RESUMO
The screening and identification of bioactive components, which are effectively resistant to metallo-beta-lactamase (MßL), were studied in the alcohol extract of Schisandra chinensis (Turcz.) Baill. by metalloenzyme-immobilized affinity chromatography. Taking bizinc metalloenzyme beta-lactamase II from Bacillus cereus (Bc II) and monozinc metalloenzyme CphA from aeromonas hydrophila (CphA) as examples, we studied the feasibility of this scheme based on the construction of metalloenzyme-immobilized chromatographic model. It was found that the Bc II- and CphA-immobilized chromatographic column could be used not only to explore the interaction between the MßL and their specific ligands, but also to screen the bioactive components from traditional Chinese medicine. The Bc II-and CphA-immobilized columns were used to screen the bioactive components from the alcohol extract of Schisandra chinensis (Turcz.) Baill. Time-of-flight tandem mass spectrometry analysis and molecular docking revealed that isobutyl 3-O-sulfo-ß-D-galactopyranoside is the effective bioactive components that could bind with metalloenzyme Bc II. It is believed that our current work may provide a methodological reference for screening MßL inhibitors from traditional Chinese medicine.
Assuntos
Proteínas de Bactérias/metabolismo , Cefalosporinase/metabolismo , Descoberta de Drogas/métodos , Extratos Vegetais/análise , Schisandra/química , beta-Lactamases/metabolismo , Proteínas de Bactérias/química , Cefalosporinase/química , Cromatografia de Afinidade , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Galactosídeos/análise , Galactosídeos/química , Galactosídeos/metabolismo , Simulação de Acoplamento Molecular , Extratos Vegetais/química , Extratos Vegetais/metabolismo , beta-Lactamases/químicaRESUMO
A combination of Angelicae Dahuricae Radix and Acori Tatarinowii Rhizoma has been widely used as the herb pair in traditional Chinese medicine to treat stroke, migraine, and epilepsy. However, the underlying synergistic mechanism of the herb pair remains unknown. This study was aimed at investigating the effects of Acori Tatarinowii Rhizoma volatile oil on the pharmacokinetic parameters of xanthotoxol, oxypeucedanin hydrate, and byakangelicin from Angelicae Dahuricae Radix in rat, and in vitro absorption behavior of the three compounds using rat everted gut sac, in situ single-pass intestinal perfusion, and Caco-2 cell monolayer models. The pharmacokinetic study exhibited clear changes in the key pharmacokinetic parameters of the three main coumarins through co-administering with Acori Tatarinowii Rhizoma volatile oil (50 mg/kg), the area under curve and the maximum plasma concentration of xanthotoxol increased 1.36 and 1.31 times; the area under curve, the maximum plasma concentration, mean residence time, half-life of elimination, and the time to reach peak concentration of oxypeucedanin hydrate increased by 1.35, 1.18, 1.24, 1.19 and 1.49 times, respectively; the area under curve, mean residence time, half-life of elimination, and time to reach peak concentration of byakangelicin climbed 1.29, 1.27, 1.37, and 1.28 times, respectively. The three coumarin components were absorbed well in the jejunum and ileum in the intestinal perfusion model, when co-administered with Acori Tatarinowii Rhizoma volatile oil (100 µg/mL). The in vivo and in vitro experiments showed good relevance and consistency. The results demonstrated that the three coumarin compounds from Angelicae Dahuricae Radix were absorbed through the active transportation, and Acori Tatarinowii Rhizoma volatile oil could promote the intestinal absorption and transport of these compounds by inhibiting P-glycoprotein (P-gp)-mediated efflux.
Assuntos
Angelica/química , Araceae/química , Furocumarinas/farmacocinética , Óleos Voláteis/farmacocinética , Administração Oral , Animais , Células CACO-2 , Furocumarinas/administração & dosagem , Humanos , Absorção Intestinal/efeitos dos fármacos , Masculino , Óleos Voláteis/administração & dosagem , Ratos , Ratos Sprague-DawleyRESUMO
Traditional Chinese medicine (TCM) represents a valuable resource for lead compounds discovery. Given the complexity of TCM components, analytical methods play a key role in novel drug development. In our study, we established a high specific and reliable bio-active components screen system, where ß2 adrenergic receptor (ß2-AR) was immobilized on silica by non-covalent bonds and packed into a stainless steel column (4.6â¯×â¯50â¯mm, 7⯵m) to form ß2-AR chromatography column. The column was further coupled with high performance liquid chromatography-time of flight tandem mass spectrometry (TOF-MS/MS). By utilizing this strategy, we successfully identified four ß2-AR-targeting compounds: tetrahydroberberine, tetrahydrocolumbamine, fumarine and corydaline from Corydalis Rhizome. The association constants between ß2-AR and tetrahydroberberine (9.04â¯×â¯104/M) as well as fumarine (4.30â¯×â¯104/M) were determined by frontal chromatography. We also found that these two compounds shared the identical binding site on immobilized ß2-AR with corresponding concentrations of 6.67â¯×â¯10-4â¯M and 5.88â¯×â¯10-4â¯M, respectively. The newly established method represents an efficient tool to identify the target specific natural compounds.
Assuntos
Alcaloides de Berberina , Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão/métodos , Medicamentos de Ervas Chinesas , Receptores Adrenérgicos beta 2/metabolismo , Alcaloides de Berberina/análise , Alcaloides de Berberina/metabolismo , Corydalis/química , Avaliação Pré-Clínica de Medicamentos/métodos , Medicamentos de Ervas Chinesas/análise , Medicamentos de Ervas Chinesas/metabolismo , Receptores Adrenérgicos beta 2/químicaRESUMO
The pursuit of drugs having improved therapeutic efficacy necessitates increasing research on new assays for screening bioactive compounds with multi-targets. This work synthesized a chromatographic stationary phase containing co-immobilized beta2-adrenergic receptor (ß2-AR) and voltage dependent anion channel isoform 1 (VDAC-1) to achieve such purpose. Specific ligands of the two receptors (e.g. salbutamol, methoxyphenamine, ATP and NADH) were utilized to characterize the specificity and bioactivity of the column. Validated application of the stationary phase was performed by screening multi-target compounds of Rhodiola crenulata using high performance affinity chromatography coupled with ESI-Q-TOF-MS. By zonal elution, we identified salidroside as a bioactive compound simultaneously binding to ß2-AR and VDAC-1. The compound exhibited the binding sites of 1.0â¯×â¯10-7 and 4.0â¯×â¯10-7â¯M on the ß2-AR and VDAC-1. On these sites, the association constants were calculated to be 3.3â¯×â¯104 and 1.0â¯×â¯104â¯M-1. Molecular docking indicated that the binding of salidroside to the two receptors occurred on Ser169 and Phe255of ß2-AR, and the channel wall of VDAC-1. Taking together, we concluded that the column containing co-immobilized receptors has potential for screening bioactive compounds with multi-targets from complex matrices including traditional Chinese medicines.
Assuntos
Descoberta de Drogas/métodos , Proteínas Imobilizadas/metabolismo , Extratos Vegetais/análise , Receptores Adrenérgicos beta 2/metabolismo , Rhodiola/química , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Medicamentos de Ervas Chinesas , Extratos Vegetais/metabolismoRESUMO
Based on the fluorescent reversible regulation, a novel sensor platform was designed for the detection of DNA intercalators utilizing the intercalation binding of DNA intercalators to DNA as an inherent exhibition and the fluorescence change of quantum dots (QDs) as an external manifestation. To prove its feasibility, acridine orange (AO) was chosen as an example of DNA intercalator. When different concentrations of herring sperm DNA (hsDNA) were added to cysteamine (CA)-capped ZnSe QDs solution, the hsDNA bound with the QDs through electrostatic interaction due to the photoinduced electron transfer from hsDNA to QDs and formed QDs-hsDNA complexes with 1:1 ratio, leading to the fluorescence quenching of the QDs; and upon addition of different concentrations of AO to the QDs-hsDNA complex system, the AO first caused the release of the hsDNA from the complexes and concomitantly bound with them through intercalation binding and formed AO-hsDNA complexes with 1:3 ratio on account of the fact that the intercalation binding constant between AO and hsDNA (1.932â¯×â¯105 L/mol) was greater than the electrostatic interaction constant between QDs and hsDNA (7.874â¯×â¯104 L/mol), resulting in the fluorescence recovery of the QDs. Therefore, the detection of AO could be achieved through the relationship between the fluorescence recovery yield of the QDs and the concentration of AO added. The results illustrated that the fluorescence recovery yield of the QDs-hsDNA system was linearly dependent to the concentration of AO in the range of 5.0-75.0â¯×â¯10-5 mol/L with a detection limit (3σ/K) of 1.5â¯×â¯10-5 mol/L. This dual-directional fluorescent regulation provided a novel method for the detection of DNA intercalators such as polycyclic aromatic hydrocarbons and drugs interfering with DNA-synthesis and possessed some potential applications in the investigation of the interactions between DNA intercalators and DNA.
Assuntos
Laranja de Acridina/análise , DNA/química , Elétrons , Substâncias Intercalantes/análise , Pontos Quânticos/química , Espectrometria de Fluorescência/métodos , Animais , Cisteamina/química , DNA/efeitos da radiação , Peixes/genética , Fluorescência , Luz , Limite de Detecção , Masculino , Selênio/química , Espermatozoides/química , Zinco/químicaRESUMO
Voltage dependent anion channel isoform 1 (VDAC-1) serves as an attractive target of anti-cancer drugs by mediating the entry and exit of metabolites between cytoplasm and mitochondria. This work reports on the preparation of a VDAC-1-based bioaffinity chromatographic stationary phase by linking the protein on lecithin modified microspheres. An assay of chromatographic methods including frontal analysis, zonal elution, injection dependent analysis and nonlinear chromatography were utilized to investigate the bindings of ATP, NADH and NADPH to VDAC-1. Electrostatic interactions were found to be main forces during these bindings. The calculated association constants of the three ligands to VDAC-1 showed good agreements between diverse chromatographic methods. Validated application of the stationary phase was performed by screening anti-cancer compounds of Rheum officinale Baill. using high performance affinity chromatography coupled with electrospray ionization-quadrupole time of flight mass spectrometry. Chrysophanol, emodin, rhein, aloe-emodin and catechin were identified as the bioactive components of the herb. These compounds targeted VDAC-1 through Thr207 and the N-terminal region of the protein. Taken together, the current stationary phase was possible to become a promising tool for protein-ligand interaction analysis and anti-cancer drug screening from complex matrices.
Assuntos
Antineoplásicos Fitogênicos/química , Ligantes , Canal de Ânion 1 Dependente de Voltagem/química , Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/metabolismo , Sítios de Ligação , Catequina/química , Catequina/isolamento & purificação , Catequina/metabolismo , Cromatografia de Afinidade , Emodina/química , Emodina/isolamento & purificação , Emodina/metabolismo , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Medicina Tradicional , Simulação de Acoplamento Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Rheum/química , Rheum/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Termodinâmica , Canal de Ânion 1 Dependente de Voltagem/metabolismoRESUMO
High throughput screening methodologies play a very important role in screening bioactive compounds from complex media. In this work, a new strategy for attaching cells onto amino microspheres using human umbilical vein endothelial cells (HUVECs) as a probe was developed. The immobilization depended on the specific affinity between integrin on the cells and the RGD peptide, which was coated on poly[oligo (ethylene glycol) methacrylate] by atom transfer radical polymerization. Validated application of the stationary phase was performed in the analysis of Ligusticum chuanxiong extraction by high performance affinity chromatography-mass spectrometry. Three compounds were screened as the bioactive compounds of Ligusticum chuanxiong. Two of them were identified as 3-butyl-hexahydroisobenzofuran-1(3H)-one and tetramethylpyrazine (TMP), whereas the other one remains indistinct. The association constant of vascular endothelial growth factor (VEGF) and TMP binding to VEGF receptor (VEGFR) on HUVECs were calculated to be (1.04 ± 0.08) × 10(11) M(-1) and (9.84 ± 1.11) × 10(8) M(-1) by zonal elution. Molecular docking showed that one hydrogen bond was formed between N atom of TMP and 3-N atom of imidazole group in histidine(223) of VEGFR. Both zonal elution and molecular docking indicated that TMP and VEGF bind to the same site of VEGFR on HUVECs. It is possible to become a promising tool for high throughput screening of the bioactive compounds binding to HUVECs through broad application of the stationary phase.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Ligusticum/química , Extratos Vegetais/farmacologia , Veias Umbilicais/efeitos dos fármacos , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Ensaios de Triagem em Larga Escala , Células Endoteliais da Veia Umbilical Humana , Humanos , Microscopia Eletrônica de Varredura , Microesferas , Simulação de Acoplamento Molecular , Extratos Vegetais/metabolismo , Pirazinas/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Propriedades de Superfície , Veias Umbilicais/citologia , Veias Umbilicais/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Demand on high-throughput methods for multi-target compounds screening continues to increase nowadays due to the decline of new drugs on the market. Two kinds of G-protein-coupled receptors, alpha1-adrenoceptor (α(1A)-AR) and beta2-adrenoceptor (ß(2)-AR), were purified and immobilized on the surface of macroporous silica gel to prepare new chromatographic stationary phases. Control drugs (e.g., prazosin, terazosin, salbutamol, and terbutaline) were used to characterize the retention behavior of the obtained α(1A)-AR and ß(2)-AR columns. This study also coupled both columns with a six-way switching valve to construct an automatic two-dimensional system for multi-target compounds screening in complex mixtures. Adrenaline hydrochloride was used as a representative drug to evaluate the chromatographic performance of the two dimensional system. The aqueous extracts from Salvia miltiorrhiza and Coptis chinensis were also analyzed by the automatic system. The compounds in S. miltiorrhiza had no binding to both α(1A)-AR and ß(2)-AR columns. But berberine, palmatine and jatrorrhizine were screened as the bioactive compounds in C. chinensis, targeting both the receptors. The proposed method is an alternative for recognizing and separating the compounds targeting different proteins from a complex matrix.