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Determination Method and Extraction Purification of Terpenoid Indole Alkaloids in Catharanthus Roseus

Author: ZhangJing
Tutor: LiuZhaoPu
School: Nanjing Agricultural College
Course: Marine biology
Keywords: RP-HPLC Vindoline Vincristhine Catharanthine Vinblastine Serpentine
CLC: R284
Type: Master's thesis
Year: 2010
Downloads: 19
Quote: 0
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Abstract


Catharanthus roseus (L.) G. Don is one of the Catharanthus in Apocynaceae. It widely grows in south of the Yangtze River of China. There are lots of alkaloids in it, of which important anti-tumor terpenoid indole alkaloid (TIAs) has abroad-spectrum anticancer activity, it can treat much kinds of cancer. The detection and purification of catharanthus various alkaloids have an important theoretical and practical significance on industrial production in Vinca.This experiment used RP-HPLC, ultrasonic-methanol extraction and resin purification, established the analytical methods of five kinds indole alkaloids in roseus and sample preparation method we established RP-HPLC determination method and sample preparation method, and study on optimizing the extraction process of four important alkaloids from Catharanthus roseus, and finally we established the new vinblastine purification process by macroeticular resins. Here are the main conclusions:The HPLC detection method of five vinca alkaloids:Vindoline, Vincristhine, Catharanthine, Vinblastine, Serpentine was established. These five ingredients were separated on mobile phase:250 mL water-triethylamine (990:10, pH adjusted with phosphoric acid is 7.2) mixture of 750 mL methanol-acetonitrile (400:100); The flow rate was 0.8 mL/min, sample load set at 20μL; detection wave length was set at 220 nm, column temperature was set at 21℃.The correlation efficients were 0.9998~0.9985 in the linear range. The RSD of themethodwas less than 3%. The average recoveries ranged from 96.4% to 98.8%. Five indole alkaloids can completely separate well in 22 min. The method is simple, rapid and accurate.The extraction method and comprehensive optimization of condition in ultrasonic-methanol method were studied, the extraction conditions are as follows:Methanol as solvent was used for extraction 150 min, which pH value was 4.0. The ratio of mass of raw material to the volume of solvent is 1:15, and extraction temperature is 45℃. The vindoline and catharanthine extraction obtained by such optimization were 0.164% and 0.081% respectively.The macroporous resin and purification process of Vinblastine method:the sample compared to the macroeticular resins= 5:1, the sample concentration is 0.5 mg/mL, eluate of 95% ethanol solution, the elution volume is 25 ml/g of the unsaturated resin, resin regeneration conditions are:first, the concentration of 5% hydrochloric acid leaching, and then the concentration of 3% sodium hydroxide wash to neutral, and then soaked in ethanol, the final wash with plenty of distilled water to the non-alcoholic flavor. Resin can be recycled four times, through the regenerated resin adsorption effects can be achieved of the original resin adsorption capacity of over 90%.

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