Study On Extraction,Characterization And Anti-human Breast Cancer MCF-7 Cells Of Gastrodia Polysaccharide

Gastrodia elata Bl.is one of precious herbal medicines in China.It has a variety of pharmacological effects?such as:sedative hypnosis,anti-depressant,anti-convulsion,anti-inflammatory,and anti-aging?.Recently,the research of Gastrodia elata polysaccharide?PGE?has attracted increasing interest.However,there are few reports on the structure-activity relationship of PGE due to the complex structure of polysaccharides.In this work,asymmetrical flow field-flow fractionation?AF4?coupled with multiangle light scattering detector?MALS?and differential refracting detector?dRI?was applied for the first time to study molecular structure of PGE.Meanwhile,fourier transform infrared spectroscopy?FTIR?and ultraviolet spectrophotometer?UV-vis?were used to characterize PGE.The relationship between the molecular structure and biological activity of PGE against human breast cancer MCF-7 cells was investigated.In the second chapter,effect of extraction methods?hot water and ultrasonic extraction methods?on the structure of PGE was studied.AF4-dRI was employed for the separation and characterization of PGE.The polysaccharide content was determined by phenol-sulfuric acid method.The results showed that the polysaccharide content of PGE extracted by the two methods was greater than 90%.The extraction rate of the ultrasonic extraction method was significantly higher than that of the hot water extraction method.The maximum extraction rate?PGE-U-120?was 53.50%.The dissolution conditions and AF4 analysis conditions of PGE were optimized.The optimal PGE dissolution conditions were 50°C,200 rpm stirring for 2 h.The optimal AF4 analysis conditions were the injection volume=100?L,sample concentration=1.0 mg/mL,V_d=1 mL/min,V_c=0.5 mL/min.The carrier liquid is water containing 10 Mm NaNO₃ and 0.02%NaN₃,and the membrane type is regenerated cellulose?RC?.Under optimized conditions,PGEs extracted with different conditions were separated and characterized.The results showed that as ultrasonic extraction time increases,the radius of gyration?R_g?and molecular weight?M_w?of the extracted PGE gradually decreases.At the same extraction time,R_g and M_w of the PGE extracted by ultrasound method were smaller than those of PGE extracted by hot water method.The AF4 results indicated that the size of PGE extracted by hot water method was large,which results in a steric transition phenomenon in AF4 analysis.In the third chapter,the influence of the crossflow rate and channel thickness on the steric transition point was investigated.The optimized operation conditions for AF4 analysis of PGE extracted by hot water method were V_d=1 mL/min,V_c decreased exponentially from 0.8mL/min to 0.05 mL/min,t_1/2=2 min,the spacer thickness=490?m.The membrane is Regenerated cellulose?RC?,and the carrier fluid is water containing 10 mM NaNO₃ and 0.02%NaN₃.Under these conditions,the extracted PGE was analyzed by AF4-UV-MALS-dRI.It was found that the resolution and signal intensity were improved.The steric transition effect in AF4analysis was weaken for the sample extracted by hot water method and was solved for the samples extracted by ultrasound method.Furthermore,the apparent density???and shape factor?R_g/R_h?of PGE extracted by ultrasound method were calculated.It was found that as the molar mass increased,?of PGE-U-20 and PGE-U-120 decreased slightly,and the?of PGE-U-40 increased accordingly,and PGE-U-20 has a loose hyperbranched chain structure,PGE-U-40 and PGE-U-120 have spherical structures.The results showed that the structure of polysaccharides can be changed by changing the ultrasound time.In the fourth chapter,effect of PGE on on the viability of MCF-7 cells was explored.FTIR results showed that PGE obtained with different extraction conditions was composed of?-D-pyranose and D-pyranose.The effect of PGE on the viability of MCF-7 cells was studied by MTT.The results showed that:1.As the concentration of PGE increased,the inhibition rate of PGE on MCF-7 cells increased.2.The inhibition rate of PGE obtained by ultrasound method on MCF-7 cells was higher than that of PGE obtained by hot water method.This may due to lower molecular weight of PGE obtained by ultrasound method.3.PGE-U-40 had the highest inhibition rate which may attribute to the spherical structure.The results of flow cytometry revealed that PGE-U-40 can block MCF-7 tumor cells in the G₂/M phase,and had an apoptotic effect on MCF-7 cells.The results showed that the polysaccharide content,molecular weight and apparent density of PGE extracted with different methods are different.The results indicated that anti-tumor activity of PGE may be related to its molecular conformation,molecular weight,and apparent density.AF4 was proved to be a promising tool for analyzing PGE.