| The planting area and output of tobacco in China stand first in the world list. There are many bioactive components in the tobacco, such as chlorogenic acid, nicotine, solanesol, etc. These bioactive compounds can be used in agriculture, medicine, food, and chemical industry. Chlorogenic acid is an important bioactive component which possesses many pharmaceutical functions, such as antibacteria, cholagogue, reducing blood pressure, increasing hemolaukocyte, exciting nerve center system and so on. In this paper, the extraction, isolation and purification of chlorogenic acid from tobacco were studied systematically. The main achievements are as following:1. The paper chromatography-ultraviolet and visible spectroscopy (PC-UV) method and high performance liquid chromatography (HPLC) were established to determine chlorogenic acid. The variation coefficient of the two methods was 1.54 % and 1.23 % respectively, and sample recovery of the two methods was 100.71 % and 100.46 % respectively. So both methods are exact and credible. The conditions of HPLC method were: Hypersil BD-C18 column (250 mm×4.6 mm i.d), mobile phase was 25 % acetonitrile + 0.5 % acetic acid, the flow rate was 1 mL/min, and the detection wavelength was at 326 nm. The content of chlorogenic acid in flue-cured tobacco C1F is 3.320 %.2. The ultrasonic assistant extraction of the main bioactive components from tobacco was investigated. The optimum conditions were obtained: 95 % ethanol as extraction solvert, at 80℃, pH 4, ratio of material to liquor 1/15 (W/V), extraction twice and each for 45 min. The yield of crude extract A was 21.18 g/100g, the content of chlorogenic acid was 13.93 g/100g, and the extraction ratio of chlorogenic acid reached 93.65 %.3. To study the separation effect of macroporous resin for the purification of chlorogenic acid, three types of macroporous resins were selected from eight kinds of resins, and their adsorption dynamics were studied. The results showed that XDA-1 resin was the best. The optimal separation conditions were as following: using crude extract A as sample, chlorogenic acid concentration of sample solution was 3.5 mg/mL, at pH 3.0, flow rate 2 bed volume (BV) per hour. Using 6 BV 40 % (v/v) ethanol as the desorption solvent to obtain the crude chlorogenic acid product B. The content of chlorogenic acid reached 39.20 g/100g in the crude product B, and the recovery of chlorogenic acid reached 80.06 %.4. Ethyl acetate was used to extract chlorogenic acid from the crude product B to increase its content. It was found that the optimal extraction condition were as follows: the chlorogenic acid concentration of initial crude product B solution was about 12 mg/mL, pH 2.0, phase ratio was 1/1 (V/V), extracting 3 times. The purity of crude chlorogenic acid C was 64.53 g/100g, the recovery of chlorogenic acid was 82.58 %.5. Finely, the purification of crude chlorogenic acid C by polyamide column chromatography was investigated. The optimal conditions of separation and purification of chlorogenic acid by 80～100 mesh polyamide column (φ30×500) were confirmed as follows: the concentration of chlorogenic acid in sampling liquor was about 10 mg/mL, 4BV 20 % (v/v) ethanol as the mobile solvent. After this procedure, the content of chlorogenic acid reached 86.71 g/100g, the recovery of chlorogenic acid reached 60.41 %. Through the further purification by 150～200 mesh polyamide column (φ30×500), white chlorogenic acid powder D was obtained, and the content of chlorogenic acid reached 92.73g/100g, the recovery reached 59.28 %.6. On the basis of melting-point determination, TLC, IR, LC-MS, the white chlorogenic acid powder D was identifited as chlorogenic acid. |
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