Study On The Chemical Constituents Of Aucklandia Lappa And Their Farnesoid X Receptor Activation

Aucklandiae radix is the dry roots of Aucklandia lappa Decne（A.lappa）.As a Traditional Chinese Medicine,A.lappa has strong taste,bitterness and warmth.It has the effects of promoting qi,relieving pain,invigorating the spleen and eliminating food.And it is mainly used for the treatment of chest depression,abdominal pain,diarrhea,emergency and other diseases.It is reported that A.lappa has cholagogic and anti liver injury effects.The ethanol extract and CO₂ supercritical extract of A.lappa showed choleretic effect by increasing bile secretion in rats.In addition,the methanol-water extract of A.lappa and costunolide have significant protective effects on LPS/D-Ga IN-induced acute liver injury in mice.Farnesoid X receptor（FXR）,also known as bile acid receptor,is one member of nuclear receptor family.FXR is distributed in the liver and intestine,and can be involved in the regulation of the biosynthesis,transport and metabolism of bile acid.It is of great significance to maintain the homeostasis of bile acid and relieve intrahepatic cholestasis.FXR is an important target for the treatment of cholestatic liver injury and other liver diseases.Therefore,the searching for FXR agonists is one of the hot spots of drug research and development in recent years.Our previous study found that the extract of Vladimiria souliei can improveα-naphthylisothiocyanate-induced cholestatic liver injury in rats by activating FXR signaling pathway,in which sesquiterpenes and their dimers may be the main active components.Based on the similarity of efficacy and chemical composition between A.lappa and V.souliei,this study mainly focused on the chemical constituents of A.lappa,hoping to find new lead compounds with FXR activation.Kinds of chromatographic methods including normal or reversed phase silica gel column chromatography,Sephadex LH-20,preparative TLC,recrystallization,semi-preparative HPLC,etc,were applied for the isolation and purification of chemical constituents of ethyl acetate extract.1D-NMR(¹H-NMR,¹³C-NMR,DEPT135),2D-NMR（HSQC,HMBC,COSY,NOESY）,HR-ESI-MS,IR,UV,CD,X-ray single crystal diffraction and other spectroscopy methods,as well as comparison with the data reported in the literature were used to identify the structure of these isolated compounds.Finally,38 compounds were obtained,including 4 sesquiterpenoid dimers,23 sesquiterpenoids,2triterpenes;1 C₂₁ steroids;3 polyalkynes;and 5 other compounds.Their structures were determined as auclappones A～C（AL-1～3）,vlamuliolide C（AL-4）,auclappones D～H（AL-5～9）,5α-hydroxy-11β,13-dihydrodehydrocostuslactone（AL-10）,dehydrocostus lactone（AL-11）,11-αH-dihydrodehydrocostus lactone（AL-12）,4-epi-arbusculin A（AL-13）,（+）-9β-hydroxyeudesma-4,11（13）-dien-12-al（AL-14）,2-one-3,4-dehydro-4,15-dihydrocostic ester（AL-15）,cnicothamnal（AL-16）,7α-hydroxycostol（AL-17）,costunolide（AL-18）,11α,13-dihydro-costunolide（AL-19）,11β,13-dihydro-costunolide（AL-20）,ptilostemonol（AL-21）,1-oxo-bisabola-2-ene-12-acetyl（AL-22）,puliglutone（AL-23）,9-oxonerolidol（AL-24）,3,7,11-trimethyldodeca-1,7（E）,10-trien-3-ol-9-one（AL-25）,3,7,11-trimethyldodeca-1,7（Z）,10-trien-3-ol-9-one（AL-26）,vernopolyanthone（AL-27）,betulinic acid（AL-28）,betulin（AL-29）,pregnenolone（AL-30）,1,4-diacetoxyltetradeca-6,12-diene-8,10-diyne（AL-31）,（4E,6E）-4,6-tetradecadien-8,10,12-triyn-1,3-diyl diacetate（AL-32）,（4E,6E,12E）-tetradecatriene-8,10-diyne-1,3-diyl diacetate（AL-33）,trans-ethyl ferulate（AL-34）,2,6-dimethoxyphenol（AL-35）,（E）-2-methoxy-4-（2-nitroprop-1-enyl）phenol（AL-36）,eugenin（AL-37）,（-）-（13S,9Z,11E）-13-hydroxy-9,11-ocatadecadienoic acid methyl ester（AL-38）.Wherein AL-1～3 are new sesquiterpenoid dimers,AL-5～9 are new sesquiterpenoids,AL-1～10,AL-12,AL-14～17,AL-21～27,AL-31～33 and AL-36 were isolated from this plant for the first time.In this study,we used the model of L02 cells induced by sterone to evaluate whether27 sesquiterpenoids could up-regulate the expression of FXR protein at low concentration（20μM）and high concentration（40μM）respectively through high content screening in vitro.The results showed that eight compounds at low concentration（AL-2,AL-11,AL-13,AL-15,AL-16,AL-23,AL-25,AL-27）and ten compounds at high concentration（AL-1,AL-2,AL-7,AL-10,AL-11,AL-14,AL-17,AL-20,AL-23,AL-26）could significantly up-regulate the expression of FXR protein.The results showed that puliglutone（AL-23）had the best activity.Among them,auclappone A（AL-1）,auclappone B（AL-2）and auclappone F（AL-7）are new compounds.In this paper,sesquiterpenoids with FXR activation were discovered from A.lappa,which has important theoretical value and practical significance for promoting the comprehensive development and utilization of A.lappa.