| Stephania kwangsiensis H. S. Lo (Menispermaceace) is a well known Chineseherbal medicine, which widely distributed in Yunnan province and the northwest andsouthwest of Guangxi province of China. The plant contains a large numbers ofalkaloid. The content of rotundine, one of the most famous analgesic medicines, inthis plant was very large. Recent years, the resource of Stephania kwangsiensis isdecreasing, due to the lack of protection awareness and irrational use of the resources.Though artificial cultivation techniques has been studied for the protection andplanting of Stephania kwangsiensis, the production still can not meet the demand ofthe market and people’s requirements, and some other similar herbs were used asStephania kwangsiensis. In the present paper, the alkaloids of Stephania kwangsiensiswere separated by HSCCC, and the alkaloids isolated were identified by spectrummethods. At the same time, the extraction process was studied. These researchesprovide basis for the further research and the development of Stephania.In this research, we mainly do the researches as follows:1. Literature reviewsAccording to the study purpose, a literature reviews relating to the source,chemical constituents, pharmacological effect, processing technique, quality ofStephania kwangsiensis were conducted.2. Preparation of sinoacutine from Stephania kwansiensis by pH-zone-refiningcounter-current chromatographyThe powder of Stephania kwansiensis was first eluted with95%ethanol for three times,2hours each time. The extraction was filtered and condensed by rotaryvaporization. The residue was dissolved in water and acidified with HCL (pH=3~4),then, the acidified phase was filtered. The aqueous phase was basified with NH3·H2Oand extracted by CHCl3. After removing of CHCl3the residue was dissolved inmethanol to obtain aturated solution of methanol. The solution was placed two daysfor the precipitation of the crude extract of sinoacutine. The sample was used for theseparation pH-zone-refining counter-current chromatography.3. Combinative application of pH-zone-refining and conventional high-speedcounter-current chromatography for preparative separation of alkaloids fromStephania kwangsiensisA method which involves the combination of pH-zone-refining counter-currentchromatography (pH-Zone-refining CCC) and conventional high-speedcounter-current chromatography (HSCCC) was established for the preparativeseparation of alkaloids from the crude extracts of Stephania kwangsiensis.pH-Zone-refining CCC was first performed with the solvent system composed ofn-hexane–ethyl acetate–methanol–water (3:7:1:9, v/v), where triethylamine (10mM)was added to the upper organic stationary phase as a retainer and hydrochloric acid (5mM) to the aqueous mobile phase as an eluter. From2.0g of crude extract,370mg ofsinoacutine and600mg of a mixture of three other alkaloids were obtained. Then, themixture was further separated by conventional HSCCC with the solvent systemcomposed of n-hexane–ethyl acetate–methanol–water (7:3:6:4, v/v), yielding42mgof (-)-crebanine,50mg of (-)-stephanine and30mg of l-romerine from150mgmixture of three other alkaloids, respectively. The purities of the four compoundswere all over98%as determined by HPLC, and the chemical structures of the fourcompounds were confirmed by positive ESI-MS and1H NMR data.4. Study on extraction of total alkaloid of Stephania kwansiensisFlash-type extractor was used for the extraction of alkaloids from Stephaniakwansiensis. The extraction parameter including time, extraction solvent andextraction ratio of solid/liquid were all optimized. By calculating the peak areas of themain alkaloids including sinoacutine, crebanine, romerine and stephanine, the optimalextraction condition of Flash-type extractor was obtained. The result showed that this technique was simple, quick and practical for the extraction of alkaloids from naturalproducts. |
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