The visualization of the spatial distribution of Cocculus orbiculatus based on air flow-assisted desorption electrospray ionization mass spectrometry imaging.

Cocculus orbiculatus (C. orbiculatus), the root of plants belonging to the Menispermaceae family, has been extensively used to treat various diseases, including malaria and rheumatism. The main chemicals in these plants are alkaloids; however, the spatial distribution of these compounds within the plant roots remains undefined. This study aimed to visualize the spatial distribution of C. orbiculatus using air flow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI). In total, the spatial distribution of four aporphine alkaloids, five benzyltetrahydroisoquinoline alkaloids, six bisbenzylisoquinoline alkaloids, and one morphinane alkaloid in the cork layer, xylem, and ray of the root of C. orbiculatus was observed; the distribution characteristics of the different compounds in C. orbiculatus were significantly different. This study provides a visualized spatial distribution analysis method for the characterization of metabolites in the root tissue of C. orbiculatus and also provides valuable information for the specificity of the root of C. orbiculatus, which is beneficial for understanding its chemical separation, biosynthesis, and pharmacological activities.

Tumor microenvironment-mediated NIR-I-to-NIR-II transformation of Au self-assembly for theranostics.

The misdiagnosis of tumors due to insufficient penetration depth or signal interference and damage to normal tissues due to indiscriminate treatment are the biggest challenges in using photothermal agents for clinical translation. To overcome these limitations, a strategy of switching from the near-infrared (NIR)-I region to the NIR-II region was developed based on tumor microenvironment (TME)-mediated gold (Au) self-assembly. Using zeolitic imidazolate framework-8 (ZIF-8) metal-organic framework-coated gold nanorods (AuNRs@ZIF-8) as a model photothermal agent, we demonstrated that only a NIR-I photoacoustic imaging signal was observed in normal tissue because ZIF-8 could prevent the aggregation of AuNRs. However, when ZIF-8 dissociated in the TME, the AuNRs aggregated to activate NIR-II photoacoustic imaging and attenuate the NIR-I signal, thereby allowing an accurate diagnosis of tumors based on signal transformation. Notably, TME-activated NIR-II photothermal therapy could also inhibit tumor growth. Therefore, this TME-activated NIR-I-to-NIR-II switching strategy could improve the accuracy of deep-tumor diagnoses and avoid the injury caused by undifferentiated treatment. STATEMENT OF SIGNIFICANCE: Photothermal agents used for photoacoustic imaging and photothermal therapy have garnered great attention for tumor theranostics. However, always "turned on" near-infrared (NIR)-I laser (700-1000 nm)-responsive photothermal agents face issues of penetration depth and damage to normal tissues. In contrast, tumor microenvironment-activated NIR-II "smart" photothermal agents exhibit deeper penetration depth and tumor selectivity. Therefore, a NIR-I-to-NIR-II switching strategy was developed based on tumor microenvironment-mediated Au self-assembly. This work provides a new strategy for developing tumor microenvironment-activated NIR-II smart photothermal agents.

A Systematic Method for the Identification of Aporphine Alkaloid Constituents in Sabia schumanniana Diels Using UHPLC-Q-Exactive Orbitrap/Mass Spectrometry

Sabia schumanniana Diels (SSD) is a plant whose stems are used in traditional folk medicine for the treatment of lumbago and arthralgia. Previous studies have revealed chemical constituents of SSD, including triterpenoids and aporphine alkaloids. Aporphine alkaloids contain a variety of active components, which might facilitate the effective treatment of lumbago and arthralgia. However, only 5-oxoaporphine (fuseine) has been discovered in SSD to date. In this study, we sought to systematically identify the aporphine alkaloids in SSD. We established a fast and reliable method for the detection and identification of these aporphine alkaloids based on ultra-high-performance liquid chromatography (UHPLC)-Q-Exactive-Orbitrap/mass spectrometry combined with parallel reaction monitoring (PRM). We separated all of the analyzed samples using a Thermo Scientific Hypersil GOLD™ aQ C18 column (100 mm × 2.1 mm, 1.9 µm). Finally, we identified a total of 70 compounds by using data such as retention times and diagnostic ions. No fewer than 69 of these SSD aporphine alkaloids have been reported here for the first time. These findings may assist in future studies concerning this plant and will ultimately contribute to the research and development of new drugs.

Screening and characterization estrogen receptor ligands from Arnebia euchroma (Royle) Johnst. via affinity ultrafiltration LC-MS and molecular docking.

Arnebiae Radix (dried root of Arnebia euchroma (Royle) Johnst.) is a traditional Chinese medicine (TCM) used to treat macular eruptions, measles, sore throat, carbuncles, burns, skin ulcers, and inflammations. The Arnebiae Radix extract can exert anti-breast cancer effects through various mechanisms of action. This study aimed to rapidly screen potential estrogen receptor (estrogen receptor α and estrogen receptor ß) ligands from the Arnebiae Radix extract. In this study, an analytical method based on affinity ultrafiltration coupled with UHPLC-Q-Exactive Orbitrap mass spectrometry was established for rapidly screening and identifying estrogen receptor ligands. Then, bindings of the components to the active site of estrogen receptor (estrogen receptor α and estrogen receptor ß) were investigated via molecular docking. Moreover, surface plasmon resonance (SPR) experiments with six compounds were performed to verify the affinity. As a result, a total of 21 ligands were screened from Arnebiae Radix using affinity ultrafiltration. Among them, 14 and 10 compounds from Arnebiae Radix showed affinity with estrogen receptor α and estrogen receptor ß, respectively. All of those ligands could have a good affinity for the multiple amino acid residues of the estrogen receptor based on molecular docking. In addition, six compounds display the great affinity by SPR. The method established in the study could be used to rapidly screen estrogen receptor ligands in Traditional Chinese medicine. The results demonstrated that the affinity ultrafiltration-UHPLC-Q-Exactive Orbitrap mass spectrometry method not only aids in the interpretation of the potential bioactive components and possible mechanisms of action of Arnebiae Radix but also provides a further effective basis for the quality control of this valuable herb medicine.

Qualitative Analysis of Multiple Phytochemical Compounds in Tojapride Based on UHPLC Q-Exactive Orbitrap Mass Spectrometry.

Tojapride is composed of Caulis Perillae, Rhizoma Cyperi, Radix Glycyrrhizae, Citrus aurantium L., Coptis chinensis Franch, Pericarpium Citri Reticulatae, Reynoutria japonica Houtt, Tetradium ruticarpum, and Cleistocactus sepium. It has the effects of inhibiting gastric acid and relieving pain. It is clinically used for treating gastroesophageal reflux disease. To further study the pharmacodynamic properties of Tojapride, the systematic characterization of the chemical constituents in Tojapride was investigated using ultra-performance liquid chromatography with Q-Exactive Orbitrap mass spectrometry combined with parallel reaction monitoring for the first time. Eventually, a total of 222 compounds, including flavonoids, alkaloids, and glycyrrhizic acid derivatives, were identified based on the chromatographic retention times, MS/MS2 information, and bibliography data; a total of 218 of these were reported for the first time as being present in Tojapride. This newly developed approach provides a powerful tool for extending our understanding of chemical constituents of Tojapride, which can be further extended to other TCMP composition research.

Identification and Quantification of Chlorogenic Acids from the Root Bark of Acanthopanax gracilistylus by UHPLC-Q-Exactive Orbitrap Mass Spectrometry.

The purpose of this study is to identify and quantify the chlorogenic acids (CGAs) from the root bark of Acanthopanax gracilistylus, which is conventionally regarded as a tonic in folk Chinese Traditional medicine. The effective methods for identification and quantification analysis of CGAs were developed based on ultra high performance liquid chromatography-Q-exactive orbitrap mass spectrometry (UHPLC-Q-Orbitrap MS) in parallel reaction monitoring (PRM) and selected reaction monitoring (SIM), which showed high sensitivity and resolution for screening and quantifying compounds. The root bark of A. gracilistylus was extracted under ultrasonication with 70% methanol. Ultimately, a for total of 70 CGAs, 64 of these were tentatively identified for the first time. Moreover, a methodological study of seven kinds of CGAs was carried out. The proposed procedure was optimized and validated in terms of selectivity, linearity of analytical curves (r 2 > 0.990), accuracy (recovery range from 96.7 to 105%), and repeatability (relative standard deviation <5%). Then it was applied to determine the content of the CGAs in A. gracilistylus roots from 66 of different batches. The total CGAs was quantified in a range between 2.150 and 33.51 mg/g, which could be considered as excellent source of natural bioactive compound. The result was extremely useful for understanding the bioactive substance and quality control of A. gracilistylus in depth.

The Application of UHPLC-HRMS for Quality Control of Traditional Chinese Medicine.

UHPLC-HRMS (ultra-high-performance liquid chromatography-high resolution mass spectrometry) is a new technique that unifies the application of UHPLC with HRMS. Because of the high sensitivity and good separation ability of UHPLC and the sensitivity of HRMS, this technique has been widely used for structure identification, quantitative determination, fingerprint analysis, and elucidation of the mechanisms of action of traditional Chinese medicines (TCMs) in recent years. This review mainly outlines the advantages of using UHPLC-HRMS and provides a survey of the research advances on UHPLC-HRMS for the quality control of TCMs.

Systematic Screening of Chemical Constituents in the Traditional Chinese Medicine Arnebiae Radix by UHPLC-Q-Exactive Orbitrap Mass Spectrometry.

Arnebiae Radix (dried root of Arnebia euchroma (Royle) Johnst.) has been used in traditional Chinese medicine (TCM) to treat macular eruptions, measles, sore throat, carbuncles, burns, skin ulcers, and inflammation. Previous studies have shown that shikonins and shikonofurans are two of their main bioactive ingredients. However, systematic investigations of their constituents have rarely been conducted. It is necessary to establish a rapid and effective method to identify the chemical constituents of Arnebiae Radix. This will help to further improve the effective resource utilization rate of this plant. In this study, a rapid and effective UHPLC-Q-Exactive Orbitrap mass spectrometry method was established to simultaneously analyze chemical ingredients in Arnebiae Radix within a short period of time. Based on the results of a full scan MS, the MS2 database (mzVault and mzCloud), the diagnostic fragment ions, the retention time, and the bibliography, a total of 188 compounds were identified, with 114 of those being reported from Arnebiae Radix for the first time. The results of this study lay the foundation for obtaining a thorough understanding of the active ingredients in Arnebiae Radix and its quality control. This method may be widely used for the chemical characterization of different samples.

Chemical characterization of extracts of leaves of Kadsua coccinea (Lem.) A.C. Sm. by UHPLC-Q-Exactive Orbitrap Mass spectrometry and assessment of their antioxidant and anti-inflammatory activities.

Kadsua coccinea (K. coccinea) has long been used as a fruit and folk medicine; however, the composition of its leaves and the activities of its constituents have been seldom studied. A total of 98 chemical constituents, including 53 phenolic acids, 41 flavonoids, and 4 lignans, were identified from the plant of kadsua coccinea by UHPLC-Q-Exactive Orbitrap Mass spectrometry. All these chemicals were reported for the first time in leaves, and 95 of them have been reported for the first time in the plant of kadsua coccinea. The biological potential of extracts of K. coccinea leaves (EKL) was evaluated by in vitro antioxidant assay and anti-inflammatory assay. EKL are composed of polysaccharides (60%), polyphenols (26%), and proteins (11%). EKL present decent potent •OH and DPPH scavenging abilities and Fe2+ chelating ability. They also inhibit the secretion of NO, reduce the level of Cox2 in proteins, inhibit the secretion of pro-inflammatory cytokines, such as IL-2 and IL-6, and promote the secretion of anti-inflammatory cytokine IL-10. These results displayed significant antioxidant and anti-inflammatory activities of EKL, which will be very beneficial for further development and investigation of kadsua coccinea leaves.

Diagnostic Fragment-Ion-Based for Rapid Identification of Chlorogenic Acids Derivatives in Inula cappa Using UHPLC-Q-Exactive Orbitrap Mass Spectrometry.

Inula cappa (Buch.-Ham. ex D. Don) DC has been used in traditional Chinese medicine to treat malaria, dysentery, and hepatitis. Previous studies have shown that chlorogenic acid is the effective ingredient of plants in this family. And the research of the chlorogenic acid in Inula cappa will help to further improve the effective resource utilization rate of this plant. Therefore, it is necessary to establish an accurate method to characterize the chlorogenic acid components in Inula cappa. In this study, a simple, fast, and sensitive UHPLC-Q-Exactive Orbitrap mass spectrometry method was established, which can simultaneously analyze known and unknown ingredients in a short time (within 30 minutes) in Inula cappa. According to the diagnosis fragmentation ions, retention time, and bibliography, 68 chlorogenic acid derivatives were identified in Inula cappa. The results of this experiment lay the foundation for the active substances and quality control of Inula cappa and provide a theoretical basis for whether Inula cappa can be an alternative to the endangered wild medicinal materials of the same family.

Rapid Identification and Systematic Mechanism of Flavonoids from Potentilla freyniana Bornm. Based on UHPLC-Q-Exactive Orbitrap Mass Spectrometry and Network Pharmacology.

Potentilla freyniana Bornm. (P. freyniana), belonging to the family Rosaceae, has been used as a folk medicine in China. However, as we know, the constituents and the systematic elucidation of the mechanism were not fully investigated. Therefore, it is necessary to develop a rapid method using LC-MS and network pharmacology for the detection and identification of constituents and the systematic mechanism of P. freyniana. Firstly, the flavonoids were detected and identified based on ultra-high-performance liquid chromatography coupled with Quadrupole-Exactive Focus Orbitrap MS (UHPLC-Q-Exactive Orbitrap MS). After that, the potential targets of those constituents were obtained by database mining. Then, the core targets were predicted by protein-protein interaction network and network analysis. Finally, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were carried out via DAVID. This finding revealed that P. freyniana possessed 43 flavonoids (40 of them were first reported) with 23 core target genes, which are associated with PI3K-Akt, MAPK, TNF signaling pathway, and pathway in cancer. This study demonstrated the multicompound, multitarget, and multimechanism of P. freyniana, which are very beneficial to develop the further study and utilization of this plant including the material basis and quality control research.

Identification of the metabolites of isochlorogenic acid A in rats by UHPLC-Q-Exactive Orbitrap MS.

CONTENT: Isochlorogenic acid A, one of the main components of Duhaldea nervosa (Wallich ex Candolle) A. Anderberg (Asteraceae), is a folk medicine used to treat a variety of diseases including fracture and rheumatoid arthritis. Despite its widespread use, the metabolism of isochlorogenic acid A in vivo has not been fully studied. OBJECTIVE: An analytical strategy based on UHPLC-Q-Exactive Orbitrap MS is proposed for the detection and identification of the metabolites of isochlorogenic acid A in rats. MATERIALS AND METHODS: Six male Sprague-Dawley rats (180 ± 20 g) were randomly divided into two groups. Then, blood and tissue samples were obtained after oral administration of isochlorogenic acid A (200 mg/kg). All the samples were pre-treated by the Solid Phase Extraction (SPE) method. Next, the samples were analysed by UHPLC-Q-Exactive Orbitrap MS. Finally, the metabolites were identified based on the metabolomic workflow template. RESULTS: A total of 33 metabolites were identified in rat plasma, with 30 of them being reported for the first time. The distribution of all metabolites in tissues was first investigated, three of them were widely distributed in liver, lungs, and kidneys. The corresponding reactions including methylation, hydrolysis, sulphate conjugation, glucuronide conjugation, as well as their composite reactions, are reported in this study. DISCUSSION AND CONCLUSIONS: This method has wide-scale application prospects in the identification of metabolites. Considering that limited research has been conducted in this area, this study proposes metabolic pathways to further understand mechanisms of isochlorogenic acid A and the forms that are truly effective in vivo.

Paris polyphylla ethanol extract induces G2/M arrest and suppresses migration and invasion in bladder cancer.

BACKGROUND: Paris polyphylla is a traditional Chinese medicinal herb with multiple antitumor activities, but the role of P. polyphylla in bladder cancer (BC) is under investigation. This study aims to examine the antitumor activities of P. polyphylla ethanol extract (PPE) on BC cells and elucidate the underlying mechanisms. METHODS: Viable cells were counted using the trypan blue exclusion assay. The cell cycle was analyzed using flow cytometry, and scratch wound-healing and transwell assays were used to evaluate cell migration and invasion abilities, respectively. The protein expression levels were determined by western blotting. A xenograft model was used to assess the in vivo inhibitory effect of PPE on BC tumor growth. RESULTS: Our results showed that PPE inhibited the growth of BC cells in vivo and in vitro. Mechanistically, PPE regulated the levels of cell cycle-associated proteins, with PPE-induced G2/M phase arrest occurring through cyclin-dependent kinase inhibitor 1 (CDKN1A) accumulation and cyclin B1 (CCNB1)/cyclin-dependent kinase 1 (CDK1) inhibition. BC tumor growth was also inhibited by PPE treatment. Moreover, the migration and invasion abilities of J82 cells were suppressed through modulating epithelial-mesenchymal transition (EMT) regulatory factors with upregulation of cadherin-1 (CDH1) and downregulation of cadherin-2 (CDH2), snail family transcriptional repressor 2 (SNAI2), and twist family bHLH transcription factor 1 (TWIST1). CONCLUSIONS: PPE inhibited cell growth, induced G2/M arrest, and suppressed the migration and invasion of J82 cells. BC tumor growth in vivo was also inhibited by PPE. Our results lay the foundation for further studies on the antitumor mechanisms of PPE.

Platelet releasate promotes breast cancer growth and angiogenesis via VEGF-integrin cooperative signalling.

BACKGROUND: Selective platelet release of pro- or anti-angiogenic factors distinctly regulated angiogenesis. We hypothesised that selective release of platelet angiogenic factors could differently regulate tumour growth. METHODS: Breast cancer cell proliferation, cancer cell-induced endothelial tube formation in vitro, and tumour growth in vivo were studied in the presence of protease-activated receptor 1-stimulated platelet releasate (PAR1-PR; rich in pro-angiogenic factors) or PAR4-PR (rich in anti-angiogenic factors). RESULTS: The PAR1-PR and PAR4-PR supplementation (10%) similarly enhanced cell proliferation of MCF-7 and MDA-MB-231 breast cancer cells. The cancer cells triggered capillary-like tube formation of endothelial cells that was further enhanced by pro-angiogenic factor-rich PAR1-PR. The VEGF, but not SDF-1α, receptor blockade abolished PAR1-PR/PAR4-PR-enhanced cancer cell proliferation. Integrin blockade by RGDS had identical effects as VEGF inhibition. The Src and ERK inhibition diminished, whereas PI3K and PKC blockade abolished platelet releasate-enhanced cancer cell proliferation. Using a model of subcutaneous implantation of MDA-MB-231 cells in nude mice, PAR1-PR enhanced tumour growth more markedly than PAR4-PR, and seemed to achieve the exaggeration by promoting more profound tumour angiogenesis. CONCLUSIONS: Platelet releasate increases breast cancer cell proliferation through VEGF-integrin cooperative signalling. Pro-angiogenic factor-rich platelet releasate enhances cancer cell-induced angiogenesis more markedly, and thus exaggerates tumour growth in vivo.

Kisspeptin permits the sexual development of female rats with normal and precocious puberty but is not a trigger for it.

OBJECTIVE: We inferred how KISS-1/GPR54 system to involved in precocious puberty by observing hormones level during the process of precocious puberty in model and normal rats during sexual development and the estrus cycle. METHOD: Female rats were divided randomly into CPP and control groups; the former were injected with NMDA twice daily, and control groups were injected with saline. Blood and tissue samples were collected and measured during the stages of prepuberty, vaginal opening, estrus, proestrus and diestrus. RESULTS: The times of onset of puberty and sexual maturity in the CPP group were significantly earlier than in the control groups. Hypothalamic levels of KISS-1 and GPR54 gene expression, kisspeptin, luteinizing hormone, and follicle stimulating hormone started to rise before puberty. In stable estrus cycles, kisspeptin levels were the lowest during proestrus, while gonadotropin-releasing hormone (GnRH) levels rose to the highest during estrus. GnRH levels increased significantly in the estrus cycle compared with the prepubertal stage, but kisspeptin levels did not change significantly. CONCLUSION: the hypothalamic KISS-1/GPR54 system might permit the onset of puberty, but is not its primary trigger. Hormone levels were lower and gonadal maturity markers in the CPP groups were worse than in the control groups.

Characterization and comparative analyses of two amphioxus intelectins involved in the innate immune response.

Intelectin is a new type of soluble galactofuranose-binding lectin involved in innate immunity. Here we report another intelectin homolog, AmphiITLN239631, obtained from amphioxus, the transitional form between vertebrates and invertebrates. AmphiITLN239631 encoded 396 amino acids with a highly conserved fibrinogen-related domain (FReD), An intelectin domain and a putative Collagen domain. AmphiITLN239631 was ubiquitously expressed in all tissues we tested and transcripts in skin increased after challenge of both Escherichia coli and Staphylococcus aureus, although in different levels. Recombinant AmphiITLN239631 expressed in E. coli system could agglutinate both Gram-positive and Gram-negative bacteria in a calcium independent manner. Furthermore, recombinant protein was able to bind to lipopolysaccharide (LPS) and peptidoglycan (PGN), the major components of Gram-positive and Gram-negative bacteria cell walls, respectively. We also compared AmphiITLN239631 with previously identified AmphiITLN71469 and found that their tissue specificities, expression patterns upon bacteria challenge, and polysaccharide-binding affinities etc vary considerably. Our results could provide insight into the evolution and function of the intelectin family.