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Studies on Chemical Constituent from Selaginella Pulvinata Maxim and Effects of Selaginellin on Senescence of Endothelial Cells

Author: TanGuiShan
Tutor: LiYuanJian;DengHanWu
School: Central South University
Course: Pharmacology
Keywords: Selaginella selaginellin endothelial cells senescence leading compound Selaginella pulvinata Maxim pulvinatadione pulvinatabiflavone pulvinataphentriol pulvinatadiol pulvinatahyde pulvinataphendiol homocysteine cell senescence reactive oxidative species telomerase activity klotho
CLC: R285
Type: PhD thesis
Year: 2007
Downloads: 283
Quote: 6
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Chapter 1Research objectives were identified on the basis of extensive overviews about activity components and pharmacologic action in genus Selaginella.Systematic studies will be taken by the means of modernistic chormotography separate methods and advanced spectrum methods.Among them,the point is the analogs of selaginellin. What’s more,the effects of selaginellin on senescence of endothelial cells will be elucidated on cells level which is induced by homocysteine.The ultimate aim is to uncover a novel leading compound which is effective to heart-blood disease such as AS,and to provide test evidences for research about creative new drugs. Chapter 2 Studies on Chemical Constituents in Selaginella pulvinata Maxim.BACKGROUNDSelaginella pulvinata Maxim.,one species of genus Selaginella,family Selaginel,was introduced in Chinese Pharmacopeia(2005 Ed)for the effectiveness of promoting blood circulation.Since 1971 when Okigawa obtained 5 flavones from S.tamariscina for the first time,34 flavones have been isolated and identified from genus Selaginella.Other chemical constituents include phenylpropanoids,alkaloids, organic acids,anthraquinones,steroids,et al.Zhang et al.isolated a compound of novel carbon structure,named as selaginellin.Pharmacological studies have revealed that biflavones from genus Selaginella exhibit activities such as anti-oxidant,anti-virus,and protective effects on cardiovascular system.Moreover,anti-inflammation and immunity-improvement have also been reported.With the purpose of elucidating the active chemical constituents of S.pulvinata Maxim.,in particular these compounds with similar structures to selaginellin,using modern chromatographic methods and spectroscopic properties.METHODSColumn chromatography and prepared HPLC were used to isolate and purify chemical constituents.Spectroscopic properties such as UV,IR,1H-NMR,13C NMR,2D-NMR,MS and HRMS were used to identify the structures.RESULTS1.Eleven compounds were obtained from 75%ethanol extracts of S.pulvinata Maxim.. 2.β-Bitostol,pulvinatadione,amentoflavone,pulvinatabiflavone,heveaflavone, isocrypromerin,selaginellin,pulvinataphentriol,pulvinatadiol,pulvinatahyde, pulvinataphendiol were identified on the basis of on the basis of spectral evidences and physicochemical properties.3.Pulvinatadione,pulvinatabiflavone,pulvinataphentriol,pulvinatadiol, pulvinatahyde and pulvinataphendiol were new compounds.CONCLUSIONS1.Eleven compounds were obtained from S.pulvinata Maxim.,among which pulvinatadione,pulvinatabiflavone,pulvinataphentriol,pulvinatadiol,pulvinatahyde and pulvinataphendiol were new compounds.2.Pulvinataphentriol,pulvinatadiol,pulvinatahyde and pulvinataphendiol were alkynyl phenols,possessing new structures Chapter 3 Effects of selaginellin on homocysteine-induced senescence of endothelial cellsBACKGROUNDAtherosclerosis(AS)is recognized as one of the major causes to threaten the life quality of people.Homocysteine(Hcy),an amino acid derived from methionine metabolism,can prompt atherogenesis,and plasma level of Hcy were positively correlated with morbidity and mortality from coronary heart diseases,suggesting Hcy is an independent risk factor for AS.Endothelial dysfunction is recognized as an early event in the pathogenesis of AS, which is closed related to senescence and damages of endothelial cells.Oxidative stress plays a key role in inducing cell senescence.Also,the decreased expressions of anti-senescent molecules contribute to progression of cell senescence.It has been reported that pro-atherogenetic properties of Hcy are related to inducing cell senescence.Selaginella Tamariscina(Beauv)spring can lower blood pressure,decrease the level of serum glucose,enhance immune function and inhibit oxidant production and so on.Selaginellin,a compound with novel chemical structure,was extracted from Selaginella Tamariscina.Our preliminary data showed that selaginellin had strong anti-oxidative activity.Based on these data mentioned above,in the present study,we investigated the effects of selaginellin on Hcy-induced senescence of endothelial cells (HUVECs)and its underlying mechanisms.METHODSHUVECs were cultured and used for all these studies.Cell viability was analyzed by using MTT assay.Cell senescence was evaluated usingβ-galactosidase staining and telomerase activity detected by telomerase repeat amplification protocol (TRAP)-silver staining.The level of intracellular reactive oxygen species(ROS) was determined using fluorescent ROS detection kit and the level of klotho mRNA was detected by real time PCR.RESULTS(1)The effects of different concentrations of selaginelin(3×10-9-10-4 Mon cell viability were observed.MTT results showed that the concentrations of selaginellin up to 10-6M had no significant effect on cell viability.(2)Treatment with Hcy(0.5 M)for 60h markedly induced the senescence of endothelial cells reflected by decrease in telomerase activity and increase inβ-galactosidase activity,which could attenuated by co-treatment with selaginellin in a concentration-dependent manner.(3)Treatment with Hcy(0.5 M)for significantly increased the level of intracellular ROS in endothelial cells.However,selaginellin could significantly inhibit Hcy-induced elevation of intracellualr ROS level.(4)The mRNA expression of klotho,an important anti-senescent protein,was markedly decreased in Hcy-incubated endothelial cells.However,selaginellin could significantly upregulate the mRNA expression of klotho in the absence of presence of Hcy.CONCLUSIONSSelaginellin attenuates Hcy-induced senescence of endothelial cells,and such effects may be related to inhibiting oxidative stress and up-regulating klotho expression.

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