Isolation, Purification and Immunological Activity of the Polysaccharides from Bee Pollen of Camellia Japonica
|School||Northeast Normal University|
|Course||Biochemistry and Molecular Biology|
|Keywords||Bee pollen Polysaccharide Fraction Immunological activity|
Camellia japonica belongs to the Camellia of Theaceae. Camellia japonica bee pollen is well known as a natural nutrition and health food. The research about polysaccharides of camellia japonica bee pollen is rare and mainly focuses on their biological activity, while the study about isolation and purification of polysaccharides from camellia japonica is very rare. In this paper, we reported the isolation and purification of camellia japonica pollen polysaccharides and immunological activity.Camellia japonica pollen polysaccharides (WCPP) was extracted with hot water,filtered, consentrated and precipitated with 80% ethanol. The monosaccharide composition of WCPP is 2.5% Man, 7.8% Rha, 3.0% GlcA, 37.0% GalA, 12.5% Glc, 15.1% Gal和22.1% Ara. The molecular weight distribution of WCPP was very wide and heterogeneous.Two methods were adopted in this paper to fractionate WCPP.First, stepwise ethanol precipitation (30%, 50%, 70% and 80%) was performed to fractionate WCPP and four fractions WCPP-30, WCPP-50, WCPP-70 and WCPP-80 were obtained. Monosaccharide composition analysis indicated that WCPP-30 was composed of 4.3% Man, 7.4% Rha, 45.4% GalA, 0.6% Glc, 10.3% Gal, 2.1% Xyl and 20.2% Ara; WCPP-50 was composed of 2.0% Man, 9.1% Rha, 42.7% GalA, 8.8% Glc, 11.5% Gal, 2.0% Xyl and 23.9% Ara; WCPP-70 was composed of 2.1% Man, 8.7% Rha, 1.9% GlcA, 15.5% GalA, 16.3% Glc, 20.6% Gal, 1.5% Xyl and 33.5% Ara; WCPP-80 was composed of 6.3% Man, 4.7% Rha, 4.8% GalA, 42.6% Glc, 12.2% Gal and 29.4% Ara. Gel permeation chromatography results implied that the molecular weight distributions of the four fractions were very wide and heterogeneous, so this method was not suitable for fractionation of WCPP.Next, anion-exchange chromatography was performed and eluted with H2O, 0.1 mol/l NaCl, 0.3 mol/l NaCl and 0.5 mol/l NaCl. Four fractions WCPP-N, WCPP-1, WCPP-2 and WCPP-3 were obtained. Monosaccharide composition analysis indicated that WCPP-N was composed of 4.1% Man, 1.0% Rha, 69.0% Glc, 13.4% Gal and 12.7% Ara; WCPP-1 was composed of 5.4% Man, 4.3% Rha, 2.5% GlcA, 45.0% GalA, 22.7% Gal, 18.0% Ara and 2.0% Glc; WCPP-2 was composed of 1.0% Man, 9.5% Rha, 1.9% GlcA, 30.2% GalA, 1.9% Glc, 19.3% Gal and 36.5% Ara; WCPP-3 was composed of 2.1% Man, 18.8% Rha, 1.1% GlcA, 33.2% GalA, 17.9% Glc, 19.1% Gal and 23.9% Ara. Gel permeation chromatography results implied that the molecular weight distributions of the four fractions were heterogeneous. In this paper, WCPP- 1 and WCPP-3 have a little content, WCPP-N and WCPP-2 were further purified mainly.WCPP-N was separated by Sephadex G-100 and two fractions WCPP-N-I and WCPP-N-II were obtained. WCPP-N-II was homogeneous and NMR results indicated that it containedα-1,4-Glucan as the main chain, andα-1,6-Glucan as side chains. So WCPP-N-II had an amylopectin structure. WCPP-2 was separated by Sepharose CL-6B and five fractions WCPP-2-I, WCPP-2-II, WCPP-2-III, WCPP-2-IV and WCPP-2-V were obtained. Among these fractions, WCPP-2-I was homogeneous and NMR results indicated that the main chain containedα-1,2-Rha andα-1,4-GalA repeating units, and the side chains ofα-1,5-Arabinan and minorβ-1,4-Galactan were linked to the C-4 of Rha. So WCPP-2-I was a RG-I type pectin.WCPP-3 could promote macrophage phagocytosis in vivo, but not in vitro. This result implied that WCPP-3 did not act directely on macrophages. It might work via stimulating other cells to produce cell factors which affected macrophage phagocytosis.