α2,6-sialic Acid on the Platelet Membrane Surface is a Novel Biomarker and Therapeutic Target in Children with Primary Immune Thrombocytopenia.

Objective: To analyze the expression of platelet membrane glycoprotein sialylation in primary immune thrombocytopenia and provide a reference for clinical diagnosis and treatment of the disease. Methods: 100 children with primary immune thrombocytopenia diagnosed and treated in the Children's Hospital of Yunhe District Central Hospital of Cangzhou City, Hebei Province from January 2020 to June 2022 were included in the study group. All children were treated with dexamethasone (DXMS) shock therapy; Another 20 healthy children who underwent routine physical examination at the same time were selected and included in the control group. The study measured the platelet membrane surface in plasma using flow cytometry in two groups: the comparative study group and the control group. The study measured the positive rates of α 2,3-sialic acid (α 2, 3-sa) and α 2,6-sialic acid (α 2, 6-sa) in both groups. The study also measured the positive rates of α 2,3-sialic acid (α 2, 3-sa) and α 2,6-sialic acid (α 2, 6-sa) before and after treatment in the comparative study group. At the same time, an enzyme-linked immunosorbent assay was used to determine the content of sialic acid and sialidase activity and content in the serum. The detection values of α2, 3-SA, and α2, 6-SA expression of children in the study group and the control group were compared, and the detection values of α2, 3-SA, and α2, 6-SA expression of children in the study group before and after treatment were compared. Results: There was no significant difference in the positive rate of α 2, 3-sa between the study group and the control group (t=0.852, P > .05); Study Group: The positive rate of α 2,6-sa was significantly lower than that of the control group (P < .05). In the study group, there was no significant difference in the positive rate of α 2,3-sa before and after treatment (P > .05). However, after treatment, the positive rate of α 2,6-sa was significantly higher than before (P < .05). The study found that the children in the study group had significantly higher levels of serum sialic acid content, sialidase activity, and content than those in the control group (P < .05). After treatment, the children in the study group showed a decrease in serum sialic acid content, sialidase activity, and content, which was statistically significant (P < .05) compared to before treatment. Conclusion: The sialylation of platelet membrane glycoprotein is abnormally expressed in primary immune thrombocytopenia. The sialylation of platelet membrane glycoprotein may be involved in the occurrence and development of the disease. It is of great practical significance to diagnose and evaluate the therapeutic effect of the disease by detecting the sialylation of platelet membrane glycoprotein.

Formulation development of a stable influenza recombinant neuraminidase vaccine candidate.

Current influenza vaccines could be augmented by including recombinant neuraminidase (rNA) protein antigen to broaden protective immunity and improve efficacy. Toward this goal, we investigated formulation conditions to optimize rNA physicochemical stability. When rNA in sodium phosphate saline buffer (NaPBS) was frozen and thawed (F/T), the tetrameric structure transitioned from a "closed" to an "open" conformation, negatively impacting functional activity. Hydrogen deuterium exchange experiments identified differences in anchorage binding sites at the base of the open tetramer, offering a structural mechanistic explanation for the change in conformation and decreased functional activity. Change to the open configuration was triggered by the combined stresses of acidic pH and F/T. The desired closed conformation was preserved in a potassium phosphate buffer (KP), minimizing pH drop upon freezing and including 10% sucrose to control F/T stress. Stability was further evaluated in thermal stress studies where changes in conformation were readily detected by ELISA and size exclusion chromatography (SEC). Both tests were suitable indicators of stability and antigenicity and considered potential critical quality attributes (pCQAs). To understand longer-term stability, the pCQA profiles from thermally stressed rNA at 6 months were modeled to predict stability of at least 24-months at 5°C storage. In summary, a desired rNA closed tetramer was maintained by formulation selection and monitoring of pCQAs to produce a stable rNA vaccine candidate. The study highlights the importance of understanding and controlling vaccine protein structural and functional integrity.

Discovery of natural multi-targets neuraminidase inhibitor glycosides compounds against influenza A virus through network pharmacology, virtual screening, molecular dynamics simulation, and in vitro experiment.

Influenza virus continually challenges both human and animal health. Moreover, influenza viruses are easy to mutate. In a certain degree, vaccines may not catch up with rapid mutant paces of viruses. Anti-influenza drugs NIs (neuraminidase inhibitors) are one of the best choices. Therefore, based on ADMET properties, eight optimal natural multi-targets NIs glycosides compounds (IC50 = 0.094-97.275 µM) are found from radix glycyrrhizae, flos sophorae, caulis spatholobi, radix astragali, radix glycyrrhizae, semen astragali complanati, and common fenugreek seed through network pharmacology, molecular docking, dynamics simulation, quantum chemistry, and in vitro experiment. Moreover, mechanism research illustrates these natural compounds treat influenza A virus through key targets TLR4, TNF, and IL6 (high fever, acute respiratory distress syndrome), MAPK1, and MAPK3 (MAPK signaling pathway, viral RNP export, and viral protein expression), IL1B (NOD-like receptor signaling pathway, suppressed maturation of pro-IL-1ß and pro-IL-18), CASP3 (apoptosis), AKT1 (inhibited premature apoptosis), and EP300 (viral myocarditis, chemoattraction of monocytes and macrophages, T-cell activation antibody response).

Screening and identification of neuraminidase inhibitors from Baphicacanthus cusia by a combination of affinity ultrafiltration, HPLC-MS/MS, molecular docking, and fluorescent techniques.

Natural products provide a new opportunity for the discovery of neuraminidase (NA)inhibitors. In this study, an affinity ultrafiltration (AUF) coupled with HPLC-MS/MS method was firstly developed and optimized for screening of NA inhibitors from natural products. The critical factors influencing the interaction of enzyme-ligand (including sample concentration, enzyme concentration, incubation time and temperature, pH of the buffer, and dissociation solvents and time) were investigated and optimized by a one-factor-at-a-time design. The method was then applied to discover NA inhibitory compounds in stems and leaves of Baphicacanthus cusia. As a result, five active alkaloids were screened out and identifiedas 2,4(1H,3H)-quinazolinedione (1), 4(3H)-quinazolinone (2), 2(3H)-benzoxazolone (3), tryptanthrin (4), and indirubin (5) through analysis of their DAD profiles, MS/MS fragments, and comparison with reference substances. These active compounds were further evaluated for their NA inhibitory activity using a fluorescence-based NA inhibition assay. The result from the fluorescent assay revealed that all the five compounds(1-5) showed pronounced NA inhibitory activities with IC50values of 98.98, 64.69, 40.16, 69.44, and 144.73 µM, respectively. Finally, molecular docking of these five alkaloids with NA showed that hydrogen bond and π-cation interactions dominated within the binding sites with binding energies ranging between -5.7 to -7.9 kcal/mol, which was supported by the results of the AUF and the fluorescence-based enzyme assay. The developed AUF method is simple and efficient for screening potential NA inhibitors from stems and leaves of B. cusia.

Inhibitory Potential of Quercetin Derivatives Isolated from the Aerial Parts of Siegesbeckia pubescens Makino against Bacterial Neuraminidase.

This study aimed to isolate bacterial neuraminidase (BNA) inhibitory O-methylated quercetin derivatives from the aerial parts of S. pubescens. All the isolated compounds were identified as O-methylated quercetin (1-4), which were exhibited to be noncompetitive inhibitors against BNA, with IC50 ranging from 14.0 to 84.1 µM. The responsible compounds (1-4) showed a significant correlation between BNA inhibitory effects and the number of O-methyl groups on quercetin; mono (1, IC50 = 14.0 µM) > di (2 and 3, IC50 = 24.3 and 25.8 µM) > tri (4, IC50 = 84.1 µM). In addition, the binding affinities between BNA and inhibitors (1-4) were also examined by fluorescence quenching effect with the related constants (KSV, KA, and n). The most active inhibitor 1 possessed a KSV with 0.0252 × 105 L mol-1. Furthermore, the relative distribution of BNA inhibitory O-methylated quercetins (1-4) in S. pubescens extract was evaluated using LC-Q-TOF/MS analysis.

A pan-influenza antibody inhibiting neuraminidase via receptor mimicry.

Rapidly evolving influenza A viruses (IAVs) and influenza B viruses (IBVs) are major causes of recurrent lower respiratory tract infections. Current influenza vaccines elicit antibodies predominantly to the highly variable head region of haemagglutinin and their effectiveness is limited by viral drift1 and suboptimal immune responses2. Here we describe a neuraminidase-targeting monoclonal antibody, FNI9, that potently inhibits the enzymatic activity of all group 1 and group 2 IAVs, as well as Victoria/2/87-like, Yamagata/16/88-like and ancestral IBVs. FNI9 broadly neutralizes seasonal IAVs and IBVs, including the immune-evading H3N2 strains bearing an N-glycan at position 245, and shows synergistic activity when combined with anti-haemagglutinin stem-directed antibodies. Structural analysis reveals that D107 in the FNI9 heavy chain complementarity-determinant region 3 mimics the interaction of the sialic acid carboxyl group with the three highly conserved arginine residues (R118, R292 and R371) of the neuraminidase catalytic site. FNI9 demonstrates potent prophylactic activity against lethal IAV and IBV infections in mice. The unprecedented breadth and potency of the FNI9 monoclonal antibody supports its development for the prevention of influenza illness by seasonal and pandemic viruses.

Antiviral effect and mechanism of Phillyrin and its reformulated FS21 against influenza.

Background: Influenza virus causes significant morbidity and mortality with pandemic threat. Oleaceae Fructus Forsythiae is a medicinal herb. This study aimed to investigate antiviral effect of Phillyrin, a purified bioactive compound from this herb, and its reformulated preparation FS21 against influenza and its mechanism. Methods: Madin-Darby Canine Kidney (MDCK) cells were infected by one of six influenza viruses: five influenza A viruses (IAVs: three H1N1 and two H3N2) and one influenza B virus (IBV). Virus-induced cytopathic effects were observed and recorded under microscope. Viral replication and mRNA transcription were evaluated by quantitative polymerase chain reaction (qPCR) and protein expression by Western blot. Infectious virus production was assessed using TCID50 assay, and IC50 was calculated accordingly. Pretreatment and time-of-addition experiments with Phillyrin or FS21 added 1 h before or in early (0-3 h), mid (3-6 h), or late (6-9 h) stages of viral infection were performed to assess their antiviral effects. Mechanistic studies included hemagglutination and neuraminidase inhibition, viral binding and entry, endosomal acidification, and plasmid-based influenza RNA polymerase activity. Results: Phillyrin and FS21 had potent antiviral effects against all six IAV and IBV in a dose-dependent manner. Mechanistic studies showed that both suppressed influenza viral RNA polymerase with no effect on virus-mediated hemagglutination inhibition, viral binding or entry, endosomal acidification, or neuraminidase activity. Conclusions: Phillyrin and FS21 have broad and potent antiviral effects against influenza viruses with inhibition of viral RNA polymerase as the distinct antiviral mechanism.

Discovery of the potential neuraminidase inhibitors from Polygonum cuspidatum by ultrafiltration combined with mass spectrometry guided by molecular docking.

Neuraminidase is an important target in the treatment of the influenza A virus. Screening natural neuraminidase inhibitors from medicinal plants is crucial for drug research. This study proposed a rapid strategy for identifying neuraminidase inhibitors from different crude extracts (Polygonum cuspidatum, Cortex Fraxini, and Herba Siegesbeckiae) using ultrafiltration combined with mass spectrometry guided by molecular docking. Firstly, the main component library of the three herbs was established, followed by molecular docking between the components and neuraminidase. Only the crude extracts with numbers of potential neuraminidase inhibitors identified by molecular docking were selected for ultrafiltration. This guided approach reduced experimental blindness and improved efficiency. The results of molecular docking indicated that the compounds in Polygonum cuspidatum demonstrated good binding affinity with neuraminidase. Subsequently, ultrafiltration-mass spectrometry was employed to screen for neuraminidase inhibitors in Polygonum cuspidatum. A total of five compounds were fished out, and they were identified as trans-polydatin, cis-polydatin, emodin-1-O-ß-D-glucoside, emodin-8-O-ß-D-glucoside, and emodin. The enzyme inhibitory assay showed that they all had neuraminidase inhibitory effects. In addition, the key residues of the interaction between neuraminidase and fished compounds were predicted. In all, this study could provide a strategy for the rapid screening of the potential enzyme inhibitors from medicinal herbs.

Bioassay guided isolation of caffeoylquinic acids from the leaves of Ilex pubescens Hook. et Arn. and investigation of their anti-influenza mechanism.

ETHNOPHARMACOLOGICAL RELEVANCE: Ilex pubescens Hook. et Arn. (Maodongqing, MDQ) is a common herbal tea ingredient in Southern China for heat clearance and anti-inflammation. Our preliminary screening showed that 50% ethanol extract of its leaves has anti-influenza virus activity. In this report, we proceed to identify the active components and clarify the related anti-influenza mechanisms. AIM: We aim to isolate and identify the anti-influenza virus phytochemicals from the extract of the MDQ leaves, and study their anti-influenza virus mechanism. MATERIAL AND METHODS: Plaque reduction assay was used to test the anti-influenza virus activity of fractions and compounds. Neuraminidase inhibitory assay was used to confirm the target protein. Molecular docking and reverse genetics were used to confirm the acting site of caffeoylquinic acids (CQAs) on viral neuraminidase. RESULTS: Eight CQAs, 3,5-di-O-caffeoylquinic acid methyl ester (Me 3,5-DCQA), 3,4-di-O-caffeoylquinic acid methyl ester (Me 3,4-DCQA), 3,4,5-tri-O-caffeoylquinic acid methyl ester (Me 3,4,5-TCQA), 3,4,5-tri-O-caffeoylquinic acid (3,4,5-TCQA), 4,5-di-O-caffeoylquinic acid (4,5-DCQA), 3,5-di-O-caffeoylquinic acid (3,5-DCQA), 3,4-di-O-caffeoylquinic acid (3,4-DCQA), and 3,5-di-O-caffeoyl-epi-quinic acid (3,5-epi-DCQA) were identified from the MDQ leaves, in which Me 3,5-DCQA, 3,4,5-TCQA and 3,5-epi-DCQA were isolated for the first time. All these eight compounds were found to inhibit neuraminidase (NA) of influenza A virus. The results of molecular docking and reverse genetics indicated that 3,4,5-TCQA interacted with Tyr100, Gln412 and Arg419 of influenza NA, and a novel NA binding groove was found. CONCLUSION: Eight CQAs isolated from the leaves of MDQ were found to inhibit influenza A virus. 3,4,5-TCQA was found to interact with Tyr100, Gln412 and Arg419 of influenza NA. This study provided scientific evidence on the use of MDQ for treating influenza virus infection, and laid the foundation for the development of CQA derivatives as potential antiviral agents.

A practical and rapid screening method for influenza virus neuraminidase inhibitors based on fluorescence detection.

A new analytical method for rapid screening of influenza virus neuraminidase inhibitors was established. The method is based on the principle that, given a certain amount of neuraminidase, the sample and the neuraminidase act in the microplate for a period of time, and the active neuraminidase that is not inhibited by the sample can generate a fluorescence value at a specific wavelength after binding to the substrate, and the rate of inhibition of neuraminidase by the sample can be calculated based on the actual detected fluorescence value. This newly developed method was used to screen and evaluate the in vitro anti-neuraminidase activity of 39 high-purity compounds contained in three traditional Chinese herbal medicines, and finally 25 compounds with strong activity were obtained. The newly established neuraminidase inhibitor analytical method has these advantages of practicality, rapidity, high sensitivity and low cost, and has a good value for promotion and application. This article newly establishes a rapid, sensitive, simple and practical screening method for influenza virus neuraminidase inhibitors, which is a great complement to the existing methods and has a good promotion and application value.

Antiviral activity of soybean GL 2626/96 (Glycine max) ethanolic extract against influenza A virus in vitro and in vivo.

Influenza viruses cause respiratory infections in humans with high morbidity and mortality rates. Neuraminidase inhibitors such as oseltamivir and peramivir are the most commonly used drugs for influenza virus infections. However, the emergence of resistant viruses necessitates the urgent need to develop next-generation anti-influenza drugs. Soybean (Glycine max L. Merr.) is widely cultivated and used as food worldwide. In addition, soybean has long been used as a nutritional supplement and herbal medicine. However, the potential anti-influenza properties of the soybean cultivar "GL 2626/96″ (SG2626) are yet to be investigated. Herein, we determined whether the ethanolic extract of SG2626 (SG2626E) has anti-viral activity through performing SG2626E pre-, co-, and post-treatment assays, using the influenza green fluorescent protein (GFP)-tagged influenza A/PR/8/34 (A/PR/8/34-GFP) virus. SG2626E showed anti-influenza virus activity in pre- and co-treated cells in a dose-dependent manner, but not in post-treated cells. SG2626E imparted a considerable inhibitory effect on influenza A virus (IAV) infection through blocking viral attachment. SG2626E inhibited the activity of viral hemagglutinin, but not viral neuraminidase of the IAV. SG2626E inhibited IAV infection by reducing intracellular calcium levels in infected human lung epithelial A549 cells. Additionally, SG2626E reduced body weight loss, decreased mortality, and increased the survival rate through reducing viral replication in the lungs of IAV-infected mice. Overall, these results suggest that SG2626E inhibits IAV infection and is a potential novel anti-influenza agent.

Immunogenicity of a recombinant hemagglutinin neuraminidase-Porcine rubulavirus produced by Escherichia coli of Porcine rubulavirus gives protective immunity of litter after challenge.

Porcine rubulavirus (PRV) is a contagious virus that affects the Mexican swine industry. This work aimed to evaluate the immunogenicity of an recombinant hemagglutinin neuraminidase-Porcine rubulavirus (rHN-PorPV) candidate vaccine on pregnant sows, and the protective efficacy afforded to their 7-day-old suckling piglets against PRV lethal challenge. Three sows were immunized with rHN-PorPV formulated with immune-stimulating complex (ISCOMs) and two sows with rHN-PorPV protein alone as well as a mock-immunized pregnant sow (negative control). Quantitative ELISA detected a high concentration of anti-rHN-PorPV Immunoglobulin G (IgG) antibodies in sow sera after the second dose of vaccine administered on day 14 until farrowing, showing viral-neutralizing and cross-neutralization activity against different variants of PRV. Sera samples from piglets of immunized sows (with or without adjuvant), showed high concentrations of IgG antibodies. As expected, piglets from the negative control sow (n=5), exhibited severe signs of disease and 100% of mortality after PRV challenge study. Conversely, 75% and 87.5% of the piglets born from the rHN-PorPV and the rHN-PorPV-ISCOMs-immunized sows (n=8), survived, respectively, showing milder PRV clinical signs. Our data indicate that rHN-PorPV candidate vaccine produced in Escherichia coli induces efficient humoral response in pregnant sows and that the maternally derived immunity provides high protection to suckling piglets against PRV lethal challenge.

Integrating Anti-Influenza Virus Activity and Chemical Pattern Recognition to Explore the Quality Evaluation Method of Lonicerae Japonicae Flos.

Lonicerae japonicae flos (LJF, Lonicera japonica Thunb.) is adopted as a core herb for preventing and treating influenza. However, the anti-influenza virus components of LJF and the impact of quality-affecting factors on the anti-influenza activity of LJF have not been systematically investigated. In this study, a strategy integrating anti-influenza virus activity, ultrahigh-performance liquid chromatography fingerprint and chemical pattern recognition was proposed for the efficacy and quality evaluation of LJF. As a result, six bioactive compounds were screened out and identified as neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 4,5-Di-O-caffeoylquinic acid, sweroside and secoxyloganin. Based on the bioactive compounds, chemical pattern recognition models of LJF were established by a linear discriminant analysis (LDA). The results of the LDA models and anti-influenza virus activity demonstrated that cultivation pattern significantly affected the anti-influenza effect of LJF and that the neuraminidase inhibition rate of wild LJF was significantly higher than that of cultivated LJF. Moreover, the quality of LJF samples with different processing methods and geographical origins showed no obvious difference. Overall, the proposed strategy in the current study revealed the anti-influenza virus components of LJF and provided a feasible method for thequality evaluation of LJF, which has great importance for assuring the clinical effect against influenza of LJF.

Urolithin M5 from the Leaves of Canarium album (Lour.) DC. Inhibits Influenza Virus by Targeting Neuraminidase.

Ganlanye (GLY), the leaf of Canarium album (Lour.) DC., is a traditional Chinese medicinal herb for warm disease treatment. We found that its aqueous extract could inhibit the influenza A virus. To find and characterize anti-influenza virus phytochemicals from GLY, we performed (1) bioassay-guided isolation, (2) a cell and animal assay, and (3) a mechanism study. Bioassay-guided isolation was used to identify the effective components. Influenza virus-infected MDCK cell and BALB/c mouse models were employed to evaluate the anti-influenza virus activities. A MUNANA assay was performed to find the NA inhibitory effect. As a result, urolithin M5 was obtained from the crude extract of GLY. It inhibited influenza virus activities in vitro and in vivo by suppressing the viral NA activity. In the MDCK cell model, urolithin M5 could inhibit an oseltamivir-resistant strain. In a PR8-infected mouse model, 200 mg/kg/d urolithin M5 protected 50% of mice from death and improved lung edema conditions. GLY was recorded as a major traditional herb for warm disease treatment. Our study identified GLY as a potent anti-influenza herb and showed urolithin M5 as the active component. We first report the in vivo activity of urolithin M5 and support the anti-influenza application of GLY.

In vitro antiviral, antioxidant and in vivo antipyretic activity of three South Africa medicinal plants crude extracts / Actividad antiviral, antioxidante y antipirética in vivo de tres extractos crudos de plantas medicinales de Sudáfrica

This study investigated anti-viral, antioxidant activity and anti-pyretic of crude extract from Artemisia afra, Artemisia absinthium and Pittiosporum viridflorum leaves. The crude extracts were prepared by maceration using aqueous, methanol and dichloromethane respectively. Antiviral studies were evaluated with influenza virus using Fluorescence based - Neuraminidase inhibitors. Antioxidant activities determined with DPPH, Nitric oxide, hydroxyl and super oxide anion radicals' Anti-pyretic activities were evaluated using rats with yeast induced pyrexia. Total phenol, flavonoids, and pro-anthocyanin contents of the plants samples were evaluated using standard protocols. The crude extracts exhibited neuraminidase inhibitory activity against the influenza virus at different thresholds. Artemisia absinthiumaqueous extract showed the best activity against A/Sydney/5/97. Whereas Artemisia afra methanol crude extract displayed highest antioxidant potential against the tested antioxidant parameters. All the crude extracts significantly reversed yeast induced pyrexia in rats, similar to paracetamol. Thus, they could serve as natural remedy for respiratory diseases such as Influenza.

Este estudio investigó la actividad antiviral, antioxidante y antipirética del extracto crudo de hojas de Artemisia afra, Artemisia absinthium y Pittiosporum viridflorum. Los extractos crudos se prepararon mediante maceración utilizando metanol acuoso y diclorometano respectivamente. Los estudios antivirales se evaluaron con el virus de la influenza utilizando inhibidores de neuraminidasa basados en fluorescencia. Actividades antioxidantes determinadas con DPPH, radicales aniónicos de óxido nítrico, hidroxilo y superóxido. Las actividades antipiréticas se evaluaron utilizando ratas con pirexia inducida por levaduras. El contenido total de fenol, flavonoides y proantocianina de las muestras de plantas se evaluó utilizando protocolos estándar. Los extractos crudos mostraron actividad inhibidora de neuraminidasa contra el virus de la influenza en diferentes umbrales. El extracto acuoso de Artemisia absinthium mostró la mejor actividad contra A/Sydney/5/97. Mientras que el extracto crudo de Artemisia aframetanol mostró el mayor potencial antioxidante contra los parámetros antioxidantes probados. Todos los extractos crudos revirtieron significativamente la pirexia inducida por levaduras en ratas, similar al paracetamol. Por tanto, podrían servir como remedio natural para enfermedades respiratorias como la Influenza.

Affinity ultrafiltration and UPLC-HR-Orbitrap-MS based screening of neuraminidase inhibitors from Angelica pubescens.

Traditional Chinese medicine is a rich source of natural products and has a long history of use because of its remarkable clinical efficacy. In the present study, the chemical constitutes of Angelica pubescens were studied by ultra high performance liquid chromatography and high-resolution Orbitrap mass spectrometry (UPLC-HR-Orbitrap-MS). A total of 78 compounds were identified and the main composition were coumarins and phenolic acids. Then, the neuraminidase was incubated with extract of Angelica pubescens to screen the neuraminidase inhibitors by affinity ultrafiltration methods. As a result, 13 small molecules were discovered to interact with neuraminidase for the first time. In vitro neuraminidase inhibitory activity of the screened compounds and extract of Angelica pubescens was tested, and isochlorogenic acid C, isochlorogenic acid B, osthole, chlorogenic acid, xanthotoxin, phellopterin and imperatorin were proved to have this activity. In addition, molecular docking analysis was conducted to predict the potential docking position. This study may provide a reference for the medical substance basis in Angelica and the clinical usage of this drug.

An affinity interaction guided two-dimensional separation system for the screening of neuraminidase inhibitors from Reynoutria japonica Houtt. roots.

Discovering bioactive compounds from medicinal herbs is crucial for drug discovery. Ultrafiltration is often used in the screening of bioactive compounds from natural herbs because of its simple and rapid operations. However, the ultrafiltration results are often disturbed by the undissolved compounds and the non-target compounds, which reduces the accuracy of the results. Herein, an affinity interaction guided two-dimensional (2D) separation system was developed. Discovery of the potential neuraminidase (NA) inhibitors from the dried roots of Reynoutria japonica Houtt. (RRJ) was used as an example. Only the small molecules showing affinity interaction with NA could be screened by the affinity interaction guided 2D separation system. Firstly, the NA and crude extract were incubated to form a sample solution (containing NA-inhibitor complexes, NA, and three types of small molecules with different polarities) by affinity interaction. Then the sample solution was separated and detected by the 2D separation system. This aimed to reduce the interference of the undissolved compounds and non-target compounds, and pick out the NA-inhibitor complexes (NA-Is). The collected NA-Is were denatured to release small molecular inhibitors (Is) for LC-MS/MS analysis. Compared with the ultrafiltration, more obvious peak area differences were observed in the results, and four potential NA inhibitors were successfully identified. In all, we provided a simple strategy with better performance in the screening of natural bioactive compounds.

New dihydrobenzoxanthone derivatives with bacterial neuraminidase inhibitory activity isolated from Artocarpus elasticus.

Artocarpus elasticus is a popular fruit tree in the tropical regions. Primary screenings of methanol extracts of the root bark confirmed its potent inhibition of bacterial neuraminidase (BNA), which plays an essential role in the pathogenesis of many microbial diseases. Assessments of the responsible phytochemicals were conducted by isolating eight compounds (1-8) and two of them (6 and 8) were identified as new compounds. Among the isolates, the dihydrobenzoxanthones attained the highest BNA inhibition with IC50 values of 0.5 âˆ¼ 3.9 µM. Further investigation of the inhibitory mechanism by Lineweaver-Burk plots revealed the phytochemicals to function as reversible noncompetitive inhibitors. Fluorescence quenching showed their binding affinities were highly correlated with their inhibitory potential dose-dependently. Molecular docking experiments suggested the dihydrobenzoxanthones (4 and 6) as noncompetitive inhibitors of BNA with unique interaction with Tyr435 of BNA in comparison with the mother flavonoid (7).

Soybean phytochemicals responsible for bacterial neuraminidase inhibition and their characterization by UPLC-ESI-TOF/MS.

Ethanol extract of soybean (Glycine max (L.) Merr.) showed good inhibitory activity against bacterial neuraminidase (BNA), which plays a pivotal role in the pathogenesis of a number of microbial diseases. The saponin portion fractionated through preparative HPLC (IC50 = 2.25 µg mL-1) was found to be responsible for the observed BNA inhibition. Estimation of the inhibitory effects by individual compounds showed that the soyasaponins of group B (Ba, Bb, Bb', Bc, and Bd) exhibited extremely high inhibitions (IC50 = 0.25-0.48 µM), whereas group A (Aa, Ab, and Ac) was almost inactive. Kinetic studies determined that group B soyasaponins were noncompetitive inhibitors. Furthermore, molecular docking experiments confirmed that soyasaponin Ba (group B) could undergo binding interactions with various residues in the binding pocket. In contrast, soyasaponin Aa (group A) failed to enter the binding pocket due to its extra scaffold structure of oligosaccharides bonded to the 22-hydroxyl position. The metabolites in the soybean extract were fully characterized using UPLC-ESI-TOF/MS.

Rapid screening of neuraminidase inhibitors with the benzoic acid skeleton from Paeonia suffruticosa Andrews by solid-phase extraction with an enzyme activity switch combined with mass spectrometry analysis.

It is meaningful for drug discovery to discover lead compounds with specific skeletons from medicinal herbs. Screening bioactive compounds with specific skeletons by a simple and rapid strategy is still a challenging task. Solid-phase extraction (SPE) is a simple and time-saving technique in the laboratory and is often used in the concentration of natural products. It is attractive to apply the SPE in the screening of bioactive compounds with specific skeletons. To achieve this goal, SPE with an enzyme activity (EA) switch combined with mass spectrometry analysis was first proposed. The screening of benzoic acid-derived neuraminidase (NA) inhibitors from the root cortex of Paeonia suffruticosa Andrews (CPSA) was used as an example. The NA and crude extract of CPSA were incubated to form a sample solution. Subsequently, the sample was separated, detected, and collected by the SPE with an EA switch. When the detected values reduced significantly, the EA switch was triggered, and the collection was stopped. The collected eluents were treated for LC-MS/MS analysis. Finally, combining diagnostic ions and mass spectrometry data, two benzoic acid NA inhibitors were successfully screened from CPSA. In this study, the separation, detection, and collection were performed on one instrument system. Compared with the traditional isolation strategy, this strategy with the simpler operation and higher experimental efficiency could be an effective tool for the rapid screening of lead compounds with specific skeletons.