Systematical Analysis Of Citrus Pectin And High Temperature Modification

Citrus affiliated to Magnoliophyta,Magnoliopsida,Sapindales,Rutaceae.As fruit,citrus is very popular to people.Citrus peel as a by-product of citrus processing process contains 20-30%of pectin,so the development and utilization of citrus pectin has become a hotspot to the scholars.In this paper,the fractionation,structural characterization,high temperature modification and antitumor activity in vitro of citrus pectin have been studied.According to the differences of charge and molecular weight,citrus pectin(CP)was fractionated on the DEAE-cellulose ion-exchange column,Sepharose Cl-6B gel chromatography column and Sephadex G-25 gel chromatography column.Then five polysaccharide fractions were obtained,they were two neutral polysaccharides:CPN-H,CPN-L,and three acidic polysaccharides:CPA-0.2,CPA-0.3 and CPA-0.4.Among them,CPN-L belongs to oligosaccharide because of its low molecular weight,at the same time it contains many impurities.So CPN-L was not further researched in this paper.The molecular weight of CPN was about 40 KD.The results of high performance liquid chromatography(HPLC)and 13C-nuclear magnetic resonance spectroscopy(13C-NMR)showed that CPN was ?-(1,4)linked galactan.The molecular weights of the three acidic polysaccharides were 85 KD,205 KD and 380 KD respectively.Their monosaccharide composition was similar.All of them contain more than 84%GalA,and a small quantity of Rha,Gal and Ara.So it was speculated that all of the three acidic polysaccharides were HG domain-rich pectin with some RG domains.Every fraction of CP was modified twice with high temperature,yielding fractions of HTCP.After being modified,except the neutral polysaccharides,all the acidic polysaccharides produced some small molecules which have ultraviolet absorption at 235 nm.In addition,the yield of the small molecules was increased with the times of modification.Take CPA-0.3 for example,when the modification times rose to 13,the yield of the small molecules reached the highest and no longer increased.This sample named HTCPA-0.3-13.HTCPA-0.3-13 was separated into macromolecules in bag and micromolecules out of bag by dialyzing against distilled water.Both parts were analysed by high performance gel permeation chromatography(HPGPC).The results showed that the macromolecules in bag were homogeneous,while the micromolecules out of bag were a mixture which was hard to separate because of similar molecular weight.The macromolecules and CPA-0.3 were both analysed by fourier transform infrared spectroscopy(FT-IR)and 13C NMR.Compare the restults,it was found that the free carboxyl groups of the macromolecules were obviously less than that of the CPA-0.3.So it was deduced that the free carboxyl groups of CPA-0.3 was destroyed seriously in the processing of high temperature modification.The growth inhibitory activities of CPA-0.3 and HTCPA-0.3-13 on two colon cancer cell lines HCT-116 and HT-29 were analysed by MTT method.The result showed that CPA-0.3 had no inhibitory activity on the growth of the cells,but after high temperature modification,the inhibitory activity was very obvious.