Cytotoxic Chemical Constituents Of Three Ligularia Medicinal Plants

The genus Ligularia(Compositae)is distributed in China with about 110 species. Most of them grow in Northwestern,Southwestern and Northeastern areas.Among them,more than 30 Ligularia species have long been used for folk medicines in China. Their roots,stems,leaves and flowers possess efficacies of antipyretic,relieving phlegm and relieving cough,invigorating the circulation of blood and soothing pain. The main components of them are sesquiterpenes and pyrrolizidine alkaloids that have mang physiological activities.Since the 1960's,about 50 plants of genus Ligularia distributed worldwide have been studied in different fields.15 species of Ligularia are distributed in Xinjiang region,and many of them have been used as folk medicine for the treatment of clearing away heat and toxic materials.Our primary bioassay showed that the ethanol extract of the roots of L. macrophylla and L.songarica distributed in Tianshan Mountains exhibited cytotoxic activity against human lung carcinoma(A549)and breast adenocarcinoma(MCF-7) in vitro.Therefore,in order to elucidate the bioactive constituents and search for cytotoxic agents,the studies on the constituents and bioassay of cytotoxicity of the two medicinal plants and their same genus L.altaica distributed in Altai mountains were carried out.Sixty compounds have been isolated commonly by means of silica gel column chromatography,preparative thin layer chromatography,recrystallization,among which twenty-one compounds were new.The structures of these compounds were elucidated by spectroscopic methods such as UV,IR,MS,CD,¹H NMR,¹³C NMR, DEPT as well as 2DNMR(HMBC,¹H-¹H COSY,ROESY),and the means of single-crystal X-ray diffraction have been used in structural elucidation.They were identified as sesquiterpenes,triterpenes,flavonoids,sterols,etc.Twenty-nine compounds isolated from L.macrophylla and L.altaica were performed cytotoxicity tests,of which 13 compounds showed activities.1.Phytochemical investigation on the petroleum ether and EtOAc soluble extract of the roots and rhizomes of L.macrophylla led to the isolation of 30 compounds, including 9 new compounds and 12 known compounds isolated from this species for the first time.Among the new compounds,there are two novel skeleton of nor-sesquitepenes,ligumacrophyllanins A-B(Wlm-1,Wlm-2);four eremophilane sesquiterpenes,ligulariatins A-D(Wlm-3,Wlm-8,Wlm-7,Wlm-12);one guaiane sesquiterpene,9β-hydroxy guaian-6,10-diene,ligulariatin E(Wlm-15);one derivative of chromone,(2S)2,3-dimethyl-6-acetyl-8-methoxychromone(Wlm-17)and one flavanone,3＇-hydroxy-5＇,7-dimethoxyflavanone(Wlm-20).The known compounds were identified as:1,10-epoxy-6-hydroxyeuryopsin(Wlm-4),6-angeloyoxy-1,10-epoxy-euryopsin(Wlm-5),1,10-epoxy-6-acetyleuryopsin(Wlm-6),1-oxo-9-des oxycacalol(Wlm-9),6β,10α-dihydroxy-1-oxoeremophila-7(11),8(9)-diene-8,12-olide (Wlm-10),10α-hydroxy-1-oxoeremophila-7(11),8(9)-diene-8,12-olide(Wlm-11),γ,3, 5-trimethy1-6-benzofuranbutanal(Wlm-13),farfugin A(Wlm-14),2-hydroxylplaty phyllid(Wlm-16),kaempferol(Wlm-21),2,4＇-dihydroxy-5＇-methoxy chalcone(Wlm-23),5-hydroxy-3,4＇,7-trimethoxyflavone(Wlm-24),2-isopropeny1-6-acety1-8-methyl oxy-1,3-benzodioxin-4-one(Wlm-18),isobutyl ester terephthalic acid(Wlm-19),4-hydroxybenzaldehyde(Wlm-22),mono(2-ethylhexyl)terephthalate(Wlm-25),lupeol (Wlm-26),3β,16β-dihydroxy-12-en-28-oleanolic acid(Wlm-27),urs-12-ene-3,28-diol (Wlm-28),ursolic acid(Wlm-29)andβ-sitosterol(Wlm-30).Sixteen compounds were screened for cytotoxicity against a panel of human tumor cell lines,of which seven eremophilane sesquiterpenes Wlm-3,Wlm-5,Wlm-7, Wlm-9,Wlm-12,Wlm-13,Wlm-14 showed significant cytotoxicity with EC₅₀values of 9.4-18.3μg/mL against A549 and MCF-7.The preliminary analysis indicated that benzofuran skeleton moiety in these sesquiterpenes was important for the cytotoxicities.2.Phytochemical investigation on the petroleum ether and EtOAc soluble extract of the roots and rhizomes of L.altaica led to the isolation of 18 compounds,including 7 new compounds.Among the new compounds,there are three oplopane sesquiterpenes,ligualtaicasins A-C(Wla-1,Wla-2,Wla-3);four bisabolane sesquiterpenes,ligualtaicasins D-G(Wla-4,Wla-5,Wla-6,Wla-7).The known compounds were identified as:1β-acetoxy-1β,8-diangloyloxy-10,11-dihydroxy-3,4-epoxybisabol-7(14)-ene(Wla-8),1β,8-diangloyloxy-3β,4β,10,11-diepoxybisabol-7 (14)-ene(Wla-9),bisabolone(Wla-10),ligulariatin C(Wla-11),1-oxo-9-desoxy cacalol(Wla-12),2-hydroxyplatyphyllid(Wla-13),1-hydroxy-6,9-dien-8-oxoeremo phi1-11-nor-ketone(Wla-14),ligulariatin D(Wla-15),tigmast-5-ene-3β,7α-diol (Wla-16),isofouquierol(â…¢)(Wla-17)and linoleic acid(Wla-18).These compounds were isolated from this species for the first time.Thirteen compounds were screened for cytotoxicity against A549,MCF-7,KB (epidermoid carcinoma of the nasopharynx),and its drugresistant KB-VIN)variant,of which four bisabolane sesquiterpenes(Wla-4,Wla-5,Wla-7,Wla-9)showed significant cytotoxicitiy,with EC₅₀values of 7.4-18.9μg/mL,and Wla-6 showed strong cytotoxicity with EC₅₀values of 0.8-3.4μg/mL.One oplopane sesquiterpenes (Wla-3),containing chlorine atom,showed significant cytotoxicity with EC₅₀values of 3.6-10.6μg/mL.3.Phytochemical investigation on the petroleum ether and EtOAc soluble extract of the roots and rhizomes of L.songarica led to the isolation of 17 compounds, including 5 new compounds and 8 known compounds isolated from this species for the first time.Among the new compounds,there are two bisabolane sesquiterpenes, ligusongarisins A-B(Wls-1,Wls-2);three oplopane sesquiterpenes,ligusongarisins C-E(Wls-3,Wls-4,Wls-5).The known compounds were identified as:bisabolone (Wls-6),8-angeloyloxy-3,4-dihydro-3,4-epoxy-β-bisabolen-5-one(Wls-7),cryptome-rione(Wls-8),7β-[3＇-ethylcrotonoyloxy]-1α-[2＇-methylbutyryloxy]-3,14-dehydro-Z-n otonipetranone(Wls-9),(1-acetoxyethyl)octahydro-6-[(2-methylbutanoyl)oxy]-4-met hylllidene-7-[2-methyloxiran-2-yl]-2-OXO-1H-inden-5-yl(2E)-4-acetoxy-3-methylpent -2-enoate(Wls-10),5β-acetoxy4β,8-diangeloyloxy-2β,3β;10,11-diepoxy-bisabola-7 (14)-ene(Wls-11),4β-acetoxy-5β,8-diangeloyloxy-2β,3β;10,11-diepoxy-bisabola-7 (14)-ene(Wls-12),ligusongaricone(Wls-13),isopetasin(Wls-14),2-hydroxyplaty-phyllid(Wls-15),2-(oleoyloxymethyl)oxirane(Wls-16)and linoleic acid(Wls-17).The results of this investigation with sixty compounds,including twenty-one new compounds and thirteen cytotoxic principles,were expected to enrich the database of natural products;to offer both phytochemical and biological activity proofs to chemtaxonomy;to afford important academic basis for the exploitation and utilization of natural resources and to supply a significant scientific basis for pharmacology,chemotaxonomy and screening of new medicines.