| Pu-erh tea is originated in Puer of Yunnan province and it has been known for a long time, which is gaining growing popularity with the world. Nowadays, it has been reported that the Pu-erh tea has the activities of antioxidant, hypolipidemic and hypoglycemic, ect. Their anti-diabetic activity was most concerned, and lots of health food were appeared in the market. It was reported that the polysaccharides and theabrownin in the Pu-erh tea could play an important part in the hypoglycemic activity. However, it’s hard to separate them. So, what is the main active component is still unknown. In this project, the polysaccharides and theabrownin were separated and purified. Then the protective effects of them on palmitic acid-induced apoptosis in pancreatic β-cell (INS-1) were studied. The results would supply the theory basis for the development of natural hypoglycemic functional food. The investigation results are as follows:The total polysaccharide (TPPS) was extracted from the Pu-erh tea leaves with hot water and precipitated by70%ethanol. After the conventional drying, the TPPS was obtained in a yield of25.4%. And there were42.5%total carbohydrates and30.9%uronic acid in it. The Ultraviolet spectroscop analysis indicated that the TPPS contained theabrownin.The appropriate method to separate and purify the tea polysaccharides and theabrownin(TB) were selected. And we chose the DEAE-Cellulose column chromatography finally. The TPPS was separated on a preparative DEAE-Cellulose column into three fractions, an unbound fraction (PPS-N) by water elution, a bound fraction(PPS-A) by0.5M NaCl elution and a stronger bound fraction (TB) by0.5M NaOH elution. The yield of them was1.4%,20.3%and40%, respectively. Total carbohydrates content of PPS-N and PPS-A was66.9%and50.3%respectively. The phenol sulfuric acid assay indicated that there were some carbohydrates in the TB and the total content of it is31.8%. Sugar composition analysis by high performance liquid chromatography indicated that PPS-N consisted of Gal(72.5%), Ara(10.2%), Glc(8.8%), Man(4%), Rha(3.4%) and GalA(0.9%). PPS-A consisted of GalA(40.4%), Gal(27.2%), Ara(15.6%), Rha(6.4%), Man(5.9%), Glc(2.8%) and GlcA(1.7%). TB consisted of Gal(24.2%), Ara(21.4%), GalA(20.1%), Glc(11.8%), Rha(10.3%), Man(9.8%) and Xyl(4.0%). According to the results of high performance liquid chromatograpy, PPS-N might contain a large amount of galactans and less arabinogalactans(AG) or arabans. PPS-A might contain homogalacturonan(HG) domains, AG and less type-I rhamnogalacturonan(RG-I) domains. And there might some AG, RG-I domains, and less glucans in TB.Then PPS-A was separated on a prepared DEAE-Cellulose column into three fractions PPS-A1, PPS-A2and PPS-A3corresponding to0.2,0.33and0.5M NaCl elution, respectively. The monosaccharide composition of PPS-A1was Man(9.8%), Rha(6.2%), GlcA(2.4%), GalA(35.4%), Glc(3.6%), Gal(29.2%) and Ara(13.4%). PPS-A2was Man(2.6%), Rha(12.6%), GalA(62.5%), Gal(11.1%), Ara(11.3%) and Xyl(4%). PPS-A3was Man(5.4%), Rha(15.2%), GalA(61.8%), Glc(2.9%) and Gal(14.7%). According to the results of high performance liquid chromatograpy, PPS-A1-might contain HG and AG domains. PPS-A2might contain a lot of HG and less AG domains. PPS-A3might contain a lot of HG domains.The protective effect of polysaccharides and TB on palmitic acid-induced apoptosis in pancreatic β-cell (INS-1) were determined. We found that the protective effects of PPS-A was much better, especially for the PPS-A2and PPS-A3. Our findings suggested that PPS-A may be the main hyperglycemic constituent, which was likely to be HG-rich pectin. |
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