Study On Extraction And Separation Of Active Components From Patinia Heterophylla Bunge And Activity Screening Of Anti-leukemia In Vitro

Objective: To study the chemical constituents of Patrinia heterophylla Bunge by macroporous resin chromatography,silica gel column chromatography,medium pressure liquid phase,high performance liquid phase and semi-preparative liquid phase,and to analyze the chemical composition and spectral data according to physicochemical properties and spectral data.The isolated monomer compounds were tested for in vitro anti-leukemia activity by CCK-8 method.Methods: The whole leaves of the leaves were harvested by refluxing with ethanol to obtain a total extract of the leaves.After adding water to a suspension,it was extracted with petroleum ether,n-butanol,chloroform and ethyl acetate in order to obtain extracts having different polarities.The n-butanol fraction was separated and purified by macroporous resin chromatography,silica gel column chromatography,medium pressure liquid phase,high performance liquid phase,semi-preparative liquid phase and other separation methods and techniques.The chemical structure of each monomer compound was determined by nuclear magnetic resonance carbon spectrum,hydrogen spectrum,and high resolution mass spectrometry.The inhibitory effect of each monomeric compound and the positive control drug cisplatin on the growth of HL-60 and K562 was determined by CCK-8 kit,and the cell inhibition rate and IC50 value were calculated,and the preliminary antitumor activity was evaluated.Results: Eight compounds were isolated from the n-butanol fraction of the leaves,identified as patrinoside(A),Heteroside A(B),Roseoside II(C),(3-O-α-L-arabinopyranosyl hederagenin 28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside(D),apigenin4’-O-β-D-glucopyranoside(E),fissoside B(F),Apigenin-7-O-β-D-lutinoside(G),rhamnocitrin 3-O-[α-L-rhamnopyranosyl(l→4)-O-α-L-rhamnopyranosyl(1→6)]-β-D-galactopyrano side(H).Compound B is a new compound;compound A is isolated from the plant for the first time;compounds C,D,E,F,and G are isolated from the genus for the first time.The anti-leukemia activity test in vitro showed that compounds A,B,C,F and G had certain toxicity inhibition effects on HL-60 cells,and compounds C,D,E,F and G had certain toxicity inhibition effects on K562 cells.Conclusion: In this study,eight compounds were successfully isolated and identified from the Patrinia heterophylla Bunge.Among them,the compounds A and B are an iridoid component,the compound C is a methylcyclohexene monoterpene glycoside,the compound D is a triterpenoid saponin component,and the compounds E to H are flavonoid components.From this,it can be seen that the chemical components contained in the different leaves are relatively rich.The inhibitory effects of compounds A to G on the proliferation of HL-60 and K562 cells were screened by CCK-8 method.The results showed that each monomeric compound had certain toxicity inhibition effect on leukemia cells,the inhibitory effects of compounds A,C,D,F,and G on HL-60 cells were dependent on drug concentration,and the inhibitory effects of compounds C,D,E,F,and G on K562 cells were dependent on drug concentration.