# Inflammation: What's There and What's New?
## 5 Methods for Assessing Anti-Inflammatory Activity
### In Vitro Assessment  
Regarding in vitro assessments, a wide range of assays are used to perform a first screening to select the most promising anti-inflammatory agents. The _\"PhagoBurst Assay\"_ consists of kits that use flow cytometry to examine the phagocytic activity of granulocytes and monocytes in the whole blood. The Phagotest allows for the quantitative evaluation of leukocyte phagocytosis (bacterial uptake). It specifies the percentage and activity (number of bacteria per cell) of phagocytes that ingest fluorescein-isothiocyanate (FITC)-labeled opsonized bacteria [107]. In the _\"Anti-inflammatory bioassay\"_, a reaction mixture consisting of egg albumin, phosphate-buffered saline, and various concentrations of the anti-inflammatory agent is incubated with egg albumin under controlled experimental conditions. Then, the absorbance is measured and the percentage inhibition of protein denaturation is calculated by using the following formula [108]. \[\%\;\text{inhibition}=100\;\times\;[\text{Vt}/\text{Vc}-1]\]  
where Vt is the absorbance of the test sample and Vc is the absorbance of the control. The _\"Cyclooxygenase inhibitors\"_, is also an in vitro approach used for more than two decades and is based on the incubation of arachidonic acid with COX-1-containing sheep seminalvesicle microsomes. However, distilled COX-2 and COX-1 were used in 2000 and 2004, respectively, by the same community. To identify the COX inhibitory effect of plant extracts, various parameters were tested. To be considered active, some researchers set a minimum COX inhibition of 50% for water extracts and 70% for organic solvent extracts. The standards where the Inhibition was below 20 percent were considered negligible. COX inhibition activity is known to be marginal between 20 and 40%, mild between 40 and 70%, and strong anti-inflammatory activity above 70 [109]. Regarding \"_Lipoxygenase inhibitors_\", it is interesting to note that leukotrienes are another class of inflammatory mediators formed by LOX enzymes from arachidonic acid metabolism. It has been documented that leukotrienes are responsible for binding white blood cells to the endothelium of weakened blood vessels and function as phagocyte chemoattractants. In addition, leukotriene development has been linked to clinical symptoms of pathological disorders, such as asthma and anaphylaxis. Leukotrienes, formed by 5-LOX in inflammatory cells, such as polymorphonuclear neutrophils, basophils, mast cells, eosinophils, and macrophages, are the most actively examined [109]. Finally, the \"_Inhibition of nitric oxide (NO)_\", potentially the most used method, is based on the ability of anti-inflammatory agents to suppress inflammatory processes through the inhibition of nitric oxide (NO) production in a cell-based model of lipopolysaccharide-accharide (LPS)-stimulated RAW 264.7 murine macrophage-like cell line [83]. Trends in the Treatment of Inflammatory Processes Targeting Glycolysis with Small Molecules to Elicit an Anti-inflammatory Effect  
Several models of infection and inflammation have shown an anti-inflammatory response after the inhibition of hexokinase by 2-DG and GAPDH by heptelidic, acid as well as tetramerization and the activation of the pyruvate kinase activity of PKM2 by TEPP-46 [27]. Targeting the glucose transporter Glut1 is one of the novel, promising targeted therapies to limit inflammation (Figure 1). Figure 1: Targeting enzymes of glycolysis as an anti-inflammatory strategy. Glut-1, glucose transporter-1; 2-DG, 2-deoxyglucose; SLE, systemic lupus erythematosus; RA, rheumatoid arthritis; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; EAE, experimental autoimmune encephalomyelitisitis; AKI, acute kidney injury. Molecular docking has been consistently used to highlight ligand-receptor molecular interactions and is thus a valuable method supporting drug discovery and development. It is one of several computational methodologies that have been developed to help researchers classify candidates for new drugs and investigate them.