| Objective:Lemonfragrant Angelica Root （LAR） is a unique plant species, with clearing heat, dispelling wind, cough relieving and sputum emlination, activing blood circulation to dissipate, promoting qi circulation and relieving pain effect, commonly used in clinical cough, chest pain syndrome, rheumatism, epigastric pain, traumatic injuries, etc.. Clinical application and literature research display that LAR has relaxation effects on the cardiovascular system, the respiratory system, the digestive system, the genitourinary system smooth muscle, especially aortic smooth muscle. However, the basic research of LAR is weak, the effective substance and mechanism of action is not clear. Our previous studies have shown that LAR ethyl acetate extraction has good active relaxation of vascular smooth muscle, this research based on our previous studies, ethyl acetate extract of LAR was isolated to obtain the active ingredients, and quality control of LAR have been studied also; and carried out its study of isolated rat thoracic aorta mechanisms of smooth muscle relaxation and explored the active ingredient of vascular smooth muscle cells of the mechanism at the cellular level. In this study, we explained the "promoting qi to relieving pain" scientific connotation of LAR from the organ level to the cellular level, and also this study can provide a reference for further research of LAR.Methods:1. The research of chemical constituents of ethyl acetate part from LAR. Experimental model of the rat thoracic aorta rings was applied, using "Bioactivity and fingerprint" as index, the active ingredients were isolated from LAR ethyl acetate extract, and the compound structures were identified. For active compounds, we used TLC and HPLC to study the quality control of LAR.2. The study of thoracic aortic rings relaxation by bioactive components from ethyl acetate. Using the rat thoracic aorta rings as the experimental model, investigate the relaxation effect of different concentration Isodillapiolglycol （Iso,200,400,800μ g/ml） on pre-contracted rat thoracic aortic rings （including endothelial and denuded aortic rings）by norepinephrine （NE） and potassium chloride （KC1）. In the absence of ca2+ free Kreb’s solution, investigate the contruction effect of removed endothelium aortic rings with Iso （800 μg/ml）, which induced by KC1 and CaCl2 investigate the relaxation effect of removed endothelium aortic rings with different concentrations Iso （200,400,800μg/ml）.which pre-contracted by NE and pre-incubated by verapamil; investigate the relaxation effect of removed endothelium aortic rings with different concentrations Iso （200,400,800 μg/ml）.which pre-contracted by NE and pre-incubation by TEA or BaCl2.3. The effects of Iso on the intracellular calcium concentration in cultured vascular smooth muscle cells. Using the laser scanning confocal microscope （LSCM） to detect the changes of calcium concentration in intracellular, investigate the calcium concentrations varition with different concentrations Iso （200,400,800 μg/ml）.which induced by KC1.Results:1. Five compounds were separated and purified from LAR ethyl acetate parts, Isodillapiolglycol,1’,2’-threo-1’,2’-dioxy-2,3-dimethoxy-4,5-methylenediox y-1-propylbenzene, Nodakenetin, （+）-Lariciresinol, Ostercitriodin A,, two of them were new compounds. We establish a thin layer chromatography （TLC） and high performance liquid chromatography （HPLC） method for the determination of isodillapiolglycol in LAR. The TLC spots of LAR were clear without interference of the negative control under the optimalchromatographic conditions.The calibration curve of isodillapiolglycol illustrated a good linearity relationship in the range of 0.0468-0.4684 μ g （r=0.9999）.The average recovery reached 101.43%（77=6）, and the relative standard deviation was 1.41%.2. Different doses of Iso had significant effects on both endothelium-intact and endothelium-denuded rat thoracic caorta vascular rings pre-contracted by NE and KC1, and showed a concentration dependence. When the concentration of Iso were 200,400 and 800 g/ml, there was no significant correlation between the endothelium-intact and endothelium-denuded rat thoracic caorta vascular rings, and the relaxation rate of the endothelium were not statistically significant.3. In the absence of calcium Kreb’s solution, after incubation with Iso （final concentration 800 mg/ml）, medicated group in different concentrations of calcium conditions, the relative shrinkage rate were significantly lower than control group.4. After the vascular rings pre-incubated with verapamil and pre-shrank with NE, medium and high dose Iso diastolic function to endothelium-denuded rat thoracic caorta vascular rings were significantly decreased in a dose-dependent manner. After the endothelium-denuded rat thoracic caorta vascular rings pre-incubated with TEA and pre-shrank with NE, medium and high dose Iso diastolic function to vascular rings were significantly decreased. After the endothelium-denuded rat thoracic caorta vascular rings were pre-incubated with BaCl2 and pre-shrank with NE, the low, medium and high dose Iso had no effects on vascular rings.5. LSCM experiment results showed that in calcium conditions, low, medium and high dose groups Iso maximum mean fluorescence intensity values were lower than CON group after KC1 stimulation, and fluorescence intensity of vascular smooth muscle cells are different degrees of inhibitory effects, the inhibition rates were 14.84%,34.82%,52.58%,43.18%, there is a certain concentration-dependent manner. In the absence of calcium Kreb’s solution, after NE stimulated and CaCl2 added, fluorescence intensity of each group were significantly increased, low, medium and high dose groups Iso maximum mean fluorescence intensity values were lower than CON group, the inhibition rates were 11.93%,24.23%,65.95%,81.33%, there is a certain concentration-dependent manner too.Conclusions:1. Compounds isolated from LAR in the majority belong to the phenylpropanoid constituents and quality standard research results show that the established method of isodillapiolglycol is qualitative and quantitative, the method is simple, accurate and reproducible.2. For the endothelium-intact and endothelium-denuded vascular rings, different concentrations Iso of the vascular ring relaxation were not statistically significant stimulated with KC1, NE, suggesting that the bioactivity of Iso on aorta rings is non endothelium dependent and might be related to endogenous vascular relaxing factor such as nitric oxide （NO） and prostacyclin. Iso has significant relaxation effect on endothelium-denuded vascular rings stimulated by NE, KC1, showing that Iso can inhibit the ROCC and VDCC to reduce the calcium concentration in the cells.3. In the absence of calcium Kreb’s solution, with the increase of calcium concentration, the caorta vascular rings tension in the control group was also increased. This confirmed that the KC1 mediated calcium internal flow leads to an increase in the intracellular calcium concentration in the vascular smooth muscle cells. After incubation with Iso （final concentration 800 g/ml）, medicated group in different concentrations of calcium conditions, the relative shrinkage rate were significantly lower than control group, this indicated that Iso can inhibit the VDCC and prevent the external calcium influx.4. After the vascular rings pre-incubated with verapamil and pre-shrank with NE, medium and high dose Iso diastolic function to endothelium-denuded rat thoracic caorta vascular rings were significantly decreased, this indicated that L-calcium channels participate in the relaxation of Iso. After the endothelium-denuded rat thoracic caorta vascular rings pre-incubated with TEA and BaCl2, and pre-shrank with NE, medium and high dose Iso diastolic function to vascular rings were significantly decreased, but the low, medium and high dose Iso have no effect on vascular rings pre-incubated with BaCl2, this proved that the calcium-activated potassium channels in the cell membrane are involved in the relaxation of Iso, whereas the inward rectifier potassium channel are not involved.5. Iso can acts upon VDCC, inhibit the intracellular calcium ion concentration induced by high concentration KC1; Iso can acts upon ROCC, which lead to intracellular calcium release and calcium influx so as to decrease calcium concentration in vascular smooth muscle cells. |
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