Extraction Of Triterpenoids Betulinic Acid Separation Process And The Determination Method

Betulinic acid (BA) and its derivatives are pentacyclic triterpenes, which can be isolated from variours plants. They have been shown to possess many advantages, such as anti-tumor and anti-HIV activities, special mechanism, low molecular weight, low toxicity and so on, so betulinic acid (BA) and its derivatives are very promising new chemotherapeutic agents for the treatment of cancer and HIV infection. It is significant to have deep research on them. Extraction, seperation technology and content analysis of the lupane-type triterpenoids have been studied in this research, so as to set up a convenient and rapid method for determination, and search the optimal technology of extraction and separation. The major contents and results of the research are as follows:Spectrophotometric method was set up to determine the lupane-type triterpenoids. The content of the lupane-type triterpenoids was quantified by Spectrometry method using betulinic acid as standard and 5% vanillin - glacial acetic acid & perchloric acid as colour - developing agents. The results showed that the mass of betulinic acid was well correlated to its absorbence at 560nm in the range of 16～96μg. The precision RSD is 0.84%.The rate of recovery is 96.06%. Ursane-type triterpenoids and oleanane-type triterpenoids had no significant influence on the determination results. So, this method is convenient, rapid, with fine reproducibility.The orthogonal design of four factors and three levels were used to select the optimal extraction method based on the single factor experiments. Four factors, such as ethanol concentration, the ratio of material to liquid, the temperature and the extracting time were investigated with the lupane-type triterpenoids yield and the content of the lupane-type triterpenoids as indexes. The best extraction process was as follows: the extrcting solvent was 50% ethanol, the ratio of material to liquid was 1 : 10, the temperature was 80℃, the extracting time was 2h and extracting 3 times. Three methods, such as the cold extraction, the heat extraction and the ultrasonic extraction, were compared in this paper. The results showed that the heat extraction was the optimal method.The purification of the lupane-type triterpenoids by using the method of macroporous adsorption resin was experimented. The results indicated that D101 fitted for the separation of lupane-type triterpenoids. The best purification process was as fllows: the concentration of sample was 0.8～0.9mg/ml, the flow rate was 1BV/h, after adsorption equilibrium, elution solvents were 4BV water, 5BV 40% ethanol, 5BV 90% ethanol in sequence, collect the 90% ethanol solution, the content of which was about 74.27%.The 90% ethanol elution part was purified by sikica gel column chromatography. Three compounds were isolated from the stings of Gleditsia sinensis Lam., and they were identified as betulinic acid 1, zizyberanalic acid 2,3β-O-trans- p -coumaroyl alphitoilic acid 3 by melting point, TLC and ¹³C NMR.