Studies On Chemical Constituents And Pharmacological Activities Of The Fresh Bark Of Ailanthus Altissima

Ailanthus altissiYma(Mill.)Swingle belongs to the genus Ailanthus,Simaroubaceae family.It can be cultivated in most areas of China.The bark,root bark and fruit of Ailanthus altissima can be used as Chinese Traditional Medicine,have the effects of heat-cleaning,damp-drying,stopping diarrhea and stopping bleeding.The ancient books about Chinese Traditional Medicine recorded that the bark is poisonous,so that can be only used for external use on the skin.Besides,the leaves are not edible.The study of the chemical composition,active and toxic components can be of great value for its effectively use.In previous studies on A.altissima,there are many similar studies on branches,roots,and fruits.As results,oils,sterols,terpenoids,alkaloids,lipids have been isolated from the bark of A.altissima.The quassinoids,which belong to both tetracyclic diterpene lactones and pentacyclic diterpene lactones,are the most characteristic components of Simaroubaceae family and also abundant in A.altissima.In order to research the undiscovered chemical constituents and pharmacological activities of the bark,we extracted and separated the dichloromethane and ethyl acetate fractions from 95%ethanol extraction of the fresh bark of A.altissima,identified the structure of the purified compound and screened the inhibitory effect of several compounds on the proliferation of several kinds of human cancer cells.Part one Studies on chemical constituents of the fresh bark of Ailanthus altissmaObjective:To study the chemical constituents of the fresh bark of Ailanthus altissima(Mill.)Swingle,chromatographic methods are used in this research to separate and purify the extraction in order to get new natural compound and provide resources for the activity experiment.The isolated pure compounds were identified by spectroscopic techniques especially by1D-and 2D-NMR spectral data.Methods:Fresh bark of Ailanthus altissima(Mill.)Swingle was smashed and extracted by 95%EtOH at room temperature.The solvent was removed under vacuum to give a crude extract,which was then subjected to extract by petroleum ether(PE),CH2C12 and EtOAc respectively.The chemical constituents were separated and purified by silica gel column chromatography,Sephadex LH-20,preparative HPLC,and were identified by TLC to assist separation and purification.The compounds were identified by spectroscopy techniques.Results:There are twenty compounds were isolated from the dichloromethane and ethyl acetate fraction of the fresh bark extract of Ailanthus altissima(Mill.)Swingle.The structures were identified as follows:gardaubryone C(1),betulafolienediolone(2),3?-hydroxy-3-decarbonyl-24-epi-piscidinol A(3),ailanthone(4),(13?,14?,17a,20R)-23,24,25-trihydroxylanost-7,9(11)-dien-3-one(5),piscidinol A(6),scopoletine(7),ailanaltiolide J(8),altissimacoumarin H(9),(12?,13?,14?,17?,20R)-12,23,24,25-tetrahydroxylanost-7,9(11)-dien-3-one(10),anastrep C(11),11?-hydroxy-23/24-epi-brumollisol A(12),amarolide(13),canthin-6-one(14),1-(4-hydroxy-3-methoxyphenyl)-2-[2-methoxy-4-(?hydroxypropyl)-phenoxy]-propane-1,3-diol(15),3,5,5'-trimethoxy-8,3'-neoligna-4,4'7,9,9'-pentol(16),stigmast-5-en-3-ol(17),(E)-1'-(5'-(7,9-dihydroxy-7-(4-hydroxy-3-methoxyphenyl)propan-2-yl)-4'-hyd roxy-5'-methoxyphenyl)acrylic acid(18),4-[3-hydroxymethyl-5-(3-hydroxypropyl)-7-methoxy-1-benzofuran-2-yl]-2-m ethoxyphenol(19),1-hydroxy-canthin-6-one(20)?Conclusions:Twenty compounds were isolated from the the fresh bark of Ailanthus altissima(Mill.)Swingle,including eight triterpenoids,two quassinoids,two steroids,two coumarins,three lignans,two alkaloids and a ferulic acid.Besides,5,10,12,16,18 are new compounds.Part two The inhibitory effects of six compounds on proliferation of four human tumor cellsObjective:In this part of the study,we choose six compounds include gardaubryone C(1),betulafolienediolone(2),3?-hydroxy-3-decarbonyl-24-epi-piscidinol A(3),ailanthone(4),piscidinol A(6),amarolide(13),test the proliferation inhibitory effect of human cervical cancer cells(HeLa),human lung cancer cells(A549)and human liver cancer cells(SMMC-7721,Bel-7402),aim to find compounds that have anticancer activity.Methods:CCK-8 assay was used to evaluate the inhibitory effects of six compounds on the proliferation of human cervival cancer cells(HeLa),human lung cancer cells(A549)and human liver cancer cells(SMMC-7721,Bel-7402).Cell survival rate was calculated from the percentage of surviving cells.The concentration effect curve of experimental compounds on the tumor cells was used to calculate the half inhibitory concentration(IC50)of the compound.Results:(1)After treated human cervical cancer cell line HeLa with concentration of 100 ?mol/L,compounds 2,3,4 and 6 showed very strong proliferation inhibitory activity,and the inhibition rates were 101.82%,91.22%,86.76%,and 98.48%,while compounds 1 and 13 have not show any activity;(2)Compounds 2,3,4,and 6 still showed strong cytotoxicity after 6 compounds of 100 ?mol/L were applied to human lung cancer cells A549.The inhibition rates were 101.30%,81.40%,82.68%and 99.15%,respectively.Compound 1 had no activity,and the inhibition rate of compound 13 was only 4.83%.(3)Six compounds of 100 ?mol/L acted on human liver cancer cell SMMC-7721 and Bel-7402,the inhibition rates of compounds 2,3,4,and 6 were still high,against SMMC-7721:104.59%,84.18%,57.25%,98.57%;for Bel-7402:100.18%,93.52%,92.42%101.52%,the inhibition rates of compounds 1 and 13 on SMMC-7721 were 9.27%and 0%,respectively;the inhibition rates on Bel-7402 were 35.24%and 25.21%;(4)Through setting different concentrations of compounds 2,3,4,and 6,the IC50 of these compounds for several cancer cells was obtained:HeLa:1.89,4.60,0.11,3.91?mol/L;A549:13.77,14.78,1.01,4.90 ?mol/L;SMMC-7721:6.50,10.32,0.21,3.24 ?mol/L;Bel-7402:1.84,3.54,0.16,2.36 ?mol/L.Conclusions:betulafolienediolone(2)?3?-hydroxy-3-decarbonyl-24-epi-piscidinol A(3)?ailanthone(4)?piscidinol A(6)has potential proliferation inhibitory activity against human ovarian cancer cell line HeLa,human lung cancer cell A549,human liver cancer cell SMMC-7721 and Bel-7402.