Study On Chemical Constituents And Cytotoxic Activity From EtOAc Fraction Of Chimaphila Japonica Miq.

In this paper,the chemical constituents and their cytotoxic activities from ethyl acetate（EtOAc）extract of Chimaphila japonica Miq.,a medicinal plant in Changbai Mountain area,were investigated.There are about five species of the genus Chimaphila in family Ericaceae in the whole world,widely distributed in China,Japan,Korea,Russia,Europe,North and Central America.Among them,there are only three species in China,which are C.japonica,C.umbellata（L.）W.Barton and C.monticola H.Andr.It has been reported that C.japonica extract has significant antioxidant,anti-tumor,antibacterial and anti-inflammatory,as well as diuretic,astringent and analgesic activities.Clinically,it can be used as a diuretic and urethral anti-inflammatory agent to treat all kinds of edema.However,there are few studies on the active components of this plant.The research on genus Chimaphila mainly focused on C.umbellata,which mainly contains terpenoids,sterols,flavonoids and quinones;and has anti-tumor,anti-inflammatory,anti-oxidation and other pharmacological effects.Therefore,it is necessary to further study on the chemical constituents and biological activities of C.japonica,which is of great significance to the development and utilization of the plant dominant resources in Changbai Mountain area.In screening the resources of cytotoxic activity from natural plant in Changbai Mountain area,it was found that the 90%of ethanol extract of C.japonica had significant inhibitory effect on three common cytoxic cell lines（human lung cancer cell A549,human cervical cancer cell He La and human hepatoma cells SK-Hep1）,and the EtOAc extract was identified as one of the main active parts.In this study,the EtOAc fraction was separated and purified by normal phase silica gel column chromatography,reversed phase silica gel column chromatography,Sephadex LH-20gel column chromatography and recrystallization.The chemical structures of the monomer compounds were determined by the physical and chemical properties,spectral analysis(¹H-NMR and ¹³C-NMR)and related literatures.A total of 18monomer compounds were isolated from the EtOAc layer of C.japonica,which were as follows:3β-taraxerol（1）,chimaphilin（2）,6-hydroxy 2,7-dimethyl-1,4-naphthaquinone（3）,β-sitosterol（4）,pyrolin（5）,ursolic acid（6）,eupatoric acid（7）,α-amaryin（8）,β-amaryin（9）,3β,27-dihydroxy-lup-12 ene（10）,oleanolic acid（11）,kaempferol（12）,quercetin（13）,kaempferol-3-O-α-L-5’’-acetyl-arabinofuranoside（14）,gallic acid（15）,corosolic acid（16）,japonica acid（17）andα-amyrenone（18）.The obtained compounds included nine triterpenes（1,6～11,16 and 18）,three flavonoids（12～14）,two quinonoids（2 and 3）,two phenols（5 and 15）,a steroid（4）and a fatty acid（17）,which indicated that triterpenes were the main chemical constituents of the EtOAc fraction of C.japonica.Among them,compounds 3,5,7,9～13,15,17 and 18 were isolated from this plant for the first time.The cytotoxic activity of the compounds isolated from the EtOAc fraction of C.japonic against A549,He La and SK-Hep1 cells in vitro was determined by MTT assay.The results showed that compounds 2～5,7,10,11 and 16 inhibited the growth of A549,compounds 3,5,10,11 and 16 inhibited the growth of He La,and compounds 1～3,5～8,10,11 and 16 inhibited the growth of SK-Hep1 at 30μM concentration.In conclusion,compounds 2,3,5,10,11 and 16 have significant inhibitory effects against the three cytotoxic cells,among which compounds 2,5,11and 16 have particularly highly cytotoxic activities.In human lung cancer cells（A549）,compounds 2,5 and 11 exhibited particularly significant cytotoxic activities,with IC₅₀values of 1.25±1.14,1.84±1.23 and 2.64±1.08μM.Compounds 2 and 4showed significant cytotoxic activities in human cervical cancer cells（He La）,with IC₅₀values of 1.21±0.47 and 1.96±0.22μM,respectively.Compounds 2,4 and 5showed significant cytotoxic activities in human hepatoma cells（SK-Hep1）,with IC₅₀values of 2.56±0.63,1.52±0.32 and 1.18±0.74μM,respectively.This study not only enriched the understanding of the chemical constituents of C.japonica,but also clarified its cytotoxic active components through the screening of pharmacological activity,which provided a scientific basis for its further research,development and utilization.