Chemical Constituents And Tumor Multidrug Resistance Reversal Activities Of Asclepias Curassavica L.

A.curassavica L.is a species of flowering plant in the milkweed family Asclepiadaceae.This species is native to the American tropics.Currently,it widely distributes in the southwest and southeast of China.Along with the rapid development of modern separation technology,multiplex structure of cardiac glycosides were found by foreign researchers from this plant,which is used to treat heart disease and anti-tumor.However,studies on chemical constituents and pharmacological activities haven′t been reported yet,especially Asclepias curassavica L.from Guangdong.Therefore,we carried out a systemic investigation on the constituents of Asclepias curassavica L.to illuminate the substance-foundation for its anti-cancer effect as well as for treating heart disease.Ⅰ Chemical constituents from A.curassavica L.1.Chemical constituents from the whole herb of A.curassavica L.This article reviewed the research progress of Asclepias L.,and studied on the chemical constituents from the 70%ethanol extract of A.curassavica L.led to the isolation of 64compounds by various chromatographic methods,fifty-three of which were elucidated by extensive spectral methods,which including 30 cardenolides:（3S,5S,8R,9S,10S,13R,14S,17R,2′S）-3-O-urarigeninlactate（M-1）,（3S,5S,8R,9S,10S,13R,14S,17R,20Z,2′R）-3-O-urarigenin-lactate（M-2）,（3S,5S,8R,9S,10R,13R,14S,17R,20Z,2″S）-19-O-coroglaucigeninlactate（M-3）,（3S,5S,8R,9S,10S,13R,14S,17R,20Z,2′R,2″R）-coroglaucigenin-3,19-O-dilactate（M-4）,（3R,5S,8R,9S,10R,13R,14S,17R,20Z,2″R）-19-O-frugoside-lactate（M-5）,（2R,3R,5S,8R,9S,10R,13R,14S,17R,20Z,1′S,2′S,3′S,5′R）-19β-hydroxycalotropin（M-6）,（2R,3R,5S,8R,9R,10S,13R,14S,17R,20Z,1′S,2′S,3′S,5′R）-19-nocalotropin（M-7）,calactin（M-8）,calotropin（M-9）,16α-hydroxycalotropin（M-10）,15β-hydroxycalotropin（M-11）,16α-acetoxycalactin（M-12）,19-dihydrocalactin（M-13）,15β-hydroxycalactin（M-14）,16α-hydroxyasclepin（M-15）,asclepin（M-16）,16β-acetoxyasclepin（M-17）,3′β-acetoxycalactin（M-18）,voruscharin（M-19）,uscharin（M-20）,calotropagenin（M-21）,frugoside（M-22）,desglucouzarin（M-23）,calactinic acid methyl ester（M-24）,ascleposide（M-25）,urarigenin（M-26）,antiaroside P（M-27）,12β-hydroxycoroglaucigenin（M-28）,19-dihydrocalotropagenin（M-29）,corotoxige-nin（M-30）;9 C₂₁ steroids:sarcostin（M-31）,12-O-benzoylsarcostin（M-32）,curassavoside B（M-33）,12-O-benzoylsarcostin（M-34）,curassavoside A（M-35）,asclepiasterol（M-36）,12-O-（E）-cinnamoyltayloron 3-O-β-digitoxopyranosyl-（4→1）-β-cymaropyranoside（M-37）,12-O-（E）-cinnamoyltayloron 3-O-β-digitoxopyranosyl-（4→1）-β-digitoxopyranosyl-（4→1）-β-cymaropyranoside（M-38）,12-O-benzoylsarcostin3-O-β-digitoxopyranosyl-（4→1）-β-cymaropyranosyl-（4→1）-β-cymaropyranoside（M-39）;7 lignans:（±）-5′-methoxylariciresinol（M-40）,（±）-yringaresinol（M-41）,ligna A（M-42）,（±）-syrmgaresmol（M-43）,isotaxiresinol（M-44）,yunnanensins A（M-45）,（+）-（6R,7S,8S）-5-Methoxyisolariciresinol（M-46）;Others areβ-sitosterol（M-47）,stigmasterol（M-48）,quercetin（M-49）,4-hydroxy-3-methoxy-benzenepropanoic acid（M-50）,esculetin（M-51）,（Z）-phytol（M-52）,pyrocatechol（M-53）。Among them,M-1～M-7,M-36～M-39 were new cardenolides,M-47,M-52,M-57,M-58were new C₂₁ steroids.2.Fingerprint analysis of A.curassavica and chemical constituents from the seed hairs.Through analyzing the chemical constituents of the tissues from Asclepias curassavica L.we had found some interesting phenomena.Firstly,chemical constituents of its root,stalk,leaves and flowers are same,but the content from flowers is four times than the others.Secondly,there isn′t cardenolide in its peel,seed capsule and seed.Thirdly,species of cardenolides from milk are not only rich,but their content is also so much,which is nearly a hundred times than the others.So cardenolides are constantly synthesized in the growth process,then they are transported to different tissues by the milk pipe.Finally,Seed doesn′t contain cardiac glycosides,but the seed silk contains high levels of cardiac glycosides.It have two ecological significance.On the one hand,it helps seeds spread further,on the other hand,the seeds can avoid prey by birds in the process of propagation.Simultaneously we studied on the chemical constituents from the 70%ethanol extract of the seed silk of Asclepias curassavica L.led to the isolation of 10 compounds by various chromatographic methods,eight of which were elucidated by extensive spectral methods,which including 5 flavonols:Isobiorobin（ZM-1）,Kaempferol 3-O-neohesperidoside（ZM-2）,quercetin（ZM-3）,Kaempferol 3-O-D-galactoside（ZM-4）,Astragaline（ZM-5）;3 cardenolides:corotoxigenin3-O-β-D-galactopyranosyl-（4→1）-6-deoxy-β-D-glucopyranose（ZM-6）,corotoxigenin 3-O-β-D-glucopyranosyl-（4→1）-6-deoxy-β-D-glucopyranose（ZM-7）,corotoxigenin 3-O-β-D-galactopyranosyl-（4→1）-6-deoxy-β-D-glucopyranose（ZM-8）.Among them,ZM-6～ZM-8were new cardenolides.Ⅱ Tumor Multidrug Resistance Reversal Activities of A.curassavica.1.The inhibitory activities against cancer cellsChemical constituents from Asclepias curassavica L.were evaluated for the inhibitory activities against MCF-7,Hep G-2 cancer cells by MTT method.The dioxane double linked cardenolide glycosides showed the most potent cytotoxic effect followed by normal cardenolides and cardenolide lactates,while the C₂₁ steroids were non-cytotoxic.And the dioxane double linked cardenolide glycosides can resverse multidrug resistance.2.The activities of resversing multidrug resistanceChemical constituents from Asclepias curassavica L.were evaluated for the inhibitory activities against multidrug resistance cancer cells by MTT method.The dioxane double linked cardenolide glycosides showed the most potent cytotoxic effect followed by cardenolide lactates and the C₂₁ steroids,which deponded their structure,while normal cardenolides were non-cytotoxic.We systematically studied on mechanism which calotropin and asclepiasterol reversesed tumor multidrug resistance.Calotropin induced cell cycle arrest at the G2/M phase through down-regulating cyclins,CDK1,CDK2 and up-regulating p53 and p21.Moreover,it mediates mitochondrial apoptosis by increasing Bax/Bcl-2 ratio and the activation of JNK in A549/CDDP cells.Asclepiasterol inhanced inhibitory activities taxol and doxorubicin against multidrug resistance cancer cells,and it increased the intracellular accumulation of doxorubicin.Asclepiasterol-mediated P-gp suppression caused inhibition of ERK1/2 phosphorylation in two MDR cell types,and EGF,an activator of the MAPK/ERK pathway,reversed the P-gp down-regulation,implicating the MAPK/ERK pathway in asclepiasterol-mediated P-gp down-regulation.3.Na⁺/K⁺-ATPase inhibitory activities of some cardenolidesBesides,Enzymatic assay and Molecular docking analysis revealed relatively strong H-bond interactions between the bottom of the binding cavity and compounds M-8 or M-9,and explained why the dioxane double linked cardenolide glycosides possessed higher inhibitory potency on Na⁺/K⁺-ATPase than the cardenolide lactate.