Chemical Constituents Identification And Monomer In Vitro Cell Antiproliferation Activity Study In Pigeon Pea Leaves

Pigeon pea leaves are the stems and leaves of Cajanus cajan（L.）Millsp.The main constituents of pigeon pea leaves were flavonoid compounds and stilbene compounds.It exhibited notable anti-inflammatory,antibacterial,antioxidant and antitumor activities.Qualitative analysis of chemical components and isomers differentiation in the ethanol extract of pigeon pea leaves were carried out in this work.Additionally,monomers were separated from the ethanol extract of pigeon pea leaves and their cell antiproliferation activities were evaluated.The specific research work was as follows:Firstly,an ultra-performance liquid chromatography coupled with tandem quadrupole time-of-flight mass spectrometry（UPLC-Q-TOF-MS/MS）method was established to qualitative analyze the chemical constituents in pigeon pea leaves.A total of 58 compounds were detected by combination of positive and negative modes.Among them,38 compounds including 16 flavones,1 flavonol,4 flavanones,9isoflavones,1 coumarin,1 lactone and 6 stilbenes were tentatively identified in view of retention time,exact molecule weight,molecular formula and fragments data.The fragment mechanisms of the above compounds were studied in detail and the main cleavage pathways of flavonoid compounds were the RDA reactions on the C ring and the neutral loss of small molecules in saccharide substituents and flavonoid aglycones,such as CO,H₂O and CO₂.The main fragment behaviors of stilbene compounds were rearrangement reactions and the neutral loss of small molecules.The qualitative analysis of the chemical composition in pigeon pea leaves could provide a scientific basis for the establishment of the related quality standard system.Secondly,the ion trap mass spectrometry was used to further investigate the twelve pairs of isomers in pigeon pea leaves.The energy-resolved breakdown curves in the light of relative abundance of characteristic fragment ions were set up by giving various collision energies to isomer compounds.The differentiation of isomers could be achieved by the energy-resolved breakdown curves and the diagnostic ions.As a result,eight pairs of positional isomers were successfully distinguished.The established method with the superiorities of simple operation,fast analysis and high sensitivity could provide relevant mass spectrometry data and method reference for the identification of constituents in other natural products.Finally,semi-preparative high-performance liquid chromatography method was further adopted to separate and purify compounds from the extract of pigeon pea leaves.As a result,four compounds were obtained and their structures were characterized as 3-methoxy-5-（2-phenylethenyl）-phenol,pinostrobin,longistylin C and cajaninstilbene acid respectively through high resolution mass spectrometry and NMR data.Additionally,the in vitro antiproliferation activities of three stilbene compounds against human hepatoma cell（HepG2）were evaluated and the results showed that all three compounds exhibited certain cell antiproliferative activities.The method could promote the in-depth pharmacological activity research of pigeon pea leaves.