Anti-rheumatoid Arthritis Constituents And Action Mechanism Of Litsea Cubeba (Lour.) Pers Root

Rheumatoid arthritis (RA) is a common, immune-mediated disease characterized bychronic progressive inflammation and destruction of joints and associated structures, aswell as systemic symptoms. Its hallmarks are persistent inflammation of the jointsynovial because of infiltration of macrophages and activated T cells, and the subsequentdestruction of surrounding bone and cartilage tissue, both of which are programs offunctional disability. In Traditional Chinese Medicine theory, RA belongs toarthromyodynia which results when genuine Qi is deficient so that Wind, Cold,Dampness, or Heat is able to invade and block the meridians, muscles and joints.Litsea cubeba (Lour.) Pers. belongs to genus Litsea (Lauraceae). It has a long usedas a folk remedy in Traditional Chinese Medicine (TCM) and Dai Ethnopharmacy for thetreatment of rheumatic diseases, common cold and stomach ache in southwestern China.Dai Ethnopharmacy is one of the ancient traditional medicines in China. The root of L.cubeba used to treat arthritis in folk medicine in southwestern China. However, relativelylittle work has been carried out to assess its anti-RA potential and the mechanism.This dissertation is therefore conducted to investigate the anti-rheumatic activity ofroot of L. cubeba, and isolate the chemical constituents from its extract. In order todetermine the chemical responsible for the anti-rheumatic activity of this species,LPS-induced inflammation in RAW264.7macrophages and osteoclast TRAP activitywere used to test the anti-rheumatic activity of the isolated compounds from CH2Cl2portion. In addition, the molecular docking technique and cellular thermal shift assay(CETSA) were applied to predict the anti-rheumatic targets of the active chemicalconstituents and validate their actual binding. Total synthesis used for large scalepreparation of LC36.1In vivo anti-rheumatic evaluation of L. cubeba root extractsThe anti-rheumatioc effects of the ethanol extract of L. cubeba root (EELC,200,50mg/kg) was investigated with Freund’s complete adjuvant (CFA) induced arthritis (AA)in rat model. The paw swelling, arthritis score, weight growt rat, index of thymus andspleen were observed, and the production of TNF-α, IL-1β, IL-6and IL-10in serum weremeasured by enzyme-linked immunosorbent assay. The expression levels ofinflammatory enzymes COX-2and5-LOX were also measured by enzyme-linkedimmunosorbent assay. Moreover, histological changes in the ankle joint were analyzed in AA rats. The results showed that the EELC significantly suppressed paw swelling andarthritic score, increased the loss in body weight and decreased the index of thymus. Theexpression levels of COX-2and5-LOX were decreased on treatment with EELC.Furthermore, the overproduction of TNF-α, IL-1β and IL-6were remarkably attenuatedin serum of all L. cubeba-treated rats, however, IL-10was markedly increased at doses of50mg/kg of EELC. Histopathological improvement in joint architecture was alsoobserved in EELC-treated AA rats. This result suggests that L. cubeba root might be usedas a therapeutic agent for the treatment of human arthritis.2Chemical constituents of L. cubebaSilica gel column chromatography, ODS, Sephadex LH-20and reversed-phaseHPLC were used to isolate the chemical compound of the L. cubeba root extracts. A total77chemical compounds isolated and63chemical compounds identified, include1newcompound and1new natural compound. The structures of these compounds, includingalkaloids, lignans, steroidals, triterpenoids, phenolic acids, isocoumarin and others whichelucidated on the basis of chemical and spectroscopic analyses.3In vitro anti-rheumatoid evaluation of the compounds isolated from L. cubebaCH2Cl2portionA total of11compounds were evaluated for the anti-inflammatory effects onLPS-induced inflammation in RAW264.7cell and the inhibition effect of TRAP activityon multinucleated osteoclasts from rat marrow cells.6of11compounds tested shownsignificant anti-inflammatory effect, which include1new compound. Only1(LC36)compound shown inhibiton effect of TRAP activity.4Anti-inflammatory mechanism of the active ingredient of L. cubeba rootWe investigated the probable anti-inflammatory mechanism of LC36and LC43. Theresults showed that the LC36and LC43show anti-inflammatory via inhibited IκBα andIKKβ phosphorylation, and inhibited the iNOS and COX-2mRNAexpression.5Predict the anti-rheumatic targets of LC36and LC43use s molecular dockingtechnique and validate their actual binding with cellular thermal shift assay(CETSA) We builded the RA targets protein database（p38MAKP，JAK，Syk，AP-1/c-Fos，c-JNK，CD80，A3AR，Cathepsin K and MEK1），molecular docking was used to dockthe LC36and LC36with the targets protein. As showed in the result the LC36shownstrong binding effect with p38MAPK, MEK1and cathepsin K. CETSA was used tocomfirn their actual binding effect of LC36with p38MAPK, MEK1and cathepsin K.The CETSA curve and ITDRFCETSAresults shown that LC36can bind with MEK1andcathepsin K.6Total synthesis of LC36The synthesis roate of LC36was designed based on the structure feature. A six stepreaction synthesized product LC36, the final yield of11.3%.