Punicalagin alleviates hepatotoxicity in rats challenged with cyclophosphamide

This study investigated the possible hepatoprotection of punicalagin in rats received cyclophosphamide (20 mg/kg/day, i.p., for 7 days). Punicalagin given at two doses, 15 and 30 mg/kg/day, p.o., for 7 days, starting the same day of cyclophosphamide administration. Punicalagin significantly and dose-dependently reduced the elevations of serum alanine aminotransferase, and liver nuclear factor-κB p65, tumor necrosis factor-α, interleukin-1β, malondialdehyde, nitric oxide, Bax/Bcl-2 ratio, inducible nitric oxide synthase, caspases 3 and 9 activities, and prevented the decrease of hepatic total antioxidant capacity. Punicalagin also attenuated the histopathological liver tissue damage, and decreased cyclooxygenase-2 expression in liver of rats received cyclophosphamide in a dose-dependent manner. It was concluded that punicalagin protected rat liver against cyclophosphamide toxicity by inhibiting oxidative/nitrosative stress, inflammation, and apoptosis.     

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...     

安石榴苷对中波紫外线诱导HaCaT细胞光损伤的预防作用研究

目的 探讨安石榴苷对中波紫外线（UVB）诱导角质形成细胞损伤的保护机制。 方法 培养的HaCaT细胞分为空白对照组、安石榴苷组、UVB组、安石榴苷 + UVB组。噻唑蓝（MTT）法检测细胞增殖能力,Hoechst/碘化丙锭（PI）染色和流式细胞仪检测细胞凋亡,RT-PCR法测定金属基质蛋白酶1（MMP1）及其组织抑制因子1（TIMP1） mRNA表达水平,Western印迹检测丝裂原活化蛋白激酶（MAPK）通路相关蛋白P38、JNK、ERK的磷酸化水平变化。 结果 MTT试验示,10 ～ 40 μmol/L安石榴苷对UVB诱导的HaCaT细胞损伤有较佳的预保护作用。UVB组HaCaT细胞强Hoechst和强PI双染细胞较空白对照组增多,安石榴苷 + UVB组较UVB组减少。流式细胞仪分析,UVB组凋亡细胞百分率（9.82% ± 0.11%）高于空白对照（1.24% ± 0.91%,P < 0.01）,而安石榴苷（10、20、40 μmol/L） + UVB组凋亡细胞百分率（分别为6.38% ± 0.14%、5.24% ± 0.17%、3.77% ± 0.11%）较UVB组低,差异有统计学意义（均P < 0.01）。UVB组MMP1 mRNA相对表达量（12.376 ± 0.602）高于空白对照组（1.007 ± 0.147,P < 0.01）,而TIMP1 mRNA相对表达量（0.103 ± 0.006）低于空白对照组（1.006 ± 0.139,P < 0.01）,安石榴苷组MMP1及TIMP1 mRNA与空白对照组比较,差异无统计学意义（均P > 0.05）。安石榴苷预处理的HaCaT细胞经30 mJ/cm2 UVB照射后MMP1 mRNA相对表达量较UVB组降低（均P < 0.01）,而TIMP1 mRNA较UVB组升高（均P < 0.01）。Western印迹示,经UVB照射后,HaCaT细胞p-ERK、p-JNK及p-p38表达升高（均P < 0.01）。安石榴苷组HaCaT细胞p-ERK、p-JNK及p-p38表达没有明显改变（P > 0.05）,而安石榴苷 + UVB组有不同程度下降（均P < 0.01）。 结论 安石榴苷对UVB引起HaCaT细胞损伤有一定的预防作用。     

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。     

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.     

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。     

优化石榴皮中安石榴苷的分离与纯化工艺

摘 要 目的：研究石榴皮中安石榴苷的提取、分离纯化和定量方法。 方法: 采用20倍量50%乙醇常温超声波辅助法提取制备石榴皮粗提物,并采用HPLC法对粗提物中安石榴苷的含量进行测定。通过静态吸附和解吸性能的研究,优选D101、A8-8、NKA-9、HPD-100和HPD 500中对石榴皮多酚富集纯化效果最佳的大孔吸附树脂,并优化大孔吸附树脂富集纯化石榴皮多酚的工艺条件,得到安石榴苷。通过反相MCI GEL CHP20P色谱柱对安石榴苷进一步分离纯化。结果: 5种树脂中,HPD 500型树脂对石榴皮中安石榴苷有较好的吸附和分离性能。最优工艺条件为：上样液质量浓度15 mg·mL^-1,上样量2BV, pH为2,乙醇洗脱剂体积分数30%,洗脱剂体积8BV,可使安石榴苷含量从10.3%提高至30.7%。反相树脂MCI GEL CHP20P 色谱柱对30%乙醇洗脱部份进一步分离纯化,纯化后安石榴苷的含量可提高至61.2%。结论:富集纯化石榴皮安石榴苷的最优大孔吸附树脂为HPD 500型树脂;反相MCI GEL CHP20P色谱柱进一步纯化可显著提高安石榴苷纯化物的含量;工艺的重复性和稳定性良好。     

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.