松果菊苷抗衰老作用机理研究

研究了中国传统药物肉苁蓉提取物松果菊苷(echinacoside,ECH)体外清除活性氧自由基和体内抗氧化、抗衰老作用的机理。运用电子顺磁共振(electron paramagretic resonance,EPR)自旋捕捉方法研究ECH对体外产生的羟自由基(^ OH)、超氧阴离子自由基(O2^-)和脂自由基(L^ )的清除能力;并以D-半乳糖衰老小鼠为实验模型,采用低温EPR技术直接检测小鼠心、肝、肾、脑组织活性氧物种(reactive oxygen species,ROS)水平;生化方法检测小鼠全血谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)和脑组织单胺氧化酶(monoaminoxidase,MAO)活性及肝组织丙二醛(malondialdehvde,MDA)含量;EPR自旋捕捉方法检测血清超氧化物歧化酶(supemxide dismutase,SOD)活力;跳台法检测小鼠记忆力。结果表明ECH能较好抑制体外^ OH,O2^-和L^ 自由基,同时能够提高GSH-Px和SOD活性,降低MDA含量,因此对D-半乳糖所致衰老引起的活性氧自由基损伤具有一定修复作用。由于抑制了MAO活性而提高小鼠的记忆力。由此可以认为ECH抗脂质过氧化及改善衰老的作用与其抗氧化活性有关。     

Separation and purification of echinacoside from Penstemon barbatus (Can.) Roth by recycling high-speed counter-current chromatography

Echinacoside is an important bioactive compound extracted from Cistanche tubulosa which was endangered by overexploitation. It is imperative to find an alternative source. Echinacoside was isolated from Penstemon barbatus (Can.) Roth for the first time. The peak contents of echinacoside are 9.09 ± 0.32 mg/g and 7.25 ± 0.36 mg/g respectively in the leaves and roots annually. The methanolic extracts from 20 g of dried powder of the roots of P. barbatus were pre-purified by AB-8 resin and the fraction containing echinacoside was further purified by conventional high-speed counter-current chromatography (HSCCC) and recycling HSCCC with the solvent system n-butanol–water (1:1, v/v). Totally 42.0 mg echinacoside with a purity of 96.3% was recovered. The recovery rate of echinacoside by recycling HSCCC reached 91.0%. The structure of our echinacoside confirmed by IR, ¹H NMR and ¹³C NMR is identical to the standard sample. This indicates that P. barbatus might be ideal source for preparation of large scale of echinacoside.     

松果菊苷对多柔比星心脏毒性保护作用及机制研究

目的:观察松果菊苷(ECH)能否减轻多柔比星(DOX)心脏毒性并初步阐明其作用机制。方法:通过单次腹腔注射大剂量多柔比星(15 mg/kg)建立急性心脏毒性小鼠模型,DOX处理后每日通过腹腔注射ECH(50 mg/kg/day)。实验分组如下:正常组(Control组);单纯松果菊苷处理组(ECH组);多柔比星处理组(DOX组);多柔比星+松果菊苷处理组(DOX+ECH组)。给药5天后检测左心室功能、心肌组织病理改变、氧化应激和心肌凋亡情况。结果:与Control组相比,DOX组小鼠心脏收缩和舒张功能明显减弱,心肌细胞出现空泡变性,心肌MDA含量、凋亡率以及促凋亡蛋白Bax和cleaved Caspase-3表达明显增加,而抑制凋亡蛋白Bcl-2表达量、SOD与GSH-Px活性明显下降。与DOX组相比,松果菊苷能明显改善心脏功能,缓解心肌空泡变性,降低MDA含量、凋亡率以及Bax和cleaved Caspase-3表达量,而提高Bcl-2表达量、SOD与GSH-Px活性(均P 0.05)。结论:松果菊苷可以通过抑制心肌组织氧化应激损伤和凋亡缓解多柔比星诱导的急性心脏毒性。     

小檗碱联合松果菊苷抗人脑胶质细胞衰老的实验研究

摘要 目的：探究小檗碱联合松果菊苷抗人脑胶质细胞衰老的作用及其分子机制。方法：采用依托泊苷诱导人脑胶质细胞衰老,使用小檗碱与松果菊苷联合干预,采用β-半乳糖苷酶染色法色观察衰老阳性细胞比例,采用CCK-8法检测细胞活力,采用流式细胞术检测细胞周期,采用Western blot法检测细胞中衰老相关蛋白FoxO1、CAT、SOD2、 Bcl-2的表达水平。结果：小檗碱联合松果菊苷可显著减少依托泊苷诱导的衰老阳性细胞比例,显著增强细胞活力,显著降低G2/M期细胞比例,显著提高FoxO1、CAT、SOD2、Bcl-2的蛋白水平。结论：小檗碱联合松果菊苷可显著抑制依托泊苷诱导的人脑胶质细胞衰老,可能与增强细胞活力、推进细胞周期进程、降低胞内氧化应激水平、抗凋亡等途径有关。     

Echinacoside's nigrostriatal dopaminergic protection against 6‐OHDA‐Induced endoplasmic reticulum stress through reducing the accumulation of Seipin

Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Recent epidemiological studies suggest that echinacoside (ECH), a phenylethanoid glycoside found in Cistanche deserticola, has a protective effect against the development of PD. However, the detailed mechanisms of how ECH suppresses neuronal death have not been fully elucidated. In this study, we confirmed that ECH protects nigrostriatal neurons against 6‐hydroxydopamine (6‐OHDA)‐induced endoplasmic reticulum stress (ERS) in vivo and in vitro. ECH rescued cell viability in damaged cells and decreased 6‐OHDA‐induced reactive oxygen species accumulation in vitro. It also rescued tyrosine hydroxylase and dopamine transporter expression in the striatum, and decreased α‐synuclein aggregation following 6‐OHDA treatment in vivo. The validated mechanism of ECH activity was the reduction in the 6‐OHDA‐induced accumulation of seipin (Berardinelli–Seip congenital lipodystrophy 2). Seipin has been shown to be a key molecule related to motor neuron disease and was tightly associated with ERS in a series of in vivo studies. ECH attenuated seipinopathy by promoting seipin degradation via ubiquitination. ERS was relieved by ECH through the Grp94/Bip‐ATF4‐CHOP signal pathway.     

Improvement of echinacoside and acteoside production by two-stage elicitation in cell suspension culture of <Emphasis Type="Italic">Cistanche deserticola</Emphasis>

At appropriate concentrations, polyamines promoted the callus growth and echinacoside content of Cistanche deserticola while Ag⁺ increased the content of echinacoside and acteoside. In a 20-day culture period, when putrescine (25 μM) and Ag⁺ (10 μM) were added on day 8 and day 16, respectively, the echinacoside production (1.7 g l⁻¹) and acteoside production (0.4 g l⁻¹) reached the maximum, which were 1.4-fold and 1.5-fold of those in single putrescine treatment, 1.6-fold and 1.4-fold of those in single Ag⁺ treatment, respectively. Exogenous putrescine enhanced cell viability and antioxidant enzyme activity markedly, so increased the final biomass. Ag⁺ addition increased H₂O₂ content and phenylalanine ammonia-lyase activity significantly which led to higher echinacoside and acteoside contents.