Experimental Study On Extraction Process And Anti-senescence Effects Of Polysaccharides From Inonotus Obliquus

Objective: To optimize the extraction process of polysaccharides isolated from Inonotus obliquus and study the protective effects and mechanism of Inonotus obliquus polysaccharides on senescence of Rat aortic smooth muscle cells induced by Etoposide.Methods:(1)Inonotus obliquus polysaccharides were extracted by water extraction and alcohol precipitation.The Box-Behnken design response surface method(BBD-RSM)was used to optimize the extraction process of Inonotus obliquus polysaccharides on the basis of single factor experiments.The effects of processing temperature,extraction time and liquid to solid ratio on the yield of Inonotus obliquus polysaccharides were studied.The ?KTA explorer 100 chromatography system was used to separate and purify Inonotus obliquus polysaccharides by DEAE-cellulose ion exchange chromatography and Sepharose CL-6B gel filtration chromatography.(2)Antioxidant model(DPPH free radical scavenging experiment,ABTS total antioxidant capacity determination experiment and FRAP iron ion reduction capacity determination experiment)in vitro were established to screen the antioxidant activities of different polysaccharides components from Inonotus obliquus.(3)To establish cellular senescence model of Rat aortic smooth muscle cells,MTT method,morphological observation,senescence ?-galactosidase staining method,DAPI staining method were used to study the protective effects of Inonotus obliquus polysaccharides on Rat aortic smooth muscle cells senescence induced by Etoposide.(4)ROS assay experiment and mitochondrial membrane potential detection experiment were used to investigate the inhibitory effects of Inonotus obliquus polysaccharides on Etoposide-induced oxidative stress in Rat aortic smooth muscle cells.(5)qRT-PCR method and Western blot method were used to detect the effects of Inonotus obliquus polysaccharides on the expression of senescence-related genes p53,p21 and p16 in Rat aortic smooth muscle cells induced by Etoposide at mRNA level and protein level respectively.Results:(1)The optimal extraction conditions of Inonotus obliquus polysaccharides were as follows: processing temperature 100?,extraction time 2.39 h,liquid to solid ratio of 35.8:1 mL/g,the maximum yield 11.21%.Inonotus obliquus neutral polysaccharides IOP-1 and acid polysaccharides IOP-2 were isolated.(2)Antioxidant experiments in vitro showed that IOP-1 and IOP-2 had antioxidant activities,including DPPH free radical scavenging capacity,ABTS total antioxidant capacity and FRAP iron ion reduction capacity.IOP-1 had stronger antioxidant activities than IOP-2 and positive control Vitamin E.In the concentration ranges of 0.025~1.6 mg/mL,DPPH free radical scavenging rate of IOP-1 at 0.2 mg/mL reached 83.39 �0.03 %,which was stronger than DPPH free radical scavenging rate 73.74�0.04 % and 39.15�0.03 % of IOP-2 and Vitamin E(p<0.05)at a concentration of 1.6 mg/mL.the ABTS total antioxidant capacity of IOP-1 was 1.39�0.08 mmoL/g at a concentration of 0.4 mg/mL,which was stronger than total antioxidant capacity 1.08�0.07 mmoL/g and 0.38�0.12 mmoL/g of IOP-2 and Vitamin E(p<0.05)at a concentration of 1.6 mg/mL.At the concentration of 1.6 mg/mL,the iron ion reduction capacity of IOP-1 was 1.15�0.10 mmoL/g,which was stronger than the iron ion reduction ability 0.75�0.07 mmoL/g and 0.22�0.04 mmoL/g of IOP-2 and Vitamin E(p<0.05)at the same concentration.(3)IOP-1 could promote the proliferation of Rat aortic smooth muscle cells,which could inhibit the proliferation arrest of Rat aortic smooth muscle cells induced by Etoposide,increase cell viability,and inhibit the cellular senescence morphological changes,senescence ?-galactosidase activities and senescence-associated heterochromatin aggregation of Rat aortic smooth muscle cells induced by Etoposide.(4)IOP-1 could inhibit oxidative stress of Rat aortic smooth muscle cells induced by Etoposide by inhibiting ROS generation and decrease of mitochondrial membrane potential.(5)IOP-1 protected the senescence of Rat aortic smooth muscle cells induced by Etoposide by inhibiting the high expression of senescence-related genes p53,p21 and p16.Conclusion: The optimal extraction process of Inonotus obliquus polysaccharides was determined.The neutral polysaccharides component IOP-1 from Inonotus obliquus was filtered out,which had strong antioxidant activities in vitro,IOP-1 could inhibit senescence phenotypes and inhibit oxidative stress on Etoposide-induced senescence of Rat aortic smooth muscle cells by reducing ROS production and balancing mitochondrial membrane potential,The molecular mechanism was to regulate the high expression of senescence-related genes p53,p21 and p16 in the classical senescence-related signaling pathways p53-p21 and p16-pRb.