| Eucommia ulmoides Oliv. is a kind of aboundant resources in China. Recent research shows that the main active chemical constituents are iridoids, lignans,flavonoids and phenylpropanoids, which are valuable in application. Eucommiol(Eu)as a secoiridoid compound, which only exists in Eucommia ulmoides Oliv., is one of the specific ingredients. In this dissertation, the leaf of Eucommia ulmoides Oliv. was used as materials, based on the establishment of the rapid detection method of three iridoids compounds by HPLC, optimized extraction process of eucommiol,established purification process by semi-preparative liquid chromatography and characterized the structural of eucommiol. The main contents were as follows:(1) The HPLC method for simultaneous determination of eucommiol, Aucubin(Au) and Geniposidic Acid(GPA) was established and the methodology was examined. The optimized determination condition of HPLC was: A Kromasil100-5C18 chromatographic column(250×4.6 mm,5 μm) was used to perform the determination, which was maintained at 35℃ throughout the analysis. Mobile phase was composed of methanol(A)-0.1% phosphoric acid solution(B) with flow rate at0.8 mL/min under gradient elution by programmed wavelength detection.Programmed gradient elution : 0~15 min,5%~7% A;15~16 min,7%~14% A;16~25min,14%~20% A;25~26 min,20%~50% A;26~35 min,50%~5% A. Programmed wavelength: 210 nm(0~12 min, Eu), 202 nm(12~18 min, Au), 238 nm(18~28 min,GPA). The results showed that the HPLC detection method has a good reproducibility and maneuverability.(2) Ultrasonic and cellulase assisted extraction technologies of eucommiol were researched. With the eucommiol yield rate as index, the results of ultrasonic-assisted extraction method were as follows: water was used as solvent, liquid-solid(mL︰g)ratio was 30︰1, ultrasonic-assisted extracting once for 20 minutes under 30℃. Under this experimental conditions, eucommiol yield rate was 0.3723%. And the optimal processing conditions for enzymatic extraction were pH 7, liquid-solid(mL︰g) ratio25︰1, quantity of enzyme 1.0%, extracting for 4 hours once under 30℃. The yield of eucommiol was 0.2357%. It turned out that ultrasonic extraction was higher than enzymatic extraction for the yield of eucommiol.(3) The eucommiol which extracted by ultrasound was purified by liquid-liquid extraction, macroporous resin adsorption and semi-preparetive HPLC preparation.The purity of obtained eucommiol was determinated by HPLC. The content of eucommiol in crude extract of eucommia leaf increased from 0.3871% to 1.154% after alcohol precipitation with ethanol and n-butanol extraction. The macroporous adsorption resin D101 was selected with the indicates of adsorption rate. Dynamic elution curves shows eucommiol mainly located in Fr-5 to Fr-14. The crude eucommiol was further purified by semi-preparetive HPLC. The purity of prepared eucommiol which calculated with the method of chromatographic peak area normalization was 95.12%.(4) The structure of isolated eucommiol was subsequently confirmed by ultraviolet spectrophotometry(UV), infrared absorption spectroscopy(IR), unclear magnetic resonance spectroscopy(NMR) and liquid chromatography-mass spectrometry(LC-MS). Its UV, IR, 1H NMR, 13 C NMR and mass spectral data are consistent with the literature of eucommiol. |
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