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A photolabile protecting group (PPG) 2-(2-nitrophenyl)-propyloxycarbonyl (NPPOC) was explored in glycosylation and applied in the consecutive synthesis of oligosaccharides. NPPOC displays a strong neighboring group participation (NGP) effect to facilitate the construction of 1,2-trans glycosides in excellent yield. Notably, NPPOC could be efficiently removed by photolysis, and the deprotection conditions are friendly to typical protecting groups. A branched and asymmetric oligomannose Man6 was rapidly prepared, and the consecutive assembly of oligosaccharides without intermediate purification was further investigated owing to the compatibility conditions between NPPPOC's photolysis and glycosylation.
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Glicósidos , Oligosacáridos , Glicosilación , FotólisisRESUMEN
How to rapidly and accurately screen bioactive components from complex natural products remains a major challenge. In this study, a screening platform for pancreatic lipase (PL) inhibitors was established by combining magnetic beads-based ligand fishing and high-resolution bioassay profiling. This platform was well validated using a mixture of standard compounds, i.e., (-)- epigallocatechin gallate (EGCG), luteolin and schisandrin. The dose-effect relationship of high-resolution bioassay profiling was demonstrated by the standard mixture with different concentrations for each compound. The screening of PL inhibitors from green tea extract at the concentrations of 0.2, 0.5 and 1.0 mg/mL by independent high-resolution bioassay profiling was performed. After sample pre-treatment by ligand fishing, green tea extract at the concentration of 0.2 mg/mL was specifically enriched and simplified, and consequently screened through the high-resolution bioassay profiling. As a result, three PL inhibitors, i.e., EGCG, (-)-Gallocatechin gallate (GCG) and (-)-Epicatechin gallate (ECG), were rapidly identified from the complex matrix. The established platform proved to be capable of enriching affinity binders and eliminating nonbinders in sample pre-treatment by ligand fishing, which overcame the technical challenges of high-resolution bioassay profiling in the aspects of sensitivity and resolution. Meanwhile, the high-resolution bioassay profiling possesses the ability of direct bioactive assessment, parallel structural analysis and identification after separation. The established platform allowed more accurate and rapid screening of PL inhibitors, which greatly facilitated natural product-based drug screening.
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Catequina , Lipasa , Ligandos , Luteolina/análisis , Catequina/química , Extractos Vegetales/química , Bioensayo , Té/químicaRESUMEN
In order to have deep insights into the mechanisms of enantiomer affinity pattern in both aqueous and non-aqueous systems, an approach combining capillary electrophoresis and molecular modeling was undertaken. A chiral ß-blocker; acebutolol, was enantioseparated in aqueous capillary electrophoresis and non-aqueous capillary electrophoresis using two anionic ß-cyclodextrin derivatives. The enantiomer affinity pattern of acebutolol was found to be opposite when an aqueous background electrolyte was replaced with non-aqueous background electrolyte in the presence of heptakis(2,3-di-O-acetyl-6-sulfo)-ß-cyclodextrin but remained the same in the presence of heptakis(2,3-di-O-methyl-6-sulfo)-ß-cyclodextrin. Molecular docking of acebutolol into two ß-cyclodextrin derivatives indicated two distinct binding modes called 'up' and 'down' conformations. After structure optimization by molecular dynamics and energy minimization, both enantiomers of acebutolol were preferred to the 'up' conformation with heptakis(2,3-di-O-methyl-6-sulfo)-ß-cyclodextrin while 'down' conformation with heptakis(2,3-di-O-acetyl-6-sulfo)-ß-cyclodextrin. The further calculation of the complex energy with solvent effect indicated that heptakis(2,3-di-O-acetyl-6-sulfo)-ß-cyclodextrin had higher affinity to S-acebutolol than R-acebutolol in non-aqueous capillary electrophoresis while it showed better binding to R-acebutolol in aqueous capillary electrophoresis. However, the heptakis(2,3-di-O-methyl-6-sulfo)-ß-cyclodextrin bound better to R-acebutolol in both aqueous and non-aqueous capillary electrophoresis, implying that the binding mode played more important role in chiral separation of heptakis(2,3-di-O-methyl-6-sulfo)-ß-cyclodextrin while the solvent effect had prevailing impact on heptakis(2,3-di-O-acetyl-6-sulfo)-ß-cyclodextrin.
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Acebutolol/química , Antagonistas Adrenérgicos beta/química , Ciclodextrinas/química , Metanol/química , Agua/química , Acebutolol/aislamiento & purificación , Antagonistas Adrenérgicos beta/aislamiento & purificación , Aniones/química , Aniones/aislamiento & purificación , Ciclodextrinas/aislamiento & purificación , Electrólitos/química , Electroforesis Capilar , Modelos Moleculares , Conformación Molecular , EstereoisomerismoRESUMEN
To evaluate the effect of the preparation strategy on the enantioseparation performance of ß-cyclodextrin-functionalized monoliths, a series of ß-cyclodextrin-functionalized organic polymeric monolithic columns were prepared through two-step, single-step, and one-pot approaches, using the same cyclodextrin, linker-spacer, and crosslinker. Physicochemical characterization of the columns was carried out by determining the morphology, ß-cyclodextrin density, permeability, and chromatographic efficiency. For each type of monolithic column, the enantioresolution of 22 chiral compounds, including mandelic acid derivatives, nonsteroidal anti-inflammatory drugs, N-derivatized amino acids, and herbicides, was comparatively studied under optimum chromatographic conditions. The ß-cyclodextrin-functionalized monolithic columns prepared through the one-pot approach exhibited higher enantioresolution for most chiral compounds, and they have the advantage of good controllability and simple preparation. On the other hand, the enantioresolution obtained on columns prepared through the single-step approach was quite unsatisfactory, and therefore the effect of using different linking spacers and crosslinkers was studied. A significant improvement of enantioresolution for 2-chloro-mandelic acid was obtained by using N,N-methylenebisacrylamide instead of ethylene dimethacrylate as the crosslinker in the single-step preparation.
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To realize the effective profile control of a heavy oil reservoir, hydrolyzed polyacrylamide (HPAM) and water-soluble phenol-formaldehyde resin (PR) were chosen to prepare the profile control system, which gelled at medium or low temperatures and existed stably at high temperatures in the meantime. The effects of phenolic ratios, PR concentration, and HPAM concentration on the formation and strength of the gels were systematically studied by the gel-strength code method and rheological measurements. And the microstructure of the gels was investigated by scanning electron microscope measurements. The results showed that the gelling time of the HPAM-PR system was 13 h at 70 °C. The formed gel could stay stable for 90 days at 140 °C. In addition, the gels showed viscoelastic properties, and the viscosity reached 18,000 mPa·s under a 1.5 s-1 shearing rate due to their three-dimensional cellular network structure. The formation of the gels was attributable to the hydroxyl groups of the PR crosslinking agent, which could undergo the dehydration condensation reaction with amide groups under non-acidic conditions and form intermolecular crosslinking with HPAM molecules. And the organic crosslinker gel system could maintain stability at higher temperatures because covalent bonds formed between molecules.
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In this study, a novel at-line nanofractionation platform was established for screening SARS-CoV-2 fusion inhibitors from natural products for the first time by combining HPLC-MS/MS with high-throughput fluorescence polarization (FP) bioassay. A time-course FP bioassay in 384 well-plates was conducted in parallel with MS/MS to simultaneously obtain chemical and biological information of potential fusion inhibitors in Lonicerae Japonicae Flos (LJF) and Lianhua Qingwen capsules (LHQW). Semi-preparative liquid chromatography and orthogonal HPLC separation were employed to enrich and better identify the co-eluted components. After comprehensive evaluation and validation, 28 potential SARS-CoV-2 fusion inhibitors were screened out and identified. Several compounds at low micromolar activity were validated by in vitro inhibitory assay, molecular docking, cytotoxicity test, and pseudovirus assay. Moreover, four potential dual-target inhibitors against influenza and COVID-19 were discovered from LJF using this method, offering novel insights for the development of future pharmaceuticals targeting epidemic respiratory diseases.
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Antivirales , Polarización de Fluorescencia , Simulación del Acoplamiento Molecular , SARS-CoV-2 , Espectrometría de Masas en Tándem , Cromatografía Líquida de Alta Presión/métodos , SARS-CoV-2/efectos de los fármacos , Espectrometría de Masas en Tándem/métodos , Antivirales/farmacología , Antivirales/química , Antivirales/análisis , Humanos , Polarización de Fluorescencia/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Medicamentos Herbarios Chinos/farmacología , Medicamentos Herbarios Chinos/química , Lonicera/química , COVID-19/virología , Cromatografía Líquida con Espectrometría de MasasRESUMEN
In this study, a novel magnetic bead-based ligand fishing method was developed for rapid discovery of monoterpene indoles as monoamine oxidase A inhibitors from natural products. In order to improve the screening efficiency, two different magnetic beads, i.e. amine and carboxyl terminated magnetic beads, were comprehensively compared in terms of their ability to immobilize monoamine oxidase A (MAOA), biocatalytic activity and specific adsorption rates for affinity ligands. Carboxyl terminated magnetic beads performed better for MAOA immobilization and demonstrated superior performance in ligand fishing. The MAOA immobilized magnetic beads were applied to screen novel monoamine oxidase inhibitors in an alkaloid-rich plant, Hunteria zeylanica. Twelve MAOA affinity ligands were screened out, and ten of them were identified as monoterpene indole alkaloids by HPLC-Obitrap-MS/MS. Among them, six ligands, namely geissoschizol, vobasinol, yohimbol, dihydrocorynanthenol, eburnamine and (+)-isoeburnamine which exhibited inhibitory activity against MAOA with low IC50 values. To further explore their inhibitory mechanism, enzyme kinetic analysis and molecular docking studies were conducted.
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Simulación del Acoplamiento Molecular , Inhibidores de la Monoaminooxidasa , Monoaminooxidasa , Inhibidores de la Monoaminooxidasa/química , Inhibidores de la Monoaminooxidasa/farmacología , Inhibidores de la Monoaminooxidasa/aislamiento & purificación , Monoaminooxidasa/metabolismo , Monoaminooxidasa/química , Ligandos , Indoles/química , Monoterpenos/química , Monoterpenos/aislamiento & purificación , Cinética , Espectrometría de Masas en Tándem/métodos , Cromatografía Líquida de Alta Presión/métodos , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Enzimas Inmovilizadas/antagonistas & inhibidores , Humanos , Extractos Vegetales/químicaRESUMEN
Human monoamine oxidase B (hMAO-B) has emerged as a pivotal therapeutic target for Parkinson's disease. Due to adverse effects and shortage of commercial drugs, there is a need for novel, highly selective, and reversible hMAO-B inhibitors with good blood-brain barrier permeability. In this study, a high-throughput at-line nanofractionation screening platform was established with extracts from Chuanxiong Rhizoma, which resulted in the discovery of 75 active compounds, including phenolic acids, volatile oils, and phthalides, two of which were highly selective novel natural phthalide hMAO-B inhibitors that were potent, selective, reversible and had good bloodâbrain permeability. Molecular docking and molecular dynamics simulations elucidated the inhibition mechanism. Sedanolide (IC50 = 103 nmol/L; SI = 645) and neocnidilide (IC50 = 131 nmol/L; SI = 207) demonstrated their excellent potential as hMAO-B inhibitors. They offset the limitations of deactivating enzymes associated with irreversible hMAO-B inhibitors such as rasagiline. In SH-SY5Y cell assays, sedanolide (EC50 = 0.962 µmol/L) and neocnidilide (EC50 = 1.161 µmol/L) exhibited significant neuroprotective effects, comparable to the positive drugs rasagiline (EC50 = 0.896 µmol/L) and safinamide (EC50 = 1.079 µmol/L). These findings underscore the potential of sedanolide as a novel natural hMAO-B inhibitor that warrants further development as a promising drug candidate.
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Anti-PD-1/PD-L1 monoclonal antibodies have displayed remarkable clinical benefits and revolutionized the treatment of multiple tumor types, but the low response rates and immune-related adverse events limit their application, which promoting the development of small molecule agents to improve the efficacy of PD-1/PD-L1 blockade therapy. Boningmycin (BON), a new small molecule belonging to bleomycin (BLM) family, exhibits potent anticancer activity in vitro and in vivo, as well as negligible lung toxicity, thereby can be an alternative of BLM. However, understandings about the anticancer mechanism of BLM-related compounds are extremely rare, it remains unclear if they affect PD-L1 level in a manner similar to that of other antitumor drugs. In this study, we discover that BON significantly reduces PD-L1 protein level in NCI-H460 and HT-1080 cells. Meanwhile, BON decreases the protein level of PD-L1 in a tumor xenograft model of NCI-H460 cells. Nevertheless, the mRNA level is not influenced after BON exposure. Furthermore, BON-induced PD-L1 reduction is proteasome- dependent. By using specific inhibitors and RNA interference technology, we confirm that the decline of PD-L1 protein by BON is mediated by AMPK-activated endoplasmic reticulum-associated degradation pathway, which is like to the action of metformin. Last but not the least, BON has synergism on gefitinib in vitro and in vivo. In conclusion, it is the first report demonstrating that BON decreases PD-L1 protein level through AMPK-mediated endoplasmic reticulum-associated degradation pathway. These findings will benefit the clinical transformation of BON and aid in the elucidation of molecular mechanism of BLM-related compounds.
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Antígeno B7-H1 , Neoplasias , Humanos , Antígeno B7-H1/metabolismo , Degradación Asociada con el Retículo Endoplásmico , Proteínas Quinasas Activadas por AMP/metabolismo , Neoplasias/tratamiento farmacológico , Bleomicina/metabolismo , Anticuerpos Monoclonales/uso terapéutico , Línea Celular TumoralRESUMEN
We herein reported a new type of S-o-(p-methoxyphenylethynyl)benzyl donor for a highly efficient glycosylation method. The donor was activated by 10% Tf2O and underwent glycosylation with various acceptors to provide the corresponding glycosides in excellent yield. Furthermore, two repetitive fragments of Polyporus umbellatus polysaccharides (PUPs), isolated from traditional Chinese medicine "Polyporus umbellatus", were prepared by combining the "single-catalyst one-pot" and "latent-active" strategies for the first time for future clear studies on the structure-activity relationship of PUPs.
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Polyporus , Glicosilación , Glicósidos , PolisacáridosRESUMEN
In this study, an at-line nanofractionation (ANF) platform was successfully fabricated in parallel with mass spectrometry and trypsin inhibitory bioactivity assessment for rapid screening of trypsin inhibitors (TIs) from natural products for the first time. After systematic optimization, the ANF platform was applied to screen and identify TIs in the extract of a traditional Chinese herb, i.e., Cotinus coggygria Scop. The semi-preparative reverse-phase liquid chromatography was used subsequently to further simplify and enrich the insufficiently separated components. After comprehensive evaluation and validation, the ANF platform successfully identified 12 compounds as potential TIs, including 8 flavonoids and 2 organic acids. Additionally, a comparison study was conducted using two other ligand fishing approaches, i.e., capillary monolithic and magnetic beads-based trypsin-immobilized enzyme microreactors, which successfully identified 8 identical flavonoids as TIs. Importantly, the molecular docking study showed the molecular interactions between enzymes and inhibitors, thus strongly supporting the experimental results. Overall, this work has fully demonstrated the feasibility of the established ANF platform for screening TIs from Cotinus coggygria Scop., and proved its great prospects for screening bioactive components from natural products.
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Anacardiaceae , Productos Biológicos , Cromatografía de Fase Inversa , Inhibidores de Tripsina , Tripsina , Simulación del Acoplamiento Molecular , Flavonoides/química , Extractos Vegetales/farmacología , Anacardiaceae/químicaRESUMEN
In this study, an advanced at-line nanofractionation based screening platform was developed to screen potential neuraminidase inhibitors (NAIs) from Lonicera japonica Thunb by involving two parallel bioassays, for determining both oseltamivir-sensitive neuraminidase (NAS) and oseltamivir-resistant neuraminidase (NAR) inhibitory activities. 20 potential NAIs with both NAS and NAR inhibitory effects were screened from Lonicera japonica Thunb and identified by mass spectrometer, including 11 phenolic acids, 8 flavonoids and one iridoid glycoside. The proposed at-line nanofractionation based screening platform for NAIs was also used to rapidly screen nine batches of water extracts of Lonicera japonica Thunb or its similar species. Clear differences in the number and content of active components were easily observed, demonstrating that the proposed method possesses great potential for the quality control of herb medicines.
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Subtipo H1N1 del Virus de la Influenza A , Lonicera , Oseltamivir/farmacología , Neuraminidasa , Antivirales/farmacología , Inhibidores Enzimáticos/farmacología , Guanidinas/farmacologíaRESUMEN
The fatigue prevalence in breast cancer survivors is high during the endocrine treatment. However, there are few evidence-based interventions to manage this symptom. The aim of this study was to investigate the effectiveness of spore powder of Ganoderma lucidum for cancer-related fatigue in breast cancer patients undergoing endocrine therapy. Spore powder of Ganoderma lucidum is a kind of Basidiomycete which is a widely used traditional medicine in China. 48 breast cancer patients with cancer-related fatigue undergoing endocrine therapy were randomized into the experimental or control group. FACT-F, HADS, and EORTC QLQ-C30 questionnaires data were collected at baseline and 4 weeks after treatment. The concentrations of TNF-α, IL-6, and liver-kidney functions were measured before and after intervention. The experimental group showed statistically significant improvements in the domains of physical well-being and fatigue subscale after intervention. These patients also reported less anxiety and depression and better quality of life. Immune markers of CRF were significantly lower and no serious adverse effects occurred during the study. This pilot study suggests that spore powder of Ganoderma lucidum may have beneficial effects on cancer-related fatigue and quality of life in breast cancer patients undergoing endocrine therapy without any significant adverse effect.
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Dilution rate is one of most important factors influencing the microstructure and performance of the laser cladding layer. In order to obtain a reasonable dilution rate in the laser cladding layer of Inconel 625 alloy, the laser cladding layers with different Fe content were prepared on the surface of 20# steel by the laser cladding technique. The influence of Fe content on the microstructure and performance of Inconel 625 alloy cladding layer was investigated. The results indicate that with the increase in Fe content in the alloy, the grain size of the cladding layer becomes coarser, the grain orientation difference increases first and then decreases, and the grain boundary angle decreases first and then increases. The hardness, high temperature wear resistance, and high temperature corrosion resistance gradually decreased. It is concluded that the dilution rate of Fe in laser cladding Inconel 625 alloy should be under 5 wt.%.
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Weak gel is a gel system formed by the mixing and crosslinking of a low-concentration polymer and a slow-release crosslinker. It can be used for profile control in deep reservoir, but its effect is greatly affected by mechanical shearing. Currently, the shearing effect on weak gel is mainly studied by way of mechanical stirring, while the effect of porous media shear on weak gel molecules and properties has been rarely discussed. In this paper, polymer solution, aluminum gel and phenolic gel were prepared. The molecular coil size, viscoelastic modulus and microscopic aggregation morphology in water solution of three systems before and after core shearing were investigated, and the injection performance of the three systems in cores with different permeabilities was tested by physical simulation experiments. The study results show that at equivalent permeability, the system with a larger equivalent sphere diameter of molecular coil is more seriously sheared and suffers greater viscosity loss. In the core with permeability of 1.0 D, polymer solution remains as the aggregation, while phenolic gel and aluminum gel cannot form network aggregations and they are inferior to polymer solution in migration capacity in the mid-deep part of the core. In the core with permeability of 1-5.8 D, the polymer solution remains as a Newtonian fluid, while phenolic gel and aluminum gel become purely viscous non-Newtonian fluids. The elastic modulus of aluminum gel and phenolic gel is four times more than that of a polymer. In the core with permeability higher than 8.5 D, aluminum gel and phenolic gel migrate with less effect by core shearing, and their profile control capacity in deep reservoir is higher than that of the polymer. In the core with permeability lower than 8.5 D, because the monomolecular activity of weak gels becomes poor, they migrate in porous media with more effect by core shearing, and their profile control and oil displacement capacity in deep reservoir is lower than that of the polymer.
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Under the background that the in-depth profile control technology is gradually applied in low-permeability fractured reservoirs, this paper regards block H of Changqing Oilfield as the research object, referring to the range of its physical parameters and field application data. Three common in-depth profile control agents (PCAs), nanosphere suspension, poly(ethylene glycol) single-phase gel particle (PEG) and cross-linked bulk gel and swelling particle (CBG-SP), are selected to investigate the compatibility between the fractured channels and the PCAs through a series of experiments. The experimental results show that the nanospheres with particle sizes of 100 nm and 300 nm have good injectivity and deep migration ability, which remains the overall core plugging rate at a high level. The residual resistance coefficient of 800 nm nanospheres decreases in a "cliff" manner along the injection direction due to the formation of blockage in the front section, resulting in a very low plugging rate in the rear section. The injection rate is an important parameter that affects the effect of PEG in the fractured channels. When the injection rate is lower than 0.1 mL/min, the plugging ability will be weakened, and if the injection rate is higher than 0.2 mL/min, the core plugging will occur. The appropriate injection rate will promote the better effect of PEG with the plugging rate > 90%. The average plugging rate of CBG-SP in fractured rock core is about 80%, and the overall control and displacement effect is good. Based on the experimental data of PCAs, the optimization criteria of slug configuration and pro-duction parameters are proposed. According to the principle "blocking, controlling and displacing", references are provided for PCAs screening and parameters selection of field implementation.
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OBJECTIVE: Magnetic resonance imaging (MRI) is increasingly used in the diagnosis of fetal malformations. The purpose of this study was to determine the diagnostic value of 1.5T MRI in fetal head and abdominal malformations. METHODS: A total of 132 pregnant women admitted to Shijiazhuang NO.4 Hospital were included and divided into a control group (CG; n=63) and a research group (RG; n=69) according to different prenatal examination methods. Patients in CG were given ultrasound, while those in RG underwent 1.5T MRI examination. The image quality of 1.5T MRI in different amniotic fluid, different gestational weeks with normal amniotic fluid and different fetal positions with normal amniotic fluid were compared. The detection rate and diagnostic value of the two methods were also compared, with the histological and pathological results as the gold standard. RESULTS: The image quality of 1.5T MRI was not affected by different gestational age with normal amniotic fluid, different fetal positions with normal amniotic fluid, or different amniotic fluid, indicating the feasibility of 1.5T MRI in fetal malformation examination. Histopathological diagnosis revealed 39 cases of head and abdominal deformities in CG and 50 cases in RG. Based on the results of ultrasound examination, there were 32 cases of deformities and 31 of non-deformities in CG. In RG, 1.5T MRI revealed 48 malformations and 21 non-malformations. The sensitivity, specificity, accuracy, missed diagnosis and misdiagnosis rates were 82.05%, 75.00%, 79.37%, 17.95% and 25.00% respectively in CG where ultrasonography was performed, and were 96.00%, 94.74%, 95.65%, 4.00% and 5.26% respectively in RG where 1.5T MRI was performed. The data identified significant differences in sensitivity, accuracy and missed diagnosis between RG and CG. CONCLUSION: 1.5T MRI is effective in diagnosing fetal head and abdominal malformations.
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Drug-induced phospholipidosis (DIPLD) represents a big concern for both regulatory authorities and pharmaceutical companies in drug discovery. Many researches pointed out that the negatively charged intralysosomal lipids play an important role in the formation of DIPLD. To better mimic this negatively charged lipid surface, a novel immobilized artificial membrane (IAM) column was prepared via in situ copolymerization of 12-methacryloyl n-dodecylphosphocholine (MDPC) and 12-methacryloyl n-dodecylphosphoric acid (MDPA). By introducing MDPA, the surface of the resulting monolithic column can be maintained negatively charged over a broad pH range. Scanning electron microscopy, elemental analysis and nano-HPLC experiments were carried out to characterize the physicochemical properties and chromatographic performance of the obtained monolithic IAM column. The results of ζ-potential and retention mechanism studies indicate that both hydrophobic and electrostatic interactions contribute greatly to the retention of cation analytes owing to the existence of the negatively charged MDPA under acidic conditions. To better assess the DIPLD potency of drug, the molar ratio between MDPC and MDPA in the monolithic column was carefully optimized. The results show that the poly(MDPC70PA30-co-EDMA) column has the best predictability with only two false-positives (donepezil, flecainide) in qualitative analysis of 61 drugs.
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Enfermedades por Almacenamiento Lisosomal/inducido químicamente , Membranas Artificiales , Preparaciones Farmacéuticas , Fosfolípidos , Química Farmacéutica , Cromatografía Líquida de Alta Presión , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/prevención & control , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Microscopía Electrónica de Rastreo , Preparaciones Farmacéuticas/análisis , Preparaciones Farmacéuticas/química , Preparaciones Farmacéuticas/metabolismo , Ácidos Fosfatidicos , Fosfolípidos/química , Fosfolípidos/metabolismo , Electricidad EstáticaRESUMEN
In this study, a novel at-line nanofractionation screening platform was successfully developed for the rapid screening and identification of α-glucosidase inhibitors from natural products. A time-course bioassay based on high density well-plates was performed in parallel with high resolution mass spectrometry (MS), providing a straightforward and rapid procedure to simultaneously obtain chemical and biological information of active compounds. Through multiple nanofractionations into the same well-plate and comparisons of the orthogonal separation results of hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC), the α-glucosidase inhibitors can be accurately identified from co-eluates. The screening platform was comprehensively evaluated and validated, and was applied to the screenings of green tea polyphenols and Ginkgo folium flavonoids. After accurate peak shape and retention time matching between the bioactivity chromatograms and MS chromatograms, ten α-glucosidase inhibitors were successfully screened out and identified. The proposed screening method is rapid, effective and can avoid ignoring low abundant/active inhibitors.
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Productos Biológicos/química , Técnicas de Química Analítica/métodos , Inhibidores de Glicósido Hidrolasas/análisis , Cromatografía Liquida , Cromatografía de Fase Inversa , Flavonoides/química , Flavonoides/aislamiento & purificación , Ginkgo biloba/química , Inhibidores de Glicósido Hidrolasas/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Espectrometría de Masas , Polifenoles/química , Polifenoles/aislamiento & purificación , Té/químicaRESUMEN
Oleanolic acid (OA) is a natural compound that can be found in a number of edible and medicinal plants and confers diverse biological actions. However, the direct target of OA in human tumor cells remains poorly understood, preventing its application in clinical and health settings. A previous study revealed that overexpression of caveolin-1 in human leukemia HL-60 cells can increase its sensitivity to OA. The present study aimed to investigate the effects of OA on the doxorubicin-resistant human breast cancer MCF-7 cell line (MCF-7/DOX), harringtonine-resistant human leukemia HL-60 cells (HL-60/HAR) and their corresponding parental cell lines. Western blotting was performed to measure protein expression levels, whilst Cell Counting Kit-8 (CCK-8) assays, cell cycle analysis (by flow cytometry) and apoptosis assays (with Annexin V/PI staining) were used to assess drug sensitivity. CCK-8 assay results suggested that MCF-7/DOX cells, which overexpress the caveolin-1 protein, have similar OA susceptibility to their parent line. In addition, sensitivity of MCF-7/DOX cells to OA was not augmented by knocking down caveolin-1 using RNA interference. HL-60/HAR cells exhibited a four-fold increased sensitivity to OA compared with that in their parental HL-60 cells according to CCK-8 assay. Both of the resistant cell lines exhibited higher numbers of cells at G1 phase arrest compared with those in their parent lines, as measured via flow cytometry. Treatment of both MCF-7 cell lines with 100 µM OA for 48 h induced apoptosis, with increased effects observed in resistant cells. However, no PARP-1 or caspase-3 cleavage was observed, with some positive Annexin V staining found after HL-60/HAR cells were treated with OA, suggesting that cell death occurred via non-classical apoptosis or through other cell death pathways. It was found that OA was not a substrate of ATP-binding cassette subfamily B member 1 (ABCB1) in drug-resistant cells, as indicated by the accumulation of rhodamine 123 assessed using flow cytometry. However, protein expression of ABCB1 in both of the resistant cell lines was significantly decreased after treatment with OA in a concentration-dependent manner. Collectively, these results suggest that OA could reduce ABCB1 protein expression and induce G1 phase arrest in multidrug-resistant cancer cells. These findings highlight the potential of OA for cancer therapy.