Antibody Production and Immunoassay Development for Authenticating Chlorpheniramine Maleate Adulteration in Herbal Tea.

Chlorphenamine maleate is a prohibited additive found in herbal teas and health foods. Excessive intake of this substance can result in adverse health effects. In this study, two novel haptens, PEM and bepotastine (PB1), mimicking chlorphenamine maleate structure were designed and synthesized based on molecular simulation for developing two corresponding polyclonal antibodies (PEM-Ab and PB1-Ab), respectively. Afterward, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed to quickly and accurately detect chlorphenamine maleate in herbal teas using PB1-Ab, which has a high sensitivity and specificity. For chlorphenamine maleate, the half-maximal inhibitory concentration (IC50) and limit of detection (LOD) of PB1-Ab under ideal circumstances were found to be 1.18 µg/L and 0.07 µg/L, respectively. Besides, an environmentally friendly sample pre-treatment strategy was employed that allowed easy and effective elimination of complex matrices. The ic-ELISA method observed the average recovery rate from 87.7% to 94.0% with the variance coefficient (CV) ranging from 2.2% to 9.4%. Additionally, the identification of 25 commercially available herbal teas using liquid chromatography-tandem mass spectrometry (LC-MS/MS) further confirmed the validity of our detection. The results of the two methods are consistent. Overall, the proposed ic-ELISA could be an ultrasensitive and reliable method for chlorphenamine maleate adulterated in foods or exposure to the environment.

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.