Studies Of Chemical Constituents And Anti-ischemic Activity Of The Essential Oil Of Syringa Pinnatifolia Hemsl．Var．Alashanensis Ma Et S. Q. Zhou

Ischemic heart disease (IHD) is one of the world's major diseases, and its incidence and mortality has been high worldwide. It is serious harmful to human health, since no sufficient therapy for this obstinate illness is available. Research and development of anti-ischemic drugs has been the focus of drug research. Attempts are made globally to get complementary and alternative medicines for IHD. In China, the application of Traditional Chinese Medicine (TCM) and folk medicines has a long history. Many of the durgs have anti-ischemic activity, rich in resources. Searching new active drug in these natural products and materials which have definite clinical effect is very effective and important in development of new drugs.Mongolian medicine is one of the most important ethnic drugs in China with its own distinctive feature, which has a long application history and is still widely used in Inner Mongolia. However, because of the late starting of modern research, many of Mongolian medicines have known exact effect but unknown material basis and pharmacological effects. Syringa pinnatifolia Hemsl.var.alashanensis Ma et S. Q. Zhou (Shanchenxiang) is one among these. The stem of Shanchenxiang is one of the best-known traditional herbal medicines frequently used to treat cardiovascular symptoms in Mongolian medicine. It is widely used in Mongolian in treatment of chest tightness, shortness of breath, myocardial ischemia and other cardiovascular diseases, basicly"all the diseases induced by hot evil invasion", and has a broad application in the prevention and treatment of IHD. However, there is few reported scientific study to support these claimed therapeutic and medicinal effects.In this paper, Syringa pinnatifolia Hemsl.var.alashanensis Ma et S. Q. Zhou essential oil (SPEO) was chosen as the major research object, and its anti-ischemic effect was studied with a variety of animal models and cell models, to confirm different levels of its cardioprotective effect. At the same time, headspace liquid phase microextraction (HS-LPME) was used in the rapid analysis of chemical components of SPEO and determination of zerumbone in the herbal material. At last, chemical constituents of ethyl acetate extract of Shanchenxiang was studied. The main results are as follows:(1) A headspace liquid phase microextraction coupled with gas chromatography- mass spectrum (HS-LPME-GC-MS) method was described for the analysis of volatile compounds from Syringa pinnatifolia Hemsl.var.alashanensis Ma et S. Q. Zhou. Parameters of optimized conditions were: sample powder 1.0 g (80 mesh), 1.0μL of benzyl alcohol extracted at 70℃for 20 min. Analysis of volatile compounds in Shanchenxiang by HS-LPME-GC-MS method was performed under the described conditions and 52 compounds were identified,and the number of the identified compounds was 50 for steam distillation (SD)-GC-MS. HS-LPME-GC-MS shared 36 same compounds with the SD method while the total content of the same components accounted for 95% of the essential oil derived by SD method. HS-LPME is simple, fast, with low energy consumption, few use of drugs and solvents, and can achieve similar results with SD method, indicating that it can be used as an alternative method to SD for the rapid analysis of volatile compounds in Shanchenxiang and other TCMs.(2) Ultrasound assisted HS-LPME gas chromatography (UA-HS-LPME-GC) was applied in determination of zerumbone in Shanchenxiang. UA-HS-LPME parameters, such as ultrasonic solvent, ultrasound time, extraction solvent, extraction temperature and extraction time were optimized. The method was comprehensively validated. The results showed that the method for determination of zerumbone was specific, accurate and reproducible.(3) Effect of SPEO on myocardial ischemia was investigated. In hypoxia tolerance test of mice, the survival time of mice under hypoxia was chosen as an index. The results showed that the SPEO 8 mg/kg and 32 mg/kg could significantly prolong the survival time of mice under hypoxia, the survival time of 32 mg/kg SPEO is close to positive drug propranolol, indicating that SPEO can enhance the body's overall tolerance of hypoxia. This effect may be related to increasing of myocardial blood flow and reduction of myocardial oxygen consumption which are critical in myocardial ischemia.The acute myocardial ischemia (AMI) model in rats was induced by ligation of left anterior descending artery, and the extent of myocardial ischemia, ischemia and damage markers in serum enzymes were tested to study the effect of SPEO on acute myocardial ischemia. Pathology slides and myocardial ultrastructure were also observed. The results showed that SPEO can reduce the deviation of ST segment in ECG, reducing the extent of myocardial ischemia. SPEO can also decreased the levels of lactate dehydrogenase (LDH), creatine kinase (CK) and cardiac Troponin T (cTn T), while increased the activity of superoxide dismutase (SOD). The protective role of SPEO was further confirmed by histopathological examination. Pretreatment with SPEO exhibited decreased degree of necrosis and less infiltration of inflammatory cells, and can protect the mitochondria, sarcoplasmic reticulum and other important structures in cardiac myocytes. The results suggest that SPEO has significant activity against acute myocardial ischemia. It can increase the ability of myocardial cells against lipid peroxidation, stabilize the cell membrane to protect the myocytes.The in vitro effect of SPEO against platelet aggregation was investigated using adenosine 5'-diphosphate (ADP) as agonist. Electrical resistance method was used in the test and aggregation was induced in the whole blood. The degree of platelet aggregation was represented by resistance quantity. The results presented that SPEO can significantly inhibit the platelet aggregation induced by ADP and high dose group (5μg/mL) exhibited the maximum inhibition rate of 47.4%, indicating good anti-platelet aggregation activity of SPEO.(4) The protective effect of SPEO on H2O2-induced oxidative damage and hypoxia/reoxygenation injury was evaluated. The results show that SPEO can significantly protect the cell injury in these models. Meanwhile, SPEO can reduced the leakage of LDH and increased SOD activity after oxidative injury, indicating a direct and significant protective effect on cardiac myocytes.(5) Effects of different polar fractions of Shanchenxiang on H2O2-induced cardiac myocytes injury were studied. The chemical composition of ethyl acetate extract was studied according to activity screening results. Using a combination of various separation methods, four compounds were finally isolated from the extract. After structural analysis and comparison, the purified compounds are identified as lariciresinol, secoisolariciresinol, 3',4'-acetal-lariciresinol and (+)-dihydrocubebin, respectively.The results of this study are the first report on effect of SPEO on myocardial ischemia in multiple levels. The role of SPEO in cardioprotection was defined. Phytochemical study and quality control index of the Materia Medica will provide basis and foundation for follow-up research of Syringa pinnatifolia Hems1. var. alashanensis Ma et S. Q. Zhou.