| ObjectiveAngelica citriodora Hance (Ostericum citriodorum (Hance) Yuan et Shan) is an endemic specie of Umbelliferae Ostericum herb in China, with a fuction "promoting blood circulation and removing blood stasis, promoting qi circulation and relieving pain". The early research conducted by our group indicated that the different polarity extraction parts (petreleum ether extraction parts, ethyl acetate extraction parts, n-butanol extraction parts) of Angelica citriodora Hance have the activity of diastolic blood vessels, and found a compound-isoapiole, isolated from the petroleum ether part, could relax the endothelium dependent vasodilatation to a certain extent. And at the same time, the found that the ethyl acetate part have excellent relaxation in vitro rat thoracic aorta with endothelial or removed blood vessels, and without difference. Prompting that there are also exists other compounds with vasodilation activity. Therefore, the experiment extended for previous studies though isolating compounds from ethyl acetate extraction part of Angelica citriodora Hance, screening active substance "Marmesin" and studying the activity and its mechanism using in vitro vascular perfusion apparatus, and combined with LSCM to detect the effects of Marmesin on the calcium ion concentration in vascular smooth muscle cells(VSMCs) in order to further study the diastolic blood vessels efficacy.In a word, the experiment studied the vasorelaxation fuction of Marmesin from the level of organs to the cellular level to enrich the scientific connotation "promoting qi circulation and relieving pain" of Angelica citriodora Hance, and to provide scientific evidence for the clinical use of it.Methods1, Isolation coumpounds from ethyl acetate extraction of Angelica citriodora Hance:reduplicative and intersectional using silica gel column, ODS column, Sephadex LH-20, preparative liquid chromatograph and combined tightly with TLC, HPLC to separation, merging, and purification. And identified the structure by ESI-MS (or HR-ESI-MS),’H-NMR,13C-NMR and DEPT spectra, and compared data with published paper.2, ① Using in vitro vascular ring perfusion experiment to observe the vasodilation effects of Marmesin (25,50,100 μg/mL) on rat thoracic aorta endothelium-intractrings and endothelium-denuded rings precontrated by norepinephrine (NE) or potassium chloride (KC1),and the influence of the vascular tension under resting state. ② The effects of marmesin on calcium inflow. Endothelium-denuded rings were incubated with marmesin(100 μg/mL) before stimulated by KC1(60 mM) in the absence of calcium Kreb’s liquid (containing 0.5 mM EGTA), and then added CaCl2 cumulately. Observing the change of calcium chloride concentration-response curves with or without marmesin; ③ Effects of marmesin on ion channels. In the Kreb’s liquid, endothelium-denuded rings were incubated with blocking agent verapamil and TEA before stimulate with NE, respectively, and compare the the relaxtion effects with that before blocking agent. ④ Influence of marmesin on internal calcium ions release:endothelium-denuded rings incubated in calcium-free Kreb’s liquid (containing 0.5 mM EGTA), and then add marmesin (100 μg/mL)or DMSO for 15 min before stimulate with NE, comparing the amplitude of contraction of medicine incubation with DMSO incubation.3, Effects of Marmesin on calcium ion concentration within the cytoplasm of A7r5 VSMCs. With calcium fluorescent probe Fluo-3, AM loaded cells, using laser scanning confocal microscope (LSCM) to real-time measure the change of marmesin of increase of [Ca2+]i in cytoplasm induced by potassium chloride (KC1) or norepinephrine (NE) and calcium, respectively.Results1, Six compounds were obtained from the ethyl acetate extraction parts of Angelica citriodora Hance and 5 compounds were identified at the moment, containing one new natural product, temporarily named as 8-(3,7-Dimethyl-octa-2,6-dienyl)-7-hydroxyl-6-methoxy-chromen-2-one, and other four compounds,9-angeloyloxy-10-senecioyloxy-9,10-dihydroxanthyletin,4,5-Dimethoxy-2,3-methylenedioxy-benzylalcohol and marmesin, first obtained from the herb.2, ① Marmesin played an concentration dependent-manner role both on relaxing endothelium-intacted and endothelium-denuded rings precontracted by NE and KC1 (P<0.05), and there were no obvious difference between rings with or without endothelium; At the same time, to the resting state rings marmesin had no effect on vascular tension. ② Marmesin could induce calcium chloride concentration-response curves down significantly (P<0.05); ③ Blocking agent-verapamil, TEA could decreases the vasodilatation function of marmesin (P< 0.05). ④ After preincubated rings with marmesin, the contraction caused by NE in calcium-free kreb’s solution was inhibited to a certain degree.3, Different concentrations of marmesin could obviously make the extremum of fluorescence density value induced by KC1, NE and calcium chloride down (P<0.05).Conclusions1, Six compounds, the majority of them were phenylpropanoids, were obtained from Angelica citriodora Hance. One of which was a new natural product, temporarily named as 8-(3,7-Dimethyl-octa-2,6-dienyl)-7-hydroxyl-6-methoxy-chromen-2-one. others were 9-angeloyloxy-10-senecioyloxy-9,10-dihydroxanthyletin,4,5-Dimethoxy-2,3-methylenedioxy-benzylalcohol and marmesin, first obtained from the herb.2, Marmesin might directly act on vascular smooth muscle to relax blood vessels in a dose-dependent manner. The vasodilative mechanisms may relate to inhibition of ROCC, VOCC activity, blocking L-type calcium ion channels, open calcium activated calcium channel (KCa), as well as the inhibition of calcium ion release.3, Marmesin could inhibit calcium ion inflow caused by KC1 and NE. |
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