Isolation, Purification And Hydrolysis Of Asiaticoside

Centella asiatica (L.) Urban, as a traditional herbal medicine, has been used in many countries and areas for hundreds of years. According to the reports, Centella asiatica and its active components can be used in treatment of various diseases such as burning wounds, depression and gastric ulcer, etc. The main active components of Centella asiatica are saponins and aglycons, including asiaticoside, madecassoside, asiatic acid and madecassic acid, and their extraction and purification methods have always been one of the main research directions. Recently, some researches showed that under the order conditions asiatic acid and madecassic acid can be synthesized to a series of aglycon derivatives, and these derivatives had better pharmacological actions than their initial saponins. Such results greatly improved the aglycons'utilization. However, due to various reasons, the lower extraction rate of these active components resulted in related products expensive and limited Centella asiatica products development and applications. Therefore, the process of extraction, separation and purification, and hydrolysis of asiaticoside was important for better utilizing Centella asiatica materials.This dissertation presented the current utilizing situation of active components of Centella asiatica, and introduced the research progress of extraction, purification and analyzing method of them. In order to use these active components efficiently, this study selected Centella asiatica for exploring extraction and purification method of the active components of asiaticoside and madecassoside, and the production progress of asiatic acid and madecassic acid. The research contents and achievements are included as following:1. Study on extraction of saponins from Centella asiaticaA colorimetric method with selected madecassoside as the standard was established to examine the content of total saponins in Centella asiatica. The optimal reaction conditions of this colorimetric method were measured and listed as follows:the content of vanillin was 5%, the content of perchloric acid was 0.8 mL, the reaction temperature was at 60℃and the reaction time was 15 min. The investigation of the stability of reaction system indicated that the absorbency had no obvious variation during 2 h after the reaction. The result showed that this method had a good linear relationship and had a wider detection range.Successively, a dipping method to extract total saponins from Centella asiatica was optimized by the single factor and the orthogonal test. The optimal extraction conditions were achieved as follows:the solvent was 70% ethanol-water solution; the ratio of material and solvent volumes was 1:10; extraction times were 3 times at room temperature and each extraction process lasted for 4h.Finally, the contents of total saponins of different part of Centella asiatica were tested. According to the results, the content of total saponins in the leaf of Centella asiatica was the highest (6.12%), then in the stick (2.43%), and the lowest in the root (1.11%).2. Enrichment and purification of madecassoside and asiaticoside with macroporous resins.The adsorption and desorption properties of five macroporous resins including HPD100, HPD300, X-5, AB-8 and D110 for the enrichment and purification of asiaticoside and madecassoside from Centella asiatica extracts have been evaluated. Equilibrium adsorption isotherms were constructed at different temperatures and the model of Langmuir and Freundlich was used to describe the adsorption behavior of the selected optimal resin. Column packed with HPD100 resin was used to separate asiaticoside and madecassoside from Centella asiatica extracts, and the optimal conditions were as follows:resin HPD100 showed the best adsorption and desorption capacity among 5 resins; 50% ethanol-water solution was selected as the eluting solvent; the adsorption and desorption flow rate was both 2 BV/h.After the treatment with gradient elution on HPD100 resin, the content of madecassoside in the product increased from 3.9 to 39.7%, with the high recovery yield of 70.4%; for asiaticoside the content increased from 2.0 to 21.5%, with the high recovery yield of 72.0%. The results showed that HPD100 resin revealed a good ability to separate madecassoside and asiaticoside.3. Hydrolysis of asiaticoside and madecassosideA HPLC method was developed to detect asiaticoside, madecassoside, asiatic acid and madecassic acid synchronously at the same time. The examination of precision, stability and average recovery indicated that this method had a good linear relationship, high stability and precision, and had a wide investigation rang. The chromatography conditions were as following:Eclipse XDB-C18 reversed-phase column (4.6 mm×250 mm,5μm, Agilent); the mobile phase was acetonitrile-acetic acid (0.3%)-water, dynamic elution; the flow rate was 0.6 mL/min:the column was 35℃; the detection wavelength was at 210 nm. The RSD values of precision of asiaticoside, madecassoside, asiatic acid and madecassic acid were 0.35%,0.24%,0.17%and 0.18%, respectively; the RSD values of stability were 0.54%,0.46%,0.44%and 0.35%, respectively; the average recovery were 101.09%,99.57%,100.86%and 99.99%, respectively. All the results above accorded with demands of pharmacopoeia, and could be used in the content detection of asiaticoside, madecassoside, asiatic acid and madecassic acid.The hydrolysis of asiaticoside and madecassoside was studied by the examination of catalyst, reaction solvent, initial concentration of saponins, reaction time and temperature. NaOH was chosen as the catalyst to hydrolyze asiaticoside and madecassoside to produce asiatic acid and madecassic acid. The ethanol concentration of reaction solution fitting for hydrolysis reaction was 50%. There was no obvious influence on hydrolysis reaction when the contents of NaOH were between 0.1-2.0 mol/mL. The influences of different initial contents of saponins in reaction solution were also investigated, and under the conditions of asiticoside (0.22～1.10 mg/mL) and madecassoside (0.40-1.20 mg/mL) the recovery of saponins varied a little. The reaction temperature had a remarkable effect on hydrolysis reaction, the reaction rate enhanced obviously with the reaction temperature increasing. Arrhenius equation was used to describe the process of saponins hydrolysis, and results revealed that the hydrolysis processes of asiaticoside and madecassoside were both first order reaction process and the active energy was 70.1 kJ/mol and 83.3 kJ/mol, respectively.In this research, a whole process was established including the production of total saponins of Centella asiatica, enrichment and purification of asiaticoside and madecassoside with macroporous resins, and the production of asiatic acid and madecassic acid. This process effectively avoided the shortcoming of high energy consumption and heavy pollution with using the conventional technique of solvent extraction to produce active components. For asiatic acid, the final purity could reach 91.3% with the recovery of 54.8% through this process. This research offered good methods for the production of new asiatic acid and madecassic acid derivatives.