الفهرس 
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Abstract
Endothelial dysfunction (ED)is defined as the inability of the endothelium to maintain vascular homeostasis, that is consider a systemic pathological state that alters endothelial cell phenotype, characterized by a shifting in the normal functions of the endothelium by reducing vasodilation, with increasing proinflammatory and prothrombic state. Endothelial dysfunction is considered one of the initial stages in the development of atherosclerosis and CVD, therefore, reversing it is considered as an attractive target for the prevention of CVD.
There is an increasing evidence that high fat-high fructose diet (HFHF) consumption causes dyslipidemia, insulin resistance, obesity, and endothelial dysfunction. All these factors are associated with increased risk of cardiovascular diseases (CVD), one of the main causes of morbidity and mortality worldwide. High fructose consumption is also reported to induce oxidative and nitrative stress in vascular tissues and that oxidative/nitrative stress seems to play a major role in endothelial dysfunction. Indeed, elevated levels of reactive oxygen species (ROS) cause an increase in production of powerful oxidant peroxynitrite that induces eNOS inactivation and a decrease in bioavailability of NO.
The present study was conducted to investigate ameliorative effect of olive seeds powder either in native or nano form against ED induced by HFHF diet in rats.
To achieve this goal this study was divided into 2 phases:
Phase (1) include chemical composition analysis of olive seeds powder, Preparation of nano olive seeds by ball milling technique and XRD was done for the native olive seed powder: -
i. Chemical composition analysis of olive seeds powder sample
The results revealed that each 100 g of olive seeds powder contains 0.5 g moisture, 28.68 g protein, 14.52 g carbohydrate, fat content was 17.42 g,
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and 23.81g crude fiber. Results of phytochemical analysis of (OSP) showed that each 100g of OSP contains 1004.9 mg total polyphenols as gallic acid equivalent (GAE%) and 24 mg total flavonoids as quercetin equivalent (QE%).
Preparation of nano olive seeds by ball milling technique.
Characterization of nano olive seeds was done by X- ray diffraction (XRD), BET surface area, Area-volume-pore, Zeta sizing and Transmission Electronic Microscope (TEM) image. The result showed that efficiency of nano olive seeds by ball milling technique which reflected that nano olive seed powder in the average size of 57.2 ± 1.2nm.
ii. XRD was done for the native olive seed powder
The result indicated that both native and nano olive seed powder have the same chemical composition without any distortion or decomposition for the origin structure of olive seeds by ball milling.
Phase (2): -
Phase 2 consist of the biological trial which investigated the ameliorative effect of olive seeds powder and its nano particles on biochemical alterations elicited by high fat high fructose diet
Experimental design:
Throughout this study a total number of 60 healthy male adult albino rats Srague-Dawely strain weighing 120 g ±5 g were subjected to experimentation. Rats were divided into 6 groups (10 rats/ group) as follows: Healthy control group (negative control): Rats fed on balanced diet for 8 weeks. High fat high fructose group (HFHF) (positive control): Rats fed on HFHF diet for 8 weeks. HFHF + 5% olive seeds group: Rats fed on HFHF diet supplemented with 5% olive seeds powder for 8 weeks. HFHF + 10% olive seeds group: Rats fed on HFHF diet supplemented with 10% olive seeds powder for 8 weeks. HFHF + 5% nano olive seeds group: Rats fed on HFHF diet supplemented with 5% nano olive seeds for 8 weeks.
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HFHF + 10% nano olive seeds group: Rats fed on HFHF diet supplemented with 10% nano olive seeds for 8 weeks.
During the experimental period, food intake was recorded daily and the animals were weighed weekly to monitor the body weight changes and calculate the biological measurements including the feed efficiency ratio (FER). At the end of experiment, all rats were fasted for 12 hours (from 7 p.m to 7a.m) but received water ad libitum, then weighed and blood samples were collected from hepatic portal vein to analyze the following parameters; Serum glucose and insulin levels and HOMA-IR was calculated for evaluating insulin resistance. For lipid determination serum TC, TAGs, LDL-C, HDL-C, VLDL-C and lipase enzyme activity were measured. Serum CRP and IL-6 as proinflammatory markers, MDA concentrations as oxidative stress marker. Serum NO, prostacyclin and eNOs as vasodilator factors and ET1 as vasoconstrictor factor, and vascular adhesion molecules VCAM-1 and E- selectin were measured
- The biological data of the study reported that there was a significant decrease in food intake in spite of the increase in body weight gain and FER of rats feed on HFHF diet for 8 weeks by about -19.59%,45.1 6 and 84.21%, respectively when compared to negative control group (p<0.05). Also, the result demonstrated that diet supplementation with olive seeds powder either native or nano form caused more significant reduction in these biological measurements (p<0.05).
-The result also showed that there was a highly significant increase in relative weight of liver and heart in rats group fed on HFHF diet .The increment ratio was 100.8% and 50.68% respectively when compared to negative control group (p<0.05). However, HFHF diet supplemented with olive seeds powder in the form native or nano showed a significant decrease in relative weight of heart and liver (p<0.05). Supplementation with 10% nano olive seeds powder caused the most significant reduction in liver and heart relative weight by about -37.12% and -34.5% when compared to positive control group (p<0.05).
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- The results revealed that there was a statistically significant increase in serum glucose level, insulin level and calculated HOMA-IR in rats fed on HFHF diet for 8 weeks. The increment ratios were 68.64%, 169.83% and 332.14%, respectively when compared to negative control group (p<0.05). Otherwise, feeding olive seeds powder either in a native or nano form had a significant effect in modulating these altered parameters and reversed them to their normal levels. HFHF fed rat group when supplemented with olive seeds powder showed a more significant reduction in serum glucose level compared to HFHF fed rat group without any supplementation. Dietary supplementation of HFHF fed rat groups with olive seeds powder either in native or nano form, showed an ameliorative effect on elevated serum glucose level. The reduction ratios in serum glucose level were -35.37%, -36.23%, -39.65% and -37.80% for HFHF groups supplemented with 5% olive seeds, 10% olive seeds ,5% nano olive seeds and 10% nano olive seeds respectively, when compared to positive control group (p<0.05).
- Dietary supplementation of HFHF fed rat groups with olive seeds powder either in nano or native form caused a significant reduction in serum insulin level. There was a significant reduction in serum insulin level were observed in both HFHF group supplemented with 5% and 10% olive seeds in native form by -31.63% and -33.23% respectively, when compared to positive control group (p<0.05).
- In addition, the results of the present work showed that the supplementation with olive seeds powder either in native or nano form showed a significant reduction in calculated HOMA-IR. The ratios were decreased by -56.28%, -57.02%, -53.72% and -52.07% in HFHF fed rat groups supplemented with 5% olive seeds, 10% olive seeds ,5% nano olive seeds and 10% nano olive seeds respectively, when compared to positive control group.
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- from the data were obtained in this study, it was obvious that feeding HFHF diet for 8 weeks resulted in a significant increase in serum TC, TAGs, LDL-C , VLDL-C levels and lipase enzyme activity , The increment ratios were 37.88%, 131% , 271.09% , 135.56% and 217.30%, respectively as compared to negative control group , this was coupled with a significant reduction in HDL-C in HFHF fed rat group by about -20.52% when compared to negative control group (p<0.05). Results also demonstrated that dietary supplementation with olive seeds powder either in native or in nano form caused a significant improvement in these altered parameters. The most significant improvement in lipid profile in the rat group supplemented with 10% nano olive seeds by decreasing the values by about -25.42%, -55.82% , -55.99 % and -55.76% for serum TC , TAGs, VLDL-C and lipase enzyme respectively when compared to positive control group (p<0.05). On the other hand, there was a significant elevation in serum HDL-C by about 23.32% in HFHF supplemented with10% nano olive seeds group as compared to positive control group (p<0.05).
-It was clear from the results that there was a statistically significant increase in serum IL-6, CRP and MDA levels in HFHF group by about 390.83% ,158.85% and 680.17% respectively when compared to negative control group (p<0.05). Meanwhile, the results demonstrated that dietary supplementation of HFHF diet with olive seeds powder either native or nano lead to significant improvement in proinflammatory markers and oxidative stress marker. Supplementation of HFHF diet with 10% nano olive seeds caused the most significant reduction in serum IL-6, CRP and MDA by -61.98%, -48.25% and -78.79 % respectively, as compared to positive control group (p<0.05).
-Results of the present study revealed that feeding rats HFHF diet for 8 weeks resulted in a significant decrease in serum nitric oxide, prostacyclin
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and endothelial nitric oxide synthase levels. The reduction ratios were -65.77%, -66.56% and -74.56%, respectively, as compared to negative control group (p<0.05). This decrease in vasodilator factors were accompanied by a significant increase in endothelin-1 level by 192.3%, when compared by negative control group (p<0.05), indicating initiation of endothelial dysfunction. Results also demonstrated that diet supplementation with olive seeds powder either in native or in nano form caused a significant improvement in these altered parameters. Supplementation of HFHF diet with 10% nano olive seeds caused the highest significant increase in serum prostacyclin, NO levels and endothelial nitric oxide synthase activity.
- The current results showed that there was a statistically significant increment in serum VCAM-1 and E –selectin ratio in rats fed on HFHF diet by about 221.95% and 331.35%, respectively when compared to negative control group (p<0.05). The increase in these parameters with vasodilator and vasoconstrictor imbalance indicating progression of ED disease. On the other hand, supplementation of HFHF diet with either native or nano olive seeds powder caused a significant reduction in the elevated levels of VCAM-1 and E –selectin compared to positive control group. The anti-adhesive effect of olive seeds powder was highly marked in10% nano olive seeds supplemented group. Serum VCAM-1 and E –selectin levels were decreased to -54.52% and -70.63%, when compared to positive control group (p<0.05). Also, E –selectin level nearly approaches that level of healthy control which confirm the ameliorative effect of olive seeds nano particles against vascular adhesion induced by HFHF diet.
- Microscopic examination of aorta tissue sections with Hematoxylin and Eosin (H&E) stain confirmed the biochemical findings showing corrugation and swelling of the endothelial cell and presence of thrombosis in HFHF group. While, olive seeds powder in native and nano form dietary
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supplementation alleviated the HFHF diet alterations exist in HFHF group showing reduction in inflammation. Hence, olive seeds powder in native and nano form has a protective effect against HFHF diet induced ED in rats, but the high efficiency returned to nano olive seeds powder than olive seeds powder in native form.
Olive seeds could be considered as an important source of bioactive phenolic compounds. In light of the obtained results, it would seem possible to use olive seeds as a source of natural antioxidants in foods, cosmetics or pharmaceutical products, thus contributing to diminishing the environmental impact of olive oil waste by-product and to its revalorization.
from the present work, it is concluded that a high-fat and high-fructose diet can cause insulin resistance, dyslipidemia and hyperglycemia and thereafter endothelial dysfunction. Olive seeds in native or nano form have a large amount of phenolic compounds that has the antioxidants ability and play an important role in the treating many diseases of CVD as ED and atherosclerosis . The positive effect of olive seeds powder and olive seeds powder -derived products consumption for human health may be due to its powerful antioxidant capacity. Olive seeds powder as a natural dietary antioxidant may act for production against oxidative damage induced by bad dietary pattern (high fat high fructose diet). Therefore, reversing endothelial dysfunction and its complications.