Assessment of the anti-inflammatory mechanism of quercetin 3,7-dirhamnoside using an integrated pharmacology strategy.

Pouzolzia zeylanica (L.) Benn. is a Chinese herbal medicine widely used for its anti-inflammatory and pus-removal properties. To explore its potential anti-inflammatory mechanism, quercetin 3,7-dirhamnoside (QDR), the main flavonoid component of P. zeylanica (L.) Benn., was extracted and purified. The potential anti-inflammatory targets of QDR were predicted using network analysis. These potential targets were verified using molecular docking, molecular dynamics simulations, and in vitro experiments. Consequently, 342 potential anti-inflammatory QDR targets were identified. By analyzing the intersection between the protein-protein interaction and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, we identified several potential protein targets of QDR, including RAC-alpha serine/threonine-protein kinase (AKT1), Ras-related C3 botulinum toxin substrate 1 (RAC1), nitric oxide synthase 3 (NOS3), serine/threonine-protein kinase mTOR (mTOR), epidermal growth factor receptor (EGFR), growth factor receptor-bound protein 2 (GRB2), and endothelin-1 receptor (EDNRA). QDR has anti-inflammatory activity and regulates immune responses and apoptosis through chemokines, Phosphatidylinositol 3-kinase 3(PI3K)/AKT, cAMP, T-cell receptor, and Ras signaling pathways. Molecular docking analysis showed that QDR has good binding abilities with AKT1, mTOR, and NOS3. In addition, molecular dynamics simulations demonstrated that the protein-ligand complex systems formed between QDR and AKT1, mTOR, and NOS3 have high dynamic stability, and their protein-ligand complex systems possess strong binding ability. In RAW264.7 macrophages, QDR significantly inhibited lipopolysaccharides (LPS)-induced inducible nitric oxide synthase expression, nitric oxide (NO) release and the generation of proinflammatory cytokines IL-6, IL-1ß, and TNF-α. QDR downregulated the expression of p-AKT1(Ser473)/AKT1 and p-mTOR (Ser2448)/mTOR, and upregulated the expression of NOS3, Rictor, and Raptor. This indicates that the anti-inflammatory mechanisms of QDR involve regulation of AKT1 and mTOR to prevent apoptosis and of NOS3 which leads to the release of endothelial NO. Thus, our study elucidated the potential anti-inflammatory mechanism of QDR, the main flavonoid found in P. zeylanica (L.) Benn.

Successful treatment with doxycycline monotherapy for human infection with Babesia venatorum (Babesiidae, Sporozoa) in China: a case report and proposal for a clinical regimen.

BACKGROUND: Human babesiosis is a worldwide disease caused by intraerythrocytic protozoa of the genus Babesia. It is transmitted by bites from ixodid ticks, and mechanically transmitted by blood transfusion. It is primarily treated with quinine and/or atovaquone, which are not readily available in China. In this study, we developed a novel treatment regimen involving doxycycline monotherapy in a patient with severe Babesia venatorum infection as an alternative therapeutic medication. The aim of our study is to provide a guidance for clinical practice treatment of human babesiosis. CASE PRESENTATION: A 73-year-old man who had undergone splenectomy and blood transfusion 8 years prior, presented with an unexplained fever, headache, and thrombocytopenia, and was admitted to the Fifth Medical Center of the PLA General Hospital. He was diagnosed with B. venatorum infection by morphological review of thin peripheral blood smears, which was confirmed by multi-gene polymerase chain reaction (PCR), and sequencing of the entire 18s rRNA and partial ß-tubulin encoding genes, as well as isolation by animal inoculation. The doxycycline monotherapy regimen (peros, 0.1 g bisindie) was administered following pharmacological guidance and an effective outcome was observed. The patient recovered rapidly following the doxycycline monotherapy. The protozoan load in peripheral blood samples decreased by 88% in hematocrit counts after 8 days, and negative PCR results were obtained after 90 days of follow-up at the hospital. The treatment lasted for 3 months without any side effects or sequelae. The nine-month follow-up survey of the patient did not reveal any signs of recrudescence or anti-babesial tolerance. CONCLUSIONS: We have reported a clinical case of successful doxycycline monotherapy for human babesiosis caused by B. venatorum, which provides an optional medical intervention for human babesiosis.

Probing the serum albumin binding site of fenamates and photochemical protein labeling with a fluorescent dye.

Human serum albumin (HSA) can bind with numerous drugs, leading to a significant influence on drug pharmacokinetics as well as undesirable drug-drug interactions due to competitive binding. Probing the HSA drug binding site thus offers great opportunities to reveal drug-HSA binding profiles. In the present study, a fluorescent probe (E)-4-(2-(5-(4-(diphenylamino)phenyl)thiophen-2-yl)vinyl)-1-propylpyridin-1-ium (TTPy) has been prepared, which exhibits enhancement of deep-red to near-infrared (NIR) fluorescence upon HSA binding. The competitive binding assay indicated that TTPy can target the HSA binding site of fenamates, a group of non-steroidal anti-inflammatory drugs (NSAIDs), with moderate binding affinity (1.95 × 106 M-1 at 303 K). More interestingly, TTPy enables fluorescent labeling of HSA upon visible light irradiation. This study provides promising ways for HSA drug binding site identification and photochemical protein labeling.

Garcinia Biflavonoid 1 Improves Lipid Metabolism in HepG2 Cells via Regulating PPARα.

Garcinia biflavonoid 1 (GB1) is one of the active chemical components of Garcinia kola and is reported to be capable of reducing the intracellular lipid deposition, which is the most significant characteristic of non-alcoholic fatty liver disease. However, its bioactive mechanism remains elusive. In the current study, the lipid deposition was induced in HepG2 cells by exposure to oleic acid and palmitic acid (OA&PA), then the effect of GB1 on lipid metabolism and oxidative stress and the role of regulating PPARα in these cells was investigated. We found that GB1 could ameliorate the lipid deposition by reducing triglycerides (TGs) and upregulate the expression of PPARα and SIRT6, suppressing the cell apoptosis by reducing the oxidative stress and the inflammatory factors of ROS, IL10, and TNFα. The mechanism study showed that GB1 had bioactivity in a PPARα-dependent manner based on its failing to improve the lipid deposition and oxidative stress in PPARα-deficient cells. The result revealed that GB1 had significant bioactivity on improving the lipid metabolism, and its potential primary action mechanism suggested that GB1 could be a potential candidate for management of non-alcoholic fatty liver disease.

Two binding epitopes modulating specificity of immunoassay for ß-agonist detection: Quantitative structure-activity relationship.

A growing number of ß-agonists are illegally using for reducing animal fat deposition in animals, but the development of analytical methods always lags behind the emergence of new illegal compounds. Therefore, class specificity antibody-based immunoassays that can detect a great many ß-agonists are important for timely supervision. In this study, a competitive inhibition enzyme-linked immunosorbent assay (ciELISA) based on a clenbuterol monoclonal antibody was developed to recognize 23 ß-agonists and analogues. Holographic and three-dimensional quantitative structure-activity relationship (HQSAR and 3D QSAR) revealed that there are two critical binding epitopes on ß-agonist hapten affecting antibody specificity, and these epitopes have been further validated using a ractopamine antibody with narrow specificity. Tert-butyl at C-2' epitope is needed to generate class specific antibodies, and different characteristics of substituents at benzene ring epitope would adjust antibody specificity. This investigation could provide reference for future design of ß-agonist haptens.

Proceedings of Chemistry, Pharmacology, Pharmacokinetics and Synthesis of Biflavonoids.

Biflavonoids, composed of two monoflavonoid residues, occur naturally in angiosperms, bryophytes, ferns, and gymnosperms. More than 592 biflavonoids have been structurally elucidated, and they can be classified into two groups of C-C and C-linear fragments-C, based on whether the linker between the two residues contains an atom. As the linker can be established on two arbitrary rings from different residues, the C-C type contains various subtypes, as does the C-linear fragment-C type. Biflavonoids have a wide range of pharmacological activities, including anti-inflammatory, antioxidant, antibacterial, antiviral, antidiabetic, antitumor, and cytotoxic properties, and they can be applied in Alzheimer's disease and Parkinson's disease. This review mainly summarizes the distribution and chemistry of biflavonoids; additionally, their bioactivities, pharmacokinetics, and synthesis are discussed.

Conformational adaptability determining antibody recognition to distomer: structure analysis of enantioselective antibody against chiral drug gatifloxacin.

Enantioselective antibodies have great potential to separate and detect chiral compounds. However, cross-reactivity of enantioselective antibodies to the distomer may limit the application. An in-depth understanding of interactions between antibodies and chiral drugs could be helpful to investigate antibody recognition to the distomer. In this study, a monoclonal antibody against chiral quinolone S-(-)-gatifloxacin (S-GAT) was produced and its Fab fragment was prepared by proteolysis. The S-GAT Fab exhibited 10% cross-reactivity against the R-enantiomer compared to that of the S-enantiomer in an indirect competitive enzyme-linked immunosorbent assay (icELISA). The crystal structures of the S-GAT Fab apo form and complex with S-GAT were analyzed, and molecular docking of the R-enantiomer was carried out. The ligand conformation was further studied using molecular dynamics simulations. The results showed that the distomer R-enantiomer could enter the chiral center recognition region of the antibody by adjusting the piperazine ring conformation. Meanwhile, the antibody binding cavity had obvious conformational adaptability during ligand binding. It demonstrated that conformational change of both ligand and antibody was the key reason why antibodies recognize the distomer. Restricting conformational adaptability could improve the enantioselective recognition ability of antibodies. This study provided a new explanation for the cross-reactivity of enantioselective antibodies to the distomer, and could help to modulate antibody enantioselectivity for immunoassay of chiral drugs.

Synthesis of Dihydroquinolines as Scaffolds for Fluorescence Sensing of Hydroxyl Radical.

A mild synthetic method to prepare dihydroquinolines has been presented. These dihydroquinolines, for the first time, showed great potential for fluorescence detection of the important biorelevant hydroxyl radicals (•OH). Sensitive and selective •OH detection and intracellular organelle-targeted fluorescence imaging of •OH have been demonstrated by using one of the synthetic dihydroquinolines. Moreover, dihydroquinoline has also exhibited promising potential to construct advanced fluorescence probes for •OH with tunable photophysical properties.

Aldol sensor-inspired fluorescent probes for measuring protein citrullination.

Protein citrullination is an important posttranslational modification on an arginine residue. However, high quality fluorescent probes for measuring the citrullination level and capturing citrullinated proteins are quite limited. Inspired by the similarity between acid-promoted citrulline-labeling reaction and aldol reaction, here we present "turn-on" and "turn-off" fluorescent probes for measuring citrulline levels based on the scaffold of aldol sensors. Further application of the modified probe showed great potential to simultaneously monitor and capture citrullinated peptides.

A new fluorescent probe for sensing of biothiols and screening of acetylcholinesterase inhibitors.

A new N2O-type BODIPY probe (LF-Bop) has been proposed for the selective and sensitive detection of biologically relevant small molecular thiols. This detection is based on the Michael addition reaction between the thiol and nitrostyrene groups in the probe, which decreases the quenching effect from the nitro group, thus resulting in the recovery of the deep-red fluorescence from the BODIPY structure. The results show that LF-Bop is able to detect all tested free thiols through a fluorescence turn-on assay. The lowest limit of detection (LOD) for glutathione was found to be down to nanomolar levels (220 nM). Based on this probe, we have developed a new fluorescence assay for the screening of acetylcholinesterase inhibitors. In total, 11 natural and synthetic alkaloids have been evaluated. Both experimental measurements and theoretical molecular docking results reveal that both natural berberine and its synthetic derivative dihydroberberine are potential inhibitors of acetylcholinesterase.

A ratiometric fluorescent probe for sensitive determination of the important glycopeptide antibiotic vancomycin.

A novel sensitive and selective probe for the important antibiotic vancomycin (Van) has been synthesized by integrating a coumarin and a fluorescein as dual fluorescence reporters and a Van binding peptide D-Ala-D-Ala. Only weak green fluorescence was initially observed, which was mostly attributed to fluorescence self-quenching induced by fluorophore stacking. Upon the binding of Van with the D-Ala-D-Ala peptide, the fluorescence turned on, probably due the disaggregation of fluorophores. The intensity ratio of the dual emission bands I519/I446 exhibited an excellent linear relationship with the concentration of Van increasing from 0-20 µM in synthetic urine. The lowest detection limit was calculated to be 92.8 nM in urine, which made the probe applicable in clinically relevant concentration ranges. The synthetic probe has also shown the potential for Van detection in human serum. More interestingly, this probe has been successfully applied for in vivo imaging of Van in zebrafish. Graphical Abstract.

Optimized Griess Reaction for UV-Vis and Naked-eye Determination of Anti-malarial Primaquine.

Primaquine (PMQ), an important anti-malarial drug, has been recommended by the World Health Organization (WHO) for the treatment of life-threatening infections caused by P. vivax and ovale. However, PMQ has unwanted adverse effects that lead to acute hemolysis in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. There is a need to develop simple and reliable methods for PMQ determination with the purpose of dosage monitoring. In early 2019, we have reported an UV-Vis and naked-eye based approach for PMQ colorimetric quantification. The detection was based on a Griess-like reaction between PMQ and anilines, which can generate colored azo products. The detection limit for direct measurement of PMQ in synthetic urine is in the nanomolar range. Moreover, this method has shown great potential for PMQ quantification from human serum samples at clinically relevant concentrations. In this protocol, we will describe the technical details regarding the syntheses and characterization of colored azo products, the reagent preparation, and the procedures for PMQ determination.

An unexpected Griess reaction on the important anti-malarial drug primaquine and its application for drug determination.

Primaquine (PMQ), a well-known anti-malarial drug, is of increasing importance as people moving toward global malaria eradication. PMQ has serious side effects that it often causes acute hemolytic toxicity in people with glucose-6-phosphate dehydrogenase (G6PD) deficiency. The development of simple and reliable approaches for quantitative dose monitoring is thus becoming important during malarial treatment with PMQ. Herein, an unexpected Griess reaction on PMQ was systematically studied. The reaction happened between substituted aniline and a primaquine molecule in the presence of nitrite. Both experimental measurements and theoretic calculation showed that UV-vis absorption of the azo products varied because of different electron contributing effects of substituents. Based on the optimized conditions, a novel colorimetric method has been developed for PMQ determination with excellent sensitivity and selectivity. The detection limits for PMQ in water and synthetic urine samples were down to nanomolar range. More importantly, this method has been successfully used to quantify PMQ from human serum samples within clinically relevant concentration ranges.

Tenacigenin B ester derivatives from Marsdenia tenacissima actively inhibited CYP3A4 and enhanced in vivo antitumor activity of paclitaxel.

ETHNOPHARMACOLOGICAL RELEVANCE: Marsdenia tenacissima (Roxb.) Wight et Arn is a vine distributed in southwest area of China and used in folk medicine for treatment of tumors. Recent decades of studies on this plant reveal its synergistic effects with certain anticancer drugs in cancer therapy. In our previous study, an extract ETA which contains total aglycones made from M. tenacissima significantly enhanced antitumor activity of paclitaxel in tumor-bearing mice. However, the effective constituents in ETA and the underlying mechanisms remain unclear. AIM OF THE STUDY: Reveal the active components in ETA as well as the molecular mechanism in enhancing antitumor efficacy of paclitaxel. MATERIAL AND METHODS: Main constituents in ETA were purified by chemical methods. Effects of the purified constituents on metabolic activity of CYP450 enzymes were evaluated in human liver microsomes. Ability of the constituents to enhance antitumor activity of paclitaxel were investigated in nude mice bearing HeLa tumors. Pharmacokinetic study was performed in SD rats. Molecular docking was carried out for investigation of drug-protein interactions. RESULTS: Three main C21 steroidal aglycones, 11α-O-tigloyl-12ß-O-acetyl-tenacigenin B (MT1), 11α-O-2-methylbutanoyl-12ß-O-tigloyl-tenacigenin B (MT2) and 11α-O-2-methylbutanoyl-12ß-O-acetyl-tenacigenin B (MT3), together with tenacigenin B (MT4) was prepared from ETA. Among them, MT1, MT2 and MT3 strongly inhibit the metabolic activity of CYP3A4. MT2 also showed inhibitory effects on CYP2C8, CYP2B6 and CYP2C19. In HeLa tumor xenografts, MT1, MT2 and MT3 (30 mg/kg) did not affect tumor growth themselves, but significantly enhanced paclitaxel-induced growth inhibition. In addition, coadministration of MT2 with paclitaxel resulted in significant reduction of liver CYP2C8. In pharmacokinetic study, MT2 significantly increased the blood concentration of paclitaxel with increased AUC value by 2.2-5.3 folds. Molecular docking analysis suggested hydrophobic interaction modes of tenacigenin B derivatives with CYP3A4, and also the essential roles of the C-11 and C-12 ester groups for effective interaction with CYP3A4. CONCLUSION: Our study proves that, 11α-O-tigloyl-12ß-O-acetyl-tenacigenin B, 11α-O-2-methylbutanoyl-12ß-O-tigloyl-tenacigenin B and 11α-O-2-methylbutanoyl-12ß-O-acetyl-tenacigenin B, which are the main constituents of ETA, are active inhibitors of CYP3A4 with potential to increase therapeutic efficacy of anticancer drugs that are substrates of CYP3A4. Tenacigenin B derivatives with C-11 and C-12 ester group substitutions, or at least a large part of them, are active components in ETA and M. tenacissima to enhance in vivo antitumor efficacies of paclitaxel.

A novel strategy for colorimetric detection of hydroxyl radicals based on a modified Griess test.

Hydroxyl radical (•OH) is the most reactive oxygen species involved in many environmental and biological processes. The development of simple and reliable methods to quantitatively determine hydroxyl radicals is desired. Herein, a colorimetric strategy based on a modified Griess test has been presented. The detection started with the nitrite release from nitroimidazoles (nIm) upon its specific reaction with •OH radicals and subsequently nitrite quantification by Griess reagent. The result showed that this nitroimidazole modified Griess test (nIm-Griess) was successfully adapted for the measurement of •OH radicals generated by Fenton reaction, water radiolysis, as well as the activation of two antimalarials artemisinin (Art) and dihydroartemisinin (DHA).

Three Constituents of Moringa oleifera Seeds Regulate Expression of Th17-Relevant Cytokines and Ameliorate TPA-Induced Psoriasis-Like Skin Lesions in Mice.

As a folk medicine, Moringa oleifera L. is used effectively to treat inflammatory conditions and skin diseases. However, its mechanism of action is not well understood, limiting its medical use. We isolated and identified three compounds, namely niazirin, marumoside A and sitosterol-3-O-ß-d-glucoside, from the seeds of Moringa oleifera, and studied their effects on the expression of Th17-relevant cytokines (IL-12/IL-23 p40, IL-17A, IL-22 and IL-23 p19) using lipopolysaccharide-stimulated THP-1 cells. Additionally, as Th17 plays a critical role in the pathogenesis of psoriasis, we used a 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced psoriasis-like skin lesion mouse model to study their potential therapeutic application in vivo. The compounds suppressed the expression of IL-12/IL-23 p40, IL-17A, IL-22 and IL-23 p19 in vitro, and in vivo they ameliorated psoriasis-like skin lesions, decreased IL-17A mRNA expression, and increased the expression of keratinocyte differentiation markers. To our knowledge, this is the first report regarding the mechanism and therapeutic application of Moringa oleifera seeds to treat psoriasis-like lesions in vivo.

Eleutheroside B1 mediates its anti-influenza activity through POLR2A and N-glycosylation.

Influenza viruses represent a serious threat to human health. Although our research group has previously demonstrated the antiviral and anti­inflammatory activities of eleutheroside B1, a detailed explanation of the mechanism by which it is effective against the influenza virus remains to be elucidated. In the present study, the transcriptomic responses of influenza A virus­infected lung epithelial cells (A549) treated with eleutheroside B1 were investigated using high­throughput RNA sequencing, and potential targets were identified using a molecular docking technique, reverse transcription­quantitative polymerase chain reaction (RT­qPCR) assay, and DNA methylation analysis. The transcriptomic data revealed that there are 1,871 differentially expressed genes (DEGs) between the cells infected with the influenza virus strain variant PR8, and the cells infected with PR8 and treated with eleutheroside B1. Among the DEGs, RNA polymerase II subunit A (POLR2A; encoding the largest subunit of RNA polymerase II) and mannosidase α class II member 1 (MAN2A1) were selected from the molecular docking analysis with eleutheroside B1. The docking score of Drosophila melanogaster MAN2A1 (3BVT) was 11.3029, whereas that of POLR2A was 9.0133. The RT­qPCR results demonstrated that the expression levels of host genes (MAN2A2, POLR2A) and viral genes (PA, PB1, PB2, HA) were downregulated following eleutheroside B1 treatment. Bisulfite­sequencing PCR was performed to investigate whether eleutheroside B1 was able to modify the DNA methylation of POLR2A, and the results suggested that the average proportion of methylated CpGs (­222­72 bp) increased significantly following treatment with eleutheroside B1. Taken together, these findings suggested that eleutheroside B1 may affect N­glycan biosynthesis, the chemokine signaling pathway, cytokine­cytokine receptor interaction and, in particular, may target the POLR2A to inhibit the production of influenza virus genes.

Four Hapten Spacer Sites Modulating Class Specificity: Nondirectional Multianalyte Immunoassay for 31 ß-Agonists and Analogues.

Immunoassay methods are important for monitoring ß-agonists illegally used for reducing animal fat deposition in livestock. However, there is no simultaneous screening surveillance immunoassay for detecting various ß-agonist chemicals that are possibly present in food. In this study, through the use of an R-(-)-salbutamol derivative as the immunizing hapten, an antibody recognizing 31 ß-agonists and analogues was generated for the first time. Three-dimensional quantitative structure-activity relationship (3D QSAR) revealed that strong steric and hydrophobic fields around the hapten spacer near C-2, as well as a chirality at C-1', dominantly modulated the class specificity of the raised antibody. However, a hapten spacer linked at C-2' or C-1 would lead to a narrow specificity, and the spacer charge at C-6 could affect the raised antibody specificity spectrum. A class specificity competitive indirect enzyme-linked immunosorbent assay (ciELISA) was established with an ideal recovery ranging from 81.8 to 118.3% based on the obtained antibody. With a good agreement to the HPLC/MS method, the proposed ciELISA was confirmed to be reliable for the rapid surveillance screening assay of ß-agonists in urine. This investigation will contribute to the rational design and control of the immunoassay specificity.

Four Specific Hapten Conformations Dominating Antibody Specificity: Quantitative Structure-Activity Relationship Analysis for Quinolone Immunoassay.

Antibody-based immunoassay methods have been important tools for monitoring drug residues in animal foods. However, because of limited knowledge about the quantitative structure-activity relationships between a hapten and its resultant antibody specificity, antibody production with the desired specificity is still a huge challenge. In this study, the three-dimensional quantitative structure-activity relationship (3D QSAR) was analyzed in accordance with the cross-reactivity of quinolone drugs reacting with the antibody raised by pipemidic acid as the immunizing hapten and compared with the reported cross-reactivity data and their hapten structures. It was found that the specificity of a quinolone antibody was strongly related to the conformation of the hapten used and that hapten conformations shaped like the letters "I", "P", and "Φ" were essential for the desired high specificity with low cross-reactivity, but that the hapten conformation shaped like the letter "Y" led to an antibody with broad specificity and high cross-reactivity. Almost all of the antibodies against quinolones could result from these four hapten conformations. It was first found that the concrete conformations dominated the specificity of the antibody to quinolone, which will be of significance for the accurate hapten design, predictable antibody specificity, and better understanding the recognition mechanism between haptens and the antibodies for immunoassays.