Safflower Yellow Pigment Anti-ischemic Mechanism Of Action

1.ObjectiveTo observe the mechanisms of ameliorating myocardial ischemia of Safflor Yellow(SY) and its effective ingredient Hydroxysafflor Yellow A.2.Methods(1)Preparation and analyze of components of Carthamus tinctorius L..The aqueous extract of Carthamus tinctorius L.was added to macroporous resin column and the SY and HSYA was prepared individually through chromatographic procedure.The HSYA and SY were analyzed with HPLC technique.(2)In vivo testsAfter isoprenaline(ISO) injection SY was given ip into rat every day.At the third day the catheter was put into animals and the blood pressure and ventricular inner pressure were measured.After experiment blood and cardiac muscle were taken and plasma free fatty acid,cardiac muscle ATP content or MDA level were assayed.(3)Cell testsCardiac muscle of sucking rat was cut and digested with trypsin then it was cultured under standard condition.At the third day the myocardial cell was put into buffer without sugar,after HSYA addition N2 was given to make hypoxic condition.After the hypoxic injury the supernate was taken to measure LDH activity and the ATP of cells was assayed. (4)Other in vitro testsThe rat cardiac muscle was taken to prepare mitochondrion.Then HSYA was put into mitochondrion suspension.After incubation OD520 was measured to test the mitochondrion swelling reduction effect of HSYA.The fluorescence was measured to assay the fluidity of mitochondrion.HSYA was added into H2O2/Fe2+ system and MDA was measured to test the mitochondrion protection effect of it.After radio ligand receptor binding assay the β receptor antagonistic effect of SY and HSYA were tested and K or IC50 was calculated with Prism 3 software.The three antioxidative effects of HSYA and SY were tested with colorimetric techniques. (5)Gene different display experimentAfter ISO and SY given the myocardial ischemia injured rat cardiac muscle was taken and RNA of it was extracted.HSYA was added into hypoxic injured sucking rat cell and its RNA taken.Then gene different display test showed the changes of above myocardium gene expression. 3.Results(1)Analyze of components of Carthamus tinctorius L.The results of HPLC analyze showed that the content of HSYA in SY was 83%.The purity of HSYA was over 98 %.(2)In vivo testsIt was showed in the results that SY was active to reduce ISO induced heart function reduction.The changes of +dp/dtmax 及-dp/dtmax and many other parameters after myocardial ischemic injury was ameliorated.And propranolol was effective to ameliorate these injuries.It was showed in the HE staining slices that the apparent denaturalization of myocardium was found after myocardial ischemia and SY was not effective to reduce these damages.SY was active to ameliorate the changes of blood pressure of myocardium ischemic rat.And cardiac muscle ATP reduction and MDA elevation of myocardium ischemic rat can be ameliorated by SY or propranolol.(3)Cell experimentHSYA were effective to reduce LDH leakage and ATP content reduction of low sugar and hypoxic injured cultured sucking rat myocardial cell.(4)Other in vitro testsIt was showed by in vitro tests that SY or HSYA was effective to scavenge hydroxyl radical and to inhibit rat myocardium mitochondrion swelling,mitochondrion membrane fluidity decrease,mitochondrion lipid peroxidation induced by hydroxyl radical,mouse liver suspension lipid peroxidation and erythrocyte broken induced by hydroxyl radical. SY or HSYA was active to inhibit specific binding of 125I-s-(-)-Pindolol to rat ventricular cell β receptor.It was shown by scatchard analyze of the results of125I-s-(-)-Pindolol binding test that Bmaxwas was 169.0 pmol/g,KD was 202.4 pmol/L。For SY, IC50 was 17.5±1.3 g/L, KiSY was 12.9 g/L respectively. For HSYA ,IC50 was 35.6±1.4 mmol/L ,KiHSYA was 26.4 mmol/L respectively.(5)Gene different display experimentExcept for some false positive results was found during experiment,no significant myocardium gene expression change was found after injury of rat myocardium ischemia induced by ISO or hypoxic injury...