The Chemical Constituents From The Leaves Of Rhododendron Amesiae And Their Anti-inflammatory Activities

Objective:To isolate and identify the compounds from the 95%ethanol extract of Rhododendron amesiae Rehd.et Wils.,and to detect their anti-inflammatory effects in enzyme level and cell level,which could provide reference for the further development and utilization of Rhododendron plantsMethods:1.The 95%ethanol extract of the Rhododendron amesiae was extracted by the petroleum ether,ethyl acetate,respectively.The compounds were isolated from the ethyl acetate extract by means of chromatography on silica gel column,ODS column,Sephadex LH-20 column,MCI column and semi-preparative HPLC.The structures were elucidated using¹H-NMR,¹³C-NMR,and MS analyses.2.All the isolates were evaluated in vitro for inhibitory activities against PDE4B.Molecular docking software AutoDock 4.2 and SYBYL were used to study the interactions between the compounds and PDE4.3.The anti-inflammatory activities of the isolated diterpenoids were studied by testing the release of NO in LPS-induced RAW264.7 cells.Results:1.Twenty compounds were isolated from the 95%ethanol exaction of the leaves of Rhododendron amesiae.Among them,six compounds were diterpenes which were determined as grayanotoxin??1?,grayanotoxin??2?,grayanotoxin??3?,grayanotoxin??4?,grayanotoxin??5?,grayanotoxin XV??6?.Six compounds were triterpenes which were determined as campanulin?7?,friedelane?8?,ursolic acid?9?,uvaol?10?,2?,3?,23-trihydroxyurs-12?30?-dien-28-oicacid?11?,30-nor-2?,3?,,23-trihydroxyurs-12-ene?12?.In addition,seven flavones were determined as epicatechin?13?,2R,3R-3,5,7,3'5'-pentahdroxyflavan?14?,cinchonain?a?15?,quercetin?16?,matteucinol?17?,methylmatteucinol?18?,farrerol?19?.And also,a fatty acid named as Oleic acid amide?20?was identified.2.The diterpenes had no inhibitory activity on PDE4.The triterpenes exhibited weak inhibitory activity on PDE4.The flavones were found to exhibit moderate inhibitory activities on PDE4 with IC₅₀0 values range of 33.7-48.3?M.The docking results show that the flavones interact with Q pocket by forming hydrogen bond?–?interactions.Additionally,the inhibitory effect of the obtained compound 17-19 on PDE4D was significantly higher than that on PDE4B.3.Compared with positive drugs,the isolated diterpenoids can effectively inhibited LPS-induced NO release from RAW264.7 cells.Conclusion:All the compounds were isolated from this plant for the first time.Compound 12and 15 were isolated from the genus Rhododendron for the first time.Compounds 1-6and 17-19 were the characteristic compounds of this genus.The flavonoids inhibited inhibitory activity against PDE4 by forming hydrogen bond and?–?interactions with Q pocket.The cell experiment results showed that the obtained diterpenoid compounds had moderate anti-inflammatory activity.