Structural Characterization And Anti-tumor Properties Of Pectin Prepared From Different Plants

Pectin, a structurally complex acidic polysaccharide, has been widely studied and developed for anti-tumor application because of its versatile biological activities. Pectin mostly exists in cell wall of the terrestrial and aquatic plants. It is of great significance to search novel pectins with potent anti-tumor activities. In this dissertation, an analysis method was established to characterize the structure of pectin in detail by the combination of multistep degradation and mass spectrometric. Eight kinds of pectin were extracted and purified from three marine plants and five Chinese medicinal materials. Four of them were fully characterized by the established method. The effect of the eight pectin on HUVECs migration and macrophage phagocytosis were tested. The research results were as follows:Firstly, an analysis method was established by using citrus pectin as material to characterize the fine structure of pectin by multistep degradation and ESI-CID-MSn analysis, and oligosaccharides derived from mild acid and pectinase hydrolysis exhibited different sequence properties. A sensitive and efficient approach had been supplied in this study for the precisely structural analysis of complex oligosaccharides. Forty-six oligosaccharides were identified and two novel linkages were found from the citrus pectin. Apart from the known O-4 of the Rha residues, the O-2 of GalA was confirmed as a novel branch site in RG-â… . Besides, a new type with multi linkage mode of galacto-tetrasccharide Galβ(1â†'3)Galβ(1â†'4)Galβ(1â†'6)Gal was identified from RG-â…  side chains.Eight kinds of pectin were extracted and purified from three marine plants and five traditional Chinese medicines, and four of them were analyzed by the established approaches including multistep degradation and mass spectrometric analysis of oligosaccharides.Pectin ZCAP was isolated from the Zoster a caespitosa Miki of Bohai bay. ZC AP was composed by AGA (70%) and RG (30%) regions with the molecular weight of 77.23 kD. AGA consisted of an â†'4-α-D-GalAlâ†' backbone substituted at O-3 position by 1,3’-linked Apif oligosaccharides (Dp≤5) and single apiose residues. RG-I was confirmed to have a backbone of [â†'4)-a-GalA-(1â†'2)-α-Rha-1(â†'] with little side chains. Pectin ZAAP isolated from Zostera asiatica of Bohai bay was detected to have a similar structure as ZCAP.Pectin ZMAP was isolated from an unidentified Zostera marina of Swan lake in Weihai. ZMAP was a mixture of HG, RG-I and AG-II components with the molecular weight of 28.21 kD. HG was composed of (1â†'4)-a-D-GalA reisdues, and RG-I was confirmed to have a backbone of repeating disaccharide unit: [â†'4)-α-GalA-(1â†'2)-α-Rha-1(â†']. The side chains included β-(1â†'4)-galactan, β-(1â†'6)-galactan substituted at O-3 position and α-(1â†'5)-arabinan substituted at O-3 position as well. AG-II contained a 1,3-linked galactan backbone with 1,6-linked galactan side chains. The galactose residues can be further substituted with 1,5/1,3,5/terminal-linked Ara residues.Pectin DGP composed of HG (20%) and RG-I (80%) regions with the molecular weight of 106.5 and 87.5 kD was isolated from Angelica sinensi. The HG and backbone of RG-I was same to ZMAP. The side chains of RG-I region constituted by Ara and Gal were determined mostly at O-4 position of Rha, and the linkage patterns included β-(1â†'6)-galactan, α-(1â†'5)-arabinan substituted at O-3 position, β-(1â†'3)-galactan substituted at O-6 position by α-(1â†'>5)-Ara and α-(1â†'3)-arabinan substituted at O-5 position by β-(1â†'6)-Gal residues, etc.Pectin RSP composed of HG (50%) and RG-I (50%) regions with the molecular weight of 26.6 kD was isolated from Panax ginseng. The HG and backbone of RG-I was same to ZMAP. The side chains of RG-I region were determined to be Ara and Gal substituted at O-4 position of Rha, which included β-(1â†'4)-galactan, β-(1â†'6)-galactan substituted at O-3 position, α-(1â†'5)-arabinan substituted at O-3 position.Antitumor activities of the eight pectin were evaluated. It was found that DGP, which has the highest RG-I content (80%), shwed a significant inhibiting activity against HUVEC cells migration and could enhance the phagocytic function of macrophage cells as well. Under the acidic degradation of polysaccharide ZMAP, the fragment derived from RG-I region showed a higher inhibiting activity against HUVEC cells migration while the backbone of HG region exhibited a higher promotion of the phagocytic function of macrophage cells, which indicated that the anti-tumor mechanisms were high depend on the pectin regions. ZCAP had been proved to be successfully binding with fibroblast growth factor (FGF-8,10), epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF), suggesting that ZCAP had an effect on tumor cells proliferation and angiogenesis.This dissertation established a sensitive and efficient analytical method to characterize the precise structure of complex polysaccharide, which was applied on the sequence analysis of pectin from different sources. Anti-tumor activity experiments provide basis and support for the structure-activity relationship and the pharmacological application of pectin.