| Paliurus ramosissimus (Lour.) Poir. are commonly distributed in southwestern China. There are the Dictionary of Medicinal Plant and local Flora and records of materia medica embodying it, and it is bitter and flat, and it has the functions of detoxicating and detumescence. So the leaves and roots of Paliurus ramosissimus have been used as traditional Chinese medicines for the treatment of rheumatism, injuries, sore throats, and so on. Pharmacological studies have indicated that the EtOH extract of the leaves of Paliurus ramosissimus possesses the effect of antitumour, and the curative effect is better.In searching for bioactive constituents, we had systematically investigated the chemical constituent of Paliurus ramosissimus. Chemical preliminary test results show that medicinal herbs extracts contain triterpenes, alkaloids, coumarins, flavonoids and other components. Related literature reports of triterpene compositions mainly include 24-hydroxyceanothic acid dimethylester,27-hydroxyceanothic acid dimethylester, ceanothic acid, ceanothic acid 28β-glucosyl ester and isoceanothic acid, 28β-glucosyl ester. Also it contains a cyclopeptide alkaloid, paliurine B. But the efficacy of the drug composition is not reported, and the quality evaluation index is not also studied.Studying on the chemical constituents from active sites of Paliurus ramosissimus for treating cancer, two compounds were isolated from its extracts. By means of UV, IR, MS,1H-NMR,13C-NMR, compound A was identified as 2α,27-2(p-hydroxy trans cinnamoyloxy)-betulinic acid. It is a new compound which was isolated from the leaves of Paliurus ramosissimus for the first time.In this paper, the method which determines the content of compound A from Paliurus ramosissimus by HPLC was established. The optimal conditions of separation and detection were achieved on a BDS HYPERSIL C18 column (4.6 mm×100 mm×2.4 μm) eluted with a gradient acetonitrile and 0.1% formic acid solution, at a flow rate of 1.0 mL/min, detected at 320 nm. The peak area and concentration of the compound showed a good linear relationship (r=0.9999) within the range of 0.0214～0.8544 μg and the recovery was 99.4% with the RSD of 1.18% (n=6). This method was simple, rapid, accurate, and reproducible, and it can be used for determining the concentration of the compound from the leaves of Paliurus ramosissimus. The variation in content of the different harvest time and its corresponding antitumor activity has certain regularity. The method can preliminarily measure the content of compound A in different regions and harvest times to make sure the quality of medicinal materials. Finally it provides a certain reference for folk cancer patients and prepares for the prophase preparation, extraction and purification of next study.Afterwards we used refluxing extraction method to study the conditions for the extraction of compound A from the leaves of Paliurus ramosissimus with the amount of compound A as index. Base on the single factor experiment, using L9(34) orthogonal design to determine the best extractive process conditions. Results indicated that the extraction technological parameters which selected were reliable and stable.Subsequently, using macroporous adsorption resin to separate and purify the extraction, and the purification process conditions are optimized by comparing the static and dynamic adsorption experiments of nine kinds of macroporous adsorption resin. The best technological parameters are following:100 g/L as experimental sample concentration, using X-5 macroporous adsorption resin as absorbent, when absorption velocity was 1 BV/h, and using 95% ethanol to elute with elution velocity of 1.5 BV/h. Using this process conditions, we got better concentration and purification, the content of the compound in the extract from the original 1.68% to 8.92%, and the transfer rate is 85%. The research results for the subsequent research laid a certain foundation. |
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