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.

GB1a Activates SIRT6 to Regulate Lipid Metabolism in Mouse Primary Hepatocytes.

Lipid accumulation, oxidative stress, and inflammation in hepatocytes are features of nonalcoholic fatty liver disease (NAFLD). Garcinia biflavonoid 1a (GB1a) is a natural product capable of hepatic protection. In this study, the effect of GB1a on anti-inflammatory, antioxidant, and regulation of the accumulation in HepG2 cells and mouse primary hepatocytes (MPHs) was investigated, and its regulatory mechanism was further explored. The result showed that GB1a reduced triglyceride (TG) content and lipid accumulation by regulating the expression of SREBP-1c and PPARα; GB1a reduced reactive oxygen species (ROS) and improved cellular oxidative stress to protect mitochondrial morphology by regulating genes Nrf2, HO-1, NQO1, and Keap1; and GB1a reduced the damage of hepatocytes by inhibiting the expression of inflammatory cytokines interleukin-6 (IL-6), interleukin-1ß (IL-1ß), tumor necrosis factor-alpha (TNF-α), and nuclear factor kappa B (NF-κB) p65. The activities of GB1a were lost in liver SIRT6-specific knockout mouse primary hepatocytes (SIRT6-LKO MPHs). This indicated that activating SIRT6 was critical for GB1a to perform its activity, and GB1a acted as an agonist of SIRT6. It was speculated that GB1a may be a potential drug for NAFLD treatment.

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.

An Integrated Pharmacology-Based Strategy to Investigate the Potential Mechanism of Xiebai San in Treating Pediatric Pneumonia.

Xiebai San (XBS) is a traditional Chinese medicine (TCM) prescription that has been widely used to treat pediatric pneumonia since the Song dynasty. To reveal its underlying working mechanism, a network pharmacology approach was used to predict the active ingredients and potential targets of XBS in treating pediatric pneumonia. As a result, 120 active ingredients of XBS and 128 potential targets were screened out. Among them, quercetin, kaempferol, naringenin, licochalcone A and isorhamnetin showed to be the most potential ingredients, while AKT1, MAPK3, VEGFA, TP53, JUN, PTGS2, CASP3, MAPK8 and NF-κB p65 showed to be the most potential targets. IL-17 signaling pathway, TNF signaling pathway and PI3K-Akt signaling pathway, which are involved in anti-inflammation processes, immune responses and apoptosis, showed to be the most probable pathways regulated by XBS. UPLC-Q/Orbitrap HRMS analysis was then performed to explore the main components of XBS, and liquiritin, quercetin, kaempferol, licochalcone A and glycyrrhetinic acid were identified. Molecular docking analysis of the compounds to inflammation-associated targets revealed good binding abilities of quercetin, kaempferol, licochalcone A and liquiritin to NF-κB p65 and of quercetin and kaempferol to Akt1 or Caspase-3. Moreover, molecular dynamics (MD) simulation for binding of quercetin or kaempferol to NF-κB p65 revealed dynamic properties of high stability, high flexibility and lowbinding free energy. In the experiment with macrophages, XBS markedly suppressed the (Lipopolysaccharides) LPS-induced expression of NF-κB p65 and the production of pro-inflammatory cytokines IL-6 and IL-1ß, supporting XBS to achieve an anti-inflammatory effect through regulating NF-κB p65. XBS also down-regulated the expression of p-Akt (Ser473)/Akt, Bax and Caspase-3 and up-regulated the expression of Bcl-2, indicating that regulating Akt1 and Caspase-3 to achieve anti-apoptotic effect is also the mechanism of XBS for treating pediatric pneumonia. Our study helped to reveal the pharmacodynamics material basis as well as the mechanism of XBS in treating pediatric pneumonia.

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.

"Ghost peaks" of olmesartan medoxomil: Two solution degradation products of olmesartan medoxomil via oxidation mediated by metal ions.

Two unknown solution degradants were found during the dissolution testing in 0.1-M HCl for olmesartan medoxomil (OLM) tablets. The structure of the degradants was identified and characterized by liquid chromatography-ultraviolet (LC-UV), liquid chromatography with tandem mass spectrometry (LC-MS/MS), and nuclear magnetic resonance (NMR) and demonstrated to be cyclization of tetrazole and benzene in the olmesartan (OL) and OLM structures. A series of studies including stress studies, simulation studies, and mechanism-based studies were performed to reveal the potential mechanisms that lead to the formation of the unknown degradants. The study results demonstrated that the degradation was catalyzed with radicals that originated from the metal ions leached from the inner surface of high-performance liquid chromatography (HPLC) glass vials with dissolved oxygen under acidic condition. Prerinsing the glass vials with acidic solution dissolved with EDTA can effectively avoid the generation of such oxidative impurities. The present work provides new insights into the understanding of degradation pathways of OLM, which might support the development of OLM tablets.

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.

Azithromycin "ghost peak": A solution degradation product of azithromycin via leaching from borosilicate glass volumetric flasks and vials.

An interference peak was found while detecting related substances of azithromycin. It is impressive that the degradation peak occurred at about 70 min in the next injection of the test solution (4 mg/mL or higher). Once the degradation peak was observed, it would keep growing. By using a strategy that Q-TOF high resolution mass spectrometry with mechanism-based stress studies, followed by preparative subsequent structure characterization by 1D and 2D NMR, the unknown peak was identified as azithromycin hydrogen borate. It apparently results from azithromycin and residual boron leaching out of the inner surface of the glass volumetric flasks and vials used in the sample preparation. By simulating the above chemical process, boric acid and azithromycin were dissolved in the same extraction diluent and a big interference peak occurred. It was found that boron-free flasks and vials, such as PMP or PP flasks and PTFE or PP vials could be used for the detection of azithromycin related substances to avoid the production of azithromycin hydrogen borate.

[Synthesis and DNA-binding properties of three compounds containing pyridinecarboxamide].

N, N'-bis(2-pyridinecarboxamide)-1,2-ethane(H2L1), N, N'-bis(2-pyridinecarboxamide)-1, 2-beneze(H2L2) and N-phenylpyridine-2-carboxamide(HL3) were synthesized, and characterized by elemental analysis, IR and HNMR spectra. UV-visible (UV-Vis) spectra, fluorescence spectra and SERS spectra to study the interaction of the three ligands with calf thymus DNA. UV-visible (UV-Vis) spectra show that with the incremental addition of DNA, the bands of H2L1, H2L2 and HL3 all show Hypochromism. Meanwhile fluorescence spectra show that the addition of the three ligands to DNA pretreated with EB causes an appreciable reduction in fluorescence intensity, indicating that the ligands compete with ethidium bromide in binding to DNA, and free ethidium bromide increases. The addition of DNA causes the SERS signals of the ligands to weakened and some bands to disappeared. Based on the above experimental results, we conclude that the three ligands bind to DNA mainly through the intercalation mode. The binding constant of the three compouds Kb was calculated, 1.20 x 10(4) for H2L1, 1.33 x 10(4) for H2L2 and is 1.52 x 10(4) for HL3. Kr was also calculated to be 0.67, 1.52 and 1.73 for H2L1, H2L2 and HL3, respectively. The value indicates that the binding of HL3 to DNA is stronger than that of H2L1 and H2L2, as HI3 has proper planar structure, smaller molecular volume and less steric hindrance. The three ligands can all induce the cleavage of plasmid pBR322 DNA. An increase in H2L1, H2L2 and HL3 concentrations causes more transformation of plasmid DNA from closed circular conformations to nicked conformations. But linear conformations have not been observed. The cleavage of plasmid pBR322 DNA caused by the three ligands is not selective.

Synthesis, characterization and DNA-binding studies on La(III) and Ce(III) complexes containing ligand of N-phenyl-2-pyridinecarboxamide.

La(III) and Ce(III) complexes containing ligand of N-phenyl-2-pyridinecarboxamide (HL) were synthesized and characterized by elemental analyses, conductivity measurement, IR spectra and thermal analysis. The general formulas of the complexes were [Ln(HL)(3)(H(2)O)(2)](NO(3))(3).2H(2)O [Ln=La(III), Ce(III)]. The results indicated that the oxygen of carbonyl and the nitrogen of pyridyl coordinated to Ln(III), and there were also two water molecules taking part in coordination. Ln(III) and HL formed 1:3 chelate complexes and the coordination number was eight. The interaction between the complexes and DNA was studied by means of UV-vis spectra, fluorescence spectra, SERS spectra and agarose gel electrophoresis. The results showed that complexes can bind to DNA. The binding ability decreased in following order: La(III) complex, Ce(III) complex, and HL. The interaction modes between DNA and the three compounds were found to be mainly intercalative.