Study On Extraction, Separation And Purification Of Polysaccharide From Punica Granatum

Pomegranate is the only one genus of plants cultivated as a fruit tree and its rind (pericarpium granati, PG) is one of China’s traditional herbal medicine. A large number of studies have confirmed that PG was composed of many chemical components, such as amino acids, polysaccharides, alkaloids. Pharmacological studies have found that the extracts of PG can reduce the risk of some diseases, such as cancer, inflammation and neurodegeneration, because they contain a variety of biologically active substances. Therefore, it has an important significance about the research of its active ingredient and the effective use for human health and the recycling of waste material.There are two parts in this thesis.Part one:introductionIt briefly summarized the reseach about pomegranate and the chemical constituents of PG firstly. Then it reviewed three sides involved the extraction technology of plant polysaccharides, separation and purification technologies and application. In addition, it elaborated the surfactant studies as a extraction solvent. Finally, high speed countercurrent chromatography (HSCCC) is outlined about separation principles, characteristics, selection of two-phase solvent system and its application.Part two:reseach reportThis section is divided into three chapters:Extraction of pericarpium granati polysaccharides (PGP); Separation and purification of PGP with HSCCC; Identification structure of PGP.1. Reseach about the extraction of pericarpium granatiPolyethylene glycol aqueous solution (PEGs), an environmentally-friendly solvent was used for the first time to develop an extraction method for crude polysaccharides from PG with ultrasonic-microwave-assisted extraction (UMAE). Compared with other extraction solvents (water and ionic liquids), PEGs, especially PEG400, displayed a higher yield. A response surface methodology was used to optimize the UMAE parameters (microwave power, concentration of PEG400solution, and the ratio of liquid to raw material) to enhance the extraction efficiency of polysaccharides. The optimum extraction conditions were microwave power of365W, PEG400concentration of30%and20mL/g ratio of liquid to raw material. Under these conditions, an extraction yield of7.94±0.3%(n=3) was obtained, which is about25%higher than that with water as solvent. The polysaccharides content in PG was6.56±0.01mg/g, expressed as glucose.2. Separation and purification of PGP with HSCCCAn aqueous biphasic system (ABS) plays a vital role for the separation of the bioactive substances. In this work, combined with the ratio of phase and the time of forming phase, phase diagram was applied in the selection of ABSs firstly, and then the ABS was determined based on the distribution of the the target compounds in the ABSs. Finally,20%PEG400-16%Na2SO4-64%H2O (w/w/w) was used to separate the PGP. The crude PGP (0.4g) was successfully separated into PGP-1(10.7mg), PGP-2(4.7mg) and PGP-3(15.6mg) by HSCCC with this ABS under the following separation conditions:800rpm of the rotate speed, flow rate of0.8mL/min and30℃of separation temperature.3. Identification of PGP’structure preliminarilyThe molecular weights of the three PGP are13210Da (PGP-1),2584Da (PGP-2) and2459Da (PGP-3), respectively, determined by gel permeation chromatography. PGP-1was composed of Xyl (12.4%), Rib (10.1%) and Glc (77.5%); PGP-2was a homogeneity polysaccharide consisted of Man; and PGP-3was consisted of Rib (51.4%), Man (26.7%) and Glc (21.9%). IR and NMR spectra prove that PGP-1is a pyran-type polysaccharide with a andβ glycosidic linkages; PGP-2is a pyran-ring polysaccharide with β-glycoside; PGP-3is a polysaccharide with β-pyran-glycoside bond, at the same time, it contains a-glycoside bond.Three kinds of PGP were achieved through the extraction, separation and purification, and their structures were identificated preliminarily for the first time. This success shows the PEG400has better extraction ability for the PGP, phase diagrams provide an excellent means to search for biphasic solvent system of HSCCC and demonstrates HSCCC with an ABS based on lower degree of PEG is an effective method for separation of polysaccharides. This work not only laid a foundation for the further research and development work about the polysaccharides of PG, but also developed an ABS composed of low degree of polyethylene glycol and inorganic salts and a separation method of HSCCC.