Study On Seperation Of Polyphenol Compounds From Leaves Of Olea Europaea L. By High-Speed Counter-Current Chromatography

High-speed counter-current chromatography originated from the early 80's,20 th century.Nowadays,HSCCC has been widely used in the field of isolation and purification of natural products and become a new and efficient purification technology.Olive is a kind of plant that has great potential value and its main active ingredient contain polyphenol compounds.At present,the researches on the Olive leaves are in rapidly developing stage in the field of foods,medicines and cosmetics.This paper mainly focused on the separation and preparation of phenolic compounds that have a variety of active from Olive leaves by high-speed counter-current chromatography and carried out related researches.Specific research contents include the following three aspects:1.In order to establish a rapid method for the rapid separation and preparation of oleuropein from olive leaves with high-speed counter current chromatography.The olive leaves collected from Longnan area of Gansu Province were took as object of study,and the separation parameters such as solvent system,flow rate and injection concentration were also investigated.The results showed that the solvent system which composed with n-butanol: ethyl acetate: methanol: water: chloroform?1:19:1:19:0.4,v/v?was selected as the suitable two-phase solvent system for the separation.The upper phase was used as the stationary phase and lower phase was used as the mobile phase.Meanwhile,the revolution speed was set at 900 rpm,the flow rate of the mobile phase was set at 1.5 m L·min^-1 and the injection2 concentration was set at 20 mg·m L^-1.Under these conditions,the oleuropein was successfully separated from olive leaves with purity over 90%.This method is simple,rapid and has high recovery rate,which provides technical support for the efficient separation of oleuropein in olive leaves.2.An appropriate solvent system is a key factor to isolate efficiently target compounds by high-speed counter-current chromatography and the screening process of perfect solvent system consumes a lot of energy and time in the whole process of HSCCC separation.In this paper,we systematically investigated the effect that two-phase solvent system composed of different proportions HEMW?1:19:1:19,v/v???1:9:1:9,v/v???1:9:2:8,v/v???1:9:3:7,v/v???1:6:1:6,v/v???1:5:1:5,v/v?on separation of the target compounds which are the main active material Oleuropein and Luteolin-7-O-?-D-glucoside in Olive leaves.Objective to investigate the correlations between polarity of target compounds and polarity of solvent systems in the process of HSCCC separation.The results show that separation effect of two compounds increased at first and then decreased with the decreasing of the polarity solvent systems.The purities of Oleuropein and Luteolin-7-O-?-D-glucoside were determined with area normalization method and were 90.8% and 92.5% under the best condition of separation,respectively.The experimental results provide the technical support for rapid screening of HSCCC solvent system and improving the efficiency of separation and preparation in the complex samples of natural products.3.This section mainly contain HSCCC separation of polyphenol compounds in Olive leaves by a new method of solvent system selection,which is composed of HEMW system and based on 9×9 table.Firstly,we choiced 4 solvent system from 9×9 table of being composed the HEMW,then we determined partition coefficient K value of the three kinds of target compounds and established a linear relationship between number of solvent system?n-hexane or methanol?and Log K.Therefore,we choiced the sweet solvent system HEMW?1.5:8.5:1.8:8.2,V/V?according to the linear relationships and the solubility of olive leaf extracts.Not only that,we also optimized the flow rate of 1.5 m L·min^-1,speed 950 rmp and sample quantity of 20 mg·m L^-1 as experimental conditions for HSCCC separation in the end.Result that we got 45.8 mg oleuropein,17.7 mg Luteolin-4'-O-?-D-glucoside and 6.8mg hydroxytyrosol from 500 mg Olive leaf extracts by HSCCC and the purities of 3 target compounds over 90%.This study verifies efficiency and feasibility of HEMWat 9 × 9map-based solvent selection strategy in the process of high-speed countercurrent chromatography separation and greatly promotes the HSCCC application in the field of natural products.