Persimmon Peel Improve Atheroscelrosis

As proposed by Ross and Glomaset response and endothelial cell injury theory on the study of atherosclerosis has undergone rapid development. Lipid metabolism in recent years on the impact of atherosclerosis and further clear. Currently accepted view is more endothelial injury and lipid metabolism is the beginning of atherosclerosis causes.Atherosclerosis of an earlier age of onset, early drug intervention can not, so by changing the life or habits, dietary intervention as the most effective means.In this experiment, Ldlr-/-mice as experimental animals given high fat diet and skin powder containing high fat diet persimmon established model of atherosclerosis was observed in mice lipids, urinary 11-dhTXB2, blood, and the main oxLDL/β2GPI artery atherosclerotic plaque area, to prove that persimmon skin can inhibit atherosclerosis in mice.Methodology:(1) In this study, three experimental diets were used. One is control high fat diet (CH) consisting of 0.2% cholesterol and 21% milk fat. Another diet is high fat diet containing 10% persimmon peel powder (PP). According to manufacturer, persimmon peel was sun-dried for 30 days and further dried with decompression machine.at 40oC for 14 h, followed by milling with milling machine. The resulting powder was added at 10% in the control high fat diet.(2) Animals and Experimental protocol:LDLR-/-mice were purchased from Jackson Laboratory. Three to five animals were housed per box in a temperature-controlled animal facility with a daily photoperiod of 12 h of light at department of animal resources, advanced science research center, Okayama University.At the age of 12-weeks old, male LDLR deficient mice were randomly assigned to CH (n=11) or PP (n=7) group and fed the test diet and water ad libitum for 12 weeks. Besides.Animal body weights were measured at the beginning of the study and every 4 weeks. At the same timing, urine was collected in metabolic cages and blood samples were obtained from animals fasted for 5-6 h by retro-orbital bleeding with EDTA as anticoagulant.(3) Plasma Lipids:Plasma cholesterol and triglyceride were determined enzymatically using commercial kits.(4) Preparation of mouse aortas and quantification of atherosclerosis:Mice were euthanized after the final blood collection. The aortic tree was perfused for with PBS containing 20 mmol/L BHT and 2mmol/L EDTA, pH7.4, by inserting a cannula into the left ventricle and allowing free efflux from an incision in the vena cava. After removal of the surrounding adventitial fat tissue, the aorta was opened longitudinally from the aortic root to the iliac bifurcation, fixed with PBS containing 4% formaldehyde, and stained with Sudan IV. The extent of atherosclerosis was determined by the"en face"method.Quantification was performed by capturing the images of aortas with Penguin 150 CL cameraconnected to SZX12 dissection microscope. The lesion percent of aorta was estimated by Scion Image analysis.(5) ELISA for mouse oxLDL/β2GPI complexes:Anti-β2GPImonoclonal antibody WB-CAL-1 (8μg/ml) was adsorbed on the wells of 96-well microtiter plate by overnight incubation at 4oC. Then the wells were blocked with BSA for 2 h. Mouse serum (100-fold diluted) or standard oxLDL/β2GPI complexes were added to the wells and incubated at 4oC overnight. The wells were subsequently incubated with peroxidase-labeled rabbit anti-mouse LDL polyclonal antibody for 3 h at room temperature. Color was developed with adding TMBUS substrate . The reaction was terminated by adding 2 N sulfuric acid and the absorbance at 450 nm was measured. Between each step, the wells were washed three times with TBS containing 0.05% Tween 20. The absorbance was measured at 450 nm with a microplate reader . The intra-assay precision CV was less than 10%.(6) ELISA for 11-dhTXB2:Urinary 11-dhTXB2 was determined using 11dhTXB2 Test Kit. The diluted samples and purified 11-dhTXB2 conjugated to alkaline phosphatase (AP), and purified mouse monoclonal antibody directed to 11-dhTXB2 were combined and incubated in microwells coated with a polyclonal anti-mouse antibody. After washing, the complex composed of monoclonal antibody and endogenous or AP-conjugated 11-dhTXB2 antibody remaining on the plate was detected by the addition of paranitrophenylphosphate (pNPP) chromogenic substrate. Color developed in the wells at an intensity inversely proportional to the sample urine concentration of 11dhTXB2, and was read at 405 nm. ConclusionPersimmon Peel has significant anti-atherosclerotic effect.(1) PP fat diet to mice increased plasma cholesterol and plasma triglyceride level is far below the CH fat diet control group.(2) Compared with the CH group, PP group oxLDL/β2GPI complex increased significantly lower than CH mice.(3) Compared with the CH group, PP group clock plaque area less than the CH group.(4) PP increased the level of urinary 11dhTXB2 less than the CH group.