Study On Cyclooxygenase-2 Inhibitors In Kadsura Coccinea Based On Affinity Ultrafiltration Coupled With High-performance Liquid Chromatography And Quadruple Time-of-flight Mass Spectrometry

Kadsura coccinea(Lem.)A.C.Smith is an evergreen liana belings to the Kadsura of the Schisandraceae family,which roots and stems are widely used for medicinal purposes,with the functions of promoting Qi,relieving pain,dispersing blood stasis,and dredging collaterals.K.coccinea is widely distributed in the southern provinces of China,which often used as the national medicine for rheumatism and pain,gynecological pain,and acute and chronic gastrointestinal diseases.Its efficacy is significant,but lack of in-depth research.In the past three decades,scholars at home and abroad have studied the chemical constituents and pharmacological activities of K.coccinea,and found that lignans and triterpenes are the main active ingredients,which have antioxidant,antiviral,antitumor,anti-inflammatory,liver protection effects.Our previous research had confirmed that 95%alcohol extract of K.coccinea had good therapeutic effect on the adjuvant arthritis(AA)of rats.However,the effective ingredients and mechanism of action of the plant for treating inflammation have not yet been clarified.Based on the literature review,this paper investigated the potential inhibitors of COX-2 for treating inflammation from K.coccinea extract by affinity ultrafiltration coupled with high-performance liquid chromatography and quadruple time-of-flight mass spectrometry(AUF-HPLC-Q-TOF/MS).(1)The target COX-2 was selected directly related to the inflammation and an analytical method based on AUF-HPLC-Q-TOF/MS was established for rapidly screening and characterizing COX-2 ligands from K.coccinea.Meanwhile,the blank group and the inactivated group were set up to exclude non-specific interference of the experimental method.As a result,a total of 21 chemical compositions were inferred,and 8 compounds were considered as COX-2 specific ligands,which relative binding degree more than 25%,namely Kadsulignan N(2),Kadsulignan L(4),N eokadsurani n(5),Kadsutherin(6),Kadsuralignan F(8),(+)-anwulignan(10),Kadsuracoccinic acid A(14),3,4-seco(24Z)-cychmrt-4(28),24-diene-3,26-dioic acid 3-methyl ester(19),among which compounds 2,4,5 and 10 not only have a large content but also have good binding affinity with COX-2.(2)The molecular docking evaluation of 8 COX-2 specific ligands was performed by AutoDock 4.2 software to identify potential inhibitors with good activities,and to understand the possible mechanism of action through binding force and binding mode.The selective inhibitor SC-558 was docked to prove the reasonableness of the parameter setting.The results of binding energy and inhibition constant shown that,Kadsulignan N(2),Kadsulignan L(4),Neokadsuranin(5),and(+)-anwulignan(10)have relatively better inhibitory activity,and interact with amino acid residues at the active site of COX-2,which identified as potential inhibitors.(3)After extracting the alcohol extract of K.coccinea with petroleum ether,the petroleum ether fraction was obtained,which chemical composition was studied.In the experiment,separation and purification methods such as silica gel column chromatography,Sephadex LH-20,and recrystallization,combining nuclear magnetic carbon spectrum(13C-NMR)and hydrogen spectrum(1H-NMR)spectroscopy techniques,the structures of 6 compounds were isolated and identified,of which were two lignans:(+)-anwulignan(Kc-1)and Kadsulignan N(Kc-6),three terpenoids:Kadsuracoccinic acid B(Kc-2),Nigranoic acid(Kc-4)and Manwuweizic acid(Kc-5),one steroidal compound:?-sitosterol(Kc-3).Compounds Kc-1 and Kc-6 were experimentally identified COX-2 potential inhibitors,and their inhibitory activity were verified by COX-2 inhibitor screening experiments in vitro,which shown their IC50 were 3.80 and 1808.13?g/mL(equivalent to 11.52 and 4204.95?M).The IC50 of the positive control celecoxib in the kit is 0.45?M.