Study Of The Structure Analysis,Antitumor Activity And Mechanism Of Polysaccharide APBPC-2 From Bee Pollen Of Carthamus

ObjectiveUsing bee pollen of carthamus tinctorius produced in Yunnan as raw material,the dilute alkali-soluble polysaccharide（APBPC）was extracted,separated and purified by dilute alkali extraction method.The homogeneous component APBPC-2with higher acquisition rate was selected as the research object.The structure of APBPC-2 was analyzed,and its anti-tumor activity in vitro was investigated.Meanwhile,the anti-tumor mechanism was also explored both on regulation of related genes expression and on the effect of the phosphatidylinositol 3-kinase/protein kinase B signaling pathway.The study may provide basic research data for screening new and promising polysaccharide drugs from bee pollen of carthamus tinctorius.Methods1.Extraction,separation and purification of APBPCPolysaccharide was extracted from the bee pollen of carthamus tinctorius by dilute alkali extraction,alcohol precipitation method.Then the polysaccharide was deproteinized by Sevag’s method until there was no obvious absorption at 280nm.Through dialysis,alcohol precipitation,centrifugation and freeze-drying,the alkali-soluble total polysaccharide of carthamus tinctorius bee pollen（APBPC）was obtained.The polysaccharide content in APBPC and the extraction rate of polysaccharide in arthamus tinctorius bee pollen raw materials were determined respectively.The APBPC was separated into different components by DEAE-52cellulose column chromatography eluted with NaCl gradient solution.Monitoring by phosphoric acid-phenol method,the different fractions of APBPC were obtained.The different fractions of APBPC with higher yield were further purified by Saphadex G75 column eluted with distilled water and were lyophilized.The purified fractions of APBPC were sealed in a sterilization tube and placed in a desiccator for later use.This study selected the fraction of APBPC-2 for subsequent experiments.2.Determination uronic acid content,molecular weight and homogeneity of APBPC-2The uronic acid content was determined by sulfuric acid-carbazole method and the high performance gel permeation chromatography（HPGPC）was used to measure the average molecular weight（Mw）,number average molecular weight（Mn）,and molecular weight distribution coefficient（D）of APBPC-2,respectively.The purity of the APBPC-2 was determined by the peak shape of HPGPC.3.APBPC-2 monosaccharide composition and infrared spectroscopy analysisThe monosaccharide composition of APBPC-2 was analyzed by high performance liquid chromatography（HPLC）with pre-column PMP derivatization.Potassium bromide tablet method was used for infrared spectroscopy analysis.4.Research on anti-tumor activity and mechanism of APBPC-2 in vitroThe CCK-8 method was used to investigate the anti-tumor activity of different dose groups of APBPC-2 on human breast cancer cells MDA-MB231,MCF-7,human prostate cancer cells DU-145,and human liver cancer cells Hep G-2.Cell morphology and scratch healing experiments were used to observe the changes of APBPC-2 on tumor cell growth.Flow cytometry was used to detect the effect of APBPC-2 on tumor cell apoptosis.RT-q PCR experiment was used to detect the effects of APBPC-2on the m RNA expression of pten,PI3K,Akt,pten,bax,bcl-2,caspase-3 and p53 in tumor cells,and the Western blot experiment to detect the effects of APBPC-2 on the expression of PTEN,PI3K,P-AKT,AKT,BAX,BCL-2,Caspase-3 and P53 protein in tumor cells.Results1.Extraction,separation and purification of APBPCThrough dilute alkali extraction,alcohol precipitation,deproteinization,dialysis and freeze-drying,the total polysaccharide（APBPC）of arthamus tinctorius bee pollen was obtained.The results showed that the conversion factor between APBPC and glucose was f=1.128（n=3）,the content of polysaccharide in APBPC was 86.25%,RSD=2.18%（n=3）.The extraction rate of polysaccharides in arthamus tinctorius bee pollen was 2.84%.Though DEAE-52cellulose column chromatography separation,five components were collected and named as APBPC-1,APBPC-2,APBPC-3,APBPC-4,and APBPC-5.Among them,the yields of APBPC-1,APBPC-4,and APBPC-5 were too low to meet the needs of the experiment.In this study,the higher yield of APBPC-2 was selected for subsequent experimental research.APBPC-2 was single peak symmetric after Saphadex G75 column chromatography purification,which indicated that APBPC-2 was a single component and could be used for subsequent experimental studies.2.Determination uronic acid content,molecular weight and homogeneity of APBPC-2The content of uronic acid in APBPC-2 was 11.78%,indicating that APBPC-2was an acidic polysaccharide.The HPGPC of APBPC-2 showed a single symmetrical peak,indicating that APBPC-2 was a homogeneous polysaccharide.3.APBPC-2 monosaccharide composition and infrared spectroscopy analysisHPLC results showed that APBPC-2 was composed of five monosaccharides,including L-RHA,D-Glu A,D-GLC and D-GAl,and L-Ara,with a molar ratio of11.93:10.06:13.37:10.29:8.79.The results of infrared spectrum analysis showed that the typical absorption peaks of APBPC-2 appeared near 3465cm^-1,2924cm^-1,2853cm^-1,1746cm^-1,1651cm^-1,1542cm^-1,1441cm^-1,1375cm^-1and 1243cm^-1.The absorption peak of APBPC-2 at855cm^-1was the C-H equivalent bond of the A-type differential isomer,while the absorption peak at 898cm^-1was the C-H vertical bond of the absorption peak of APBPC-2 at 855cm^-1was the C-H equivalent bond of the a-type differential isomer,while the absorption peak at 898cm^-1was the C-H vertical bond of theβ-type differential isomer.type differential isomer.It showed that there were both a-andβ-configurations in APBPC-2 molecule.There was an absorption peak at 967cm^-1,which was a rolling absorption peak caused by the methylation of deoxy-sugar,suggesting that APBPC-2 could contain rhamnose or fucose.4.Research on anti-tumor activity and mechanism of APBPC-2 in vitroThe results of CCK-8 showed that APBPC-2 inhibited MDA-MB231,DU-145and HepG-2 in different degrees of time-dose dependence.Cell morphology and scratch healing experiments also showed that APBPC-2 inhibited the growth and proliferation of these tumor cells.The results of flow cytometry showed that APBPC-2had higher apoptosis rate than the negative control group.RT-q PCR results showed that APBPC-2 promoted the mRNA expression of pten,bax,caspase-3 and p53,and inhibited the m RNA expression of PI3K,Akt and bcl-2 compared with the negative control group.Western blot results showed that compared with the negative control group,APBPC-2 could promote the expression of PTEN,BAX,Caspase-3 and P53proteins,and inhibit the expression of PI3K,P-AKT,AKT and BCL-2 proteins.Conclusions1.APBPC-2 is an acid polysaccharide with a glucuronic acid content of 11.78%.The Mw of APBPC-2 is 2.1106×10⁵Da,the Mn is 1.3652×10⁵Da,and the distribution coefficient is D=1.5460.APBPC-2 is composed of five monosaccharides:L-Rha,D-Glu UA,D-Glc,D-Gal,and L-Ara,with a molar ratio of11.93:10.06:13.37:10.29:8.79.APBPC-2 infrared spectroscopy detection shows typical characteristic absorption peaks of polysaccharides,and APBPC-2 molecules have both a-andβ-configurations.2.APBPC-2 has significant anti-tumor activity in vitro.The mechanism of anti-tumor activity is closely related to its regulation of pten,bax,caspase-3,p53,PI3K,Akt,bcl-2 genes expression,which in turn inhibts the PI3K/AKT signaling pathway and induces cell apoptosis.