| Objective: To extract antioxidants from pomegranate peel and investigate their antioxidant activities and anti-atherogenic effects in vitro and in vivo, in comparison with pomegranate pulp extract, including free radical scavenging abilities, protection against low-density lipoprotein (LDL) oxidation, inhibition against the formation of atherosclerotic plaque and the oxidative damage to cultured vascular endothelial cells (VEC) induced by oxidized-LDL (Ox-LDL).Methods:Part 1 : Extraction of antioxidants from pomegranate peel and analysis of themajor componentsThe peels were cut into pieces and extracted by different solvents, including methanol, ethanol, acetone and their combinations. All extracts were pooled together and concentrated under vacuum at 60 C, and the concentrates were powdered and stored in a desiccator. The antioxidants extracting efficiency was evaluated by FRAP assay. The influences of temperature, time, pH value and ratio of solvent to peel (V/W) were also studied. The pulps were similarly extracted. The contents of polyphenols, flavonoids, proanthocyanins and ascorbic acid of the extracts were determined, and the possible toxicity of the extracts was examined by acute toxicity test.Part 2 : The antioxidant activity in vitro of pomegranate peel extractThe extract obtained was assessed for superoxide anion, hydroxyl and peroxyl radicals scavenging abilities, as well as the inhibitory action against CuSO4-induced LDL oxidation in vitro.Part 3 : The anti-atherogenic effect in vivo of pomegranate peel extract1. Effects of pomegranate peel extract on blood lipids and atherosclerosis inC57BL/6J mice fed a high fat dietThe C57BL/6J mice were divided randomly into four groups, i.e. control group, high fat diet group (HF), high fat diet plus pomegranate peel extract group (EPE), and high fat diet plus pomegranate pulp extract group (EPU). EPE and EPU groups received peel or pulp extract supplemented in drinking water respectively. Control and HF groups received plain drinking water.At the end of the study (4 mo later), blood, liver, heart and aorta were sampled. Serum cholesterol, triglyceride, LDL-ch, HDL-ch, FRAP values, GSH-Px activity, paraoxonase activity, SOD activity and malondialdehyde (MDA) were determined. Contents of fat and cholesterol in livers, cholesterol in heart and aorta were also assayed. Part of the aorta was rapidly removed and fixed in 6% glutaraldehyde for electron microscope examination, and the remainder were immersed in 4% neutral formaldehyde buffer solution for light microscope examination.2. Effects of pomegranate peel extract on atherosclerosis in rabbits fed a high fat dietForty Japanese white rabbits were divided randomly into four groups. Control group was fed the standard diet and HF group was fed the diet high in lipid and cholesterol, and EPE and EPU groups were fed the diet high in lipid and cholesterol, supplemented with peel or pulp extract respectively. Blood was obtained to evaluate dynamic changes of blood lipids. At the end of the study (3 mo later), blood and aorta were sampled. Serum FRAP value, GSH-Px activity, SOD activity and MDA were determined. Aortas were collected for microscope examination and determination of atherosclerotic plaque area.Part 4 : In vitro effects of pomegranate peel extract on cultured VEC exposed to Ox-LDL1. The oxidative injury model of VEC induced by Ox-LDL in vitroOx-LDL was made by copper-mediated oxidation and human ECV304 cells were used to establish the model. The Ox-LDL was added to damage VEC oxidatively. LDH activity in the culture medium and MTT value of VEC were determined. The morphologic changes were observed by light microscope.2. Protective effect of pomegranate peel extract on VEC exposed to Ox-LDL VECs were divided into 9 groups, i.e. Control, Ox-LDL, Ox-LDL+VE (VE+),Ox-LDL+EPE groups (EPE-L+, EPE-M+, EPE-H+: 50, 100, 200mg/L), Ox-LDL+EPU groups (EPU-L+, EPU-M+, EPU-H+: 50, 100, 200mg/L), respectively. VECs were incubated with Ox-LDL for 24 hours after being preincubated wit...
|
PDF Full Text Request