Study On Separation And Pharmacological Activity Of An Unknown Component In The Seed Of Stizolobium Cochinchinensis

Objective: To establish the preparation method of levodopa analog (abbreviate: LDA) reference substance by AKTA Explorer 100 Preparative Chromatography System; Research the technology of extraction, separation and purification of LDA from the seed of Stizolobium cochinchinensis and optimize the process; Confirming the chemical structure of LDA; Study on the physicochemical constants and the method of identification and content determination; Develop the pharmacological research on Sedative-hypnotic action of LDA.Methods: The AKTA Explorer 100 Preparative Chromatography System was used to separate and purify of LDA from the seed of Stizolobium cochinchinensis, and the purity of LDA was determined by HPLC; The orthogonal test was adopted to optimize the best percolation and separation technologies of LDA by cationic resin and anion resin, and the content of LDA was taken as the index; The crude LDA was purified by the method of water solution and precipitate in ethanol; The structure of LDA was confirmed by Ultraviolet Spectrum(UV), Infrared Spectrum(IR), Mass Spectrum(MS), ¹H- and ¹³C-Nuclear Magnetic Resonance(NMR); The physicochemical constants of the compound were determined, the methods of identification and content determination by HPLC were established; The method of autonomic activities on mice, and the sleep time-prolonging test of LDA with threshold dosage and sub-threshold of pentobarbital sodium were applied for hypnotizing tests, and the convulsion induced by strychnine nitrate was observed to study the sedation, hypnosis and anti-convulsion effects of LDA.Result: To establish the method of separation and purification of LDA from the seed of Stizolobium cochinchinensis by AKTA Explorer 100 Preparative Chromatography System, and the purity of the product is more than 99.0% which is determined by HPLC. Optimize the percolation technology of LDA is: 50 times of 0.1mol/L hydrochloric acid as percolate solution, percolate velocity is 15ml/min; The best separation process by cationic resin is: 150 times of 1% ammonia as elution solution, elution velocity is 5mL/min, and the elution rate is 60.94%; The separation process by anion resin is: 50 times of 1% ammonia as elution solution, elution velocity is 3mL/min, and the elution rate is 63.46%. After crystallized in ethanol, the purity of LDA is more than 98%. Based on the analysis of UV, IR, MS, NMR spectrum, the chemical name of LDA is: 3-guanidino-6-hydroxymethyl-l-methylpyrazin-2-one. Then the methods of identification and content determination by HPLC were established. The results of pharmacology research indicate that with the dosage of 375mg/kg, LDA possesses sedative effects and the dosage of 500mg/kg,625mg/kg possesses hypnotic effects on mice.Conclusion: A new chemical constituent was found in the seed of stizolobium cochinchinensis; The preparative method of LDA reference substance, the method of identification and content determination was established; The process of extraction, separation and purification was optimized. LDA possesses sedative-hypnotic effects in a certain dose.