Studies On The Chemical Constituents And Antioxidant Effect In Vitro Of Flavonoid From Pyrethrumta Tsienense

Pyrethnmta tsienense is the aerial part of genus Pyrethrumta,which belongs to compositae family.It is mainly distributed in Qinghai,Sichuan,Yunnan and Tibet eastern part.As a commonly used Tibetan medicinal herb,it is often used to clear away the heat-evil and expell superficial evils,dispell wind and eliminate,dampness,analgetic,dephlogisticate and so on.Because of its notable therapeutic effect,it used widely in folk and clinic.But so far there are less research on the chemical constituents and pharmacological activities of this species;even the study on pharmacodynamic substance foundation is bare.In this paper,a systematic research in chemical constituents and the pharm-acodynamic substance foundation of P.tsienense were carried out.On the basis of the study chemical constituents,the antioxidant effect in vitro and the interaction between the four flavonoid and human serum albumin were further studied.These may help to further study the pharmacodynamic material foundation of hepatoprotective effect and the mechanism of action of this medicine.The research results were following:1.According to using various chromatographic techniques,25 compounds were isolated from methanol extract of P.tsienense.And their structures were identified on the basis of spectroscopic analysis.It included 4 coumarins:scopoletin(PT-1),umbelliferone(PT-2),aesculetin(PT-3),scopoletin-7-O-?-D-glucosied(PT-4);14 flavonoids:tricin(PT-5),4'-meth--oxy-tricin(PT-6),apiolin(PT-7),luteolin(PT-8),quercetin(PT-9),axillarin(PT-10),luteolin-7-O-?-D-glucoside(PT-11),apiolin-7-O-?-D-glucoside(PT-12),apigenin-7-O-?-D-glucuronic acid methyl ester(PT-13),quercetin-3-O-?-D-gIucoside(PT-14),6-hydroxy-luteolin-7-O-?-D-glucoside(PT-15),luteolin-7-O-?-D-glucosideaid(PT-16),scopoletin-7-O-?-D-glucosie aid(PT-17),chrysoeriol-7-O-?-D-glucoside aid(PT-18).5 small molecular phenolic acids:cinnamic acid-2-O-?-D-glucoside(PT-19),3-hydroxy-4-methoxybenzoic acid(PT-20),p-hydroxybenzoic acid(PT-21),protocatechuic acid(PT-22),caffeic acid(PT-23),two sterol and its glycoside:?-sitosterol(PT-24),daucosterol(PT-25).And the compounds PT-(1-4),12-18,20-23 were all isolated from genus Pyrethrumta for the first time.2.According to the prophase researches about the pharmacological activities of P.tsienense,the result was shown that the total flavonoids in the ethanol extract of this herb can help to protect the hepatic injury to the mouse.Thus,on the basic of chemical constituents,we studied the pharmacodynamic substance foundation.It included the antioxidant effect of vitro and the interaction between the main flavonoid and human serum albumin(HSA)by spectroscopic methods.They may help to further study the pharmacodynamic substance foundation of hepatoprotective effect of this medicine.Firstly the DPPH and TEAC to study the antioxidant effect of vitro with the main substance in P.tsienense.The two methods were frequently used in antioxidant effect of vitro.In this case,the 12 chemical constituents were chosed,which were confirmed to flavones.The results indicated that the flavones had well antioxidant activity.This may help to further study the mechanism of action hepatoprotective effect and the ralation between flavone structure and activity.In the part of investigation of the interaction between the four flavones drugs(PT-11,PT-12,PT-14,PT-15)and human serum albumin(HSA),the optical spectroscopy methods,such as fluorescence,UV-vis absorbance and circular dichroism(CD)were used.After the experiment measurements,we calculated the drugs-HSA quenching constants,binding constants and combining sites at different temperatures,energy transfer parameters,thermodynamic parameters and the binding constants and sites in the presence of mental ions(Cu2+,Mg2+,Zn2+).At 25?,the binding constants,Ka was found as 7.74×105 mol/L,6.56×105mol/L,5.97×105 mol/L,2.81×10 mol/L,and n was 1.2146,1.2024,1.1968,1.2944 for the drugs-HAS,respectivily.From the results of thermodynamic parameters?G,?H,?S,they indicated the hydrophobic force and Vander Waals forces played a major role in the interactions of luteolin-7-O-?-D-glucoside-HSA(PT-11 to HSA),apiolin-7-O-?-D-gluco-side-HSA(PT-12 to HSA),quercetin-3-O-?-D-glucoside-HSA(PT-14 to HSA),6-hydroxy-luteolin-7-O-?-D-glycoside-HSA(PT-15 to HSA).And according to the Foroster theory of nonradiactive energy transfer,we calculated the distances r between the HSA and the acceptor of drugs,which were 1.265,1.2155,1.2911 and 1.2012nm,respectivively.The results indicated that the fluorescence quenching mechanism between the luteolin-7-O-?-D-glcoside-HSA(PT-11),apiolin-7-O-?-D-glycoside-HSA(PT-12),quercetin-3-O-?-D-glu--coside-HAS(PT-14),and 6-hydroxy-luteolin-7-O-?-D-glycoside-HSA(PT-15)were static quenching and nonradioactive energy transfer;Besides,the effect of the metal iron(Cu2+,Mg2+,Zn2+)to the drugs-HAS were investigated.In addition,the UV-vis absorbance,circ--ular dichroism(CD)and synchronous fluorescence spectrashowed the binding of the four flavones to the HSA can lead to conformational changes in HSA molecular.