Studies On Purification And Activities Assay Of Pectin And Pectic Oligosaccharides

In this thesis, four pectin polysaccharides were sequentially extracted from apple peel by water, ammonium oxalate, HCl and KOH. Pectin polysaccharides hydrolyzed by pectinase had been isolated and purified, the obtained oligogalacturonic was analyzed by ESI-MS. The biological activity of pectin and oligogalacturonic acid had been studied. The main results were summarized as follows:1. Physicochemical properties and biological activity of apple pectinWater-soluble pectin(WSP), chelate-soluble pectin(CSP), acid-soluble pectin(ASP) and base-soluble pectin(BSP) were sequentially extracted from apple peel by water,0.5%ammonium oxalate,0.05M HCl and1M KOH, the yield were3.49%(WSP),5.23%(CSP),2.77%(ASP),2.35%(BSP), respectively. Gas chromatography analysis showed their mainly composed and molar ratio as follows:WSP(Rha:Ara:Xyl:Glc:Gal:GalA=1.51:58.5:2.13:19.51:11.88:6.44), CSP(Ara:Glc:Gal:GalA=54.61:9.81:13.43:22.16), ASP(Rha:Ara:Xyl: Glc:Gal:GalA=1.11:23.28:1.4:16.51:14.14:13.56), BSP(Rha:Ara:Xyl:Glc:Gal=3.15:28.33:4.51:49.70:14.32); Phenol-sulphuric acid analysis results indicated that the total sugar content of pectin were WSP:85.00%, CSP:96.20%, ASP:83.48%, BSP:80.15%; Carbazole-sulphuric acid analysis results indicated that the uronic acid content of pestin were CSP:24.5%, ASP:15.7%, WSP:7.3%, BSP:0; The protein content were WSP:2.58%, CSP:0.41%, ASP:1.48%, BSP:6.45%; The pectin molecular mass exceed400KDa; All of the four pectins could inhibit the growth of HT-29cells.2. Preparation and purification of apple pectin oligosaccharidesA low degree of esterification pectic acid was obtained by sodium hydroxide treatment of CSP. We optimized the degradation condition and combined the best enzyme solution conditions:enzyme concentration (0.5mg·mL-1), time (60min), the temperature(40℃), pH4.5. Pectic acid was digested with pectinase to obtain a mixture of oligo-GalA (DPI-11) and neutral oligosaccharides (DP1～DP4), the mixture were separated respectively by a ion-exchange chromatography and Bio-Gel-P4to obtain pure oligo-GalA and neutral oligosaccharide. The purification of enzyme mixture from500mg pectin acid could obtain390mg oligo-GalA and10mg neutral oligosaccharides, oligosaccharides through separation analyzed by ESI-MS.3. Effect of oligogalacturonic acid (DP2-DP5) on the growth of tumour cellsThe MTT method was used for studying the effect of oligogalacturonic acids on the growth of tumour cells. The results showed that oligogalacturonic acid could inhibit the growth of A549, HT-29, H1299and LoVo cells. In suitable concentration, the effect of oligogalacturonic acid on the growth of tumour cells as same as cancer chemotherapeutic drugs (5-FU).4. Effect of oligogalacturonic acid (DP2～DP6) on lymphocyte proliferationTo detect the immunomodulatory activities of oligosaccharide acids, mouse splenocytes were stimulated with oligosaccharide acids at serial concentrations. Lymphocytes proliferations were analyzed with3H-TdR incorporation method using a β-scintillation counter. The results showed that oligogalacturonic acid could induce splenocyte proliferation at a low dose and could be an inhibiter in a high dose. Effects of oligogalacturonic acid on ConA-or LPS-induced lymphocyte proliferation, DP2, DP4and DP6showed synergistic effect when the uronic acid and Con A combination, but, when the uronic acid and LPS combination, their activities reduce obviously.