Study On Anti-inflammatory Active Substances Of WuweiGanLu Extract And Preparation Of Self-heating Gel Sticker

Wuwei Gan Lu（WGL）is a classic prescription in the Four Medical Classics of Tibetan medicine,which treats rheumatoid arthritis（RA）and joint swelling,which are defined by Tibetan medicine as"true cloth disease".The basic formula for WGL is composed of ephedra,juniper,artemisia macro seed,cypress branch,and rhododendron,and its medicinal bath has a particularly significant clinical effect on"true cloth disease".With the simplification of modern life,the clinical use of medicinal baths has begun to be limited.The study found that WGL anti-inflammatory active substances and pharmacological studies were rarely reported.Therefore,this study explores the active substances of the drug by clarifying the core targets of drug action and tracking pharmacological activity.After determining the anti-inflammatory active substance,WGL aqueous extract was prepared for WGL self-heating gel paste,and the quality evaluation was carried out with the active substance,which provided an experimental basis for improving the scientificity of medication and also provided convenience for arthritis patients.The following three parts of this paper are mainly carried out:Part I:Prediction and verification of potential targets and related pathways based on network pharmacology.Through network pharmacological analysis,a total of 125 anti-inflammatory components of WGL were obtained,271 potential anti-inflammatory targets were obtained,and 52 core targets were obtained,including TNF-α,IL-6,IL-1β,IL-10,and other important targets.The KEGG pathway enrichment analysis involved 52 genes and enriched 17related pathways,including lipid and atherosclerosis,and rheumatoid arthritis pathways.In vitro,anti-inflammatory activity studies have shown that WGL can inhibit the ROS production of macrophages RAW264.7 induced by lipopolysaccharides（LPS）,and inhibit the expression of NLRP3,IL-1βand TNF-αm RNA and proteins.Fluorescence microscopy observed the expression of i NOS and autophagy lysosomes in LPS-induced RAW264.7 cells,and found that WGL treatment could reduce the production of both and reduce the inflammation level of LPS-induced RAW264.7 cells.Part II:Analysis of anti-inflammatory active substances in WGL and its different extracted phases（petroleum ether,ethyl acetate,and n-butanol）based on the pharmacodynamic-profile model.Cell experiments showed that WGL and ethyl acetate phase could significantly inhibit the expression of NLRP3 m RNA in LPS-induced RAW264.7 cells.The ethyl acetate phase was found to be the total flavonoid enrichment site by full-wavelength microplate reader colorimetric method（Na NO₂-Al NO₃-Na OH as the color developer）.The atlas analysis was carried out by UPLC-Q-Exactive MS technology,and it was found that the content of various flavonoids in the ethyl acetate phase was significantly higher than that of WGL and single flavor medicinal materials.Using DS Lib Dock molecular docking technology,five flavonoid monomers with high content in the map were selected as ligands,and it was found that the parent nuclear structure of their C₆-C₃-C₆showed a certain affinity in the active site of NLRP3receptor protein.This section shows that the flavonoids in Wumian Manna act as anti-inflammatory actives.PartⅢ:Preparation of WGL self-heating gel patch based on orthogonal experimental design and Box-Behnken design response surface method.L₉（3⁴）orthogonal experimental design optimizes the WGL water extraction process;Box-Behnken designed the response surface method to optimize the matrix prescription of gel stickers;sensory evaluation and rheometer analysis determined that the plaster loading amount was 2.5%;the mouse pinna swelling model found that the anti-inflammatory effect of self-heating gel patch was better than that of gel patch.The prepared WGL self-heating gel patch had good heat resistance,formability,adhesion,and stability,and the transdermal rate of flavonoids at 6 h was 23.01%,and the dissolution rate of flavonoids in hydrochloride buffer reached 26%.The content of total flavonoids per gram of gel was 0.52～0.65 mg.Animal experiments showed that WGL gel pretreatment significantly inhibited the increase of serum IL-1βand TNF-αlevels of carrageenan-induced foot swelling,and HE staining observed that its pretreatment could reduce neutrophil infiltration in the plantar of mice.In summary,the anti-inflammatory active substance in WGL aqueous extract has a significant blocking effect on the activation of NLRP3 inflammasome induced by LPS264.7cells,and it has potential application value in the treatment of NLRP3-targeted inflammatory diseases.WGL self-heating gel patch prepared from WGL aqueous extract has the advantages of stable process,high transdermal efficiency of drug dissolution,and good anti-inflammatory effect,which provides a reference for the development of new varieties of Tibetan medicine preparations.