Preparation, Structure Analysis And Anti-tumor Activity Of Apple Oligosaccharide

The natural polysaccharide possesses a variety of biological activities. At present,more than30polysaccharides are in clinical trials for treatment of cancer, HIV infectionand diabete. Some polysaccharides have been approved to be used in clinic, such aslentinan in Japan, achyranthan, coriolus versicolor polysaccharide and ganoderma lucidumpolysaccharide in China. Our research shows that the low molecular apple polysaccharideinhibited the cancerization of colitis by influencing the expression of TLR-4and Gal-3.This reveals the potential application of apple polysaccharide in treatment colon/rectumcancer. However, there are some problems in the R＆D of natural polysaccharides:⑴Natural polysaccharides exist as the macromolecule forms, it has broad molecular weight range, poor homogeneity. It is hard to control the quality of polysaccharide.⑵It isdifficulty to elucidate the action mechanism of polysaccharide at the molecular level.⑶Itis very difficulty for natural macromolecular polysaccharide to transport in the body, andits pharmacokinetic characteristics in vivo is unclear.⑷It is difficult to extracthomogeneous polysaccharide. So, decrease the molecular weight and homogenization isan important strategy to realize the quality control of natural polysaccharide.Objective:⑴to establish a new method to separate, purify and identify the appleoligosaccharides;⑵to investigate the antitumor activities and its mechanism of appleoligosaccharide and lay a foundation for the development of apple oligosaccharide.Methods:1. The procedure of water extract-alcohol precipitation was used to isolate applepolysaccharide from apple pomace. Then the protein, pigment and small molecularsubstances were removed. The apple polysaccharide component1(AP-1) and component2(AP-2B) were obtained by dialysis, ion exchange chromatography and gel columnchromatography. The physiochemical properties were determined.2. Alkali and enzymic hydrolysis were coherently used to preparation appleoligosaccharides. The monosaccharide and oligosaccharides were obtained by using anioncolumn chromatography. The structure of OS-5was investigated by MS and NMR.3. MTT assay was adopted to investigate the effects of apple polysaccharides andoligosaccharides on the cell vitality. Apoptotic cells were measured with an AnnexinV-FITC staining, and cell cycle was determined by flow cytometry. Morphologicalchanges were examined by electron microscopy. The proteins and mRNA associated withapoptosis and cell cycle were demonstrated with western blot and real time PCRrespectively.4. The colorectal cancer model (CACC) and tumor bearing nude mice model wereused to evaluate the anti-tumor activity of OS-5in vivo. Colon cancer occurrence wasobserved by pathological histology method. TNF-α and Galectin-3levels in serum andapoptosis proteins were observed through ELISA and Western Blot. The antitumor effects of OS-5on tumor bearing nude mice were evaluated by tumor volume and the survivaltime on nude mice model.Results:1. AP-1and AP-2B were showed as a single sharp peak by using HPSEC, whichsuggested they were homogeneous polysaccharides. The monosaccharide composition ofapple polysaccharide was Rha, GalA, Glc, Gla, Arb in molar ratio of1.00:17.67:8.50:4.01:2.03. The molecular weight of AP-1was10kD, and the monosaccharide composition wasRha, GalA, Glc in the molar ratio of1.20:10.00:5.25. The molecular weight of AP-2B was1900kD, and the monosaccharide composition was Rha, GalA, Glc, Gal in the molar ratioof1.00:20.30:8.50:8.03.2. The purity of OS-5was99.21%. OS-5was an oligogalacturonide, its molecularweight was898. The structure of OS-5was α-D-GalAp-(1→6)-α-D-GalAp-(1→6)-α-D-GalAp-(1→6)-β-D-alAp-(1→4)-α-D-GalAp.3. The proliferation rate of HIEC cell was37.3±1.8%after it was treated with9μg/mL OS-5for36h. OS-5showed low inbibitional effect to A549, H1299, SMMC-7721,HCT116and SW480cells.36h after treatment, the inhibition rate on H1299, SW480cellswere24.99±0.6%,23.4±1.4%respectively. For A549cells and HCT116cells, theinhibition rate was less than10%. After SMMC7721cells were treated with differentconcentration of OS-5for24h, cells inhibition rate was about25%without anyconcentration-dependent relationship. The inhibition effect of OS-5on HT29cells wassignificant and showed time and concentration dependent manner. Flow cytometry showedthat OS-5could induce HT29cells apoptosis and S phase arrest. The apoptosis ratio ofHT29cells after treated with10μmol/L OS-5was45.9%(P<0.01). The percentages of Sphase was60.86±3.44%after treated with10μmol/L OS-5, and it was significantlyhigher than normol cells (P<0.01). Bax protein of HT29cells expression was significantlyhigher after it was treated with OS-5, while the expression of anti-apoptotic proteins Bcl-2and Bcl-xl was significantly lower than normol cells. OS-5significantly reduced theexpression of Cdk2and cyclin B1proteins in HT29cells, while increased the expressionof cyclin A1. 4. After treated by OS-5, the thymus index was significantly higher than the normalgroup and the model group (P<0.05) in colon cancer model. HE staining showed thatOS-5can reduce colon cancer cells invasion and growth. In OS-5treatment group, TNF-αlevel in serum was significantly higher than the model group (P<0.01), Galectin-3level inserum was lower than the model group, but there was no significant difference. WBshowed that the expression of Bax was significantly increased and expression of Bcl-2andBcl-xl decreased in OS-5treatment group. The expression of Bax significantly decreasedin model group (P<0.01). The experiment of nude mice showed that OS-5couldsignificantly inhibit the proliferation of transplanted tumors in dose-depenent manner. On35day, the tumor volume in5mg/kg dose group,10mg/kg dose group and20mg/kg dosegroup was529.84±44.01mm~3,443.81±38.94mm~3and358.96±28.68mm~3respectively.The median survival time of nude mice model group was38.5d, it was prolonged to39and47days in10mg/kg and20mg/kg group respectively.Conlusion：1. A controllable method to degrade apple monosaccharide was established, and OS-1,OS-2, OS-3, OS-4and OS-5were obtained.2. The structure of OS-5is α-D-GalAp-(1→6)-α-D-GalAp-(1→6)-α-D-GalAp-(1→6)-β-D-alAp-(1→4)-α-D-GalAp, which has not been reported.3. OS-5showed effectively antitumor activities on HT29cells. The possiblemechanism is associated with overexpression of Bax and decreased the levels of of Bcl-2and Bcl-xl. Apple oligosaccharide induced cell cycle arrest in S phase, which correlatedwith the decreased expression of Cdk2and cyclin B1on HT29cells.4. OS-5shows anti-tumor activities both on DMH/DSS-induced colon cancer modeland tumor bearing nude mice model, indicating OS-5has potential in colon cancertreatment and be worthy of further development.