| Tea tree has been planted for thousands of years. Tea flower (TFPS) is one of its reproductive organs. While, we only take advantage of tea leaf, and tea flower is ignored, which caused a large waste. In recent years, studies, at home and abroad, have shown that the ingredients of tea flower are similiar to tea leaf. It contains lots of active biological ingredients, leading to a variety of physiological functions. While the research of tea flower isn’t comprehensive enough. In this research, tea flower has been studied, including optimization of the extraction conditions, purification, and detemiining its structure.1. Research on extraction condition of TFPSTemperature, time and ratio of water to raw material were selected in single-factor experiment. Based on the single-factor experiment, the extraction parameters were optimized by using three-factor-five-level Central Composite design (CCD). Then the data was analyzed by Design-Expert Software, integrating actual operability, the optimum extraction parameters were obtained as follows:extracting temperature 96℃, extracting time 230 min, ratio of water to raw material 25 mL/g. Under optimum extraction conditions, the real extraction yield of TFPS was 13.43±3.17%, consistent with the predicted values.After filtered by 0.1μm, tea flower polysaccharide extraction was divided into two fractions, which were retentate liquid (TFPS-1) and permeate liquid (TFPS-2). The recovery rate were 10.84% and 3.09%, respectively.2. Isolation, purification and physicochemical characterization of TFPSThe retentate liquid and permeate liquid of TFPS were purified by DEAE-cellulose anion-exchange column chromatography and Sephadex G-100 gel-filtration column chromatography successively. Six fractions were obtained (TFPS-1-1, TFPS-1-2, TFPS-1-3, TFPS-2-1, TFPS-2-2, TFPS-2-3). While TFPS-1-1 fraction was ignord because of few of it.The recovery rate of the remaining five fractions based on the amount of crude TFPS were 19.62%,35.86%,13.75%,18.55%,30.28%, respectively.Basic physicochemical characterization of crude TFPS, TFPS-1-2, TFPS-1-3, TFPS-2-1, TFPS-2-2, TFPS-2-3 were determined by methods of physics and chemistry. The results were as follows:carbohydrate was 23.1%,14.4%,20.3%,85.0%,39.8%,18.5%, respectively; protein content was 0.25%,0.08%,0.10%,0.11%,0.09%,0.10%, respectively; sulfuric radical content was 1.36%,1.63%,2.06%,2.04%,1.26%,1.08%, respectively.3. Structure analysis of TFPSUsing HPLC to detect the purity of each fraction. The results showed that all five fractions were homogenerous polysaccharides and the average molecular weight for TFPS-1-2, TFPS-1-3, TFPS-2-1, TFPS-2-2, TFPS-2-3 is 215.419 kD,142.731kD,3.716kD, 2.518kD,2.087kD.Monosaccharide composition and molar ratio of TFPS-1-2, TFPS-1-3, TFPS-2-1, TFPS-2-2 and TFPS-2-3 were analyzed by HPLC. They were Mannose:Galacturonic acid: Galactose:Arabinose=66.71:22.71:3.98:6.59, Mannose:Galacturonic acid:Galactose: Arabinose=61.89,26.68,5.13,6.3; Mannose:Glucuronic acid:Glucose:Galactose:Xylose: Fucose=58.95:8.61:8.74:8.14:5.31:10.25, Mannose:Galacturonic acid:Glucose: Galactose:Arabinose=56.50:10.81:2.74:14.29:15.65, Mannose:Galacturonic acid: Glucose:Galactose:Arabinose=54.88:24.38:2.84:5.82:12.09.The infrared absorption spectroscopy (IR) of TFPS-1-2, TFPS-1-3, TFPS-2-1, TFPS-2-2 and TFPS-2-3 showed that they were polysaccharide.The shape of the five fractions were observed by Atomic Force Microscope (AFM). The results showed that they are aggregation of some sugar chains.Using scanning electron microscope (SEM) to observe the surface morphology of TFPS-1-2, TFPS-1-3, TFPS-2-1, TFPS-2-2 and TFPS-2-3. We found that TFPS-1-2 and TFPS-2-2 had holes at surface at high magnification, explaining the weak of intermolecular forces. While TFPS-1-3, TFPS-2-1 and TFPS-2-3 showed smooth surface, explaining the strong of intermolecular forces.Methylation and GC-MS analysis of TFPS-1-3 showed that Mannose connection were (1â†'2)-linked, (1â†'4)-linked and (1â†'3)-linked, Arabinose connection were (1â†'2,3)-linked mainly, Galactose connection were (1â†'3)-linked and terminal-linked. |
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