| Objective:Lemonfragrant Angelica Root (LAR) (Ostericum citriodorum (Hance) Yuan et Shan)is the fragrance of endemic plants in China, with " promoting qi circulation and relieving pain" effect, clinically used in the treatment of angina pectoris due to smooth muscle spasm, stomach pain and abdominal pain and other diseases, and the treatment is effective, but that its material basis and mechanism of action is not clear. Our previous studies have found a lower moiety of LAR - petroleum ether and a representative of the active ingredient-isoapiole,which have vasodilating action in isolated rat thoracic aorta endothelial vascular rings, and isoapiole can endothelium-dependent relaxed the isolated aortic rings. Continuation of pre-study, this subject further investigates vasorelaxation effect induced by an n-butanolic fraction of LAR in rat thoracic aorta and its mechanism of action, it carries out vasorelaxation effect induced by an n-butanolic fraction of LAR in rat thoracic aorta, and the effect of an n-butanolic fraction of LAR on intracellular calcium level in cultured vascular smooth muscle cells; the chemical compositions were isolated from an n-butanolic fraction of LAR and the pharmacodynamic activity of isolated compounds; and it investigates activity of coarse sub-parts.which got from an n-butanolic fraction of LAR macroporous resin. From the organ to the cellular level it discusses its related mechanism, aiming to further illustrate the scientific connotation of high polar fractions of LAR, and to provide a theoretical basis for further clinical development and application.Methods:1.The contraction activity and possible action mechanisms of an n-butanolic fraction of LAR on rat thoracic aorta vascular rings with or without endothelium in vitro. Experimental model of the rat thoracic aorta rings was applied to investigate the related effects of an n-butanolic fraction of LAR (0.34、0.65、1.22mg/mL) on both endothelium-intact and endothelium-denuded aortic rings tensions, which induced by potassium chloride (KCl) and Norepinephrine (NE). In the absence of Ca2+ Kreb’s solution, it investigated the related effects of different concentrations of an n-butanolic fraction of LAR (0.34、0.65、1.22mg/mL) on endothelium-denuded aortic rings tensions, which induced by Cacl2 and Norepinephrine (NE).2. The effect of an n-butanolic fraction of LAR on the concentration of intracellular calcium level in cultured vascular smooth muscle cells. By using laser scanning confocal microscope (LSCM), the [Ca2+] i Fluorescence signal changes was investigated in VSMC loaded with Ca2+indicator Fluo-3/AM. In Hank’s solution (calcium concentration was 2.Ommol/L), to investigate the effect of an n-butanolic fraction of LAR (0.65mg/mL) on the calcium ion concentration increases in cytoplasm, which induced by potassium chloride (KCl); In D-Hank’s solution (calcium concentration was 0. Ommol/L) to investigate the effect of an n-butanolic fraction of LAR (0.65mg/mL) on the calcium ion concentration increases in cytoplasm.which induced by Norepinephrine(NE)and thapsigargin(TG).3. The contraction activity of monomer components isolated and purified from an n-butanolic fraction of LAR on rat thoracic aorta vascular rings with or without endothelium in vitro. Extracting、separating、purificating the compounds in an n-butanolic fraction of LAR by the phytochemical separation method, and experimental model of the rat thoracic aorta rings was applied to investigate the related effects of compound Ⅰ, compound Ⅱon on both endothelium-intact and endothelium-denuded aortic rings tensions, which induced by potassium chloride(KCl) and Norepinephrine(NE).4. The contraction activity of coarse sub-parts, which got from an n-butanolic fraction of LAR macroporous resin on rat thoracic aorta vascular rings with or without endothelium in vitro. Experimental model of the rat thoracic aorta rings was applied to investigate the related effects of the water solution and 30%、60%、95%alcohol solution of an n-butanolic fraction of LAR via macroporous resin on both endothelium-intact and endothelium-denuded aortic rings tensions, which induced by potassium chloride(KCl) and Norepinephrine(NE).Results:1. An n-butanolic fraction of LAR caused concentration relaxation of rat thoracic aorta vascular rings preconstricted with potassium chloride (KC1) in both endothelium-intact and endothelium-denuded rat thoracic aorta vascular rings, and the relaxation had not effect in endothelium-denuded rat thoracic aorta vascular rings;An n-butanolic fraction of LAR caused concentration relaxation of rat thoracic aorta vascular rings preconstricted with Norepinephrine (NE) in both endothelium-intact and endothelium-denuded rat thoracic aorta vascular rings, and the relaxation had not effect in endothelium-denuded rat thoracic aorta vascular rings; Denuded aortic rings in the absence of Ca2+conditions, after the concentration of Ca2+ in bath solution gradually increased (Maximum concentration was3. OmM), aortic rings further contracted correspondingly, compared with the control group, An n-butanolic fraction of LAR could cause contraction intensity to be lowered significantly;Denuded aortic rings in the absence of Ca2+ conditions, an n-butanolic fraction of LAR (0.34、0.65、1.22mg/mL) could cause significant decrease of contraction induced by Norepinephrine (NE) at calcium-free solution.2. Compared with control group, an n-butanolic fraction of LAR on VSMC cytopasm had no significant impact at resting state ((1.06±0.09) vs. (1.07 ±0.14), P>0.05).An n-butanolic fraction of LAR had a certain inhibition effect on the calcium ion concentration increases in cytoplasm by potassium chloride(KCl) 、 Norepinephrine(NE) and thapsigargin(TG)[(3.76 土1.58) vs.(1.76±0.28);(4-53±1.57) vs.(0.89±0.13);(4. 48土0.83〉vs.(1.66±0.46),P<0. 01]. The peak inhibition rates of an n-butanolic fraction of LAR were(48. 18± 15_ 12)%、(49.67 ± 16. 57)%、(52. 90 ±9. 57)% respectively induced by potassium chloride(KCl)、Norepinephrine(NE) and thapsigargin(TG) at the concentration of 0. 65mg/mL3.From the n-butanolic fraction of LAR, 2 compounds were isolated, which called compound I、compound II. Compound I、compound II presented poor relaxant effects in both endotheliunrintact and endothelium-denuded aortic rings by potassium chloride(KCl) and Norepinephrine(NE)(P>0,05).4.The water solution、30% alcohol solution and 60% alcohol solution of an n-butanolic fraction of LAR via macroporous resin presented poor relaxant effects in both endothelium-intact and endothel ium-denuded aortic rings by potassium chloride (KCl)、Norepinephrine(NE) and thapsigargin(TG)[(3.76± potassium chloride (KC1) and Norepinephrine(NE) (P> 0.05).95% alcohol solutionan of an n-butanolic fraction of LAR via macroporous resin had a certain vasorelaxant effect in both endothelium-intact and endothelium-denuded aortic rings by potassium chloride (KCl),and the relaxation had not effect in endothelium-denuded rat thoracic aorta vascular rings.95% alcohol solution of an n-butanolic fraction of LAR via macroporous resin had a certain vasorelaxant effect in both endothelium-intact and endothelium-denuded aortic rings by Norepinephrine(NE), and the relaxation had not effect in endothelium-denuded rat thoracic aorta vascular rings.Conclusions:1. The n-butanolic fraction of LAR had a certain vasorelaxant effect in both endothelium-intact and endothelium-denuded aortic rings, which was without endothelium dependent manner, and the effect of which was with concentration-dependent manner. And its possible mechanisms have relation with rat aorta rings through by blocking voltage-dependent calcium channels (VDCC) and receptor-operated calcium channels (ROCC), and furtherly inhibiting IP3 sensitive calcium release, which lead to decrease intracellular calcium release and calcium influx so as to decrease calcium concentration in cytoplasm.2. An n-butanolic fraction of LAR could inhibit the increase of calcium concentration in vascular smooth muscle cells, which induced by potassium chloride (KCl)、Norepinephrine(NE) and thapsigargin(TG). The possible mechanisms were:1, by blocking voltage-dependent calcium channels (VDCC) on the cell membrane; 2, by blocking the sarcoplasmic reticulum IP3-sensitive Ca2+ channels on the retina reduce the endoplasmic reticulum Ca2+ release and inhibits receptor-operated calcium channels (ROCC) on the cell membrane; 3, through the activation of sarcoplasmic reticulum Ca2+ -ATPase, antagonizing calcium store depletion, and antagonizing store-operated calcium channels (SOCC).3. Compound Ⅰ、compound Ⅱpresented poor relaxant effects in aortic rings. And it implied that compound Ⅰ、compound Ⅱ was not the n-butanolic fraction of LAR vasodilation representative ingredients.4. The water solution of an n-butanolic fraction of LAR via macroporous resin presented poor relaxant effects in aortic rings. And 95% alcohol solution of an n-butanolic fraction of LAR had a certain vasorelaxant effect in both endothelium-intact and endothelium-denuded aortic rings, which was without endothelium dependent manner. It implied that 95% alcohol lotion can be further isolated and purified, and the monomers isolated from 95% alcohol lotion could represent an n-butanolic fraction of LAR, which a certain vasorelaxant effect in both endothelium-intact and endothelium-denuded aortic rings, which was without endothelium dependent manner... |
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