Journal of pharmaceutical and biomedical analysis最新文献

Saposhnikovia divaricata (SD) is a traditional Chinese medicine (TCM) which has been commonly used for the treatment of rheumatoid arthritis (RA). However, its active components and mechanism of anti-RA are still unclear. Targeting rheumatoid arthritis-fibroblastoid synovial (RA-FLS) and synovial macrophages are promising strategies for RA treatment, and their membrane receptors are important targets for anti-RA active substances. A dual channel 3-mercaptopropyltrimethoxysilane (MPTS) modified 2D cell membrane chromatography (CMC) system was established to characterize dual-cell membrane binding active components in SD. Nine components retained on RAW-CMC column and 8 components retained on FLS-CMC column were screened out. Among them, 8 components retained well on both CMC columns. We further validate the pharmacological activity of 5-O-methylvisammioside, 3'-O-angeloylhamaudol, imperatorin, phellopterin and anomalin. They could efficiently target both inflammatory macrophages and fibroblast synovial cells, reduce the release of inflammatory factors, inhibit abnormal cell proliferation, and promote cell apoptosis. 5-O-methylvisammioside, which exhibited the best pharmacological ability on both target cells, inhibited the NF-κB pathway. Our results showed that the dual channel MPTS modified 2D CMC system is a practical method for rapid screening the active components in TCM binding on multiple target cells' membrane protein of a disease. The method is very suitable for elucidating the mechanism of TCM with multiple-components and targets, and rapid screening of lead compounds.

Hura crepitans (Euphorbiaceae), is widespread in the Amazon rainforest and on plantations in sub-Saharan Africa. This tree produces an irritating milky latex rich in secondary metabolites, notably daphnane-type diterpenes and cerebrosides. Previous studies have shown that huratoxin, the main daphnane in the latex, significantly and selectively inhibited the growth of colorectal cancer cells through a unique mechanism involving the activation of PKCζ. One major challenge in isolating active molecules from natural products is the accessibility of the resource. This study explores the phytochemical composition and cytotoxic activities of latexes collected in Peru, Benin, and Togo using UHPLC-MS and metabolomics tools to identify a renewable source of bioactive compounds. Significant inter- and intra-continental differences in chemical composition have been highlighted, with daphnanes being concentrated in the Peruvian samples. Extracts form latexes collected in Peru showed cytostatic activity on Caco-2 cells, correlated with the presence of daphnanes, while some African samples exhibited cytotoxic activity on Jurkat and Hela cancer cell lines, leading to the identification of potential other new bioactive compounds such as elasterol and cerebrosides. OBJECTIVE: To compare the composition of different Hura crepitans latex samples and determine their cytotoxic activity in order to identify new bioactive compounds CONCLUSIONS: Inter- and intra-continental variations in the phytochemical composition of latex were observed, leading to significant cytotoxic activities on different cell lines. Daphnanes were identified as responsible for the activity on Caco-2 cells, while elasterol and cerebrosides were putatively associated with the activity on Hela cells.

Qingwen Zhike prescription (QWZK), a traditional Chinese medicine (TCM) hospital prescription developed in response to the corona virus disease 2019 (COVID-19) pandemic, has demonstrated efficacy in clinical practice. Nevertheless, its specific antiviral components and mechanisms of action remain unclear. This study screened the antiviral compounds against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from Qingwen Zhike prescription and explored the underlying mechanism through chemical composition analysis, serum and lung exposure profiles analysis, high-throughput screening, and transmission electron microscopy (TEM) observation. Utilizing the UPLC-Q-Exactive Orbitrap MS system, a total of 279 components were identified from Qingwen Zhike. Among these, 49 components were detected in the serum and lungs of dosed rat, with 26 components distributed abundantly in the lungs. Subsequently, a SARS-CoV-2 pseudovirus-based assay and a main protease (M^pro) enzymatic assay were used to screen for viral entry inhibitors and M^pro inhibitors. The results showed that two alkaloids (ephedrine and pseudoephedrine) and five polymethoxy-flavonoids (3,5,6,7,8,3',4'-heptamethoxyflavone, nobiletin, isosinensetin, tangeretin, and sinensetin) exhibited potent inhibitory effects on viral invasion. Further observation by TEM indicated that these two alkaloids could dissolve the viral envelope, while these five polymethoxy-flavonoids could cause leakage of virus contents, deformation of viral envelope or decomposition of the virus. Collectively, these seven compounds may serve as key antiviral components of QWZK.

Controlling the spread of bacterial infectious diseases is a major public health issue, particularly in view of the pandemic of bacterial resistance to antibiotics. In this context, the detection and identification of pathogenic bacteria is a prerequisite for the implementation of control measures. Current reference methods are mainly based on culture methods, which generate a delay in obtaining a result and requires equipment. Consequently, focusing on the detection of the whole bacterium represents a very attractive alternative, since no culture is required. Several techniques have already been deployed to identify whole-cell bacteria. In recent decades, growing interest in nucleic acid aptamers has emerged as a viable alternative to antibodies as recognition elements, offering preferable stability, cost-efficiency, good specificity and affinity. This review explores current alternative methods for the detection of whole-cell bacteria, with particular emphasis on aptamer-based assays. These assays have shown promising results in various transduction mechanisms, including optical, electrochemical, and mechanical approaches, enhancing their versatility in different diagnostic platforms. The integration of aptamers in these detection methods offers rapid, sensitive, versatile and portable solutions for pathogen identification, positioning them as valuable tools in the fight against bacterial infections.

The low bioavailability of insoluble flavonoids in the total flavonoids of Epimedium brevicornu Maxim. (TFE) severely hindered its clinical efficacy exertion. This research attempted to evaluate the promoting effects of pharmaceutical strategies, including nanosuspensions (NS), cyclodextrin inclusion complexes (CD), and solid dispersions (SD), on the oral absorption of active components in TFE. A rapid ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was established to quantify ten pentenyl flavonoids of TFE in rat plasma. Good linearity was presented within the expected ranges (0.1 ∼ 10 ng/mL) in the calibration curves for ten analytes with an acceptable intra- and inter-day precision and accuracy of < 11.34 % and ± 11.91 %, respectively. By employing this selective UPLC-MS/MS method, the full-scale concentration-time curve for icariin (ICA), icariin II (ICA II), and epimedin C (EPI C) were drawn after oral administration of the crude TFE and its formulations. The results showed that the relative bioavailability (F_rel) of ICA and ICA II in the NS and CD formulations were 228-295 % when the crude TFE was as a reference, whereas the F_rel of ICA, ICA II, and EPI C in SD formulation were 416 %, 234 %, and 112 %, respectively. The findings suggest that SD technology holds significant promise for enhancing the oral bioavailability of various poorly soluble ingredients in herbal extracts, such as TFE, and for augmenting their therapeutic capabilities in clinical practice.

Shenhua compound, composed of ginseng, hawthorn and sophora flower, has been shown to improve hyperlipidemia. However, the main ingredients, their metabolic pathways in vivo, and pharmacokinetic characteristics. In this study, ultra-high-performance liquid chromatography coupled with electrospray ionization quadruple time-of-flight mass spectrometry (UHPLC-Q-TOF) was used to qualitatively analyze the main ingredients in ethanol extract of Shenhua compound and to investigate the metabolites in serum, bile, feces, and urine of rats following oral administration. The pharmacokinetics of ginsenoside Rg₁, ginsenoside Re, ginsenoside Ro and rutin in rats were analyzed using triple-quadrupole liquid chromatography combined with electrospray ionization mass spectrometry (QQQ-LC/MS). The results indicated that 48 compounds were present in Shenhua compound, including saponins, flavonoids and organic acids. Metabolites were comprehensively analyzed after oral administration of Shenhua compound, and 24 prototype ingredients and 92 metabolite ingredients were identified or characterized. By analyzing the pharmacokinetic parameters of ginsenoside Rg₁, ginsenoside Re, ginsenoside Ro and rutin from 0 to 72 h after oral administration of various dose of Shenhua compound, it was observed that the concentration of ginsenosides in blood remained below 2 ng∙mL¹, but the metabolic excretion time was prolonged. Meanwhile, the blood concentration of rutin was significantly higher than ginsenosides and showed a double absorption peak. In conclusion, this study provides valuable insights into compound ingredients metabolism regularities in vivo and pharmacokinetics after oral administration of Shenhua compound.

A simple, fast, sample-saving, and sensitive liquid chromatography-tandem mass spectrometry method was established with a linear range adjusted by in-source collision-induced dissociation. Notably, this could simultaneously determine busulfan, fludarabine, phenytoin, and posaconazole in plasma from children, each having unique physical and chemical properties. The procedure necessitated only 20 μL of plasma and involved a simple protein precipitation process. Chromatographic separation was accomplished on a reversed-phase column (C18, 50 × 2.1 mm, 2.6 μm) through gradient elution utilizing water (containing 0.1 % formic acid and 2 mM ammonium acetate) and acetonitrile (containing 0.1 % formic acid) as the mobile phase. An injection volume of 2 μL was utilized, with a total run time of 3.6 min. Mass spectrum acquisition was performed on a Triple Quad™ 4500MD tandem mass spectrometer with an electrospray ionization source in positive mode. Moreover, in-source collision-induced dissociation was used to adjust the linear range of phenytoin due to its excessive response. The calibration curves ranged from 20 to 2560 ng/mL for busulfan, 10-1280 ng/mL for fludarabine, 0.4-51.2 μg/mL for phenytoin, and 0.1-12.8 μg/mL for posaconazole, with mean r² greater than 0.997. In addition, the method underwent rigorous validation following the European Medicines Agency guidelines, demonstrating exceptional accuracy (90.5 %-106.7 %) and precision (2.0 %-13.0 %). Furthermore, its applicability to atypical matrices, including hemolytic and hyperlipidemic plasma, was thoroughly assessed. As such, this approach was effectively utilized for the therapeutic drug monitoring of busulfan, fludarabine, phenytoin, and posaconazole for children undergoing hematopoietic stem cell transplantation.

Alkaptonuria (AKU) is a rare autosomal-recessive disease which is characterized through black urine and ochronosis. It is caused by deficiency of the enzyme Homogentisate 1,2-dioxygenase in the Phenylalanine/Tyrosine degradation pathway which leads to the accumulation of Homogentisic acid (HGA). Urine was provided by AKU patients and healthy controls. Several different methods were developed in this study each with a specific goal. Firstly, a simple and inexpensive RP-UHPLC-UV method for routine monitoring of HGA as a key metabolite employing a Phenylhexyl stationary phase chemistry. Validation was performed in accordance to FDA guidelines and method selectivity was further evaluated via on-line high-resolution sampling 2D-LC-QToF-MS, coupling the Phenylhexyl phase in the first dimension with a C18 phase in the second dimension. Secondly, a targeted and accurate RP-UHPLC-MRM-QTRAP assay, providing quantitative analysis of the relevant pathway metabolites based on a Phenylhexyl stationary phase, and lastly an untargeted HILIC-UHPLC-QToF-MS/MS method with SWATH (sequential window acquisition of all theoretical mass spectra) acquisition employing a sulfobetaine-type HILIC-Z superficially porous particle column, with the aim of uncovering more details about the metabolic profile of this genetic disorder. By untargeted analysis 204 metabolites could be detected and annotated in positive and negative ESI mode in total. Two separate LC methods were employed, differing in their conditions depending on the ionization mode (20 mM ammonium formate as buffer additive adjusted to a pH = 3.5 with formic acid in ESI⁺ mode and 20 mM ammonium acetate adjusted to a pH = 7.5 with acetic acid in ESI^- mode). By effectively combining the aforementioned methods, a comprehensive workflow was developed, allowing the effective analysis of both patient and control urine samples.

Background: The incidence of Parkinson's disease (PD) increases with age. Previous pharmacological studies have shown the potential of Huatan Jieyu Granules (HGs) for the treatment of PD, but the exact mechanisms remain unclear. This study aimed to explore the effects of herbal treatment on PD using mouse models and single-cell sequencing.

Methods: In this study, we established in vivo 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD models in mice. Motor function was assessed through behavioral tests. Immunofluorescence was used to examine dopaminergic neuron loss. Single-cell sequencing was performed on mice from the blank, PD model and medication groups. After quality control and dimensionality reduction of the single-cell data, cells were clustered, and different cell types were identified. We then identified the intersection of differentially expressed genes (DEGs1) in the blank and model groups and DEGs2 in the model and medication groups, yielding intersected DEGs. Key drug targets were identified by intersecting these DEGs with the drug targets of active ingredients in TCM. Topological analysis of the PPI network was used to identify key genes. Cell types exhibiting high expression of these genes were designated as key cells. These key cells were subjected to cellular communication analysis and temporal analysis, after which they were classified into subtypes.

Results: HGs significantly improved motor function and prevented dopaminergic neuronal loss in the substantia nigra (SN) of MPTP-treated mice. A total of 34 cell clusters were delineated, with 9 cell types identified, including oligodendrocytes (oligo), neurons, and T cells. We identified 758 intersected DEGs and 13 key drug targets, including Egfr, Ntrk2, Grm5, Htr2c, Bcl2l1. Oligo and neuronal cells were identified as key cells due to higher expression levels of these key genes. In the cellular communication analysis, oligo-neuronal interactions in the blank and model groups, and oligo-OPC and oligo-T cell interactions in the medication group, exhibited the most receptor-ligand interactions. In temporal analysis, both oligo and neuronal cells were differentiated into 9 states, with C1 being the most differentiated.

Conclusion: HGs demonstrate neuroprotective effects in MPTP-treated mice. Using single-cell sequencing, we identified five key genes (Egfr, Ntrk2, Grm5, Htr2c, Bcl2l1) and two key cell types (oligo and neuronal) related to HGs in PD. These findings provided a foundation for understanding the molecular mechanisms by which HGs treat PD.

Agastache rugosa (AR), a traditional edible and medicinal herb, is often used for treating gastrointestinal (GI) motility disorder. But little effort has been done on its gastrointestinal motility modulation (GMM) efficacy-related components and quality control of AR. In this study, a novel strategy was proposed to find GMM efficacy-related chemical markers for the quality control of AR. Firstly, network pharmacology and serum pharmacochemistry were applied to predict potential GMM efficacy-related marker components. Secondly, the GMM efficacy-related marker components were verified through literature matching, target isolation/identification and activity evaluation. Lastly, a quantitative analysis of multiple components by a single marker (QAMS)-based method for simultaneous quantification of marker components was established and validated by HPLC-DAD. The results showed that nine components in AR were screened as potential GMM related components, five of which (rosmarinic acid, tilianin, apigenin, acacetin, and cirsimaritin) were matched by literatures, and four (acacetin-7-O-(6''-O-malonyl)-β-D-glucopyranoside, agastachoside, acacetin-7-O-(2''-O-acetyl-6''-O-malonyl)-β-D-glucopyranoside, and isoagastachoside) were chemically identified and newly evaluated on zebrafish model. The nine components were used as marker compounds to develop an effective QAMS-based method for the quantitative evaluation of 26 batches of commercial AR samples.