| Kudingcha, which has rich resources and a long drinkable history in China, is significant medicinal efficacy and health function. In recent years, the researches about kudingcha have been concentrated on processing made tea and tea drinks, while it remains to further discussion on physiological and biochemical mechanism, finish machining and deep processing of kudingcha, especially on the research of functional components in kudingcha. It's known that kudingcha of aquifoliaceae contains a series of pentacyclic triterpenoids, such as ursolic acid, oleanolic acid, betulinic acid, lupeol, pomolic acid, kudinlactone, ilexgenin and kudinchagenin etc.. Ursolic acid which is the uppermost medicinal component in kudingcha of aquifoliaceae, has evident clinic efficacy on hypertension, hyperlipidemia and sore throat etc., and has showed good pharmacological characters on anti-tumor, anti-diabetic, anti-HIV, anti-inflammatory, protecting liver and organism immunoregulation etc.. In this paper, in order to put more vitality into kudingcha of aquifoliaceae industry through improving added value of kudingcha products by means of promoting production and utilization of pentacyclic triterpenoids, quality analysis and seperation-purification technique of pentacyclic triterpenoids based mainly on ursolic acid in kudingcha of aquifoliaceae has been studied, which aims at screening out excellent germplasm resources suitable for ursolic acid industrialized extraction, and finding a technological process for production in an industrialized scale. The research expresses that kudingcha of aquifoliaceae with high content of ursolic acid and lupeol are important medicine source plant, the processes of separating and purifying ursolic acid and lupeol are short, low cost and environmentally friendly, the purity and recovery yield of product come to higher level.1. The determination of ursolic acid and oleanolic acid in Ilex kudingcha C. J. Tseng with reversed-phase high performance liquid chromatography (RP-HPLC) has been developed. The chromatographic conditions are as bellow:Eclipse XDB-C18 column(5μm, 4.6mm×150mm), elution influent methanol-0.2% aqueous solution of phosphoric acid(88:12), detection wavelength 220 nm, flow rate 0.8ml/min. The method is accurate and reliable, also suitable for analyzing other species of aquifoliaceae kudingcha. The determination of betulinic acid in Ilex kudingcha C. J. Tseng by RP-HPLC has been developed for the first time. The chromatographic conditions are following:Eclipse XDB-C18 column(5μm, 4.6mm×150mm), elution influent methanol-0.2% aqueous solution of phosphoric acid(84:16), detection wavelength 210nm, flow rate 0.8ml/min. The average contents of betulinic acid in llex kudingcha C.J. Tseng is 0.28%. The method is accurate and reliable. Besides, the determination of lupeol in L cornuta Lindl. by RP-MPLC has been developed. The chromatographic conditions are following:Eclipse XDB-C18 column(5μm,4.6mm×150mm), elution influent methanol-water(84:16), detection wavelength 210 nm, flow rate 1.0ml/min. The average contents of lupeol in L cornuta Lindl. is 0.28%. The method is accurate and reliable.2. The contents of ursolic acid in different species kudingcha of aquifoliaceae are compared for the first time. Significant differences are observed in 6 Kudingcha species in Aquifoliaceae, and the results are as follows:/. cornuta Lindl. (1.64%)>L. huoshanesis Y. H. He (1.63%)>L latifolia Thunb. (1.54%)>L pentagona S. K. Chen, Y. X. Feng et C. F. Liang (1.23%)>L kudingcha C. J. Tseng (0.96%)>L centrochinesis X. Y. Hu (0.69%)> the marketed shoots products of Ilex kudingcha C. J. Tseng(<0.28%).3. The first examination has beem made of urolic acid in Ilex kudingcha C. J. Tseng during different seasons and in different parts. Functional leaves of Ilex kudingcha C. J. Tseng collected in april and July have equivalent ursoiic acid levels, but much higher urolio acid than october and the january materials which is the lowest. So, it can ensure material supply of high ursolic acid contents to select functional leaves of Ilex kudingcha C. J. Tseng during spring and summer. Ursoiic acid is not found in the root and stem of Ilex kudingcha C. J. Tseng, the highest amount in its functional leaves(0.95%), the lower contents in the tender bud(0.52%), stem bark(0.35%) and fruit(0.33%). However, the whole plant can be used to produce urolio acid, for fully utilizing plant materials.4. The contents of ursolic acid in Ilex kudingcha C. J. Tseng from different areas are also determined. There is diversity in Ilex kudingcha C. J. Tseng from different areas or from the same area. As for the quality of ursolic acid, Ilex kudingcha C. J. Tseng from different areas of Guangdong province which has less differences is higher than that from Guangxi province and Hainan province which is rather different from Guangdong province, and the highest contents of 0.99% is from Dabu of Guangdong province. Even from the same area, the determination of ursolic acid in Ilex kudingcha C. J. Tseng have obvious difference, such as Ilex kudingcha C. J. Tseng from nanning of Guangxi province(0.42%～1.33%) and from the central south of Hainan(0.7%-1.19%).5. The alcohol refluxing extraction method of ursoiic acid is simple, easy to operate and suitable to large-scale production, and the optimum extraction process is as follows:adding 15 times amount of 95%alcohol into kudingcha materials, reflux extracting 3 times at 85℃,5 hours per time. The results show the maximum extraction rate of ursolic acid is 93.83% under this conditions. 6. Before further purification of ursolic acid by column chromatography, three Methods, generating precipitation with acid and alkali, decoloring with active charcoal and extracting impuritiy with petroleum ether, are used to preliminarily sublimate the crude extract of Ilex kudingcha C. J. Tseng. The 50%alcohol extract concentrated from the crude extract, is treated to pH 10 with 10% sodium hydroxide solution, and the filtrate by filtering is adjusted pH value to 4.0 by 5% hydrochoric acid solution, then precipitation is obtained through standing, filtering, water washing to neuter, fully drying. The precipitation is dissolved in 95%alcohol, then the solution is returned to cooking for 1 hour under 80℃, after adding activated carbon according to the amount of 3g/100ml. The filtrate by filtering is concentrated to dry under reduced pressure, and the dried products is put in petroleum ether with 20 times to remove lesser polarity fat-soluble impurities, by extracting 3 times and emptying the petroleum ether after 12 hour each time. The remaining solids made from airing petroleum ether is dissolved in 75% ethanol solution as the solution for Column Chromatography.7. The study of using X-5 macroporous adsorption resin as filler of column chromatography to purify ursolic acid, has a good effect, and is completely adapted for mass production. The optimum purification process by first sequential elution is as follows: water-20%ethanol-40%ethanol-50%ethanol-80%ethanol(pH=10), the purity of ursolic acid can reach above 64%. The purification process by second elution is:adjusting the first 80% ethanol eluent to 75%ethanol solution, keeping pH=10, flow rate 2.5BV/h, the purity of ursolic acid can reach above 77.3%. The purity is above 85%after ursolic acid being crystallized, and recovery is higher than 50%.8. The purification of lupeol is as follows:adding 15 times amount of 95%alcohol into powder of L cornuta Lindl., reflux extracting 3 times at 85℃,5 hours per time, then filtering, the dried products concentrated under reduced pressure is put in water to extract lupeol by an equal volume petroleum ether, continuous extracting 4 times. The extract dissolved in 90% ethanol solution is adsorbed by X-5 macroporous adsorption resin, and the sequential elution of lupeol by flow rate 2BV/h is as follows:70%(6BV)-80%(2BV)-85%, the 85%alcohol eluant was collected for as parted, mergeing all the part of lupeol. The eluant is concentrated to dry under reduced pressure, finally lupeol of 90% purity is obtained by twice recrystallization with 95% alcohol. The recovery rate is more than 72%. |
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