Study On Recombination Of Antioxidants In The Chaenomeles Speciosa And Its Anti-atherosclerotic Effects In ApoE^-/- Mice

Today it is widely believed that the development and progression of atherosclerosis is caused by a chronic inflammation in the vessel wall. Cells found in early atherosclerotic lesions are typically inflammatory cells (monocytes/ macrophages and T-lymphocytes). There is convincing clinical and experimental evidence that formation of reactive oxygen species (ROS) is augmented during this chronic inflammatory process due to an imbalance between synthesis of ROS and neutralizing antioxidative defense mechanisms. Biological molecules such as lipids, proteins and DNA may be reacted with ROS, which causes oxidative-stress induced damage. The evolving inflammatory reaction and ROS (any reactive organic or inorganic molecules with one or more unpairedelectrons) is instrumental in the initiation of atherosclerotic plaques and their destabilization Given that oxidative stress plays a pivotal role in the pathogenesis of cardiovascular diseases, it is not surprising that antioxidant therapies are one of the most effective and promising strategies against atherogenesis. Inhibition of ROS generation and function is a potential therapy to attenuate the extent of various cardiovascular diseases with nature antioidants ,such as bioflavonoids, which are polyphenolic compounds, are believed to be beneficial for the prevention and treatment of atherosclerosis and cardiovascular diseases because not only they possesses high antioxidant property and decreases the susceptibility of LDL to oxidation, but downregulate inflammatory factors (such as TNF-α) and attenuate accumulated macrophage-induced foam cells. There are abundant antioxidants, such as Vitamin C, polyphenolic compounds and superoxide dismutase in chaenomeles. With mice fed by extract of chaenomeles , Comparing to the model control group, the contents of MDA in serums and tissues of each dosages groups all decreased, and SOD activity in serums increased in irradiation peroxidation damage animal models. In vitro tissue factor inhibitory triterpene from the fruits of Chaenomeles sinensis was confirmed. Consuming a diet rich in natural antioxidants or drug of synthesized antioxidants(probucol) have been associated with prevention from and/or treatment of atherosclerosis. Bioactive components of food with antioxidation, which are of special interest, include the Vitamins E and C, polyphenols, carotenoids and so on. Based on the'oxidation theory'for atherosclerosis, dietary antioxidants have attracted considerable attention as preventive and therapeutic agents. There is adequate evidence from observational, in vitro, ex vivo, controlled intervention and animal model studies that consumption of certain foods results to a reduction in oxidative stress and myocardial infarction biomarkers. The effects of dietary antioxidants assessed in vitro, ex vivo and in vivo was shown below: (1) Increase in plasma oxidative resistance;(2) Decrease in plasma MDA levels;(3) Increase in LDL oxidative resistance;(4) Decrease in foam cell formation and extend of the atherosclerotic lesion; (5) Upregulation of GSH-Px, SOD, catalase gene expression; (6) Decrease in macrophage accumulation and severity of the lesion in the aorta;(7) Suppression of TNF-αmRNA expression. The antioxidant properties of plant components, such as carotenoids, vitamin C, flavonoid, are individually examined by epidemiological studies or supplementation trials. However, the results are not as clear-cut as those obtained for fruit and vegetables and are often disappointing. Since there is an agreement that not one antioxidant alone can lead to health benefits but the combination, as found for example in fruits and vegetables, is the active principle, transferred the main antioxidative composition from the fruit of Chaenomeles speciosa into maltodextrin (Chaenomeles speciosa powder, CSP) by process with a combination of enzyme-aided extraction and fluid bed spray-drying techniques, to preserve their antioxidant activity. Subsequently, we determined the contents of antioxidants and antioxidant capacity, and further to evaluated the potential anti-atherosclerotic effects of CSP in apolipoprotein E deficient (ApoE-/-) mice. Method:Chaenomeles (Cultivated in Qijiang county of Chonqing, China) that have been frozen, pulped with distilled water, cured with enzymes (the pectinase and cellulase), spray-dried with maltodextrin and reground. To assess the effects of enzymolysis on the juice yield of the extraction, the conditions for enzymolysis, including concentrations of pectinase and cellulase, temperature and time, were investigated through an orthogonal design of L9(34) on juice yield. The aqueous extract by optimal enzymatic technique was processed to CSP by techniques of inclusion and delayed release powder granulated. Subsequently, the contents of antioxidants (VitaminC, total phenols, tannin) and antioxidant capacity(TAC, SOD and FRAP ) were determined. Polyphenol compunds were analyzed by TLC for the qualitation of the monomers and tannin. Microplate quantification of DPPH?was used to estimate the change of antioxidant system in chaenomeles and CSP by process with comparison of kinetic curve.Apoe-/- mice of age 6 wk and weight 20±3 g were kindky provided by Pr. Ling at Sun Yat-Sen University. All animal procedures were followed in accordance with the approved protocol for use of experimental animals set by the standing committee on animal care at The Third Military Medical University. Mice were averagely divided into five groups and then fed with 5g CSP/100g high-fat diet (low dosage, 5%CSP group) ,with 10g CSP/100g high-fat diet (high dosage, 10%CSP group) , with 3 g green tea powder high-fat diet (Tea group), with 0.02% Vitamin C high-fat diet (VC group)for 16 wk and fed high-fat diet without CSP were used as control . All mice were then killed by decapitation under anesthesia. The blood samples were harvested for serum lipid analysis and antioxidant capacity determanation, and the descending aorta, aortic sinus and aortic arch were collected for quantification analysis of lipidoses, atherosclerotic plaque area and intima-media thichness by staining with oil red and hematoxilin-eosin, respectively. The expression of TNF-α, CD68 and the amount of macrophagocyte were determined by immunohistochemistry. The histopathological studies of blood vessel endothelium were carried by electron microscope scanning.ResultsEnzymolysis technique was optimized as 2.0 ml/100g of pectinase and 2.0 ml/100g of cellulase in water bath at 40℃for 60 min. The technique of Fluid Bed Granulation is that: Temperature control in batch at 45℃, frequency of aerator at 4~5kHz, peristaltic pump at 3~5Hz, the frequency of buffeting at 35~40 times/min There were 2.05±0.09mg Vitamin C, 22.70±1.57mg total phenols and 9.64±0.65 mg tannin in 1g CSP. There are rutin, catechin, quercetin, chlorogenic acid and procyanidine. The ferric reducing antioxidant power of CSP was (172.9±6.7)μmol Fe2+/g and the scavenging activity on DPPH?(TAC) and O2 ? (SOD) were (945±20)μg DPPH?/g and (700±21) U, respectively. there was no much change of antioxidant compound and property by process with confirmation of MQ method. The associated binding constants of enzymatic extract withγ-cyclodextrin.Comparing with control group, A dose-dependent inhibitory effect of CSP on reduction of serum LDL cholesterol and TC levels (P<0.05). There was a reduction of TG found in10%CSP and tea groups. Comparing with control group Tea, 5% and 10% CSP dietary supplement significantly increased glutathione peroxidase activity and total anti-oxidative capacity, and subsequently achieved a reduction of lipidoses on descending aorta by 29.2%, 40.8% and 67.3%; reduction of IMT by 35.1%, 30.1% and 48.2%; reduction of relative atherosclerotic plaque area of aortic sinus by 17.4%, 23.9% and 38.1% in ApoE-/- mice. Concerning about inflammatory factor, tea and CSP significantly downregulated the expression TNF-αand CD68, especially a dose-dependent effect of CSP. There were also amounts of macrophagocyte decreased significantly by tea and CSP supplement(P<0.05).Conclusion:We successfully transferred antioxidants in Chaenomeles to the powder composed of cyclodextrin with good shelf life, achieving abundant Vitamin C and polyphenols, which achieved scavenging superoxide radical and DPPH?. CSP significantly increase antioxidant status of plasma, which would enhance LDL oxidative resistance; The decrease of serum TG, TC, LDL would reduce lipidoses and subsequently attenuate foam cell formation and extend of the atherosclerotic lesion; The downregulation of expression of TNF-αand CD68 would reflect lower level of inflammation, which decrease in macrophage accumulation and severity of the lesion in the plague. In summary, as reported that increased levels of LDL together with decreased antioxidant capacity of blood may accelerate the formation of atherosclerosis by increasing LDL-oxidation and ox-LDL is atherogenic, the anti-atherosclerotic of the powder processed by Chaenomeles Speciosa was maybe due to the enhanced antioxidative status in circulation, anti-inflammation in the aorta endothelium and the decreased serum cholesterol levels, especially LDL.