Isolation And Purification Of Pentacyclic Triterpenes From Centella Asiatica

Centella asiatica (L.) Urban has been used as a medicinal herb for more than two thousand years. It has been used to treat skin wounds, depression, gastric ulcer, etc. C. asiatica mainly consist of total glucosides of C. asiatica, and those constituents are claimed to be C. asiatica total triterpenoid saponins, which mainly consist of asiaticoside, madecassoside (asiaticoside A) and terminoloside (asiaticoside B), etc. Asiaticoside, a triterpene glycoside from C. asiatica, has been claimed to possess various physiological effects, such as wound healing, hypertrophic scar healing and antidepressant-like effects. And it has been widely used in pharmaceutical and cosmetic fields.Many constituents have been reported from C. asiatica, such as pentacyclic triterpenes, polyacetylenes, flavones and sterols. Pentacyclic triterpenes from C. asiatica, which mainly consist of asiaticoside, madecassoside, asiatic acid and madecassic acid, differ from each other only by the lacation or count of hydroxyl, methyl or double bond. There are also many isomers claimed to be found in extracts of C. asiatica, such as asiaticoside A (Madecassoside, urs-12-en) and asiaticoside B (Terminoloside, Olea-12-en) differing from each other by the location of C-29. These two components possess the same sugar chain units but different aglycone skeletons, it was observed that successful separation and isolation of the pure components was difficult by their similar polarity, due to the highly polar sugar chain units which lead to weak polar difference between them. The conventional separation method of these saponins is lead to be an inefficient process, consuming longer time and more waste.Therefore, the process of separation and purification of triterpene glycosides from C. asiatica was important for better utilizing C. asiatica materials, which could be referenced for the separation of other triterpenoid saponins.This dissertation presented the current utilizing situation of active components from C. asiatica, and introduced the research progress of separation and purification methods of them. In order to reveal the separation rule of pentacyclic triterpenes, and develop a efficient preparation method of asiaticoside, this study selected C. asiatica total triterpenoid saponins for exploring separation and purification method of the active components. Preparation of triterpene glycosides from C. asiatica, HPLC separation method development and chromatography behavior analysis of the isomers, crystallization thermodynamic property research of asiaticoside and madecassoside, and crystal separation method development of asiaticoside from C. asiatica total triterpenoid saponins was carried out. The research work was summarized as below.1. Preparation of triterpene glycosides from C. asiaticaThe prep-HPLC was used to prepare the triterpene glycosides from C. asiatica, and 12 monomers were obtained.1H-,13C-, DEPT 90-NMR, DEPT 135-NMR, 2D-NMR spectra, ESI-MS and HRFTMS was used to elucidate those monomers. Besides the widely known compounds asiaticoside, madecassoside and terminoloside, four known compounds were characterizded as centellasaponin A, centellasaponin B, 2a,3 p,23-trihydroxyurs-20-en-28-oic acid O-a-L-rhamnopyranosyl-(1â†'4)-O-β-D-glucopyranosyl-(1â†'6)-O-β-D-glucopyranosyl ester, scheffoleoside A. As we known, there has been no similar 13C-NMR data reported to the other five compounds, which are probably new compounds found from C. asiatica.2. HPLC separation and chromatography behavior analysis.Many isomers were preparaed from the C. asiatica total triterpenoid saponins, which could be used as model substances of the isolation of complex pentacyclic triterpenes. The separation methods of madecassoside and terminoloside, centellasaponin A and asiaticoside were developed and the chromatography behavior of these pairs of isomers were investigated. As applied to samples of C. asiatica, a specific HPLC method for the determination of asiaticoside in C. asiatica products was presented.3. Determination of crystallization thermodynamic propertiesThe solubilities of asiaticoside and madecassoside in water, methanol, ethanol, n-propanol, n-butanol and a methanol+water mixture were determined over the temperature range from (278.15 to 343.15) K, which was carried out by a static analytical method. The induction period of asiaticoside and madecassoside in a methanol+water mixture were determined at the tempeture of 298.15 K, which was carried out by laser scattering method. And the interfacial tension (surface energy) data of asiaticoside was obtained, which is in the range of 0.55-0.86 mJ/m2.4. Development of asiaticoside crystallization processThe crystallization thermodynamic property differences between asiaticoside and madecassoside were analysised. And the 3D molecular structure differences between asiaticoside and madecassoside were explored, including the conformation transformation, intramolecular hydrogen bond. Finally, a crystallization separation process for preparing asiaticoside from C. asiatica total triterpenoid saponins was developed.