Target-guided isolation and purification of cyclooxygenase-2 inhibitors from Meconopsis integrifolia (Maxim.) Franch. by high-speed counter-current chromatography combined with ultrafiltration liquid chromatography.

Meconopsis integrifolia (Maxim.) Franch. is used extensively in traditional Tibetan medicine for its potent anti-inflammatory properties. In this study, six cyclooxygenase-2 (COX-2) inhibitors were purified from M. integrifolia using high-speed counter-current chromatography guided by ultrafiltration liquid chromatography (ultrafiltration-LC). First, ultrafiltration-LC was performed to profile the COX-2 inhibitors in M. integrifolia. The reflux extraction conditions were further optimized using response surface methodology, and the results showed that the targeted COX-2 inhibitors could be well enriched under the optimized extraction conditions. Then the six target COX-2 inhibitors were separated by high-speed countercurrent chromatography with a solvent system composed of ethyl acetate/n-butanol/water (4:1:4, v/v/v. Finally, the six COX-2 inhibitors, including 21.2 mg of 8-hydroxyluteolin 7-sophoroside, 29.6 mg of 8-hydroxyluteolin 7-[6'''-acetylallosyl-(1→2)-glucoside], 42.5 mg of Sinocrassoside D3, 54.1 mg of Hypolaetin 7-[6'''-acetylallosyll-(l→2)-3''-acetylglucoside, 30.6 mg of Hypolaetin 7-[6'''-acetylallosyll-(l→2)-6''-acetylglucoside and 17.8 mg of Hypolaetin were obtained from 500 mg of sample. Their structures were elucidated by 1 H-NMR spectroscopy. This study reveals that ultrafiltration-LC combined with high-speed counter-current chromatography is a robust and efficient strategy for target-guided isolation and purification of bioactive molecules. It also enhances the scientific understanding of the anti-inflammatory properties of M. integrifolia but also paves the way for its further medicinal applications.

Synthesis and Biological Evaluation of Gentiopicroside Derivatives as Novel Cyclooxygenase-2 Inhibitors with Anti-Inflammatory Activity.

Purpose: Nonsteroidal anti-inflammatory drugs cause a series of adverse reactions. Thus, the search for new cyclooxygenase-2 selective inhibitors have become the main direction of research on anti-inflammatory drugs. Gentiopicroside is a novel selective inhibitor of cyclooxygenase-2 from Chinese herbal medicine. However, it is highly hydrophilic owing to the presence of the sugar fragment in its structure that reduces its oral bioavailability and limits efficacy. This study aimed to design and synthesize novel cyclooxygenase-2 inhibitors by modifying gentiopicroside structure and reducing its polarity. Materials and Methods: We introduced hydrophobic acyl chloride into the gentiopicroside structure to reduce its hydrophilicity and obtained some new derivatives. Their in vitro anti-inflammatory activities were evaluated against NO, TNF-α, PGE2, and IL-6 production in the mouse macrophage cell line RAW264.7 stimulated by lipopolysaccharide. The in vivo inhibitory activities were further tested against xylene-induced mouse ear swelling. Molecular docking predicted that whether new compounds could effectively bind to target protein cyclooxygenase-2. The inhibitory activity of new compounds to cyclooxygenase-2 enzyme were verified by the in vitro experiment. Results: A total of 21 novel derivatives were synthesized, and exhibit lower polarities than the gentiopicroside. Most compounds have good in vitro anti-inflammatory activity. The in vivo activity results demonstrated that 8 compounds were more active than gentiopicroside. The inhibition rate of some compounds was higher than celecoxib. Molecular docking predicted that 6 compounds could bind to cyclooxygenase-2 and had high docking scores in accordance with their potency of the anti-inflammatory activity. The confirmatory experiment proved that these 6 compounds had significant inhibitory effect against cyclooxygenase-2 enzyme. Structure-activity relationship analysis presumed that the para-substitution with the electron-withdrawing groups may benefit the anti-inflammatory activity. Conclusion: These gentiopicroside derivatives especially PL-2, PL-7 and PL-8 may represent a novel class of cyclooxygenase-2 inhibitors and could thus be developed as new anti-inflammatory agents.

Bioactivity-guided isolation of cyclooxygenase-2 inhibitors from Saussurea obvallata (DC.) Edgew. Using affinity solid phase extraction assay.

ETHNOPHARMACOLOGICAL RELEVANCE: Saussurea obvallata (DC.) Edgew. is a traditional Tibetan medicine used for the treatment of inflammation-related diseases, but the scientific validation was very limited. AIM OF THE STUDY: This study aimed to rapid screen and targeted isolate cyclooxygenase-2 (COX-2) inhibitors from S. obvallata extract. MATERIALS AND METHODS: An efficient ligand-fishing method based on affinity solid phase extraction (A-SPE) combining with HPLC was developed. The identified COX-2 inhibitors were separated using preparative liquid chromatography. In vitro COX-2 inhibition assays were employed to confirm the inhibitory activities of the isolated compounds. In addition, the effect of the isolated compounds on the production of prostaglandin E2 (PGE2) and the expression of COX-2 in LPS-induced RAW 264.7 were evaluated. RESULTS: A total of four phenylpropanoids, isolariciresinol, syringaresinol, pinoresinol and balanophonin were targeted isolated as COX-2 inhibitors with IC50 values of 36.4 ± 2.6 µM, 23.1 ± 1.8 µM, 3.6 ± 0.3 µM and 12.1 ± 0.9 µM, respectively. The isolated compounds significantly inhibited LPS-induced NO production in a dose-dependent manner. And, the results of the inhibitory effect on the release of PGE2 and the expression of COX-2 in LPS-induced macrophages were consistent with A-SPE analysis. CONCLUSION: The present work demonstrated that the developed A-SPE-HPLC method could successfully targeted isolated COX-2 inhibitors from S. obvallata extract. And, the isolation results indicated that the therapeutic effect of S. obvallata on inflammation-related diseases was partly based on the COX-2 active ingredients.

Targeted Separation of COX-2 Inhibitor from Pterocephalus hookeri Using Preparative High-Performance Liquid Chromatography Directed by the Affinity Solid-Phase Extraction HPLC System.

Pterocephalus hookeri, as a kind of popular traditional Tibetan medicine, is reputed to treat inflammatory related diseases. In the present work, a cyclooxygenase-2 functionalized affinity solid-phase extraction HPLC system was developed and combined with preparative-HPLC for rapidly screening and separating cyclooxygenase-2 ligand from P. hookeri extracts. Firstly, ligands of cyclooxygenase-2 were screened from extracts by affinity solid-phase extraction HPLC system. Then directed by the screening results, the recognized potential active compounds were targeted separated. As a result, the major cyclooxygenase-2 inhibitor of P. hookeri was obtained with a purity of >95%, which was identified as sylvestroside I. To test the accuracy of this method, the anti-inflammatory activity of sylvestroside I was inspected in lipopolysaccharide-induced RAW 264.7 cells. The results show that sylvestroside I significantly suppressed the release of prostaglandin E2 with dose-dependent, which was in good agreement with the screening result of the affinity solid-phase method. This method of integration of screening and targeted separation proved to be very efficient for the recognition and isolation of cyclooxygenase-2 inhibitors from natural products.

Comprehensive screening and separation of cyclooxygenase-2 inhibitors from Pterocephalus hookeri by affinity solid-phase extraction coupled with preparative high-performance liquid chromatography.

Pterocephalus hookeri, a classical Tibetan herb, is mainly used to treat rheumatoid arthritis (RA) and contains various constituents potentially with cyclooxygenase-2 (COX-2) selective inhibition. A novel strategy for screening and target separating COX-2 inhibitors from the extracts of P. hookeri based on affinity solid-phase extraction (ASPE) column combined with preparative high-performance liquid chromatography (pre-HPLC) was successfully developed. The potential COX-2 inhibitors of P. hookeri were screened and recognized by the ASPE-HPLC system, which strategy is to analyze the compounds isolated by the ASPE column. Then, the active compounds were targeted separated by pre-HPLC according to real-time chromatograms. The control drugs celecoxib and glipizide were analyzed to verify the specificity and accuracy of the developed method. As a result, two pure compounds with COX-2 binding affinities were successfully separated from P. hookeri. They were characterized as swertisin and scopoletin using 1H- and 13C NMR spectroscopy, and the in vitro COX-2 inhibitory activities were verified. Compounds with COX-2 inhibitory activities could be screened and targeted separated from crude extracts by this strategy, which indicated that the proposed method was feasible, robust and effective for rapid separation of COX-2 inhibitors from natural products.

Characterization and screening of cyclooxygenase-2 inhibitors from Zi-shen pill by affinity ultrafiltration-ultra performance liquid chromatography mass spectrometry.

ETHNOPHARMACOLOGICAL RELEVANCE: Zi-shen pill (ZSP) is a classical Chinese herbal formula used for treatment of benign prostatic hyperplasia (BPH). AIM OF THE STUDY: To characterize and screen cyclooxygenase-2 (COX-2) inhibitors from ZSP extract. MATERIALS AND METHODS: The ZSP extract was incubated with COX-2 and the potential ligands were screened out by affinity ultrafiltration. Celecoxib and glipizide were chosen as positive control and negative control drug, respectively. Affinity ultrafiltration-ultra performance liquid chromatography-mass spectrometry (UPLC-MS) method was used. In addition, in vitro COX-2 inhibitory assay and in silico molecular docking technique were used for further validation. RESULTS: A total of 20 components were discovered and identified from ZSP ultrafiltrate by high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), among which 8 compounds were deduced as potential COX-2 inhibitors by their high specific binding values (>1.5). Inhibitory activities of demethyleneberberine, palmatine, berberine and timosaponin A-I were confirmed by an enzyme assay of COX-2, which validated the reliability of our approach. Molecular docking simulation investigated potential mechanism of action for these compounds. CONCLUSION: The results revealed that affinity ultrafiltration UPLC-MS could successfully screen out the potential COX-2 inhibitors from complex Chinese herbal formula ZSP extract, indicating that its therapeutic effect on BPH was partly based on the enzyme active ingredients.

Screening and isolation of cyclooxygenase-2 inhibitors from the stem bark of Phellodendron amurense Ruprecht by ultrafiltration with liquid chromatography and tandem mass spectrometry, and complex chromatography.

Nonsteroidal anti-inflammatory drugs appear to reduce the risk of developing cancer. One mechanism through which nonsteroidal anti-inflammatory drugs act to prevent carcinogenesis is inhibition of the activity of the enzyme cyclooxygenase-2. The cyclooxygenase-2 inhibitors are widely used to reduce the risk of developing cancer. Natural products are considered to be a promising source of several novel cyclooxygenase-2 inhibitors. Ultrafiltration with liquid chromatography and mass spectrometry is an efficient method that can be applied to rapidly screen and identify the ligands from the barks of Phellodendron amurense Ruprecht. A continuous online method comprised of pressurized liquid extraction, countercurrent chromatography, and semi-preparative liquid chromatography was developed for the efficient scaled-up production of eight compounds with high purities. The bioactivities of the separated compounds were assessed by an in vitro enzyme inhibition assay. The use of bioactivity screening method combined with preparation method of bioactive compounds and an in vitro enzyme inhibition assay facilitated the efficient screening and isolation of the cyclooxygenase-2 inhibitors from complex samples. This could be used as an efficient method for the large-scale production of functional ingredients.

Putative neuroprotective pharmacotherapies to target the staged progression of mental illness.

AIM: Neuropsychiatric disorders including depression, bipolar and schizophrenia frequently exhibit a neuroprogressive course from prodrome to chronicity. There are a range of agents exhibiting capacity to attenuate biological mechanisms associated with neuroprogression. This review will update the evidence for putative neuroprotective agents including clinical efficacy, mechanisms of action and limitations in current assessment tools, and identify novel agents with neuroprotective potential. METHOD: Data for this review were sourced from online databases PUBMED, Embase and Web of Science. Only data published since 2012 were included in this review, no data were excluded based on language or publication origin. RESULTS: Each of the agents reviewed inhibit one or multiple pathways of neuroprogression including: inflammatory gene expression and cytokine release, oxidative and nitrosative stress, mitochondrial dysfunction, neurotrophin dysregulation and apoptotic signalling. Some demonstrate clinical efficacy in preventing neural damage or loss, relapse or cognitive/functional decline. Agents include: the psychotropic medications lithium, second generation antipsychotics and antidepressants; other pharmacological agents such as minocycline, aspirin, cyclooxygenase-2 inhibitors, statins, ketamine and alpha-2-delta ligands; and others such as erythropoietin, oestrogen, leptin, N-acetylcysteine, curcumin, melatonin and ebselen. CONCLUSIONS: Signals of evidence of clinical neuroprotection are evident for a number of candidate agents. Adjunctive use of multiple agents may present a viable avenue to clinical realization of neuroprotection. Definitive prospective studies of neuroprotection with multimodal assessment tools are required.

Vitex negundo inhibits cyclooxygenase-2 inflammatory cytokine-mediated inflammation on carrageenan-induced rat hind paw edema.

BACKGROUND: Vitex negundo L. (Verbenaceae) is a hardy plant widely distributed in the Indian subcontinent and used for treatment of a wide spectrum of health disorders in traditional and folk medicine, some of which have been experimentally validated. In present study, we aimed to investigate the anti-inflammatory effects of V. negundo in carrageenan-induced paw edema in rats, and to investigate the probable mechanism of anti-inflammatory action. MATERIALS AND METHODS: Paw edema was produced by injecting 1% solution of carrageenan, and the paw volume was measured before and after carrageenan injection up to 5 h. V. negundo leaf oil was extracted using a Clevenger apparatus and administered by a trans-dermal route to Wistar rats and the percentage of inhibition of inflammation was observed using a Plethysmometer by comparing a compound aerosol-based formulation with 1 mg diclofinac diethylamine BP and 7 mg methyl salicylate IP/kg body weight served as a standard drug whereas paraffin oil served as the placebo group. After withdrawing of blood, serum was separated and cyclooxygenase (COX)-1 and COX-2 inhibitory activities were measured by the enzyme immuno assay (EIA) method by using a COX inhibitor screening assay kit. RESULTS AND DISCUSSION: V. negundo leaf oil significantly (P < 0.05) reduced the carrageenan-induced paw edema as compared to the placebo group (paraffin oil) and 1 mg diclofinac diethylamine BP and 7 mg methyl salicylate IP showed the maximum inhibition of paw edema as compared to the V. negundo leaf oil treated group and the control group. Also in the present study V. negundo leaf oil showed significantly (P < 0.05) inhibits COX-1 pathways rather than COX-2 pathways as compared to the V. negundo leaf oil treated group. CONCLUSION: It is suggested that the V. negundo leaf oil is a potent anti-inflammatory agent and acts via inhibition of COX-2 without much interfering COX-1 pathways.

Uso combinado da laserterapia de baixa potência e da inibição da ciclooxigenase-2 na reepitelização de ferida incisional em pele de camundongos: um estudo pré-clínico / Combined use of low level laser therapy and cyclooxygenase-2 selective inhibition on skin incisional wound reepithelialization in mice: a preclinical study

FUNDAMENTOS: A laserterapia de baixa potência e os inibidores seletivos da ciclooxigenase-2 (ICOX2) vem sendo muito utilizados para modular a resposta inflamatória, entretanto, os seus efeitos na reepitelização de feridas não são bem compreendidos. OBJETIVO: Avaliar os efeitos isolados e combinados da laserterapia de baixa potência e da ICOX2 na reepitelização de ferida incisional na pele de camundongos. MÉTODO: Foi induzida uma ferida de 1 cm no dorso de cada camundongo, que foram divididos em quatro grupos (N=20): Controle, Laserterapia, Tratados com celecoxib e Terapia conjugada. Os animais dos grupos celecoxib e Terapia conjugada foram tratados com celecoxib por 10 dias antes da incisão cutânea. As feridas experimentais foram irradiadas com laserterapia de baixa potência He-Ne (632nm, dose: 4J/cm2) em varredura, por 12 segundos durante três dias consecutivos nos grupos Laserterapia e Terapia conjugada. Os animais foram sacrificados no 3º dia de pós-operatório. Amostras das feridas foram coletadas e coradas (Tricromio de Masson) para análise histomorfométrica. RESULTADOS: Tanto o grupo Laserterapia, quanto o grupo celecoxib, mostrou aumento da reepitelização cutânea em relação ao grupo Controle, entretanto, o grupo Terapia conjugada não apresentou diferenças. Quanto à queratinização o grupo Laserterapia e Terapia conjugada apresentaram redução dos queratinócitos, comparados com o grupo Controle. CONCLUSÕES: Os resultados mostram que o uso da laserterapia de baixa potência e da ICOX2 isoladamente aumentam as células epiteliais, mas somente a laserterapia de baixa potência reduziu os queratinócitos cutâneos. O tratamento conjugado restabelece a reepitelização inata e dimunui a queratinização, embora ocorra uma acelerada contração da ferida com melhora na organização da ferida na pele de camundongos.

BACKGROUND: Low level laser therapy and cyclooxygenase-2 (ICOX2) selective inhibitors have been widely used to modulate inflammatory response; however, their effect on wound reepithelialization are not well understood. OBJECTIVE: To evaluate the isolated and combined effects of low level laser therapy and ICOX2 in the reepithelization of skin incisional wounds in mice. METHODS: We induced a 1-cm wound on the back of each mouse, which were divided into four groups (N = 20): control, laser therapy, treated with celecoxib and combined therapy. The animals in the celecoxib and combined therapy groups were treated with celecoxib for 10 days before skin incision. The experimental wounds were irradiated with He-Ne low power laser (632nm, dose: 4J/cm2) in scanning for 12 seconds during three consecutive days in the laser therapy and combined therapy groups. The animals were sacrificed 3 days after surgery. Samples of the wounds were collected and stained (Masson's Trichrome) for histomorphometric analysis. RESULTS: Both the laser therapy group and the celecoxib group showed an increase in skin reepithelialization compared to the control group; however, the combined therapy group showed no differences. As for keratinization, the laser therapy and combined therapy groups showed a reduction in keratinocytes compared with the control group. CONCLUSION: The results show that the use of low level laser therapy and ICOX2 in isolation increases epithelial cells, but only low level laser therapy reduced skin keratinocytes. The combined treatment restores innate epithelialization and decreases keratinization in spite of accelerating wound contraction with improvement in the organization of the wound in the skin of mice.