Study Of Flavonoids Semen In Chemical Composition And Quality Of Analytical Methods

Semen zizyphi spinosae (SZS), the mature seed of Zizyphus jujuba var. spinosa (Bunge) Hu.et H.f. Chou, is a well-known Traditional Chinese Medicine for the treatment of insomnia and anxiety. Previous chemical investigations resulted in the isolation of bioactive components including flavonoids, saponins, fatty oil and organic acids. Modern pharmacological studies have demonstrated that flavonoids from SZS are the main bioactive compounds responsible for the sedative and hypnotic effects. Therefore, the separation and structural elucidation of the flavonoids from SZS, and the quantitation of multicomponents from SZS are important for its quality control and the safety of clinical utilization.Five flavonoid glycosides were isolated from SZS and identified on the basis of their UV spectral data, NMR spectral data, accurate molecular weights and mass fragmentation behavior as spinosin,6'''-feruloylspinosin,6'''-p-hydroxybenzoylspinosin, 6'''-p-coumaroylspinosin and isovitexin. The results provide material basis and references for the further study of the isolation and identification of flavonoids from SZS.In this paper, a novel high-performance liquid chromatography coupled with photo diode array detector/Fourier Transform Ion Cyclotron Resonance Mass Spectrometry/MSn (HPLC-PDA/FTICRMS/MS") method was established to separate and identify flavonoids from the extract of SZS. The chromatographic separation was performed on a HYPERSIL C18 column (4.6*250 mm,5μm) maintained at 25℃. A linear gradient elution of acetonitrile and water containing 0.1% acetic acid were used. The sample injection volume was 20μL. UV spectra were recorded between 200-400 nm and the detection wavelength was set at 270 nm. The solvent flow rate was 0.8 mL/min with 30% of the eluent being split into the inlet of the mass spectrometer. The optimized parameters in the positive ion mode were:ion spray voltage,3.5 kV; capillary temperature,275℃; capillary voltage,45 V; and tube lens,94.5 V. Mass spectra were recorded in a mass range of m/z 90-1000. UV spectra, accurate molecular weights and multi-stage MS/MS fragmentation information were obtained for the nineteen compounds. Ten flavonoids were identified based on multi-stage MS/MS data analysis of known falvonoids, mass fragmentation rules summary and comparison with reference data. Other nine compounds were tentatively characterized as flavonoids that have not been reported previously. The method is useful for separation and identification of the flavonoid components from SZS or other complex samples, especially for minor constituents, and is important for its active mechanism and quality control.The pharmacodynamic action of Traditional Chinese Medicines are based on the multicomponents existed in them. Rapid, precise and sensitive analytical technology and method are significant for components analysis and quality evaluation for Traditional Chinese Medicine. A novel rapid and suitable UHPLC method coupled to diode array detection has been developed and validated for the fingerprint analysis of the flavonoids from SZS and quantitation of isovitexin,spinosin,swertisin,6'''-p-hydroxybenzoylspinosin,6'''-p-coumaroylspinosin and 6"'-feruloylspinosin. The analysis was performed on a Agilent ZORBAX Eclipse Plus C18 RRHD column (2.1*100 mm,1.8μm) connected with Agilent ZORBAX Eclipse Plus C18 RRHD column (2.1*50 mm,1.8μm), both contained at 40℃. A linear gradient elution of acetonitrile and water containing 20 mM NaH2PO4 was used. The solvent flow rate was 0.4 mL/min. The sample injection volume was 1μL. UV spectra were recorded between 200-400 nm and the detection wavelength was set at 270 nm. The method showed good linearity within test ranges of 0.39-195.00μg/mL for isovitexin,0.38-191.00μg/mL for spinosin, 0.40-201.00μg/mL for swertisin,0.33-166.00μg/mL for 6'''-p-hydroxybenzoylspinosin, 0.39-198.00μg/mL for 6'''-p-coumaroylspinosin and 0.39-196.00μg/mL for 6"'-feruloylspinosin. The RSDs of intra-day and inter-day precision were less than 1.9% and 4.0% respectively for the six components. The repeatabilities were good with RSD less than 4.1%. It's also showed good intra-day and inter-day stability by RSD less than 4.7% and 4.6% respectively. The recoveries were in the range of 97.4%-104% with RSD less than 4.7% for the six components. The method is rapid, accurate and suitable for both fingerprint analysis and quantitation of the six components. It can provide reference information for quality evaluation of SZS.