Study On The Structural Characteristics And Anti-tumor Effects Of Low-molecular Polysaccharides Extracted From Atractylodes Macrocephala Koidz

Baizhu is a herbaceous plant belonging to the family of Composite.Atractylodes macrocephala Koidz,the dry rhizomes of baizhu,is a common Chinese herbal medicine which has many pharmacological effects.The main active substances of Atractylodes macrocephala Koidz include esters and sugars,etc.The carbohydrates in Atractylodes macrocephala Koidz have drawn increasing attention from many scholars for their biological activities,such as anti-tumor activity,anti-inflammatory activity,immune-enhancing activity.At present,the research on carbohydrates in Atractylodes macrocephala Koidz mainly focuses on the polysaccharides of Atractylodes macrocephala Koidz obtained by ethanol precipitation after hot water soaking,but high temperature will change the structure of carbohydrates and affect its biological activity.Besides,our previous studies found that the macromolecular Atractylodes macrocephala Koidz polysaccharides had weak anti-tumor activity,while the small molecule polysaccharides showed good anti-tumor effects.Therefore,in this research,Atractylodes macrocephala Koidz was soaked in cold water at 4℃ and precipitated by different concentrations of alcohol to remove the high-molecular polysaccharides.Two kinds of low-molecular Atractylodes macrocephala Koidz polysaccharides were prepared,purified,analyzed the structure,and investigated the anti-tumor activity.First of all,two kinds of Atractylodes macrocephala Koidz polysaccharides were extracted,separated,purified and characterized.The dried rhizomes of Atractylodes macrocephala Koidz were soaked in 4℃ water,then added the absolute ethanol until the final concentration of ethanol was 60%.Discarded the precipitate and added absolute ethanol into supernatant until the final concentration of ethanol was 90%.Then,the protein was removed by the Sevag method,and polysaccharides were purified by dialysis and ultrafiltration.The purified alcohol-precipitated part of Atractylodes macrocephala Koidz polysaccharide was named PAMK-90 and the purified alcohol-soluble part of Atractylodes polysaccharide Koidz was named APA.The sugar content of PAMK-90 and APA are 95.24% and 92.16%,respectively.High performance gel permeation chromatography(HPGPC)analysis found that the average molecular weights of PAMK-90 and APA were about 4.1 KDa and 2.1 KDa,both of which are neutral polysaccharides with uniform molecular weight distribution and high purity.Results of infrared chromatography(FT-IR),gas chromatography(GC),nuclear magnetism(NMR),periodic acid oxidation and Smith degradation experiments and methylation analysis showed that PAMK-90 was mainly composed of α-galactose,α-arabinose and α-glucose(the molar ratio is about 1.0:1.5:5.0).The main chain was mainly linked by1,6-Linked-Glcp and the branched chain were linked by 1,5-Linked-Araf and1,6-Linked-Galp.While APA was mainly composed of α-arabinose and α-glucose(the molar ratio is about 1.0 : 4.6).The main chain of APA was mainly linked by1,6-Linked-Glcp and the branched chain was linked by 1,5-Linked-Araf.Comparing the two polysaccharides we can find that galactose in the monosaccharide composition of PAMK-90 was not in APA.In addition,the spatial configurations pictured by SEM showed that PAMK-90 was generally a rod structure with numerous voids and some sheet-like structure with a small number of pore-like structures.APA showed a dense pore sheet-like morphology,and many clusters of cotton-like structures were attached to the sheet-like structure.Then the anti-tumor effects of PAMK-90 and APA on H22 tumor-bearing mice were studied.The data showed that both 200 mg/kg PAMK-90 and 200 mg/kg APA could effectively inhibit the growth of H22 tumors in mice,and the tumor inhibition rates reached 53.01% and 58.41%,respectively,which were similar to the 20 mg/kg cyclophosphamide intraperitoneal injection group.HE staining of H22 cells showed that there were obvious apoptotic cells and lymphocytes in the H22 tumor tissue and tumor cell grinding fluid of PAMK-90 and APA groups.The results of Annexin V-FITC/PI double staining,Rh123 single staining and reactive oxygen content detection showed that the apoptosis rate of tumor cells increased,the mitochondrial membrane potential decreased and ROS content increased significantly in the PAMK-90 and APA groups compared with the model group.Besides,the number of infiltrating lymphocytes in the tumor tissue of mice in the PAMK-90 and APA groups were significantly higher than that in the model group,indicating that PAMK-90 and APA could stimulate lymphocytes to kill tumor cells.Cell cycle results confirmed that both PAMK-90 and APA could block tumor cells in the S phase to stop normal growth and proliferation of tumor cells.In summary,both PAMK-90 and APA have good inhibitory effects on the growth and proliferation of solid tumors in H22 tumor-bearing mice.To further study the anti-tumor mechanism of PAMK-90 and APA in vivo,a series of immune indicators of H22 tumor-bearing mice were tested to study their immune-enhancing activity and antioxidant effect.The experimental results indicated that PAMK-90 and APA could protect the thymus,spleen and liver tissues without toxic side effects,and maintain the balance of blood cells in the blood of tumor-bearing mice.Furthermore,they could effectively reduce inflammation,anemia caused by tumors and increase body immunity.Results of antioxidant experiments showed that PAMK-90 and APA could increase SOD,CAT and GSH-Px activities,and reduce MDA content to enhance the antioxidant capacity of mouse liver,kidney and serum.The above data indicated that both PAMK-90 and APA can exert anti-tumor effects through immune enhancement without obvious side effects.Finally,we aim at researching the inhibition of cellular proliferation and induction of apoptosis in Eca-109 cells by APA.MTT results revealed that PAMK-90 had no obvious inhibitory effect on Eca-109 cells,while APA could effectively inhibit the proliferation of Eca-109 cells in a dose-and time-dependent manner.Under the microscope,it was observed that Eca-109 cells gradually became smaller,rounded,and shrank.The edges of the cells were curled and cell nuclear condensation and apoptosis bodies could be detected in APA-treated groups.Results of Annexin V-FITC/PI double staining and Tunel staining further confirmed that APA had a significant pro-apoptotic effect on Eca-109 cells.Cell cycle results showed that APA treatment led to an accumulation of cell numbers in S phase,which meant that APA could arrest cells at S phase to suppress proliferation on Eca-109 cells.The results of Rh123 staining and the detection of ROS level reflected that APA treatment could decrease the mitochondrial membrane potential and increase the content of ROS in Eca-109 cells.Then,western blot analysis suggested that APA induced apoptosis by changing the expression of Bcl-2 and Bax and decreasing mitochondrial membrane potential which resulted in the release of Cytochrome C and the increasing of Cleaved-caspase 9 and Cleaved-caspase 3.In conclusion,APA had growth inhibitory effect on Eca-109 cells by mitochondrial pathway.This paper provides the experimental basis for improving the economic value of Atractylodes macrocephala in the food and drug industry,as well as provides theoretical support for its development as a new anti-tumor drug.