Isolation And Purification Of Flavones From Murraya Exotica L By High-speed Counter-current Chromatography

Traditional Chinese medicine is a treasure with thousands of brilliant culture, its effect has been accepted by Chinese people, but compared to western medicine, most material basis of the unilateral and compound preparation is not yet clear, the research and development of pharmacological effects is relatively weak, limiting the development and utilization of traditional Chinese medicine. Therefore, extracting the active ingredients or further separation and purification of effective monomer from Chinese medicine is also an important component in the field of traditional Chinese medicine research. As in different Chinese herbal medicine, the composition with a wide range is very complex, and the content of the same composition is various. So extraction, separation and purification of the active ingredients from Chinese medicine is a very difficult and delicate task. The high-speed counter-current chromatography (HSCCC) is a continuous and efficient liquid - liquid partition chromatography separation technique, Its benefits are as follows: separation speed, high sample recovery rate, low cost, and so on. HSCCC is very suitable for separation and preparation of the active ingredient from Chinese medicine and natural productsAs the traditional Chinese medicine, Murraya exotical L is a species of Murraya, Rutaceae, its leaves and shoots with leaves both are rich in coumarins, flavones. Flavones is one of the active ingredients of natural products. It is able to play an important role at reducing blood lipid, hypoglycemic effect, anti-oxidation, anti-aging effect, and so on. In this paper, establishing a method of separation and purification of flavones from Murraya exotical L is significant for the study of natural flavones products and provides a technical basis for the isolation and purification of flavones from other Chinese medicines and natural products.In this study, heat reflux by organic solvents was used in extraction of Murraya exotical L total flavones, then the total flavones crude extract was further purified by macroporous resin to obtain the weak polar and medium polarity crude extract of Murraya exotical L. High-speed counter-current chromatography was used to isolate and purify the two kinds of polar extracts, Finally seven high purity flavones were obtained. They were all analyzed by high performance liquid chromatography (HPLC), and identified by mass spectrometry (MS), ¹H-nuclear magnetic resonance (NMR) and 13C NMR.High-speed counter-current chromatography (HSCCC) was used to isolate and purify flavones from the weak polar crude extract of Murraya exotica L. The optimum separation conditions were as follows: A two-phase solvent system was petroleum ether-ethyl acetate-methanol-water (5:5:4.8:5, v/v/v/v). The lower phase as the mobile phase was operated at a flow rate of 2.0mL/min, while the apparatus rotated at 800r/min. 200mg sample was loaded for one time. Under these conditions, 54.31 mg of recrystallized 5,7,3′,4′,5′-penta Methoxyflavone, 107.68mg of 5-hydroxy-6,7,3'4'-tetramethoxyflavone, 215.54 mg of 5-hydr oxy-6,7,8,3'4'-pentamethoxyflavone,84.36mg of 5-hydroxy-6,7,8,3',4',5'-hexamethoxyflavone ,and with their purities respectively 97.28%,97.56%,98.75%,95.05% were successfully obtained from 4.0g of the weak polar crude extract of Murraya exotica L. The four compounds were analyzed by high performance liquid chromatography (HPLC), and identified by mass spectrometry (MS), ¹H-nuclear magnetic resonance (NMR) and 13C NMR. The known compound 5-hydroxy-6,7,3'4'-tetramethoxyflavone was isolated and purified from Murraya exotica L for the first time.High-speed counter-current chromatography (HSCCC) was used to isolate and purify flavones from the medium polarity crude extract of Murraya exotica L. The optimum separation conditions were as follows: A two-phase solvent system was n-hexane-ethyl acetate-methanol-water (4:6:4:6, v/v/v/v). The lower phase as the mobile phase was operated at a flow rate of 2.0mL/min, while the apparatus rotated at 800r/min. 300mg sample was loaded for one time. Under these conditions, 30.45mg of recrystallized 5,7,3'4'-tetramethoxy flavone, 145.63 mg of 5,7,3′,4′,5′-pentamethoxyflavone, 34.28mg of 5,7,8,3',4',5'-hexamethox yflavone, 62.17mg of5,7,3′,4′,5′-pentamethoxyflavanone,47.95mg of 5-hydroxy-6,7,3',4'-hexa methoxyflavone, with their purities respectively 97.34%,97.69%,98.12%,96.78%,95.16% were successfully obtained from 4.0g of the medium polarity crude extract of Murraya exotica L. The five compounds were analyzed by high performance liquid chromatography (HPLC), and identified by mass spectrometry (MS), 1H-nuclear magnetic resonance (NMR) and ¹³C NMR. Among the seven compounds, 5,7,3′,4′,5′-pentamethoxyflavone and 5-hydroxy -6,7,3',4'-hexamethoxyflavone were both isolated and purified from two kinds of polar crude extract, but under different conditions, the weight and the purity of the two compounds were completely different.