Purification And Identification Of Tea Saponin From Cold Pressing Oleifera Seed Cake

Tea Saponin, a natural non-ionic surfactant, consists of hydrophilicglycosylation and hydrophobic aglycone, as well as organic acids. Tea Saponin ismainly extracted from tea seed meal, which is abundant in China. The developmentof tea saponin has been a good prospect and economic benefits.This paper is consist of five parts: the preparation method of the standard teasaponin, quantitative methods of tea saponin, extraction process, purificationprocess, monomer separation and structural identification. A new detectionmethod for tea saponin had been established, the extraction and purification processhad been optimized to reduce production costs and environmental pollution, andimprove the yield and purity of the tea saponin, the tea saponin monomer separationand basic structure identification were studied.Firstly, by comparing the advantages and disadvantages of several preparationof the standard methods. the preparation method of the standard tea saponin wasconfirmed. Organic solvent and macroporous resin method could remove most ofthe impurities. Ethanol recrystallization purification of tea saponin were up to therequirements of the standard of tea saponin, except flavonoids and other impurities,on basis of the organic solvent, most of the flavonoids and other impurities wereremoved by reverse phase column chromatography, and the UV and IR spectra wereconsistent with the literature.Secondly, the as-prepared standard was adopted for the quantitativedetermination of tea saponin in the tea seed cake by using high performance liquidchromatography(HPLC),the HPLC analysis was performed on ODS column(Phenomenex,4.6mm×250mm,5μm),by use of the mixture of acetonitrile andwater as the mobile phase at flow rate of1.0mL/min,215nm as detectionwavelength, the gradient elution was processed as an beginning condition of20%acetonitrile for5min, with a linear increase to100%acetonitrile within5min, andthen return to the beginning condition (20%acetonitrile) via a linear decrease overanother5min, the total run time was15min for each sample. The tea saponin peak showed great symmetry, moderate retention time and perfect resolution with otherimpurities, this assay demonstrated a great advantage of simpleness, speediness andefficiency, it would provide a good new way for the detection of tea saponin in teaseed cake.Thirdly, tea saponin extraction process was studied. Aqueous ethanol wasselected as the extraction agent after screening experiments,. The orthogonal L16（45）test was used to optimize the parameters of extraction of tea saponin whichwas on the basis of single-factor test. The effects of ethanol concentration,temperature, ratio of solid to liquid, time, pH value were investigated. The optimumresults showed the ratio of solid to liquid1:3, ethanol concentration70%,temperature at55℃, time of3.0h, pH value of8.5, the tea saponin rate was11.55%,the purity was71.74%under this condition.Fourthly, tea saponin purification had been studied. D4020macroporous resinhad good adsorption and desorption effects on tea saponin through static anddynamic adsorption experiment. The orthogonal test was used to optimize theparameters of purification of tea saponin on basis of single-factor tests. The resultsshowed that the optimum adsorption conditions were the feeding rate of0.9mL/min,alcohol concentration of20%, sample concentration of27mg/mL, Under theseconditions: the tea saponin adsorption rate was83%, the optimum desorptionparameters at the eluent alcohol concentration of60%, the flow rate of1.8mL/min,the eluent volume2.0BV (Bed Volume). As a result, tea saponin desorption ratewas99%, purity was81%, the recovery rate was more than82%. All the datasshows that the optimization was feasible. The purified tea saponin had significantlyimproved the purity. The experimental results provided a theoretical basis for thefuture industrial production of purified tea saponin.Fifthly, the tea saponin monomer separation method and the structure identification had been studied. Tea saponin monomer was separated by HPLCmethod, performing on an ODS column (Phenomenex,4.6mm×250mm,5μm), themixture of acetonitrile (A) and water (B)was as the mobile phase at the flow rate of1.0mL/min,215nm detection wavelength, the gradient program used as follows:initial0–5min, A–B (25:75, v/v) remains the same;5–10min, linear change to A–B(28:72, v/v);10–15min, linear change to A–B (32:68, v/v);15–20min, linearchange to A–B (90:10, v/v);20–25min, A–B (90:10, v/v) remains unchanged;25–30min, linear change to A–B (25:75, v/v), the sample injection volume was20μL and the column temperature was maintained at25℃. The6kinds of componentswere collected and their structure was analyzed.