Constituents Investigation By UPLC-QTOF-MS And Compounds Isolation By HSCCC From Pigeon Pea Leaves

In this thesis,multi-constituents of pigeon pea leaves have been investigated by using UPLC-QTOF-MS.Seven components in pigeon pea leaves were isolated and purified by counter-current chromatography separation system.Two main parts were investigated in this study:Firstly,a validated method based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was established to separate and identify compounds in pigeon pea leaves.A total of twenty-eight compounds were detected,and twenty-one compounds,mainly flavonoids were unequivocally identified or tentatively assigned based on retention time,ultraviolet maximum absorption,molecular formula and fragments.The method validation results demonstrated the UPLC method possessed desirable precision,reproducibility and accuracy.Secondly,a validated method was established to separate seven compounds from pigeon pea leaves via high speed counter-current chromatography(HSCCC).In this work,many solvent systems were screened.It was found that the acidic solvent system can significantly affect the chromatographic peaks.The preferred solvent system were composed of n-hexane/methanol/formic acid aqueous solution of pH=3(10:6:4,v/v/v).The isolation was completed in two steps,the first step was,five compounds were obtained gained by the conventional one-step elution.The other two compounds were obtained by a two-step elution.The purities of all the compounds were over 95% as determined by high-performance liquid chromatography.Their structures were identified by UV,high resolution mass spectrometry,and NMR spectroscopy.The compounds included a flavanone,a chalcone,and five stilbenes.