Study On The Extraction And Separaction Of Geniposide And Geniposidic Acid By Aqueous Two-phase Extraction

The fruits of Gardenia jasminoides Ellis(named as Zhizi) and the leaves of Eucommia ulmoides(named as Duzhongye) are used as typical traditional Chinese medicines, which are widespreading in China. They contain many kinds of iridoids substances, and geniposide(GP) and geniposidic acid(GPA) are two typical iridoids active ingredients. The content of GP in Zhizi is higher, and the content of GPA in Duzhongye is higher. GP and GPA have been extensively applied in the fields of medicine, health care products, food, chemical dyes and so on, and the price of GPA is higher than GP in the market. Up to now, several methods have been reported for extraction and separation of GP and GPA, but there are some remarkable shortcomings in these procedures, such as lengthy process, high cost of organic solvents, low recovery, and toxic solvent residuals in the products, etc. So it is very important to establish a new extraction and separation method for selective extraction and separation of GP and GPA form Zhizi and Duzhongye.As a novel liquid-liquid fractionation technique, aqueous two-phase extraction(ATPE) has many advantages include scale-up potential, continuous operation, ease of process integration, low toxicity, etc. Furthermore, ATPE has been used to extract and separate biomacromolecules, metal ions, natural active components and other contents. In order to further improve the selectivity and efficiency of separation, ATPE need to combine some modern separation techniques, such as ultrasonic technology, solvent sublation technology, and so on. Extract and separate natural products by combined ATPE can achieve the extraction and separation synchronization, simplified separation steps, and the application ranges of ATPE were also been further expanded.In this dissertation, ATPE combined ultrasonic technology and solvent sublation technology had been selected for the extraction and separaction of GP and GPA from Zhizi and Duzhongye, respectively. And the process of GP transform in GPA in alkaline condition was also been studied.1) GP from Zhizi was extracted by ultrasound-assisted ATPE. The factors of the concentration and kinds of alcohol, the concentration and kinds of salt, temperature, time and sample loading were investigated. Based on the single factor experimental results, we had optimized the extraction process by box-Behnken design and response surface methodology(BBD-RSM). The results showed that aqueous two-phase system was composed of 31.19%(w/w) ethanol/15.00%(w/w)Na H2PO4, extraction temperature was 30℃, extraction time was 30 min, sample loading was 0.03 g, the yield of GP was 51.77 mg/g. The extraction efficiency is superior to the traditional methods.2) Aqueous two-phase flotation(ATPF) used for separation and enrichment of GPA from Duzhongye. In the ATPF experiment, the effects of the concentration and kinds of alcohol, the concentration and kinds of salt, air flow, time and crude extraction loading were investigated. The optimal conditions for ATPF were obtained by BBD-RSM, the concentration of ethanol was 28%(w/w), the concentration of Na H2PO4 was 23%(w/w), crude extraction loading was 5 g, air flow was 40 m L/min, time was 20 min.Under optimal conditions, the flotation efficiency and enrichment times of GPA were 97.88% and 27.34 respectively. At the same time, two parallel experiments of 100 times scale-up experiment was empolyed, the flotation efficiency and enrichment times of GPA were 95.60% and 8.40 respectively, and RSD was 0.77%. The result of 100 times scale-up experiment was almost the same with small scale experiment. Compared with the traditional liquid-liquid extraction method, ATPE has obvious advantages.3) Optimization of the preparing processing of GPA from Gardenia extract with alkaline hydrolysis using orthogonal test design. The influencing parameters of temperature, the adding amount of Na OH, solid to liquid ratio and time were investigated. The optimal conditions were obtained by four factors and three levels orthogonal test design, tempurature was 70℃, time was 10 min, the adding amount of Na OH was 14 m L, solid to liquid ratio was 1:30 g/m L. The yield of GPA was 11.53%. The purity of geniposidic acid was 45.33% after static adsorption by activated carbon.