Computational screening of potential anti-inflammatory leads from Jeevaneeya Rasayana plants targeting COX-2 and 5- LOX by molecular docking and dynamic simulation approaches.

Inflammation plays a pivotal role in various pathological processes, ranging from routine injuries and infections to cancer. Cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) are two major enzymes involved in the formation of lipid mediators of inflammation, such as prostaglandins and leukotrienes, through the arachidonic acid pathway. Despite the frequent use of nonsteroidal anti-inflammatory drugs for managing inflammatory disorders by inhibiting these enzymes, there is a wide spectrum of adverse effects linked to their usage. Jeevaneeya Rasayana (JR), a polyherbal formulation traditionally used in India, is renowned for its anti-inflammatory properties. The present study aimed to identify the potential phytocompounds in JR plants against COX-2 and 5-LOX, utilizing molecular docking and dynamic simulations. Among the 429 identified phytocompounds retrieved from publicly available data sources, Terrestribisamide and 1-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine have shown potential binding affinity and favorable interactions with COX-2 and 5-LOX arachidonic acid binding sites. The physicochemical properties and ADMET profiles of these compounds determined their drug-likeness and pharmacokinetics features. Additional validation using molecular dynamics simulations, SASA, Rg, and MM-PBSA binding energy calculations affirmed the stability of the complex formed between those compounds with target proteins. Together, the study identified the effectual binding potential of those bioactive compounds against COX-2 and 5-LOX, providing a viable approach for the development of effective anti-inflammatory medications.

Enhancing therapeutic efficacy in triple-negative breast cancer and melanoma: synergistic effects of modulated electro-hyperthermia (mEHT) with NSAIDs especially COX-2 inhibition in in vivo models.

Triple-negative breast cancer (TNBC) is a leading cause of cancer mortality and lacks modern therapy options. Modulated electro-hyperthermia (mEHT) is an adjuvant therapy with demonstrated clinical efficacy for the treatment of various cancer types. In this study, we report that mEHT monotherapy stimulated interleukin-1 beta (IL-1ß) and interleukin-6 (IL-6) expression, and consequently cyclooxygenase 2 (COX-2), which may favor a cancer-promoting tumor microenvironment. Thus, we combined mEHT with nonsteroid anti-inflammatory drugs (NSAIDs): a nonselective aspirin, or the selective COX-2 inhibitor SC236, in vivo. We demonstrate that NSAIDs synergistically increased the effect of mEHT in the 4T1 TNBC model. Moreover, the strongest tumor destruction ratio was observed in the combination SC236 + mEHT groups. Tumor damage was accompanied by a significant increase in cleaved caspase-3, suggesting that apoptosis played an important role. IL-1ß and COX-2 expression were significantly reduced by the combination therapies. In addition, a custom-made nanostring panel demonstrated significant upregulation of genes participating in the formation of the extracellular matrix. Similarly, in the B16F10 melanoma model, mEHT and aspirin synergistically reduced the number of melanoma nodules in the lungs. In conclusion, mEHT combined with a selective COX-2 inhibitor may offer a new therapeutic option in TNBC.

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.

Rosmarinic acid in combination with ginsenoside Rg1 suppresses colon cancer metastasis via co-inhition of COX-2 and PD1/PD-L1 signaling axis.

Metastasis of colorectal cancer (CRC) is a leading cause of mortality among CRC patients. Elevated COX-2 and PD-L1 expression in colon cancer tissue has been linked to distant metastasis of tumor cells. Although COX-2 inhibitors and immune checkpoint inhibitors demonstrate improved anti-tumor efficacy, their toxicity and variable therapeutic effects in individual patients raise concerns. To address this challenge, it is vital to identify traditional Chinese medicine components that modulate COX-2 and PD-1/PD-L1: rosmarinic acid (RA) exerts striking inhibitory effect on COX-2, while ginsenoside Rg1 (GR) possesses the potential to suppress the binding of PD-1/PD-L1. In this study we investigated whether the combination of RA and GR could exert anti-metastatic effects against CRC. MC38 tumor xenograft mouse model with lung metastasis was established. The mice were administered RA (100 mg·kg-1·d-1, i.g.) alone or in combination with GR (100 mg·kg-1·d-1, i.p.). We showed that RA (50, 100, 150 µM) or a COX-2 inhibitor Celecoxib (1, 3, 9 µM) concentration-dependently inhibited the migration and invasion of MC38 cells in vitro. We further demonstrated that RA and Celecoxib inhibited the metastasis of MC38 tumors in vitro and in vivo via interfering with the COX-2-MYO10 signaling axis and inhibiting the generation of filopodia. In the MC38 tumor xenograft mice, RA administration significantly decreased the number of metastatic foci in the lungs detected by Micro CT scanning; RA in combination with GR that had inhibitory effect on the binding of PD-1 and PD-L1 further suppressed the lung metastasis of colon cancer. Compared to COX-2 inhibitors and immune checkpoint inhibitors, RA and GR displayed better safety profiles without disrupting the tissue structures of the liver, stomach and colon, offering insights into the lower toxic effects of clinical traditional Chinese medicine against tumors while retaining its efficacy.

A high throughput method for detection of cyclooxygenase-2 enzyme inhibitors by effect-directed analysis applying high performance thin layer chromatography-bioassay-mass spectrometry.

A high throughput method was developed to detect bioactive molecules with inhibitory activity over cyclooxygenase (COX-2) enzyme applying effect-directed analysis and planar chromatography hyphenated with bioassay and mass spectrometry. The assay was based on the indirect measurement of arachidonic acid transformation into prostaglandin with the colorimetric co-substrate N,N,N',N'-tetramethyl-p-phenylenediamine. Inhibitory zones were observed as colorless bands over a blue background. Using a central composite design the critical factors like substrate concentration, enzyme: substrate ratio, reaction time, and co-substrate concentration were optimized. Optimal conditions were achieved with 0.03 mg/mL of arachidonic acid, 0.15 U/mL of COX-2, and 8.21 mg/mL of chromogenic reagent. Method usefulness was challenged analyzing fresh Chiloe's giant garlic (Allium ampeloprasum L) ethanol: water (8:2 v/v) extract, finding COX-2 inhibitors that were preliminarily identified as the isomers γ-glutamyl-S-allyl-l-cysteine and γ-glutamyl-S-(trans-1-propenyl)-L- cysteine.

Effect of a Cyclooxygenase-2 Inhibitor in Combination with (-)-Epigallocatechin Gallate or Polyphenon E on Cisplatin-Induced Lung Tumorigenesis in A/J Mice.

We investigated the effects of celecoxib combined with (-)-epigallocatechin-3-gallate (EGCG) or polyphenon E in a cisplatin-induced lung tumorigenesis model. Four-week-old female A/J mice were divided into seven groups: (i) Control, (ii) 150 mg/kg celecoxib (150Cel), (iii) 1,500 mg/kg celecoxib (1500Cel), (iv) EGCG+150 mg/kg celecoxib (EGCG+150Cel), (v) EGCG+1,500 mg/kg celecoxib (EGCG+1500Cel), (vi) polyphenon E+150 mg/kg celecoxib (PolyE+150Cel), and (vii) polyphenon E+1,500 mg/kg celecoxib (PolyE+1500Cel). All mice were administered cisplatin (1.62 mg/kg of body weight, i.p.) 1×/week for 10 weeks and sacrificed at week 30; the numbers of tumors on the lung surface were then determined. The tumor incidence and multiplicity (no. of tumors/mouse, mean±SD) were respectively 95% and 2.15±1.50 in Control, 95% and 2.10±1.29 in 150Cel, 86% and 1.67±1.20 in 1500Cel, 71% and 1.38±1.24 in EGCG+150Cel, 67% and 1.29±1.38 in EGCG+1500Cel, 80% and 1.95±1.36 in PolyE+150Cel, and 65% and 1.05±0.10 in PolyE+1500Cel. The combination of high-dose celecoxib with EGCG or polyphenon E significantly reduced multiplicity in cisplatin-induced lung tumors.

Racemic norneolignans from the resin of Ferula sinkiangensis and their COX-2 inhibitory activity.

Five new norneolignans sinkianlignans G-K (1-5), one phenolic compound ferulagenol A (6) and seven known compounds (7-13) were isolated from Ferula sinkiangensis. All the norneolignans were racemic mixtures, and chiral HPLC was used to further separate them. Their structures were assigned, including absolute configurations, using spectroscopic and computational methods. Biological evaluation showed that compounds 1-9 had significant COX-2 inhibitory activity with IC50 values ranging from 3.00 µM to 23.19 µM.

Upregulation of Beta 1 and Arachidonic Acid Metabolizing Enzymes in the Mouse Hearts and Kidneys after Sub Chronic Administration of Rofecoxib.

BACKGROUND: An imbalance in the levels of arachidonic acid (ARA) metabolites in cardiovascular disorders and drug-induced cardiotoxicity have been previously described. AIMS: This study aimed to investigate the influence of cyclooxygenase-2 (COX-2) selective inhibitors on the gene expression of ARA-metabolizing genes and beta1 gene in the hearts and kidneys of experimental mice. METHODS: Thirty-five balb/c mice were divided into five groups with seven mice per group. The groups were then given two distinct types of COX-2 selective inhibitors, rofecoxib and celecoxib, in two different doses equivalent to those used in human treatment for 30 days. The mRNA expression of beta1, ace2, and ARA-metabolizing genes, coxs, lipoxygenases (aloxs), and cytochrome p450 (cyp450s) in mice heart and kidneys were assessed. Genes were analyzed using real-time polymerase chain reaction analysis. In addition, rofecoxib-induced histological alterations were examined. RESULTS: It was found that only the high dose of rofecoxib (5 mg/kg) caused toxicological alterations, a finding that was indicated by a significant increase (P < 0.05) in the relative weight of the mouse hearts and increase in the ventricle wall thickness as observed through pathohistological examination. This increase was associated with a significant increase in the mRNA expression level of the beta1 receptor in both the heart and kidneys of the mice (53- and 12-fold, respectively). The expression of both cox1 and 2 genes was increased 4-fold in the kidneys. In addition, the expression of the alox12 gene increased significantly (by 67-fold in the heart and by 21-fold in the kidney), while alox15 gene expression was upregulated in the heart by 8-fold and 5-fold in the kidney. The genes responsible for synthesizing 20- Hydroxyeicosatetraenoic acid (cyp4a12 and cyp1a1) were significantly upregulated (P < 0.05) in the hearts of high-dose rofecoxib-treated mice by 7- and 17 -fold, respectively. In addition, the expression of epoxyeicosatrienoic acid-synthesizing genes, cyp2c29 and cyp2j5, was increased significantly (P < 0.05) in the hearts of high-dose rofecoxib-treated mice by 4- and 16-fold, respectively. CONCLUSION: Rofecoxib caused upregulation of the mRNA expression of the beta 1 gene in association with increased expression of ARA-metabolizing genes in mouse hearts and kidneys. These findings may help us understand the molecular cardiotoxic mechanism of rofecoxib.

Fimbristylis aestivalis Vahl: a potential source of cyclooxygenase-2 (COX-2) inhibitors.

Cyclooxygenase-2 (COX-2) is an inducible enzyme that accelerates the biosynthesis of PGs during inflammation and has emerged as an important therapeutic target for anti-inflammatory drugs. Natural compounds may serve as a source of inspiration for pharmaceutical chemists and a foundation for developing innovative COX-2 inhibitors with fewer side effects. Therefore, the objective of this study was to identify the potent COX-2 inhibitor and anti-inflammatory activity of the Fimbristylis aestivalis whole plant extract (FAWE). The plant extract was found dominant with rosmarinic acid followed by catechin hydrate, syringic acid, rutin hydrate, (-) epicatechin, quercetin, myricetin, and catechol. FAWE exhibited considerable dose-dependent analgesic efficacy in all analgesic test models. FAWE also showed promising anti-inflammatory potential in carrageenan-induced inflammations in mice. This result was corroborated by molecular docking, revealing that the aforesaid natural polyphenols adopt the same orientation as celecoxib in the COX-2 active site. On the other hand, molecular dynamics (MD) simulations were performed between the most abundant components (rosmarinic acid, catechin hydrate, and syringic acid) and COX-2. Based on hydrogen bonding, RMSD, RMSF, radius of gyration, PCA, and Gibbs free energy landscape analysis, the results demonstrated that these compounds are very stable in the active site of COX-2, indicating substantial COX-2 inhibitory activity.

Design, Synthesis, and Biological Evaluation of Dual-Target COX-2/CYP51 Inhibitors for the Treatment of Fungal Infectious Diseases.

The design of novel dual-target (COX-2/CYP51) inhibitors was proposed in the study, and three series of compounds were constructed though the pathway of skeleton screening and combination; their molecular structures were synthesized and evaluated. Most of the compounds exhibited significant antifungal ability. Among them, potential compounds (10a-2, 16b-3) with excellent antifungal and anti-drug-resistant fungal ability (MIC50, 0.125-2.0 µg/mL) were selected for the subsequent mechanistic study. On the one hand, these compounds could block the ergosterol biosynthesis pathway by inhibiting CYP51 and influence the internal physiological function of fungal cells, which included the increase of the ROS level, the anomaly of ΔΨm, and the emergence of an apoptotic state. On the other hand, these compounds also effectively showed COX-2 inhibition ability, eliminated the inflammatory reaction of the infected region, and activated the body's immune function. In summary, this study not only provided a novel antifungal drug design pathway but also discovered excellent target compounds.

[Virtual screening and antiepileptic activity evaluation of COX-2 inhibitory components in Trachelospermi Caulisetfolium].

This study investigated the potential active components against cyclooxygenase-2(COX-2) from Trachelospermi Caulisetfolium and explored the pharmacodynamic material basis.A pharmacophore-based virtual screening method was adopted to establish a COX-2 ligands-based HipHop pharmacophore model on the basis of the information on compounds with COX-2 inhibitory activity reported in published research articles.The reported components in Trachelospermi Caulisetfolium were collected to establish the compound library and matched with the pharmacophores.Subsequently, the matched small molecule compounds underwent molecular docking with COX-2 targets(PDB ID: 3 LN1), and the interaction of potential active monomers and COX-2 was further explored by molecular dynamics.The antiepileptic effect of active monomer arctigenin(15) was determined based on the pentylenetetrazole(PTZ)-induced seizure model, and its modulatory effect on the COX-2 level was evaluated.A compound library containing 118 chemical constituents in Trachelospermi Caulisetfolium was established by literature retrieval.The preferred pharmacophore 04 was selected through test set verification for virtual screening of the compound library of Trachelospermi Caulisetfolium.After matching, six potential constituents with COX-2 inhibitory activity were obtained.The interaction of five compounds with COX-2 and COX-1 was analyzed by molecular docking, and 10 ns molecular dynamics was performed on two compounds.Compound 15 could prolong the latent time of PTZ-induced seizures at medium and high doses, improve the anxiety-and depression-like behaviors induced by PTZ, reduce the expression level of COX-2, and decrease the number of COX-2 immuno-posi-tive cells in the hippocampal CA1 region.The results showed that it was reasonable to investigate the components in Trachelospermi Caulisetfolium with COX-2 inhibitory activity based on virtual screening and activity evaluation.

Computational Analysis and Biological Activities of Oxyresveratrol Analogues, the Putative Cyclooxygenase-2 Inhibitors.

Polyphenols are a large family of naturally occurring phytochemicals. Herein, oxyresveratrol was isolated from ethanolic crude extracts of Artocarpus lacucha Buch.-Ham., and chemically modified to derive its lipophilic analogues. Biological screening assays showed their inhibitory potency against cyclooxygenase-2 (COX-2) with very low cytotoxicity to the MRC-5 normal cell lines. At the catalytic site of COX-2, docking protocols with ChemPLP, GoldScore and AutoDock scoring functions were carried out to reveal hydrogen bonding interactions with key polar contacts and hydrophobic pi-interactions. For more accurate binding energetics, COX-2/ligand complexes at the binding region were computed in vacuo and implicit aqueous solvation using M06-2X density functional with 6-31G+(d,p) basis set. Our computational results confirmed that dihydrooxyresveratrol (4) is the putative inhibitor of human COX-2 with the highest inhibitory activity (IC50 of 11.50 ± 1.54 µM) among studied non-fluorinated analogues for further lead optimization. Selective substitution of fluorine provides a stronger binding affinity; however, lowering the cytotoxicity of a fluorinated analogue to a normal cell is challenging. The consensus among biological activities, ChemPLP docking score and the binding energies computed at the quantum mechanical level is obviously helpful for identification of oxyresveratrol analogues as a putative anti-inflammatory agent.

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.

Alkaloids of Adhatoda vasica Nees. as potential inhibitors of cyclooxygenases - an in-silico study.

Eicosanoid pathways play a crucial role in the progression and resolution of inflammation. NSAIDs act as anti-inflammatory agents by inhibiting both the isoforms of cyclooxygenases (COXs) whereas, COXIBs act as specific COX-2 inhibitors. Excessive usage of the same is linked with gastrointestinal bleeding and increased cardiovascular risk, respectively. The current in-silico study was aimed at evaluating the potential of major alkaloids of A. vasica (vasicine (VAS), vasicinone (VAE), and Deoxyvasicine (DOV)) as inhibitors of COXs. The results of the computed binding energy (ΔG) indicate that Celecoxib (CEL), DOV, and VAS have a higher affinity to COX-2, while VAE has a higher affinity to COX-1, and Mefenamic acid (MEF) was not selective. Among the alkaloids, VAE exhibited the best ΔG (of -8.2 kcal/mol) with COX-1, while VAS exhibited the best ΔG (of -8.2 kcal/mol) with COX-2. This was comparable to the ΔG exhibited by Mefenamic acid (-8.7 kcal/mol with both the COXs). With their potential to remain gastroprotective while having the ability to inhibit enzymes of both the prostaglandin and leukotriene pathways, the alkaloids of A. vasica could be promising leads for the design of Eicosanoid pathway modulators/inhibitors.Communicated by Ramaswamy H. Sarma.

Evaluation of in vitro and in silico anti-inflammatory potential of some selected medicinal plants of Bangladesh against cyclooxygenase-II enzyme.

ETHNOPHARMACOLOGICAL RELEVANCE: Medicinal plants are sources of chemical treasures that can be used in treatment of different diseases, including inflammatory disorders. Traditionally, Heritiera littoralis, Ceriops decandra, Ligustrum sinense, and Polyscias scutellaria are used to treat pain, hepatitis, breast inflammation. The present research was designed to explore phytochemicals from the ethanol extracts of H. littoralis, C. decandra, L. sinense, and P. scutellaria to discern the possible pharmacophore (s) in the treatment of inflammatory disorders. MATERIAL AND METHODS: The chemical compounds of experimental plants were identified through GC-MS analysis. Furthermore, in-vitro anti-inflammatory activity was assessed in human erythrocytes and an in-silico study was appraised against COX-2. RESULTS: The experimental extracts totally revealed 77 compounds in GC-MS analysis and all the extracts showed anti-inflammatory activity in in-vitro assays. The most favorable phytochemicals as anti-inflammatory agents were selected via ADMET profiling and molecular docking with specific protein of the COX-2 enzyme. Molecular dynamics simulation (MDS) confirmed the stability of the selected natural compound at the binding site of the protein. Three phytochemicals exhibited the better competitive result than the conventional anti-inflammatory drug naproxen in molecular docking and MDS studies. CONCLUSION: Both experimental and computational studies have scientifically revealed the folklore uses of the experimental medicinal plants in inflammatory disorders. Overall, N-(2-hydroxycyclohexyl)-4-methylbenzenesulfonamide (PubChem CID: 575170); Benzeneethanamine, 2-fluoro-. beta., 3, 4-trihydroxy-N-isopropyl (PubChem CID: 547892); and 3,5-di-tert-butylphenol (PubChem CID: 70825) could be the potential leads for COX-2 inhibitor for further evaluation of drug-likeliness.

Screening cyclooxygenase-2 inhibitors from Allium sativum L. compounds: in silico approach.

Inflammation is a natural protective response toward various simulators, including tissue damage or pathogens. The cyclooxygenase-2 (COX-2) is a very important protein in triggering pain and inflammation. Previous studies have claimed that Allium sativum offers a wide range of anti-inflammatory therapeutics for human consumption. Drug discovery is a complicated process, though in silico methods can make this procedure simpler and more cost-effective. At the current study, we performed the virtual screening of eight Allium sativum-derived compounds via molecular docking with COX-2 enzyme and confirmed the binding energy by docking score estimate followed by ADMET and drug-likeness investigation. The resulting highest-docking scored compound was exposed to molecular dynamics simulation (MDS) for evaluating stability of the docked enzyme-ligand complex and to gauge the oscillation and conformational alterations for the time of enzyme-ligand interaction. The factors of RMSD, RMSF, hydrogen bond interactions, and Rg after 100 ns of MDS proved the stability of alliin in the active site of COX-2 in comparison with celecoxib (CEL) as the control. Moreover, we investigated the binding affinity analysis of all compounds via MM/PBSA method. The results from this study suggest that alliin (a sulfuric compound) exhibits a higher binding affinity for the COX-2 enzyme compared to the other compounds and CEL. Alliin showed to be a possible anti-inflammatory therapeutic candidate for managing the inflammatory conditions.

Activity-Guided Characterization of COX-2 Inhibitory Compounds in Waltheria indica L. Extracts.

Inflammation is the body's response to infection or tissue injury in order to restore and maintain homeostasis. Prostaglandin E2 (PGE-2) derived from arachidonic acid (AA), via up-regulation of cyclooxygenase-2 (COX-2), is a key mediator of inflammation and can also be induced by several other factors including stress, chromosomal aberration, or environmental factors. Targeting prostaglandin production by inhibiting COX-2 is hence relevant for the successful resolution of inflammation. Waltheria indica L. is a traditional medicinal plant whose extracts have demonstrated COX-2 inhibitory properties. However, the compounds responsible for the activity remained unknown. For the preparation of extracts with effective anti-inflammatory properties, characterization of these substances is vital. In this work, we aimed to address this issue by characterizing the substances responsible for the COX-2 inhibitory activity in the extracts and generating prediction models to quantify the COX-2 inhibitory activity without biological testing. For this purpose, an extract was separated into fractions by means of centrifugal partition chromatography (CPC). The inhibitory potential of the fractions and extracts against the COX-2 enzyme was determined using a fluorometric COX-2 inhibition assay. The characterizations of compounds in the fractions with the highest COX-2 inhibitory activity were conducted by high resolution mass spectrometry (HPLC-MS/MS). It was found that these fractions contain alpha-linolenic acid, linoleic acid and oleic acid, identified and reported for the first time in Waltheria indica leaf extracts. After analyzing their contents in different Waltheria indica extracts, it could be demonstrated that these fatty acids are responsible for up to 41% of the COX-2 inhibition observed with Waltheria indica extract. Additional quantification of secondary metabolites in the extract fractions revealed that substances from the group of steroidal saponins and triterpenoid saponins also contribute to the COX-2 inhibitory activity. Based on the content of compounds contributing to COX-2 inhibition, two mathematical models were successfully developed, both of which had a root mean square error (RMSE) = 1.6% COX-2 inhibitory activity, demonstrating a high correspondence between predicted versus observed values. The results of the predictive models further suggested that the compounds contribute to COX-2 inhibition in the order linoleic acid > alpha linolenic acid > steroidal saponins > triterpenoid saponins. The characterization of substances contributing to COX-2 inhibition in this study enables a more targeted development of extraction processes to obtain Waltheria indica extracts with superior anti-inflammatory properties.

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.

A Cornflower Extract Containing N-Feruloylserotonin Reduces Inflammation in Human Skin by Neutralizing CCL17 and CCL22 and Inhibiting COX-2 and 5-LOX.

Thymus and Activation-Regulated Chemokine (TARC/CCL17) and Macrophage-Derived Chemokine (MDC/CCL22) are two key chemokines exerting their biological effect via binding and activating a common receptor CCR4, expressed at the surface of type 2 helper T (Th2) cells. By recruiting Th2 cells in the dermis, CCL17 and CCL22 promote the development of inflammation in atopic skin. The aim of this research was to develop a plant extract whose biological properties, when applied topically, could be beneficial for people with atopic-prone skin. The strategy which was followed consisted in identifying ligands able to neutralize the biological activity of CCL17 and CCL22. Thus, an in silico molecular modeling and a generic screening assay were developed to screen natural molecules binding and blocking these two chemokines. N-Feruloylserotonin was identified as a neutraligand of CCL22 in these experiments. A cornflower extract containing N-feruloylserotonin was selected for further in vitro tests: the gene expression modulation of inflammation biomarkers induced by CCL17 or CCL22 in the presence or absence of this extract was assessed in the HaCaT keratinocyte cell line. Additionally, the same cornflower extract in another vehicle was evaluated in parallel with N-feruloylserotonin for cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) enzymatic cellular inhibition. The cornflower extract was shown to neutralize the two chemokines in vitro, inhibited COX-2 and 5-LOX, and demonstrated anti-inflammatory activities due mainly to the presence of N-feruloylserotonin. Although these findings would need to be confirmed in an in vivo study, the in vitro studies lay the foundation to explain the benefits of the cornflower extract when applied topically to individuals with atopic-prone skin.

Insights into cyclooxygenase-2 inhibition by isolated bioactive compounds 3-caffeoyl-4-dihydrocaffeoyl quinic acid and isorhamnetin 3-O-ß-D-glucopyranoside from Salicornia herbacea.

BACKGROUND: Cyclooxygenase-2 (COX-2) is an important enzyme with numerous biological functions. Overexpression of COX-2 has been associated with various inflammatory-related diseases and therefore, projected as an important pharmacological target. PURPOSE: We aimed to investigate the inhibitory potential of isolated bioactive compounds, 3-caffeoyl-4-dihydrocaffeoyl quinic acid (CDQ) and isorhamnetin 3-O-ß-d-glucopyranoside (IDG), from Salicornia herbacea against COX-2 using both computational and in vitro approaches. METHODS: Computational analysis, including molecular docking, molecular dynamics (MD) simulations, and post-simulations analysis, were employed to estimate the binding affinity and stability of CDQ and IDG in the catalytic pocket of COX-2 against Celecoxib as positive control. These predictions were further evaluated using in vitro enzyme inhibition as well as gene expression mediation in macrophages cells. RESULTS: Molecular docking analysis revealed substantial binding energy of CDQ (-6.1 kcal/mol) and IDG (-5.9 kcal/mol) with COX-2, which are lower than Celecoxib (-8.1 kcal/mol). MD simulations (100 ns) and post simulation analysis exhibited the substantial stability and binding affinity of docked CDQ and IDG compounds with COX-2. In vitro assays indicated significant COX-2 inhibition by CDQ (IC50 = 76.91 ± 2.33 µM) and IDG (IC50 = 126.06 ± 9.44 µM). This result supported the inhibitory potential of isolated bioactive compounds against COX-2. Also, a cellular level study revealed a downregulation of COX-2 expression in tumor necrosis factor-alpha stimulated RAW 264.7 macrophages treated with CDQ and IDG. CONCLUSION: Computational and experimental analysis of CDQ and IDG from S. herbacea established their potential in the inhibition and mediation of COX-2. Hence, CDQ and IDG can be considered for therapeutic development against COX-2 linked disorders, such as inflammation and cancer. Furthermore, CDQ and IDG structures can be served as a lead compound for the development of advanced novel anti-inflammatory drugs.