| At present,malignant tumor have become the severe disease that threaten mankind health seriously.In the world,the death rate of the tumor is the first or the second in all of the death causes and the lung cancer has been the first in China.Thus it is becoming a focus in medical research to search chemotherapy for the less side effect.The anthocyanins have some pharmacologic effects including antibiosis, anti-virus,analgesia,anti-oxydation,anti-tumor and et al.Especially anti-tumor effect is paied closed attention by much researcher.In our research,the method for extracting anthocyanin pigment from purple plant of new breed of maize was researched,and we used A549 cells to identify the functions of the maize purple plant pigment.Orthogonal experimental design is used,in which it chooses concentration of ethanol,volume of extration solvent,temperature, and time in extraction as 4 factors to be studied and selects 4 different levels for each one.Then do research according to orthogonal table,regarding absorbance as the index of extraction ratios.The optimal conditions to extract the pigment by ethanol was obtained by orthogonal experiment:to adjust acidity to pH=3 by citric ,the ratio between material and 80 % ethanol solvent was 1:50,the extracting time was 4h,temperature was 25℃,the average extracting rate was 10 % and color value was 4.92.In vitro,exponential phase human lung cancer cell line A549 was treated by the maize purple plant pigment in various concentration(0mg/ml,0.1mg/ml,1mg/ml,10mg/ml,20mg/ml,30mg/ml,40mg/ml,50mg/ml,60mg/ml,100mg/ml).Cell morphology changes were observed by optical microscopy.Using MTT assay to detect absorbance value and calculate inhibition rate and then the effect of cell growth inhibition was assayed by MTT method.The induction of cell cycle and apoptosis by the maize purple plant pigment was assessed by flow cytometry.Human lung cancer cell line A549 were treated with various concentrations of the maize purple plant pigment for 24 hours.The results confirmed high anti-proliefration activity in all test cells and the inhibitory effect was dose dependent.The maize purple plant pigment with dosage at 1mg/ml has inhibition effects on A549 cell(P<0.05),and it with dosage at 20mg/ml has significant inhibition effects(P<0.01).The inhibition rate was the maximum under concentration of the maize purple plant pigment was 50mg/ml which was 85.63%.The typical apoptosis morphology was identified under inverted microscope.It was observed that enlarged cell space,poor adherence, pyknosis,abnormal shape,membrane blebbing,and more dead cell floating in medium.Flow cytometery analysis results showed that the cells in G1/G0 stage descended gradually at 40mg/ml,cells in S stage multiplied, while cells in G2/M stage have no variation.In addition,there was an apoptotic peak in A549 cells after incubating with the maize purple plant pigment detected by flow cytometry,and the maize purple plant pigment promoted the apoptosis of A549 cells.Quantitative analysis result showed that cell apoptotic ratio of 0mg/ml,10mg/ml,20mg/ml,40mg/ml density was 0.00%,1.32%,7.86%,41.52%.In this research,the best technical process of extraction is: with the concentration of 80% for extraction solvent ethanol,the ratio of 1:50 for the weight of corn to the volume of extraction solvent, temperature of 25℃,treatment for 4 hours,the highest extraction ratio could be acquired.Cell crowth inhabition could be carried out by the pigment from corn purple plant in a dose dependent manner.In addition,the maize purple plant pigment arrests the cell cycle of A549,it inhibits the S cell cycle to enter the G2/M cell cycle. |
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