Isolation And Identification Of Pronuciferine Monomer And Its Effects On Functions Of Cultured Human Umbilical Vein Endothelium Cells

Endothelial cells located between blood and vascular smooth muscle cells. Due to its unique position in the vessel wall, impairment of its structure or dysfunction plays pivotal role in the pathogenesis of many cardiovascular diseases, especially being an initiating event in atherogenesis, and contributing to plaque initiation and progression of peripheral artery disease. Endothelial dysfunction is characterized by a shift of the actions of the endothelium toward reduced vasodilation, and prothrombic properties, which was caused by the decrease of endothelium-derived relaxing factor(EDRF), such as Nitric oxide(NO), and the increase of endothelium-derived growth factor(EDGF), such as endothelin(ET). Apoptosis of endothelial cells plays an important effect in the endothelium detriment. There were many motivations to the apoptosis. Angiotensin â…¡ may induce apoptosis of endothelial cells by causing disequilibrium of NO ,which have been concentrated recently.Because traditional medicine has mild effect and lower side-effect than modern medicine, it is important to discover a traditional drug which can protect cardiovascular function and reverse the progress of cardiovascular disease. The seed of Nelumbo nucifera is one of the important traditional medicine. It had effects on easing mental anxiety, and restoring normal coordination between heart or kidney and haemostasis. More than twenty isoquinoline alkaloids have been isolated from theseed of Nelumbo nucifera so far. Pronuciferine was a pro-aporphine alkaloid which was also isolated from plumula nelumbinis by a Japanese scholar about twenty years ago. No reserch about its pharmacological function has been performed so far. However, many researches revealed Neferine and Lensinine both of which were also isolated from plumula nelumbinis could block calcium channel, and inhibit platelet aggregation, and protect the endothelial cells. We supposed that Pronuciferine might have the same effect on endothelial protection.So, the isolation and characterization of Pronuciferine and its pharmacological function on HUVEC have been performed in this study. The detailed protocols and results are as follows:1 Isolation and identification of Pronuciferine monomer. MethodsProtocol 1: The primary compositions in the seed of Nelumbo nucifera were extracted by supercritical CO2 technique, and were assayed by GC-MS. Protocol 2: Pronuciferine monomer were isolated by column chromatography fractionation from the total extraction of SFE and identified by HPLC, TLC, GC-MS. Results Under the pre-set parameters of SFE, the total extraction coefficient was 2.5%. There are fourteen of different components, including Pronuciferine, in the extraction. Pronuciferine monomer was further isolated by column chromatography fractionation, and an exclusive component was identified by HPLC, TLC, GC-MS. Conclusion The Pronuciferine monomer could be isolated from the seed of Nelumbo nucifera by supercritical CO2 way and column chromatography fractionation, which will be available for further functional study. 2.Effects of Pronuciferine on cultured Human Umbilical Vein Endothelium Cells. Methods The experiments were performed on the culture of Human Umbilical Vein Endothelium Cells(HUVECs) in vitro.Protocol 1: Control group, nifedipine group(10umol/L), Pronuciferine group (100umol/L,10nmol/L,lumol/L,0.1umol/L and O.Olumol/L respectively) were assigned randomly.The cases of each group were 6. After being coincubated for 24 hours, cell- morphous was observed by light microscope. Cells viability wereassessed by MTT assay. NO, total nitric oxide synthase(tNOS) and inducible nitric oxide synthase(iNOS) were measured by Colorimetry.Protocol 2: Control group, angiotensin II(AngII)group(lumol/L), captopril +AngII group (after being incubated with lOumol/L captopril for 30 minuters, then lumoI/L Ang II were coincubated), Pronuciferine +Ang II group(after being incubated with 100umol/L,10umol/L,l^mol/L,0.1umol/L and O.Olumol/L respectively for 30minutes, lumol/L of Ang II were then coincubated). The cases of each group were 6. Being coincubated for 24 hours, cell-morphous was observed by light microscope. Cells viability were assessed by MTT assay. NO, tNOS and iNOS were measured by Colorimetry. The percentage of apoptosis was measured by flow cytometer(FCM). Results1. Compared with control group,both cell-morphology and viability weren't affected by Pronuciferine. 0.01~10umol/L Pronuciferine and lOumol/L nifedipine could significantly increase the level of NO and the activity of tNOS, but had no effect on the activity of iNOS. Moreover, no effect was observed in lOOumoI/L Pronuciferine group.2. Compared with control group, Ang II could decrease cell viability and induce typical endothelial cell apoptosis, but had no effect on the level of NO, the activity of both tNOS and iNOS. The positive apoptotic cell percentage in Ang II group was significantly higher than that of the control group. 0.01~10umol/L Pronuciferine and lOumol/L captopril could significantly increase the level of NO and the activity of tNOS, but had no effect on the activity of iNOS. Compared with control group.both cell viability and the positive apoptotic cell percentage were significantly inhibited by 0.01~10umol/L Pronuciferine and 10umol/L captopril, but no effect was observed in 100umol/L Pronuciferine group.Conclusion Pronucifeine in dose range of 0.01~10umol/L could decrease thepositive apoptotic cell percentage induced by Ang II through increasing the activity ofeNOS and the level of NO. Pronucifeine may have the protection on endothelialfunction.