Research On Optimal Extraction And Anti-retinoblastoma Activity Of Andrographis Paniculata Polysaccharide

Objectives:In this paper,the extraction,purification and antioxidant activity of Andrographis paniculata polysaccharide(APP)were studied,and its anti-retinoblastoma(RB)activity and mechanism in vitro were explored.Methods:1.The study of extraction,purification and antioxidant activity of APP: Petroleum ether and anhydrous ethanol were used in turn for reflux.Using single factor test,response surface methodology(RSM)was employed to optimize APP by water extracting-alcohol precipitating method.After deproteinization by Sevage and depigmentation by activated carbon,the purity and composition of APP were identified by ultraviolet(UV)and infrared(IR)spectra respectively.The antioxidant activity of APP was detected by DPPH· scavenging experiment.2.Study on the activity and mechanism of APP anti-RB: CCK8 method was used to detect the inhibitory activity of APP on Y79 cells.Apoptosis promoting effect of APP on Y79 cells was detected by Annexin V-FITC / PI cell apoptosis kit.The effect of APP on the cell cycle of Y79 cells was detected by cell cycle test kit.Real-time PCR and western blot assays were used to detect the effects of APP on the expression of five genes(Caspase-3,Caspase-8,caspase-9,CDK1 and cyclin B1)in Y79 cells.Results:1.The study of extraction,purification and antioxidant activity of APP: The result showed that the best extraction process of APP were: the ratio of material to liquid1:36.71(g/ml),at 63.73 ℃ and extracting for 7.38 h.In order to facilitate the operation,the extraction process was modified as follows: the ratio of material to liquid was 1:40(g/ml),the extraction time was 7.5 h,the extraction temperature was 65 ℃,and the extraction rate of polysaccharide was 3.48 ± 0.96%.The results of UV spectrum showed that the purity of the extract was very high.The results of IR spectrum showed that the extract was polysaccharide.The antioxidant experiments showed that APP had certain antioxidant activity within the experimental range,and the scavenging capacity of DPPH· free radical was dose-dependent with a maximum clearance rate of 24.01%.2.Study on the activity and mechanism of APP anti-RB: The results of CCK8 inhibition experiments presented that Y79 cells showed different degree of proliferation inhibition after APP treatment at different concentrations for different time,and the results were both concentration-dependent and time-dependent.Flow cytometry showed that the apoptosis rate of Y79 cells increased significantly after APP treatment,and the number of cells blocked in G2/M phase significantly increased.The results of Real-time PCR and western blot presented that Caspase-3,Caspase-8 and Caspase-9 in APP groups were significantly higher in transcription and translation level than those in control groups,while CDK1 and cyclin B1 were significantly lower.Conclusion:In this paper,polysaccharide was extracted from Andrographis paniculata for the first time.On the basis of single factor experiment,response surface methodology(RSM)was used to optimize the extraction conditions of APP,and APP with single component and high purity was obtained.At the same time,APP was proved to have antioxidant activity.Through vitro cell experiments,we found that APP has the activity of inhibiting the proliferation and inducing the apoptosis of Y79 cells,and its possible mechanism is to up-regulate the expression of Caspase-3,Caspase-8 and Caspase-9 in the cell apoptosis signaling pathway and down-regulate the expression of CDK1 and Cyclin B1 in the cell cycle signaling pathway.The results of this study can expand the comprehensive development value of Andrographis paniculata and provide a new direction for the treatment of RB.