Study On Porcine Coronary Vasodilatation Mechanism And Pharmacology Of Ligustilide

This study separation, identification, determination of the content of ligustilide in volatile oil of Ligusticum Chuanxiong; The porcine isolated coronary artery rings experiment to study the mechanism of ligustilide on coronary artery rings; To observe the ligustilide protective effect of ischemia reperfusion injury in rat myocardium by SD isolated rat heart ischemia reperfusion model. Through the experimental study to provide reference for clinical practice and further research of ligustilide.Part One The extraction and separation of qualitative and quantitative analysis of ligustilidePurpose:Establishment of an experimental method of ligustilide extract from Chuanxiong volatile oil, And the ligustilide extracts were analyzed by qualitative and quantitative analysis.Method:Silica gel G thin layer chromatography determine the silica gel column chromatography conditions of extraction and separation of ligustilide from volatile oil of Ligusticum Chuanxiong; UV absorption of HPLC and Mass spectra of LC-MS were used for qualitative analysis of ligustilide; Area normalization method of gas chromatography for were used the determination of ligustilide.Results:To establish a method for separating and extracting ligustilide from the essential oil of Ligusticum chuanxiong Hort,and determining the extraction material is ligustilide, Area normalization method of gas chromatography determine the content of extracted ligustilide was95.25%.Conclusion:Analysis of ligustilide by this method for extraction and separation, qualitative and quantitative, high extraction rate, strong specificity, can get high purity of ligustilide.Part two Study on porcine coronary vasodilatation mechanismPurpose:By observing the effect of ligustilide relaxation on porcine coronary artery rings, probes into the action mechanism of ligustilide in porcine coronary arteries.Method:By using the method of the isolated porcine coronary arterial rings experiment, observe the relaxation effect of ligustilide on resting state and prostaglandin (PGF2a) pre contraction on porcine coronary artery; Compared the relaxation rate of coronary artery rings of ligustilide alone and pretreatment with endothelial cells, nitric oxide synthase inhibitor N-nitro-L-arginine (L-NNA), prostaglandin synthetase inhibitor indomethacin (Indo), guanylate cyclase inhibitor methylene blue (MB), calcium activated K+channel inhibitor (tetraethylammonium, TEA), inward rectifier K+channel inhibitor barium chloride (BaCl2), ATP sensitive K+channel inhibitor glibenclamide (Glyburide, Gly), non selective beta adrenergic receptor antagonist propranolol (pro), beta1-adrenergic receptor antagonist Ati Carol (atenolol), beta2-adrenergic receptor inhibitor ICI118551; And observe the influence of effect curve of ligustilide on the potassium chloride (KCl), calcium chloride (CaCl2), histamine induced contraction of porcine coronary artery rings.Results:The larger dose of ligustilide have diastolic effect on isolated porcine coronary arterial rings in resting state;Ligustilide was dose dependent vasodilation effect on PGF2a pre contraction porcine coronary artery ring; Removal of the endothelium, L-NNA, MB, Indo, all of them reduce the relaxation of ligustilide on coronary artery rings; TEA,BaCl2,Gly,pro,atenolol,ICI118551, was not inhibited vasodilation of ligustilide on coronary artery rings; Ligustilide could inhibit the KC1, CaCl2and histamine induced contractions on coronary artery rings; Ligustilide has significant inhibitory effect calcium free Krebs solution the first systolic coronary artery rings induced by histamine and the second systolic coronary artery rings induced by Ca2+.Conclusion:Vasodilation effect of ligustilide on porcine coronary artery rings is mainly through the calcium to prevent the cell membrane influx and sarcoplasmic reticulum calcium release;and prostacyclin (PGI2) pathway and endothelial-NO-cGMP pathway are also partly reason on ligustilide induces porcine coronary arterial rings diastolic; But the beta adrenergic receptor and potassium channel do not participate in the diastolic of ligustilide on porcine coronary artery rings. Therefore, Diastolic function mechanism of ligustilide on porcine coronary artery rings are endothelium-dependent and non endothelium dependent ways combined action result.Part three Protective effect of ligustilide on isolated rat heart ischemia-reperfusion injuryPurpose:Establish the Langendorff model of in vitro myocardial ischemia reperfusion, to observe the protective effects of ligustilide on isolated myocardial ischemic reperfusion injury in rats.Method:18male SD rats were randomly divided into three groups:Blank perfusion group, ligustilide1group and ligustilide2group,6rats in each group. Establish the Langendorff model of the isolated rat heart, recording the heart function index data of each time point of control group, ligustilide1group, ligustilide2group on constant temperature and constant pressure perfusion,and the time point is perfusion30minutes, reperfusion5minutes,10minutes,20minutes,30minutes,and the heart function index data is the left ventricular diastolic pressure(LVDP)、The internal pressure of left ventricle increased the maximum rate of change (+dp/dtmax) and left ventricular pressure drop maximum rate of change (-dp/dtmax); collect and measure cardiac perfusion30minutes after reperfusion fluid volume of5minutes,10minutes,20minutes,30minutes; collect the perfusate in each group of reperfusion20min, determinate the CK and LDH activity.Results:Compared with the blank group, ligustilide was dose dependently increased the heart of the LVDP,+dp/dtmax and absolute value of-dp/dtmax; compared with the blank group, increase of ligustilide dose-dependent cardiac reperfusion fluid volume; compared with the blank group, the contents of LDH and CK in blood of ligustilide dose dependently reduced.Conclusion:Ligustilide can dilate coronary artery, increased blood flow in the coronary artery,improve blood flow in myocardial ischemic area, enhance increased left ventricular systolic function and diastolic function, Remove enzymes of myocardial metabolism, The damage of myocardial ischemia reperfusion is in a certain degree repair.