Study On Analyusis And Preparation Of Natural L-Citrulline

Citrulline is a non-essential amino acid with multi-medical and health function, which is first identified from the juice of watermelon. Citrulline has been isolated in other cucurbitaceous fruits including bitter melon, cucumber, muskmelon, pumpkin, bottle gourd, dishrag gourd, and wax gourd. High content of free L-citrulline was found in the root of Trichosanthes kirilowii Maxim also. Citrulline plays important role in the urea cycle. Recent years many foreign researches show citrulline has a lot of important physiological functions such as radical-scavenging hydroxyl, indicator of the foreign exclusive effect, vasodilatation, stabilizing blood pressure, diagnosing rheumatoidal arthritis, antioxidation and so on. The thesis investigated the citrulline content in many kind of plants with HPLC method. Specially, free citrulline content in watermelon and the root of Trichosanthes kirilowii Maxim was specified, the method of L-citrulline purification was established accordingly. The thesis investigated the analysis and purification of natural L-citrulline in plants in order to find a new way of high quality natural L-citrulline production.The thesis adopted an optimized HPLC method for analyzing free citrulline content in different kinds of plant by2,4-DNFB pre-column derivatization. The contents of citrulline in different kinds of watermelon and different tissues were variable. Citrulline contents in watermelon flesh/rind/leaf/cane range between2.5473-5.7219mg/g,1.7615-5.3470mg/g,1.4748-5.6922mg/g,3.3350-8.4652mg/g, respectively. The citrulline content in watermelon of red flesh is universally higher than that in watermelon of yellow flesh. The analysis of free L-citrulline content(triplicate analyses) in the root of Trichosanthes kirilowii Maxim of three major producing area show the highest concentration of free L-citrulline in the root of Trichosanthes kirilowii Maxim was found in Shandong producing area with content17.646mg/g.Using the root of Trichosanthin as raw material, the optimum extraction conditions were obtained by investigating the extraction solvent, time and temperature of extraction and extraction mode. Deionized water as extraction solvent, extraction processed once for2hrs at room temperature with the proportion of extraction solvent and material8:1. Extraction mode is agitation.The thesis studied on the separation and purification of citrulline and established the optimum process. The optimum condition as follow:hollow fiber module, MWCO=6000, membrane’s area is1.5m, membrane’s material is PS, flow velocity of ultra-filtration is150mL/min, pressure is0.05MPa. The pH value of filtrate was adjusted to4after ultra-filtration. The filtrate was loaded on cationic-exchange column at6BV/h flow rate on room temperature. Elution mode is Linear gradient elution, Eluent type is ammonia, eluent concentration range is0-1%. The eluate was evaporated then crude citrulline product was crystallized by dissolved in alcohol liquor. The recovery of elution was98%, the purity of the product was60.3%and the yield of citrulline in the whole process was38%.Different extraction methods were investigated when watermelon cane/peel and flesh was raw material respectively. Watermelon cane/peel extraction condition as follows:Deionized water as extraction solvent, extraction processed once for2hrs at room temperature with the proportion of extraction solvent and material8:1. Extraction mode is agitation. Watermelon flesh extraction condition as follows:juice extractor squeeze juice, then the juice was centrifuged at4000rpm rotate rate. The supernatant fluid was filtrated. The results indicate that three kinds of tissues as citrulline raw materials is feasible.Cationic-exchange resin was used to purify citrulline from crude extraction. The filtrate from extraction was centrifuged at4000rpm, supernatant fluid was ultra-filtrated. Ultra-filtration condition as follows: flow velocity150mL/min, pressure of watermelon cane/peel extraction0.05MPa, pressure of watermelon flesh extraction is0.01MPa. The optimum condition of cationic-exchange purification as follows:flow rate of loading is6BV/h, pH value of filtrate is4.0, elution mode is Linear gradient elution, eluent type is ammonia, eluent concentration range is0～1%, elution flow rate is1BV/h. The eluate was evaporated then crude citrulline product was crystallized by dissolved in alcohol liquor. The purity of the product was65%and the yield of citrulline in the whole process was36%.