| Pinus sylvestris L. var. mongolica is rich in active ingredients including polysaccharides, flavonoids, phenolic substances, organic acids, tannins, and so on. It has medicinal development value. With Pinus sylvestris L. var. mongolica bark as raw materials, our research studied the influence of different extraction conditions on polyphenols, purification and composition analysis respectively, and evaluated the polymerization between polyphenols and protein of Pinus sylvestris L. var. mongolica bark polyphenols. The experiments results provided references for Pinus sylvestris L. var. mongolica development and utilization in future. Main research results are listed below:1. Extraction Technology of polyphenol of Pinus sylvestris L. var. mongolica. In order to search an effective extraction of polyphenols from Pinus sylvestris L. var. mongolica., the solvent extraction, ultrasonic-assisted extraction and ultrasonic-compound enzymatic assisted extraction were explored.The optimum condition of solvent extraction were as follows:time was4h, ethanol concentration was60%, temperature was60℃, ratio of material to solvent was1:25. The optimal conditions to obtain the highest yield of polyphenols for ultrasonic extraction were determined as follows:ultrasonic time was2.5h, ultrasonic power was300W, ultrasonic temperature was60℃, pH value was3.0. The best technical conditions for ultrasonic-compound enzymatic assisted extraction were as follows:enzymatic hydrolysis time was40min, temperature was45℃, enzyme concentration was4%, pH value was4.0. The polyphenol yields of the solvent extraction, ultrasonic-assisted extraction and ultrasonic-compound enzymatic assisted extraction method were12.56,23.01,30.12mg/g respectively. Ultrasonic-compound enzymatic assisted extraction method not only greatly shortened processing time, but also produced higher yield of polyphenols components.2. Purification of polyphenol of Pinus sylvestris L. var. mongolica. Static absorption-desorption and dynamic adsorption-desorption experiment were carried out to select ideal resin (AB-8) from9different macroporous resins. The optimum purification conditions of polyphenols from Pinus Sylvestris L. var. mongolica were optimized by Plackeett-Burman and Box-Behnken designexperiment were sample solution concentration2.5mg/mL,28mL sample solution,eluting speed4.25BV/h,the concentration of ethanol51.6%and the ratio of diameter to height of resin colum1:27.3. The primary pine polyphenols were purified by Sephadex LH-20column chromatography, refined the pine polyphenols and the purity of polyphenols was85%. 3. Structure analysis of polyphenol.The polyphenols had peaks in the240-280nm and300-400nm region by Ultraviolet Spectrum Analysis, which preliminary indicated there were flavonoids in the polyphenols.The strong and broad absorption peak at3400cm-1generated from stretching and vibration of phenolic hydroxyl Ar-OH.Characteristic of in-plane bending vibration absorption peaks at1390-1315cm-1indicated there were hydroxyl groups belongs to polyphenols by Fourier Transform Infrared Spectrum. Analysised the material can be inferred that it might be sinapic acid-dextrose glucoside at52.317min, at63.344min might be the peony-3-glucoside or Centaurea-3-(6-0-acetyl)-glucose glycoside,69.963min might be the sinapic acid peonidin-3-sophoroside by liquid chromatography-mass spectrometry.4. Study on polymerization of polyphenols and protein. The optimum conditions of polyphenols-gelatin polymerization were as follows:the ratio of polyphenols and gelatin was2:l,temperature was40℃, time was30min and pH value was7.0. The optimum conditions of polyphenols-bovine serum protein polymerization were as follows:the ratio of polyphenols and bovineserumalbumin was2:1,temperature was30℃, time was40min,pH value was5.0.The optimum conditions of polyphenols-isolated soy protein polymerization were as follows:the ratio of polyphenols and isolated soy protein was1:4, temperature was30℃, time was20min, pH value was5.0. |
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