Study On The Quality Evaluation Of Grifola Frondosa And The Mechanism Of Its Extracts On The Apoptosis Of PANC-1 Cells

Objective Grifola frondosa is one of macro fungi with great development value and market potential,which was not recorded in“Chinese Pharmacopoeia”,resulting in the lack of an accurate quality standard for Grifola frondosa.Therefore,this subject intends to identify the characters,measure the contents of water,Total ash,extract,trace element and active components from Grifola frondosa in different harvest periods by using Modern Detection Technology.And a preliminary study was conducted on the quality standards of Grifola frondosa in Zhejiang based on the above indicators.At present,there are relatively many studies on the composition and pharmacological effects of polysaccharides of Grifola frondosa,but there are few studies on its alcohol extracts.And it is found that the ethanol extracts have certain biological activity based on the preliminary experiments,the subject separated and purified the ethyl acetate fraction of Grifola frondosa,which had a mechanism of inhibiting the action of PANC-1 cells.Methods（1）Describe the shape,color,size,odor,and other aspects by observing the Grifola frondosa in different harvest time.Referring to the 2015 edition of“Chinese Pharmacopoeia”,this paper measured the contents of water,Total ash,extract and active components from Grifola frondosa in different harvest periods.The contents of elements in Cd,Pb,Se,Hg,As,Co,Cu,Mn,S,Zn,Mo,Ca,Fe,Mg were determined by inductively coupled plasma atomic emission spectrometry,in order to make sure it meet the relevant regulations.（2）The extracting technology of polysaccharide was optimized and the contents of polysaccharide from Grifola frondosa in different periods were determined by UV-Vis spectrophotometry.The extracting technology of ergosterol was optimized and the contents of ergosterol from Grifola frondosa in different periods were detected by using high-performance liquid chromatography method.（3）The active constituents of Grifola frondosa were extracted by alcohol abstraction method,and were separated by gradient elution with petroleum ether and ethyl acetate after being extracted by ethyl acetate.The obtained fractions were acted on pancreas cancer cells（PANC-1）respectively.The best component was selected by MTT method,and its chemical composition were analyzed by GC-MS.The optimal effect time and concentration were screened by observing the cell morphology.Flow cytometry was used to analyze the effect of different concentrations of the best components on the apoptosis of PANC-1 cells.The expression of related genes Bcl-2,BAX and MMP9was detected by RT-PCR after PANC-1 cells were treated with different concentrations of the best components.Based on the above experiments,this paper studied the initial anti-tumor mechanism of Grifola frondosa..Results（1）Put forward the characteristics of Grifola frondosa identification in different harvest periods.The test data of Grifola frondosa is as follows:moisture contents range from 7.947～10.519%,total ash contents range from 4.876～5.561%,water-soluble extractives contents by hot dipping method range from 45.868～52.370%,ethanol-soluble extractives content by cool dipping method range from 27.761～32.658%,ethanol-soluble extractives content by hot dipping method range from 20.659～25.749%,the content of each element basically comply with a standard to the"Medicinal Plants and Preparations for the Import and Export of Green Industry Standard".（2）Optimum extraction process of polysaccharide from Grifola frondosa is as follows:extraction temperature 79.6℃,extracting duration 3.4h,liquid-solid ratio 50:1,the contents of polysaccharide are 6.147～9.660%.Optimum extraction process of ergosterol from Grifola frondosa is as follows:extraction temperature 70℃,extracting duration 50 min,liquid-solid ratio 27:1,the contents of ergosterol are 0.300～0.525%.（3）Six constituents of A,B,C,D,F and G were obtained after the extraction and separation of Grifola frondosa.The G component showed the best inhibitory effect on PANC-1 cells by acting comparison,with IC₅₀performed 355.12μg·mL^-1.The analysis of its composition by GC-MS shows he main components of G-component were organic acids and esters.The optimum actuation duration of G components to PANC-1 cells was 48h,optimum concentration was 160、320、640μg·mL^-1.Flow cytometry showed that the G component promoted the apoptosis of PANC-1 cells.The results of real-time quantitative PCR showed that G-components acted on PANC-1 cells by up-regulating BAX and down-regulating the expression of Bcl-2 and MMP9.Conclusion（1）It is suggested that Grifola frondosa in Zhejiang,the moisture content does not exceed 11%,total ash content should not be more than 6%,water soluble extract content should not be less than 40%,ethanol soluble extract content should not be less than 20%,polysaccharide content should not be less than 6%,ergosterol content should not be less than 0.3%.（2）The alcohol extracts of Grifola frondosa were acted on PANC-1 cells after extraction and separation.The G component had the best inhibition of cell proliferation effect among others.（3）The main components of G-component were organic acids and esters.（4）G-component of Grifola frondosa could up-regulate the expression of BAX gene and down-regulate the expression of Bcl-2 gene,which inducing the apoptosis of PANC-1 cells and leading to anti-tumor effect.