Studies On The Separation, Purification And Bioactivity Of Oleiferasaponin

The Camellia plant belonging to Theacea, Camellia oleifera Abel, is the specificwoody oil tree species which seed is rich in oil and saponin. It’s difficult to makequalitative and quantitative analysis for oleiferasaponin because there are wide varieties and nostandard sample. At present, the purity of oleiferasaponin that can be purchased at themarket is low and its application scope is limited, so it is helpful for improving the value oftea seed pomace and refined byproduct by studying the industrial purification technologyof the high purity oleiferasaponin.The crude oleiferasaponin was taken as the material in this research, and HPLCdetection method was studied. The industrial purification technology was established, andthe monomer compound was separated and identified, then the bioactivity was investigated.The main results were as following:1. The determination method of oleiferasaponin was established usingHPLC-UV/ELSD. The determination condition was as follows: A fow rate of1.0mLmin1and sample size of5μl were used. The mobile phase consisted of0.2%acetic acidin water （A） and100%acetonitrile （B）; and the gradient of B increased linearly from35%to40%（v/v） at15min, to60%at45min, to80%at55min, and return to35%at60min.The UV detection wavelength was set at280nm for real-time monitoring of the peakintensities. To the evaporative light-scattering detector, a drift tube temperature of80°C, adrying gas pressure of25psi, gain of50, and the sensitivity of0.01were used. For theseries connection of UV-detector and evaporative light-scattering detector, the sensitivitycould be complementary for each other, and the key component of oleiferasaponin couldbe detected. Take oleiferasaponin A1as standard substance, at280nm under HPLC-UVdetection, the result showed that at0.0096～6mg/mL, the peak intensities linearlyresponded to the concentration of oleiferasaponin. The correlation equation wasy=553272x-2357.1with the correlation coefficient of R²=0.9997. HPLC analysis suggestedthat the varieties of saponin in seed were identical with pomace. And the stable chemicalproperties could provide convenience for the comprehensive development ofoleiferasaponin.2. The qualitative determination method of oleiferasaponin was builded utilizingHPLC/TOF-MS. It was suggested that the molecular weight of oleiferasaponin was mainly1039～1379D, which was consistent with that in Camellia sinensis. A oleiferasaponin compound have been conjectured through HPLC-MS/MS analysis, with m/z1201（[M-H]-）and molecular formula C₅₈H₈₉O₂₆.The structural formula was as the same as camelliasaponinB1in the seed of Camellia japonica L, which illustrated that saponins in Camellia havehigh identity. It could be speculated that there were many compounds with2and30difference of molecular weight in oleiferasaponin by HPLC-MS/MSn. Compounds with2difference of molecular weight could because-CHO was replaced by-CH₂O in C4position,and compounds with30difference could owing to-CHO supplanted by-CH₃in C4positionand-OH supplanted by–H in C21position.3. The industrial purification technology for the high purity oleiferasaponin wasdeveloped. The oleiferasaponin was separated fristly by the0.2μm ceramic membranescombined with the30000-Da and5000-Da organic membranes, and then the macroporousresin was used for further purification. The high purity oleiferasaponin was obtained afterthe spray drying and the yield was30.0%, the purity was94.2%. The oleiferasaponin afterpurification was white powder and the stickiness was lower and also the foamability hasbeen improved significantly.The membrane module can be reused. The macroporous resinis cheap and can be used multiple times.The adsorption rate and the desorption rate are notchange after loading sample8times. Only ethylalcohol was used during the purificationprocess, which can be recycled. So the purification technology was low-carbonenvironment and suitable for industrial development.4. A new saponin fron tea seed pomace （Camellia oleifera Abel） was separated andpurified by silicagel column, C18column and HPLC. The molecular formula, C₅₉H₉₂O₂₆,was determined from the [M H] ion at m/z1215.57975by high-resolution negative-ionelectrospray ionization mass spectroscopy （ESI-MS/MS）. It was identified as a newcompound, oleiferasaponin A1, on the basis of IR,¹H NMR,¹³C NMR and2-d spectrum.5. The bacteriostatic bioactivities of oleiferasaponin was studied preliminarily. Theresults showed that100μg·mL-1oleiferasaponin had higher inhibitory activity on plantdisease pathogenic hyph and yeasts, while10000μg·mL-1oleiferasaponin had lessinhibitory activity on the experimental treatment groups of bacteria. Thus oleiferasaponincan be used for the exploitation of fungal inhibitors. Among the experimental treatments,oleiferasaponin had prominent inhibitory effect on Helminthosporium Maydis with a MICvalue of250μg/mL and slight inhibitory effect on Gloeosporium theae-sinensis Miyakewith a MIC value of2100μg/mL. When observing the hypha of Helminthosporium maydistreated with125μg/mL oleiferasaponin with optical microscope, we discovered that itappeared inflated cryptomere which hindered the growth of hyphae. But only by the concentration of oleiferasaponin reached1400μg/ml, can it had relatively obviousinhibitory effect on Gloeosporium theae-sinensis Miyake, which illustrated that inhibitoryeffect of oleiferasaponin on fungus had a certain selectivity. At the same time,oleiferasaponin was proved to have cell protective effects on H₂O2-induced cytotoxicity in PC12cells, when compared the differences with H₂O₂group, both purified total oleiferasaponinand monomer compounds oleiferasaponin A₁of25μM,125μM had significant difference,which provide the basis for its exploitation in drug.