Studies On Purification, Preparation And Activities Assay Of Pectic Oligosaccharides From Apple Peel

In this paper, pectin oligosaccharides hydrolyzed by pectinase from pectin extracted from apple peel as a material had been isolated and purified, the obtained oligogalacturonic acid was analyzed by electrospray ionization mass(ESI-MS), and its biological activity had been studied. The main results are as follows:1. Extraction and physicochemical properties of apple pectinApple pectin polysaccharides were obtained by acid-extracted, alcohol precipitation, and deproteinazation from apple peel, the raw yield was 15.8%. Gas chromatography analysis showed that it mainly composed of GalA, Gal, Rha, Ara, Glc, Xyl with a molar ratio of 78.54:6.29:4.71:6.21:2.27:1.98; Phenol-sulphuric acid analysis results indicated that the total sugar content of pectin was 91.75%; the protein content was 2.33%(Bradford method), Its uronic acid content was 76.4%(m-hydroxydiphenyl method); high performance liquid chromatography analysis indicated that the degree of methylation(DM) was 80.7%, the degree of acetylation (DAc) was 10.6%.2. Preparation and purification of apple pectin oligosaccharidesA low degree of esterification pectic acid was obtained by sodium hydroxide treatment of apple pectin. Pectic acid was digested with a commercially available pectinase (EC.3.2.1.15)to obtain a mixture of oligo-GalA (DP2～8), the mixture was separated by a low-pressure ion-exchange chromatography to obtain pure oligo-GalA. The degradation conditions were optimized as enzymatic reaction at 40℃for 1 h, oligo-GalA was obtained (yield of 37%) with the range of DP 2～5 through separation and preparation and show purity over 92% analyzed by TLC and ESI-MS.3. ESI-MS analysis of oligogalacturonic acidIn the ESI-MS analysis process for oligogalacturonic acid, we found that oligogalacturonic acid monomer was easily to form a dimmer in the process of ionization, compared with oligogalacturonic acid standard, the results of ESI-MS analysis show that the dimmer which formed in the ionization process exist in the analysis of acidic oligosaccharides. The dimmer formation is moderately stable, which performed that the collision energy required is greater than the energy needed to break down the glycosidic bond in the MS2 analysis process. The results showed that the dimmer was combined by a non-covalent bond (hydrogen bonding) after reduced by NaBH4.4. Biological activities assay of oligogalacturonic acidThe MTT method was used for studying the effect of oligogalacturonic acid,5-FU, and their combination on the growth of SW-620 cell line. The results showed that oligogalacturonic acid and cancer chemotherapeutic drugs (5-FU) had synergies tendencies. The cell cycle measured and analyzed by FCM, results indicate that the DNA synthesis phase(S phase) cells increased significantly after the oligogalacturonic acid treatment, which indicating oligogalacturonic acid had contributed to significantly promoting SW-620 cell line to enter S phase, it can make the cell cycle arrest in S phase, block the process of cells from S phase to G2/M phase, and block cell mitosis.