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单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,Introduction,Cancer is the worlds second biggest killer after cardiovascular disease.In 2005,7.6 million people died from cancer,more than HIV/AIDS,malaria and tuberculosis combined.In addition,this number is expected to rise to 9 million in 2015 and increase further to 11.5 million in 2030(WHO,2007).Clearly,there is a great need to improve current cancer therapies and to search for new therapies.,Data from Statistics Canada,Daphne altaica,Pall.,locally known as,uwsoyq,or,qasqr jiydek,is a deciduous herb of the Thymelaeaceae family.It is endemic to the north of Jungar Basin of Xinjiang,China(the Tacheng and Habahe areas),Altai,Manrak and Tarbagatai Mountains of Kazakhstan and Altai region of Russia as well as northwest Mongolia,D.altaica,have long been used in TKM to treat esophagus cancer,gastric cancer,tracheitis,common cold,soar throat,rheumatism,snakebite,and for their antitussive and diaphoretic properties(Xu et al.,2009).Its medicinal use was firstly recorded in a Kazakh medical classic,Shipagerlik Bayan,which was written by Oteyboydak Tleukabyluly in 15th century(Tleukabyluly,1994).,Oteyboydak Tleukabyluly（,1388-？）,Shipagerlik Bayan,Translation：,Prescription 4573:add the bark of Daphne into the meat broth and boil them.As soon as the broth comes to a full boil,remove the residue,drink the broth and eat the meat.It can cure asthma and cough.,At page 420 of this book,there is the following prescription：,Although many benefits of this plant have been claimed,no scientific datum is available so far about its biological properties,especially its anticancer activity.Therefore,this prompted us to investigate the antiproliferative activity of this plant on cancer cells.For these purposes,six extracts were prepared and tested for their potential antiproliferative properties with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)bioassay on different cancer cell lines.To our knowledge,this is the first time that the anticancer activity of,D.altaica,is evaluated.,MATERIALS AND METHODS,Plant material,The barks of,D.altaica,were collected from Altai Mountain,Xinjiang,China in July 2008.The plant was identified and authenticated by Bahargul Kongirkhan,a herbalist at the herbarium of Altay Institute for Drug Control,Xinjiang,China.A voucher specimen(No.050036)was deposited at the same place.,Tested material,Dried barks of the plant(150 g)were chopped and extracted with 95%EtOH by maceration for 2 weeks in a dark place at a room temperature of 20 2.The procedure was repeated for twice.The extracts were combined,concentrated under reduced pressure and freeze-dried to yield the EtOH extract(DA-Et,12.10 g).0.5 g of this extract was kept for MTT assay and the rest was submitted to a sequential liquid-liquid extraction with solvents of increased polarity to yield petroleum ether(DA-Pt，1.7064 g),chloroform(DA-Ch,0.6915 g),ethyl acetate(DA-Ea，1.8237 g),n-butanol(DA-Bu，2.5242 g)and aqueous(DA-Aq，2.5998 g)fractions.,Cell culture,human esophageal squamous cell carcinoma(Eca-109)cells,gastric carcinoma(AGS)cells,hepatoma(SMMC-7721)cells,cervical carcinoma(HeLa)cells,All cell lines were cultured at 37C in a humidified atmosphere of 5%carbon dioxide.,Cell proliferation assay,All the samples were tested at 6.25,12.5，25，50，100 g/ml concentrations.The samples were dissolved in DMSO and further diluted with cell culture medium.Cells incubated with the same concentration of DMSO were used as a control.The DMSO final concentration was adjusted to 1%of the total volume of medium in all treatments,including the control.For MTT assay,1 10,5,cells/well were plated into 96-well plates(Nunclon,Denmark)and incubated for 24 h before the addition of drugs.After 48 h of incubation for all cells,20 l of MTT(Sigma,USA)reagent(5 mg/ml)in phosphate buffered saline(PBS)was added to each well.The plates were incubated at 37C for 4 h.At the end of the incubation period,the medium was removed and pure DMSO(150 l)was added to each well.,The metabolized MTT product was quantified by reading the absorbance at 490 nm on a Beckman Coulter-AD340(Beckman Coulter,Fullerton,CA,USA).Results were expressed percentage of cell viability(%).All assays were performed in triplicate.The median growth inhibitory concentration values(IC,50,)were used to compare the antiproliferative activity of extracts on cancer cells.,Statistical analysis,The results of percentage of cell viability were presented as means SD.Statistical comparisons between treatment groups and the control group were performed using the one-way ANOVA followed by,post hoc,Tukey(in case of equal variance)or Dunnetts T3(in case of unequal variance)test.These tests were performed using SPSS 13 for Windows.p 0.05 was considered statistically significant.IC,50,values and their 95%confidence interval(IC95)were calculated using sigmoidal dose-response model with variable slope in the Graphpad Prism software(version 4.03).,RESULTS AND DISCUSSION,Figure 1.Antiproliferative activity of DA-Aq,DA-Bu,DA-Ea,DA-Ch,DA-Pt and DA-Et on esophageal squamous cell carcinoma(Eca-109)cells.,Figure 2.Antiproliferative activity of DA-Aq,DA-Bu,DA-Ea,DA-Ch,DA-Pt and DA-Et on gastric carcinoma(AGS)cells,Figure 3.Antiproliferative activity of DA-Aq,DA-Bu,DA-Ea,DA-Ch,DA-Pt and DA-Et on hepatoma(SMMC-7721)cells.,Figure 4.Antiproliferative activity of DA-Aq,DA-Bu,DA-Ea,DA-Ch,DA-Pt and DA-Et on cervical carcinoma(HeLa)cells.,Table 1,.Antiproliferative effect of six extracts from the barks of,D.altaica,on four different tumor cell lines.Data are presented as IC50(g/ml)values,Indeed,some species of genus,Daphne,were reported to have significant antitumor activity.For example,it was described that extracts and compounds of,D.genkwa,(Zhan et al.,2005),D.mucronata,(Mahdavi et al.,2007)and,D.odora,var.,marginata,(Zhang et al.,2006)markedly inhibits the proliferation of several tumor cells,in vitro,.Crude extracts and isolated compounds from,D.tangutica,showed potent antitumor activity,in vivo,(Zhang et al.,2007).From these species,different classes of natural compounds have been isolated,including coumarins,diterpenes,flavonoids,biflavonoids,lignans,simple phenylpropanoids and steroids.Among these reported secondary metabolites,only daphnane type diterpene esters,biflavonoids,coumarins and lignans were reported to be the major bioactive compounds responsible for the antiproliferative activity of,Daphne,species(Zhan et al.,2005;Mahdavi et al.,2007;Zhang et al.,2006;Kasai et al.,1981;Yazdanparast et al.,2004;Zheng et al.,2007;Ma et al.,1994;Park et al.,2008).,
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