Target protein identification of andrographolide based on isomer approach.

The target identification of natural products is one of the most challenging issues in the standardized application of traditional Chinese medicine. It is widely recognized that magnetic nanoparticles (MNPs) could function as a tool that capture the target proteins of active molecule. However, the false positives caused by non-specific adsorption should not be ignored. Here, we reported a functionalized MNPs technique that could enrich the target proteins of andrographolide (AG) based on isomers approach. We designed and characterized MNPs and isomers of AG. The combination of the two could be used as an ideal coupling, which provides a feasible method for the target proteins enrichment of AG. In addition, the target proteins were identified by HPLC-MS/MS. Moreover, bioinformatics analysis and systematic computational dockings were performed to search for the interactions between target proteins and AG. Six inflammation-related proteins, including CD4, IKBKB, PKN1, PKN2, YWHAB and YWHAH were proved to be the anti-inflammatory targets of AG. All of the results indicated this integrated system could benefit target identification of bioactive natural products.

Biodistribution of andrographolide to assess the interior-exterior relationship between the lung and intestine using microPET.

BACKGROUND: One classic traditional Chinese medicine theory is that the "lung and intestine are exterior-interiorly related"; however, this has not been confirmed experimentally. The aim of this study was to provide a biological basis for the theory by measuring the tissue distribution of andrographolide. METHODS: Acute pneumonia was induced in a mouse model by repeated stimulation with lipopolysaccharide. The distribution of andrographolide in mice was observed by positron emission tomography (PET) imaging with [18 F]-labeled andrographolide, and changes in the in vivo distribution before and after modeling were compared. Subsequently, the consistency of pathological changes in lung and intestine was confirmed by observation of pathological sections. Finally, the results were verified by cytokine detection. RESULTS: The value of organ uptake, pathological changes and inflammatory factor expression of the lung and intestine were consistent. The concentration of andrographolide in the lung and intestine increased significantly, and was confirmed by pathology and enzyme-linked immunosorbent assays (ELISA). CONCLUSIONS: Micro-positron emission tomography (microPET) can be used to visually observe the distribution of medicinal ingredients in vivo, and [18 F]-andrographolide can be used as a tool to assess the interior-exterior relationship between the lung and intestine.

Micro-PET Imaging Demonstrates 3-O-ß-D-Glucopyranosyl Platycodigenin as an Effective Metabolite Affects Permeability of Cell Membrane and Improves Dosimetry of [18F]-Phillygenin in Lung Tissue.

Platycodon grandiflorum, as a traditional medicinal plant, is commonly used in the treatment of pulmonary disease. Platycodon saponins are proposed as active ingredients. However, the role of secondary saponin metabolites (SSM) in the traditional use of Platycodon has not yet been fully clarified. In this study, [18F]-phillygenin ([18F]-PH) probe was synthesized and thereby used as a tracer for micro-positron emission tomography scanning to explore the effects of platycodon saponins. The membrane permeability with different SSM was evaluated in vitro based on the dye-carrying capacity of fluorescein isothiocyanate. The results showed that total platycodon saponins improved the dosimetry of [18F]-PH in the lung tissue, and an SSM named 3-O-ß-D-glucopyranosyl platycodigenin (GPD682) appreciably changed the distribution of drugs both in vitro and in vivo. We propose that GPD682 could be utilized as an important ingredient to help drug delivery to the lung tissue and improve the treatment of respiratory disease.

Efficacy evaluation of Qingyan formulation in a smoking environment and screening of anti-inflammatory compounds.

Qingyan formulation (QF) is a common preparation that is often used to control inflammation in the haze environment. However, the efficacy and effective constituents of QF are still uncertain and difficult to identify. This paper aims to evaluate the efficacy by simulating a haze environment and determine its anti-inflammatory compounds by UPLC/Q-TOF-MS/MS combing with bioactivity screening. The therapeutic effect of QF in the simulated haze environment was confirmed from the aspects of lung histomorphology and inflammatory factor expression levels. QF showed strong anti-inflammatory activity with the minimum effective concentration reaching 1.5 g/kg. Potential anti-inflammatory components were screened by the NF-κB activity assay system and simultaneously identified based on mass spectral data. Then, the potential active compounds were verified by molecular biological methods, the minimum effective concentration can reach 0.1 mg/L. Six structural types of NF-κB inhibitors (phenolic acid, scopolamine, hydroxycinnamic acid, flavonoid, dihydroflavone and steroid) were identified. Further cytokine assays confirmed their potential anti-inflammatory effects of NF-κB inhibitors. This strategy clearly demonstrates that QF has a significant therapeutic effect on respiratory diseases caused by haze, so it is necessary to promote its commercialization and wider application.

An Optimized MicroPET Imaging Method for the Distribution and Synergies of Natural Products.

Purpose: Understanding the distribution and interaction of the Traditional Chinese Medicines (TCMs) is an integral source of herbal drug discovery. An optimized radio-labeled method was explored that could conduct in situ biodistribution studies in animals. We evaluated the feasibility of the method and applied glycyrrhetinic acid and platycodon (PG) polysaccharides as models. Procedures: [18F]-GA is a novel radiotracer which was performed positron emission tomography (PET) studies to assay the biodistribution of GA in mice. In addition, PG polysaccharide was used to intervene the biodistribution and dosimetry of GA. Scanning data were analyzed with professional software. Results: Record the time-activity curves for all organs then use the normalization method to calculate the area under the curve as a dosimetry for each organ. Moreover, the addition of PG polysaccharides can significantly improve the dosimetry of GA in the lungs, and its effect was related to the administration time. Conclusion: MicroPET imaging opens up a new avenue for the application of drug interactions between the TCMs.

Combining UPLC/Q-TOF-MS/MS With Biological Evaluation for NF-κB Inhibitors in Uyghur Medicine Althaea rosea Flowers.

The Althaea rosea (Linn.) flower is a common plant that is often used to control inflammation in Uyghur ethnic medicine. However, its active ingredients remain uncertain and difficult to identify, severely limiting its use as a valuable crop. This paper aims to establish a rapid assay strategy for the integration of ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS/MS) and a biologically active (NF-κB inhibitor) luciferase reporter detection system to explore various anti-inflammatory compounds of A. rosea (Linn.) flowers. Potential anti-inflammatory components were screened using the NF-κB activity assay system and simultaneously identified based on mass spectrometry data. Four structural types of NF-κB inhibitors (phenolic acid, hydroxycinnamic acid, flavonoid, and dihydroflavone) were identified. Further cytokine assays confirmed their potential anti-inflammatory effects as NF-κB inhibitors. Compared with traditional chromatographic separation, integrated UPLC/Q-TOF-MS/MS identification compounds, and biological activity verification are more convenient and more reliable. This strategy clearly demonstrates that fingerprinting based on MS data not only can identify unknown components but also is a powerful and useful tool for screening trace active ingredients directly from complex matrices. A. rosea (Linn.) exhibits great health and pharmaceutical value and may contribute to the development of new anti-inflammatory drugs.

Biodistribution of arctigenin-loaded nanoparticles designed for multimodal imaging.

BACKGROUND: Tracking targets of natural products is one of the most challenging issues in fields ranging from pharmacognosy to biomedicine. It is widely recognized that the biocompatible nanoparticle (NP) could function as a "key" that opens the target "lock". RESULTS: We report a functionalized poly-lysine NP technique that can monitor the target protein of arctigenin (ATG) in vivo non-invasively. The NPs were synthesized, and their morphologies and surface chemical properties were characterized by transmission electron microscopy (TEM), laser particle size analysis and atomic force microscopy (AFM). In addition, we studied the localization of ATG at the level of the cell and the whole animal (zebrafish and mice). We demonstrated that fluorescent NPs could be ideal carriers in the development of a feasible method for target identification. The distributions of the target proteins were found to be consistent with the pharmacological action of ATG at the cellular and whole-organism levels. CONCLUSIONS: The results indicated that functionalized poly-lysine NPs could be valuable in the multimodal imaging of arctigenin.

Active fragments-guided drug discovery and design of selective tropane alkaloids using ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry coupled with virtual calculation and biological evaluation.

Tropane alkaloids (TAs), rich in the plant of Physochlaina infundibularis Kuang, which is named Huashanshen (HSS) in China, showed good effects on types of spasms. However, no data were collected to explore the relationship between the specificity for muscarinic receptor subtypes and the structures of these TAs. To address this issue, an extracted ion chromatogram (EIC) strategy using ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-Q/TOF-MS) based on the fragmentation behavior of the TA standards was established to rapidly capture the varied TAs from HSS. Based on the provided structural information of diagnostic ions or neutral loss, 29 TAs were efficiently profiled, especially some trace ingredients. In additional, via virtual validation combined with molecular dynamic simulation, approximately a dozen alkaloids were found with high selectivity for muscarinic receptors. In additional, N-acetyl convolicine was chosen for selectivity evaluation of M2 or M3 receptors through the use of a dual-luciferase reporter assay system at the cellular level and an ACh-induced constricted strip test in vitro. After summarizing the active fragments and the structure-activity relationship (SAR) information, a new modified TA that takes advantage of both the high affinity and high selectivity for M3 receptors was proposed and evaluated successfully. This study provided an effective approach for the discovery and design of natural products based on highly selective drugs by UPLC-Q/TOF-MS coupled with virtual calculation and biological evaluation. Graphical Abstract Active fragments-guided strategy for selective inhibitors from HSS.

A strategy for fusion expression and preparation of functional glucagon-like peptide-1 (GLP-1) analogue by introducing an enterokinase cleavage site.

KGLP-1, a 31-amino acid glucagon-like peptide-1 (GLP-1) analogue, has a great therapeutic potential for anti-diabetes. In this work, a strategy for expression and purification of functional KGLP-1 peptide has been established. KGLP-1 cDNA was fused with glutathione S-transferase (GST), with an enterokinase cleavage site in the fusion junction. The recombinant fusion protein GST-KGLP-1 was affinity purified via the GST-tag, and then digested with enterokinase. The resulting GST part as well as the enzymes were eliminated by ultra-filtration followed by size exclusion chromatograph. The yield of purified KGLP-1 was approximately 12.1 mg/L, with purity of 96.18 %. The recombinant KGLP-1 was shown to have similar bioactivity as native GLP-1 when evaluated in a Chinese hamster ovary cell line expressing a GLP-1 receptor-egfp reporter gene.

Absorption, metabolism and effect of compatibility on absorption of qishenyiqi dropping pill.

Qishenyiqi dropping pill (QSYQ), is a traditional Chinese medicine (TCM) prescription for treating heart diseases in China. Knowledge concerning the systemic identification of active compounds and metabolic components of QSYQ is generally lacking. Therefore, it is essential to develop a valid method for the analysis of active compounds of the combined prescription and determination of interactions among the herbs. The absorbable compounds and metabolites of QSYQ were profiled using computational chemistry prediction, an improved everted gut sac in vitro experiment, the Caco-2 cell monolayer in vitro test, a rat in vivo experiment and ultra-performance liquid chromatography/diode array detection/quadrupole-time of flight mass spectrum (UPLC/DAD/Q-TOF MS). In total, 42 prototype compounds were recognized as absorbable compounds, and eight metabolites were identified by UPLC/DAD/Q-TOF MS. The absorption rates of phenolic acids and saponins were significantly improved and the absorption of isoflavone was inhibited after compatibility. The volatile oil component had an improved effect on the absorption of other compounds, while its own absorption was inhibited. In conclusion, the present study established a rapid and effective strategy for demonstrating the absorption and metabolism of QSYQ and revealing the compatible relationship among herbs. This investigation can provide a reference for the compatibility of prescriptions and the modernization of TCM.

An integrated strategy of ultra-high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry and virtual screening for the identification of α-glucosidase inhibitors in acarviostatin-containing complex.

We propose a strategy that integrates ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS) and virtual docking to identify inhibitors of multiple human -glucosidases. UPLC yielded AIB656, an acarviostatin-containing complex, which was analyzed by Q-TOF-MS to acquire structural information and was tested for inhibition of N-terminal (MGAM-N), C-terminal (MGAM-C) catalytic domain of maltase-glucoamylase, and human pancreatic -amylase (HPA).A systematic computational study was performed to evaluate the inhibition activity for 51 identified acarviostatins with various sizes, including trace or difficult-to-prepare ingredients. We evaluated the selectivities of three -glucosidases to acarviostatins and revealed the strong inhibition of MGAM-Nby acarviostatin I0-1. The high accuracy of the dual-screening was validated using enzyme inhibition assays, and docking was suggested as a possible mechanism for the strong inhibition of MGAM-N by acarviostatin I0-1 and of MGAM-C by acarbose (acarviostatin I01). No compound in AIB656 was suitable for inhibiting all three -glucosidases. Compared with conventional chromatographic separation and inhibitory activity detection, integrating UPLC/Q-TOF-MS identification and virtual validation was more convenient and more reliable. This strategy clearly demonstrates that MS data-based fingerprinting is a meaningful tool not only in identifying constituents in complex matrix but also in directly screening for powerful trace ingredients in natural products.

Identification and comparison of anti-inflammatory ingredients from different organs of Lotus nelumbo by UPLC/Q-TOF and PCA coupled with a NF-κB reporter gene assay.

Lotus nelumbo (LN) (Nelumbo nucifera Gaertn.) is an aquatic crop that is widely distributed throughout Asia and India, and various parts of this plant are edible and medicinal. It is noteworthy that different organs of this plant are used in traditional herbal medicine or folk recipes to cure different diseases and to relieve their corresponding symptoms. The compounds that are contained in each organ, which are named based on their chemical compositions, have led to their respective usages. In this work, a strategy was used to identify the difference ingredients and screen for Nuclear-factor-kappaB (NF-κB) inhibitors with anti-inflammatory ability in LN. Seventeen main difference ingredients were compared and identified from 64 samples of 4 different organs by ultra-performance liquid chromatography that was coupled with quadrupole/time of flight mass spectrometry (UPLC/Q-TOF-MS) with principal component analysis (PCA). A luciferase reporter assay system combined with the UPLC/Q-TOF-MS information was applied to screen biologically active substances. Ten NF-κB inhibitors from Lotus plumule (LP) extracts, most of which were isoquinoline alkaloids or flavone C-glycosides, were screened. Heat map results showed that eight of these compounds were abundant in the LP. In conclusion, the LP extracts were considered to have the best anti-inflammatory ability of the four LN organs, and the chemical material basis (CMB) of this biological activity was successfully validated by multivariate statistical analysis and biological research methods.

Preparation of functionalized alkynyl magnetic microspheres for the selective enrichment of cell glycoproteins based on click chemistry.

Functionalized alkynyl polyvinyl alcohol magnetic microspheres (PVA MMs) were developed for the specific enrichment of sialic acid-rich glycoproteins by click chemistry. The capture capability for proteins was evaluated through a novel dual-labeled bovine serum albumin (BSA) that utilizes fluorescence resonance energy transfer (FRET). The PVA MM parameters, including the size and coverage of functionalized groups, were optimized by response surface methodology. The optimal parameters obtained were 1.25-6.31 µm in size and 48.53-73.05% in coverage. Then, the optimal PVA MMs were synthesized, and the morphology and surface chemical properties were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FT-IR). To capture glycoproteins from the cell surface, a bioorthogonal chemical method was applied to metabolically label them with an azide group. The functionalized alkynyl PVA MMs showed a high specificity and strong binding capability for glycoproteins through a [3 + 2] cycloaddition reaction. The results indicated that the functionalized alkynyl PVA MMs could be applied to the enrichment of cell glycoproteins, and the merits of the MMs suggested an attractive and potential way to facilitate glycoprotein research.

Liquid phase nitration of benzene over supported ammonium salt of 12-molybdophosphoric acid catalysts prepared by sol-gel method.

A mild and clean liquid nitration of benzene with 65% nitric acid as nitrating agent over silica supported ammonium salt of 12-molybdophosphoric acid catalysts has been investigated. These catalysts with different loadings were prepared by sol-gel method and characterized by X-ray diffraction (XRD) and FTIR spectra. The acidity of these catalysts was measured by the potentiometric titration method. The XRD and IR analysis revealed that supported catalysts possess the Keggin structure which is similar to 12-molybdophosphoric acid. And it can be found that the supported catalysts had high nitration reaction catalytic activity and selectivity over nitrobenzene. The effects of various parameters such as nitric acid/benzene volume ratio, temperature and time of reaction have also been systematically studied.

Molecular cloning, sequence and expression analysis of ZmArf2, a maize ADP-ribosylation factor.

A full-length cDNA encoding a maize GTP-binding protein of the ADP-ribosylation factor family was cloned by suppression subtractive hybridization and an in silico cloning approach. The cDNA was 938 bp in length and contained a complete ORF of 612 bp, which encodes a protein of 203 amino acid residues. Its deduced amino acids sequence had an 83% identity with that of a GTP-binding protein in rice. The gene was designated ZmArf2. The ZmArf2 gene consists of G1, G2, G3, G4 and G5 boxes, and Switch I and Switch II regions. Eight nucleotides differed and five amino acids changed between the popcorn inbred N04 and the dent corn inbred Dan232. One changed amino acid was in the G1 box. RT-PCR analysis showed that ZmArf2 expression increased in the early stages of endosperm development and was not tissue-specific.