Investigation On The Material Base Of Shaoyao-Gancao Decoction Against Acute Asthma By β₂-Adrenoceptor Affinity Chromatography

The discovery,identification and evaluation of material base of traditional Chinese medicine(TCM)is one of the major bottlenecks that restrict the process of the modernization of TCM.In previous study,metabonomics study of Shaoyao-Gancao decoction(SGD)in a rat model of acute bronchial asthma was conducted.On this basis,using β2-AR as a target protein,the target-drug screening model was established to separate and recognize the bioactive components in SGD water-extracts which are specifically binded to β2-AR,in order to explore the material base and mechanism of SGD against acute asthma.The main content of this dissertation includes the following four aspects:The β2-AR column was prepared and characterized.β2-AR was purified by affinity chromatography and immobilized on amino microspheres by mild chemical method.The bioactivity of β2-AR was characterized by the agonist of the receptor.The results showed thatβ2-AR still had the ability of recognizing its specific ligands after immobilization and packing procedures.The bioactive ingredients was separated and identified in SGD water-extracts.The bioactive ingredients in SGD water-extracts were screened by β2-AR column,and were identified as paeoniflorin(PF),albiflorin(AF)and liquiritin(LQ)by quadrupole time-of-flight mass spectrometry(Q-TOF-MS).The binding interaction between LQ,AF,PF and β2-AR.Frontal analysis was utilized to investigate the interaction between the three bioactive ingredients.The results showed that they had only one type of binding site in β2-AR.The association constants were 2.95×104 L/mol,2.53×104 L/mol and 2.16×104 L/mol,the binding-site number 1.0×1016 mol/L 1.0×10-6 mol/L and 2.0×10-7 mol/L,respectively.The affinity rank order of the ligands binding to β2-AR was LQ>AF>PF.Effects of PF,AF and LQ on SD rats bronchial smooth muscle.High concentration of potassium solution was used to stimulate the trachea contraction of SD rats.Using ICI 118551 as β2-AR antagonist,we investigated the changes of rat tracheal smooth muscle under the intervention of PF,AF and LQ.PF and LQ displayed a concentration-dependent relaxation of tracheal smooth muscles following potassium-induced contraction,and the relaxation effects were abrogated by ICI 118551,while AF without the same function.