-monophosphate (UMP), guanosine-5'-monophosphate (GMP), and adenosine-5'-monophosphate (AMP), and the nucleobases and nucleosides hypoxanthine, adenosine, cytosine, inosine and cytidine from Cordyceps sinensis, Lentinus edodes and plasma samples. Graphical abstract Schematic presentation of the workflow for the extraction of nucleobases, nucleosides and nucleotides using phytic acid-Ti(IV) functionalized magnetite@graphene oxide nanocomposites under two distinct modes.

In vitro anti-platelet aggregation effects of fourteen fruits and vegetables.

In the present study, the anti-platelet aggregation activity of 14 vegetables and fruits was tested in vitro. The aqueous, 90% ethanol and ethyl acetate extracts, as well as concentrated juices of 14 foods (fruits and vegetables) were prepared, and the anti-platelet aggregation activity of those extracts was analyzed on a platelet aggregation analyzer in vitro with adenosine 5'-diphosphate (ADP), bovine thrombin (THR) and arachidonic acid (AA) as aggregation inducers, respectively. Aspirin (ASP) was used as the positive control. A number of the tested foods had inhibitory effects in concentration-dependent manner on platelet aggregations induced by various agonists. Especially, some foods such as lemon, leek, garlic, scallion, ginger, tomato and grapefruit showed good anti-platelet aggregation effect similar or higher than that of positive control group i.e. aspirin (ASP). The results of present study provide scientific reference for reasonable selection of daily dietary with supplementary curative effects or prevention of cardiovascular diseases (CVD).

Preparation of titanium ion functionalized polydopamine coated ferroferric oxide core-shell magnetic particles for selective extraction of nucleotides from Cordyceps and Lentinus edodes.

In this study, a titanium ion (Ti4+) functionalized polydopamine coated ferroferric oxide (Fe3O4@PDA@Ti4+) core-shell magnetic particle was prepared for the selective extraction of nucleotides. Firstly, different metal ions including Ti4+, Zr4+, Fe3+, Al3+, Cu2+, Zn2+, Ni2+ and Mg2+ were respectively immobilized onto Fe3O4@PDA particles and their extraction efficiency for five nucleotides [cytidine-5'-monophosphate (CMP), uridine-5'-monophosphate (UMP), guanosine-5'-monophosphate (GMP), thymidine-5'-monophosphate (TMP) and adenosine-5'-monophosphate (AMP)] were compared. Among these prepared materials, Fe3O4@PDA@Ti4+, which exhibited the highest extraction efficiency for nucleotides, was further characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy and energy dispersive X-ray spectroscopy. After being optimized of the extraction parameters including adsorbent amounts, extraction time, extraction temperature, type and concentration of the eluent, the prepared Fe3O4@PDA@Ti4+ magnetic particles were successfully applied for the selective extraction and determination of CMP, UMP, GMP, TMP and AMP in Cordyceps and Lentinus edodes. Good linearity (varying from 0.063 to 19.000 µg/mL, R2 > 0.999) and low limit of detection (LODs) (ranging between 0.0047 and 0.0141 µg/mL) for target analytes were achieved. These results demonstrated that the synthesized material in this study had potential for selective extraction of phosphorylated small molecular compounds in complicated matrix.

Potential target-related proteins in rabbit platelets treated with active monomers dehydrocorydaline and canadine from Rhizoma corydalis.

BACKGROUND: Dehydrocorydaline (DHC) and canadine (THB) are two active alkaloid compounds in Corydalis yanhusuo (Y.H. Chou & Chun C. Hsu) W.T. Wang ex Z.Y. Su & C.Y. Wu (Papaveraceae) (Rhizoma Corydalis). DHC and THC were previously shown to exert anti-platelet aggregation effect dose-dependently, but their exact mechanisms had not yet been addressed. Therefore, it is essential to study the mechanisms of DHC and THB affecting on platelet's function. PURPOSE: To investigate the anti-platelet effects and corresponding signal cascades of DHC and THB on platelet aggregation. METHODS: Firstly, in vitro anti-platelet aggregation of DHC and THB induced by different agonists including thrombin (THR), adenosine diphosphate (ADP) and arachidonic acid (AA) were determined through turbidimetry method. Then the possible target-related platelet proteins after treated with DHC/THB were separated and identified by two dimensional gel electrophoresis (2-DE) and MALDI-TOF-MS/MS analysis, respectively. Finally, the signal cascades network induced by DHC/THB were predicted through functional analysis of these proteins along with the determination of platelet DAG concentration. RESULTS: The platelet aggregation stimulated by THR, ADP and AA were inhibited by DHC and THB dose-dependently to a certain degree. Meanwhile, DHC and THB had the strongest effect on ADP- and THR-induced platelet aggregation respectively. In addition, treatment of these two compounds caused regulations of about sixty proteins in platelet, including cytoskeleton proteins, cell signaling proteins, proteins related to material energy metabolism, etc. CONCLUSIONS: Using proteomic analysis combined with platelet aggregation test and ELISA, this study was successful in exploring the possible mechanisms of DHC/THB on platelet aggregation. DHC might inhibit platelet aggregation by a mechanism involving the ADP receptors P2Y1 and P2Y12, and the effect of THB on platelet function may be related to its binding to THR receptor PAR1 for mediated Gi signaling pathway. These results provide fundamental information for the anti-thrombotic effect of RC.

Differential proteomics for studying action mechanisms of traditional Chinese medicines.

Differential proteomics, which has been widely used in studying of traditional Chinese medicines (TCMs) during the past 10 years, is a powerful tool to visualize differentially expressed proteins and analyzes their functions. In this paper, the applications of differential proteomics in exploring the action mechanisms of TCMs on various diseases including cancers, cardiovascular diseases, diabetes, liver diseases, kidney disorders and obesity, etc. were reviewed. Furthermore, differential proteomics in studying of TCMs identification, toxicity, processing and compatibility mechanisms were also included. This review will provide information for the further applications of differential proteomics in TCMs studies.

Applications of polydopamine modifications in capillary electrophoretic analysis.

Mussel-inspired polydopamine has been widely used in capillary electrophoresis as a facile and universal tool for the surface modification of capillary, mainly due to its versatility, stability, strong adhesiveness, and biocompatibility properties. In this review, the recent development of mussel-inspired surface chemistry with rapid deposition of polydopamine was introduced, and the recent applications of polydopamine in capillary electrophoresis (2011-July 2018) were summarized into four main aspects, namely, sample pretreatments, functional coatings, codeposition coatings, and intermediate coatings. Further study may be focused on clarifying the mechanisms of polydopamine formation and polydopamine-assisted codeposition, and expanding coatings methods to plant polyphenols.

An ultrafiltration and high performance liquid chromatography coupled with diode array detector and mass spectrometry approach for screening and characterizing thrombin inhibitors from Rhizoma Chuanxiong.

Thrombin (THR) plays a significant role in thromboembolic diseases, direct THR inhibitors are a class of important clinical anticoagulant drugs. This study established a THR in-solution based biospecific extraction combined with ultrafiltration and high performance liquid chromatography coupled with diode array detector and mass spectrometry analysis (TUA) method to screen and identify ligands for THR in Rhizoma Chuanxiong. After evaluating the reliability of the present TUA method using positive (argatroban) and negative (adenosine, tirofiban, ticagrelor) control drugs, this method was successfully applied to detect eight potential active compounds in Rhizoma Chuanxiong. Two new THR-targeted compounds isochlorogenic acid C and senkyunolide I with high THR inhibitory activity (IC50 206.48 and 197.23µM, respectively) were identified by liquid chromatography/mass spectrometry and enzyme inhibitory activity test finally. They were reported with direct THR inhibition activity for the first time and their ligand-THR interactions were explored by in silico molecular docking research. In addition, based on the TUA screening result, four compounds gained similar structure with the two hit compounds were also investigated as promising candidates targeting THR with high binding energy (>5.0kcal/mol). These results may prove that the proposed method could effectively screen THR inhibitors in complex mixtures.

Biotransformation and metabolic profile of anemoside B4 with rat small and large intestine microflora by ultra-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry.

Pulsatilla chinensis (Bunge) Regel is commonly used in Asia, and anemoside B4 (AB4) is its major saponin, with diverse pharmaceutical effects. Previous studies showed that intestinal flora plays an important role in the metabolism of herbs administered orally. In this study, the metabolic profile of AB4 with microflora in rat small and large intestines in vitro was investigated. Gut microflora was collected from different intestinal segments and anaerobically incubated with AB4 at 37°C for 24, 48, 72 and 96 h, respectively. A total of 10 metabolites were detected and identified by ultra- performance liquid chromatography/quadrupole time-of-flight mass spectrometry, involving the products of oxygenation and deglycosylation reactions. Gut microflora in the large intestine generated more comprehensive metabolic pathways, which appears to be attributable to the wider range of bacterial types and numbers of bacteria. Human cancer cell lines SMMC-7721, Hela and MCF-7 were treated with metabolite pools by MTT assay, together with M6 as the greatest deglycosylation product. As a result, M6 exhibited a reduction in cell viability of SMMC-7721 with an IC50 value of 22.28 ± 1.26 µg/mL. The present study provided scientific evidence for AB4 metabolism in small and large intestines, which is helpful to reveal the active forms of AB4 in vivo.

Evaluation of interactions between RAW264.7 macrophages and small molecules by capillary electrophoresis.

In this study, the affinity interactions between RAW 264.7 macrophages and three small molecules including naringin, oleuropein and paeoniflorin were evaluated by affinity capillary electrophoresis (ACE), partial filling affinity capillary electrophoresis (PFACE) and frontal analysis capillary electrophoresis (FACE), respectively. The result indicated that ACE (varying concentrations of cell suspension were filled in the capillary as receptor) may not be suitable for the evaluation of interactions between cell and small molecules due to the high viscosity of cell suspension; PFACE can qualitatively evaluate the interaction, but the difference in viscosity between RAW264.7 suspension and buffer effects on the liner relationship between filling length and injection time, which makes the calculation of binding constant difficult. Furthermore, based on the PFACE results, naringin showed stronger interaction with macrophages than the other two molecules; taking advantage of the aggregation phenomenon of cell induced by electric field, FACE was successfully used to determine the stoichiometry (n = 5×109 ) and binding constant (Kb = 1×104 L/mol) of the interaction between RAW264.7 and naringin.

Differential proteomic analysis of platelets suggested target-related proteins in rabbit platelets treated with Rhizoma Corydalis.

CONTEXT: Corydalis yanhusuo W.T. Wang (Papaveraceae) (Rhizoma Corydalis) showed inhibitory effects on rabbit platelet aggregation induced by ADP, thrombin (THR) or arachidonic acid (AA). OBJECTIVE: This study separates and identifies the possible target-related platelet proteins and suggests possible signal cascades of RC antiplatelet aggregation. MATERIALS AND METHODS: Based on comparative proteomics, the differentially expressed platelet proteins treated before and after with 50 mg/mL RC 90% ethanol extract (for 15 min at 37 °C) were analyzed and identified by two dimensional gel electrophoresis (2-DE) and MALDI-TOF-MS/MS. To further verify the possible signalling pathways of RC antiplatelet aggregation function, the concentration of calcium (Ca2+) was measured by Fura-2/AM fluorescence (Ex 340/380 nm, Em 500 nm) (RC final concentrations of 0.0156-0.1563 mg/mL), the levels of P-selectin and cyclic guanosine monophosphate (cGMP) were quantified by ELISA (OD. 450 nm) (RC final concentrations of 0.0156-1.5625 mg/mL), and the 5-hydroxytryptamine (5-HT) level was measured using ortho-phthalaldehyde (OPT) fluorescence (Ex 340 nm, Em 470 nm) (RC final concentrations of 0.3125-1.5625 mg/mL). RESULTS: The expression of 52 proteins were altered in rabbit platelets after the treatment and the MALDI-TOF-MS analysis indicated that those proteins include 12 cytoskeleton proteins, 7 cell signalling proteins, 3 molecular chaperone proteins, 6 proteins related to platelet function, 16 enzymes and 7 other related proteins. Furthermore, RC extract could decrease the levels of 5-HT [inhibition rate of 96.80% (p < 0.05, vs. THR-activated group) treated with 0.7813 mg/mL of RC], Ca2+ [172.73 ± 5.07 to 113.56 ± 5.46 nM (p < 0.001, vs. THR-activated group) treated with 0.0313 mg/mL of RC] and P-selectin [13.48 ± 0.96 ng/3 × 108 to 11.64 ± 0.17 ng/3 × 108 (p < 0.05, vs. THR-activated group) treated with 0.0156 mg/mL of RC], and increase in cGMP level [38.93 ± 0.57 to 50.26 ± 4.05 ng/3 × 108 (p < 0.05, vs. THR-activated group) treated with 1.5165 mg/mL of RC] in ADP (10 µmol/L), THR (0.25 u/mL) or AA-(0.205 mmol/L) activated rabbit platelets. DISCUSSION AND CONCLUSION: The present study indicated that P2Y12 receptor might be one of the direct target proteins of RC in platelets. The signal cascades network of RC after binding with P2Y12 receptor is mediating Gαi proteins to activate downstream signalling pathways (AC and/or PI3K signalling pathways) for the inhibition of platelet aggregation.

Analysis of Eight Isoflavones in Radix Puerariae by MEEKC: Comparison on Three Different Oil Phases.

In this study, three different oil phases include 1-butyl-1-methylpyrrolidinium bis(trifluoromethanesulfonyl)imide (BMPy[NTf2]), 1-butyl-3-methylimidazolium hexafluorophosphate (BMImPF6) and n-octane were compared for the MEEKC analysis (SDS as surfactant and n-butanol as co-surfactant) of eight isoflavones from pueraria. The investigated isoflavones can be well separated by all of those three microemulsion systems after careful optimization, and the MEEKC with n-octane as the oil phase was the best choice (good symmetry and high resolutions of peaks with short analysis time) for the analysis. The optimum conditions of MEEKC method were as follows: 70 mM SDS, 0.7 M n-butanol and 0.5% (w/v) n-octane in 10 mM sodium tetraborate (STB) at pH 8.5, applied voltage was 23 kV and cassette temperature was set at 30°C. And then the developed method was fully validated (limit of detection, limit of quantification, intraday precision, interday precision and recovery) and successfully applied to determine the eight analytes in three Radix Puerariae samples. In addition, although the MEEKC with classic oil phase (n-octane) showed better results for isoflavones analysis in this study, the MEEKC with ionic liquids (BMPy[NTf2] and BMImPF6) also showed great separation potential for analytes, which may be further applied in the analysis of other natural products.