Punica granatum果实皮甲醇提取物化学成分的分离和标准化(英文)

AIM:The present study was undertaken to isolate and standardize the various active phytochemical constituents present in the fruit rinds of Punica granatum.METHODS:Fruit rinds of Punica granatum were dried and extracted with methanol in a static extractor;the percentage yield of the methanolic extract (MEPG) was found to be 26%;the methanolic extract was partitioned using n-butanol,ethyl acetate and water;the percentage yield of the fractions were found to be 17.16%,26.88% and 47.72% respectively.HPLC was c...     

安石榴苷在MDCK细胞单层模型上的转运机制研究

摘 要 目的：采用MDCK细胞单层模型考察安石榴苷跨膜转运特性。方法: CCK8法筛选安石榴苷对MDCK细胞作用的安全浓度，Millicell ERS测量细胞单层的TEER值确定细胞单层的完整性及致密性，考察给药浓度、方向、时间、温度、维拉帕米和EDTA Na2不同条件下安石榴苷的转运情况，采用HPLC法测定安石榴苷浓度，并计算表观渗透系数( Papp ) 和外排率（ER）。结果:安石榴苷在MDCK模型上累积转运量具有时间和浓度依赖性，在100~300 μg·mL^-1浓度范围内测得的顶侧（AP）到基底侧（BL）的Papp值为（6.13±0.12）×10-7cm·s-1、（6.96±0.26）×10-7cm·s-1、（5.94±0.10）×10-7cm·s-1，未随浓度升高而增大，4℃时转运量降低，P gp 抑制药维拉帕米可以促进AP BL方向的转运，加入EDTA Na2后Papp(AP BL)显著增大。结论:安石榴苷以被动转运为主兼有主动转运参与，是P 糖蛋白（P gp）的底物受到P gp的外排作用，同时有细胞旁路转运途径。     

安石榴甙在机械力诱导成纤维细胞凋亡中的作用及其机制研究

目的:探索安石榴甙(PUN)在机械力诱导成纤维细胞凋亡中的作用及其可能机制。方法:不同浓度(0、2.5、5、10、25、50、100μmol/L)的PUN处理L929细胞不同时间(12、24、48小时)后,采用CCK8法检测细胞增殖活性;采用Western blot检测不同浓度PUN(0、2.5、5、10、25μmol/L)处理24小时后Nrf2蛋白表达;将L929细胞根据是否行PUN(10μmol/L,24小时)预处理和是否采用四点弯曲力学加载系统进行机械力干预(5333μstrain,1 Hz,4小时)分为4组:空白对照组、加力组、PUN组及PUN加力组,采用流式细胞术检测各组细胞凋亡率;采用Western blot检测凋亡相关蛋白Bax、Bcl-2及caspase3的表达;采用H2DCF-DA荧光探针检测各组细胞内活性氧簇(ROS)水平的变化。结果:PUN在浓度10μmol/L、作用时间24小时对细胞增殖活性及Nrf2蛋白表达的促进作用最显著;与对照组相比,加力组细胞凋亡率明显增加、ROS水平显著上升、Bax表达和Active-caspase3/caspase3比率明显增加(P0.05);与加力组相比,PUN加力组细胞凋亡率显著减少、ROS水平明显降低、Bcl2表达显著增加、Bax表达和Active-caspase3/caspase3比率降低(P0.05)。结论:PUN可明显减少机械力诱导的成纤维细胞凋亡,其机制可能是通过上调抗氧化蛋白Nrf2的表达,从而抑制机械力诱导ROS水平上升所导致的氧化损伤。     

Punicalagin reduces H2O2-induced cytotoxicity and apoptosis in PC12 cells by modulating the levels of reactive oxygen species

Background: Oxidative stress has long been linked to neuronal cell death in many neurodegenerative diseases. Antioxidant conventional supplements are poorly effective in preventing neuronal damage caused by oxidative stress due to their inability to cross the blood brain barrier. Hence the use of molecules extracted from plants and fruits such as phenolics, flavonoids, and terpenoids compounds constitute a new wave of antioxidant therapies to defend against free radicals.

Objective: In this study we examined the effects of punicalagin, a ellagitannin isolated from the pomegranate juice, on a rat adrenal pheochromocytoma cell line, treated with hydrogen peroxide, evaluating the viability, oxidation potential, mitochondrial function, and eventual apoptosis.

Methods: This study was performed on PC12 cells pretreated with punicalagin (0.5, 1, 5, 10 e 20?µM) 24 hours before of the damage by hydrogen peroxide (H₂O₂). H₂O₂ concentration (300?µM) used in our study was determined by preliminary experiments of time course. The cell viability and ROS production were evaluated by MTS assay and cytofluorometry assays, respectively. Subsequently, the number of apoptotic-positive cells and mitochondrial transmembrane potential, were measured by flow cytometry, in the same experimental paradigm. Finally, the expression of Bax and enzymatic activity of Caspase 3, some of the principle actors of programmed cell death, were investigated by semiquantitative PCR and utilizing a colorimetric assay kit, respectively.

Results: We found that pretreatment with punicalagin protected the cells from H₂O₂-induced damage. In particular, the protective effect seemed to be correlated with a control both in radical oxygen species production and in mitochondrial functions. In fact the cells treated with H₂O₂ showed an altered mitochondrial membrane integrity while the pretreatment with punicalagin retained both the cellular viability and the mitochondrial membrane potential similar to the control. Furthermore, the punicalagin, modulated the apoptotic cascade triggered reducing Bax gene expression and Caspase 3 activity.

Discussion: Results of the present study demonstrated a neuroprotective effect of punicalagin on H₂O₂-induced PC12 cell death, including mitochondria damage and expression of apoptotic gene Bax; therefore we hypothesize a possible prevent role for this molecule in neurodegenerative diseases related to oxidative stress.     

β-Secretase(BACE1) inhibitors from pomegranate (Punica granatum) husk

In the course of screening for anti-dementia agents from natural products, two β-secretase (BACE1) inhibitors were isolated from the husk of pomegranate(Punica granatum) by activity-guided purification. They were identified as ellagic acid and punicalagin with IC₅₀ values of 3.9 x10^-6 and 4.1x10^-7 M andKi values of 2.4x10^-5 and 5.9x10^-7 M, respectively. The compounds were non-competitive inhibitors with a substrate in the Dixon plot. Ellagic acid and punicalagin were less inhibitory to α-secretase (TACE) and other serine proteases such as chymotrypsin, trypsin, and elastase, thus indicating that they were relatively specific inhibitors of BACE1.