| At present, with the changes of life-style and the development of economy, human disease spectrum have obviously changed. Morbidities of some diseases such as obesity, cardiovascular disease, diabetes and etc. are increasing, which become the biggest threat to human health. Many more studies indicated endothelium is one of the significant target organs of angiopathy. Some stimulates, such as high glucose and high fat etc., could induce the imbalances of biological active compositions secreted in the endothelium, which forms the common pathophysiological basis of cardiovascular and diabetes. Vascular endothelial dysfunction is the important pathologic features of chronic diseases in the early phase of these diseases, and endothelial hyperpermeability is one of the main significant performances. Therefore, looking for the effective intervention measures to protect endothelial hyper-permeability, even endothelial function, is the key job foe us to reduce the incidences of chronic diseases.Resveratrol (Res) is a compound of polyphenols, and its chemical name is 3,5, 4'-trihydroxystilbene. In 1992, resveratrol was detected in wine and people thought it was strong linked to French paradox, which attracted the great interest of researchers. Especially in 1997, an article about resveratrol's anticancer properties was published in Science, then researchers began to focus attentions on it and done a lot of researches. So far, there have been numerous studies shown that resveratrol has strong pharmacological activities to prevent cancer, cardiovascular disease, mutation, oxidation, antibacterial, anti-inflammation, regulating apoptosis and estrogen and so.The western developed countries have researched the main sources, dietary intake of phytochemicals, and the relationship of phytochemicals and human health, which supported lots of important data. Although Chinese are primary eating plant food everyday, Intake values of trans- and cis-resveratrol and piceids are not available since there is no complete food composition database, and recently studies about the content of resveratrol were focuses on the foods which contain high level of resveratrol, such as grapes, wines and peanuts and etc. So setting up an integrated analysis method used for detecting resveratrol contents of diary vegetable foods, establishing a Chinese database of resveratrol, evaluating accurately major food sources and their daily intake in Shenzhen adult population, and knowing about the metabolism of resveratrol in vivo is an important project, which deserves more attention of researchers.A lot of research confirmed plant-based diet (vegetables, fruits and complete grains) is negative with obesity, cardiovascular disease, diabetes and cancer etc., which shows that plant foods play a very important role in prevention and treatment of chronic non-communicable diseases, so is resveratrol. Recently studies showed resveratrol is effective to protect cardiovascular disease and diabetes. However, the exact mechanism of the effect of resveratrol on endothelial dysfunction is unclear at present.According to our country's dietary habits, we collected the samples from four regions, which were the South, North, West and Midland in China, analyzed the contents of resveratrol and piceid in all samples, so that we can establish a database of resveratrol and piceid, and preliminarily evaluated major food sources and their daily intake in Shenzhen adult population, which are beneficial to establish better dietary standards of human daily recommended intake level and safe doses. Meanwhile, we investigated preliminarily the bioavailability of resveratrol in vivo, combining cells and animal studies, explored the effects of resveratrol on preventing metabolic disease. Now the results are as follows:Part I Database of resveratrol and assessment of resveratrol intake in Shenzhen adult populationObjective:To set up a integrated method used for detecting trans-resveratrol and trans-piceid contents of diary vegetable foods, and to investigate the levels of trans-resveratrol and trans-piceid in daily vegetable foods of China, compile a composition database of resveratrol and evaluate major food sources and their daily intake in Shenzhen adult population, which will be the important foundation of resveratrol for efficiency studies.Methods:Collected the samples from four regions, which were the South, North, West and Midland in China, cut them into pieces and then freeze-dried. We compared the pretreatment methods of ultrasonication and maceration to gain the optimum treatment conditions. The samples were separated on a Hypersil ODS-C18 column with gradient elution of A (Methanol/acetic acid/water=10/2/88) and B (Methanol/acetic acid/water=90/2/8)at a flow rate of 1.Oml/min, and detected atλex 33Onm,λem 374nm. Samples were gained by the way of ultrasonic extraction and determined using high performance liquid chromatography (HPLC) method with fluorescence detection (FLD). According to the database, we preliminarily evaluate major food sources and their daily intake in Shenzhen adult population.Results:Samples in pieces contained higher resveratrol than powders, ultrasonication was better than maceration, and trans-resveratrol and trans-piceid were detected maximum when ultrasonication extraction for 40 min in sample-to-solvent ratios of 1:8. The database showed there were difference of the contents of trans-resveratrol and trans-piceid in same or different varieties and regions with the same pretreatments. Trans-piceid was the major form existing in most vegetable foods, and most of samples contained higher trans-piceid than trans-resveratrol. Moreover, peculiar vegetable foods to some region were also one of the most important sources of trans-resveratrol and trans-piceid. The one that had the highest level of trans-piceid and trans-resveratrol was skin of Hongti Grape; which were more than 40ug/g. Celery contained the highest level of trans-piceid among vegetables, which was up to 7.86μg/g. The dietary survey of Shenzhen city showed that the residents'daily intake of vegetable was about 332. 1g, fresh fruit was 159.4g, and rice was priori to other grains, which was up to 236.6g. The total daily resveratrol intake of Shenzhen adult residents from vegetable foods was about 196.99μg, and this equated to 3.28μg/kg.d for an adult.Conclusion:We set up an integrated method used for detecting trans-resveratrol and trans-piceid contents of diary vegetable foods, and investigated the levels of trans-resveratrol and trans-piceid in daily vegetable foods of the four regions of China, and established a database of resveratrol for the first time. Vegetable foods were the significant sources of trans-resveratrol and trans-piceid in China. The Shenzhen residents'intake of resveratrol from vegetable foods was first assessed. The data showed the level was very low, and it was only located in the range of lower doses of the biological concentrations of animals. It's not sure whether the level was up to the effective concentration of human biological effect. This investigation was very beneficial to further research about the effect of residents'dietary on regional disease and the epidemiological corelationship of both of dietary and chronic diseases.PartⅡResveratrol ameliorates high glucose induced hyperpermeability in bovine aortic endothelial cellsObjective:The aim of present study is to investigate the effects of resveratrol on high glucose-induced endothelial hyperpermeability and potential mechanisms involved in Bovine Aortic Endothelial Cells (BAECs).Methods:BAECs were incubated with high glucose (33 mmol/L) and resveratrol (0.1, 1.Oμmol/L) for 72h. The endothelial permeability was measured by the FITC-dextran fluorescent method, and nitric oxide contents were determined by measuring nitrate/nitrite levels in the supernatant. Endothelial nitric oxide synthase (eNOS) protein and phosphorylation expressions were examined using Western blotting. The message RNA and protein expressions of Caveolin-1 were tested by Real-Time PCR and Western blotting, respectively.Results:Resveratrol stimulated eNOS phosphorylation and NO production, and obviously ameliorated high glucose-induced endothelial hyperpermeability (p<0.05). The mRNA, protein expressions and phosphorylation (tyrosine 14) of Cav-1 induced by high glucose were all down-regulated after treatment with resveratrol (p<0.05). Pretreatment with eNOS inhibitor (L-NAME) blocked NO production and increased endothelial hyperpermeability. Beta-Cyclodextrin ((3-CD), an inhibitor of Cav-1, exerted the same effect of resveratrol on endothelial permeability to some degree while pretreatment with it.Conclusion:The study provided a novel insight of resveratrol, which prevented high glucose-induced endothelial hyperpermeability by regulating Cav-1/eNOS pathway and stimulating NO production. Cav-1 played a key role in this process. Part III Protective effects of resveratrol on high-fat/sucrose fed rats and related mechanismsObjective:The aim of the study was to investigate the protective effects of resveratrol on tissue endothelial permeability, insulin resistance and to explore the possible molecular mechanisms in cardiovascular system.Methods:45 male Wistar rats were divided into 5 groups and fed with normal diet, high-fat/sucrose diet, and high-fat/sucrose diet with 50,100 and 150 mg/kg-bw intragastric resveratrol respectively. Tissues permeability was assessed by using Evan Blue dye. The message RNA and protein level of endothelial nitric oxide synthase (eNOS) and caveolin-1 (Cav-1) in the cardiovascular system were tested. Fasting plasma glucose was measured monthly. OGTT and ITT were also tested in all the groups after 12th week. After 13th week, the rats were killed, fasting plasma glucose and fasting plasma insulin were measured, and HOMA-IR index of each group were calculated.Results:①Compared with the high-fat/sucrose group, rats body weight, plasma glucose, total cholesterin and total triglyceride were significantly lowered after resveratrol treatments (p<0.05).②Resveratrol protected aorta, heart, liver and kidney from hyperpermeable injuries and up-regulate eNOS, down-regulate Cav-1 mRNA and protein expressions in aorta and heart.③Compared with HFS group, rats'fasting plasma insulin and HOMA-IR index were significantly decreased after treatment of resveratrol. OGTT and ITT were obviously ameliorated in resveratrol groups.Conclusion:Resveratrol down-regulated the rat body weight and plasma glucose induced by high-fat/sucrose. High-fat/sucrose diet caused an increase in cardiovascular endothelial permeability, which was ameliorated by resveratrol through Cav-1/eNOS pathway. Resveratrol also markedly improved insulin sensitivity of high-fat/glucose rats, and decreased the incidence of insulin resistance.Part IV Metabolism studies of resveratrol in vivoObjective:The bioavailability, metabolic pathways and distribution, transformation and excretion of metabolites play a very significant role in biological effects of phytochemicals. The aim of the study was to investigate the bioavailability of resveratrol and the tissue distribution.Methods:10 male Wistar rats were divided into 10 metabolic cages after acclimatization to the laboratory environment for 1 week.50mg/kg resveratrol was given to rats by intragastric with normal diet under overnight fasting. The levels of trans-resveratrol and its glucuronide conjugates were detected in the plasma, urine, brain, liver, lungs, heart, spleen, intestines, kidney and artery using high performance liquid chromatography (HPLC) method with fluorescence detection (FLD), and plasma samples were further determined with GC-MS, and the condition of HPLC was same to the Part I.Results:The maximum concentration of trans-resveratrol in plasma was 4.79μg/mL after consumption of resveratrol 1h, while the maximum concentration of trans-piceid was up to 23.78μg/mL after 3h. Resveratrol and its glucuronide conjugates were almost excreted by urine during 24h, and resveratrol was over 90% of all the excrement, more than 50% of intake. Resveratrol were widely little cumulated in all the tissues studied. The levels were obvious different between 24h and 30d which cumulated in the tissues. The cumulations in 24h was liver>lung>brain>intestine > kidney> spleen> aorta> heart, and 30d was:liver> lung> kidney> intestine> spleen>brain> aorta>heart. The highest concentrations were found in liver, and the lowest in heart. Metabolites in most tissues were increasing with the intake time increasing, but brain was opposite to other tissues. The kinds and levels of metabolites were different in different tissues. Resveratrol was the major metabolites in liver and lung, and piceid was the primary metabolites in aorta, heart and spleen.Conclusion:Piceid was a major form existed in plasma. To our knowledge, this is the first time to observed that resveratrol and its glucuronide conjugates were almost excreted by urine during 24h, and resveratrol was over 90% of all the excrement, more than 50% of intake. Resveratrol were widely little cumulated in all the tissues studied. The levels were obvious different between 24h and 30d which cumulated in the tissues. But the highest concentrations were found in liver all the time, and the lowest in heart. Metabolites in most tissues were increasing with the intake time increasing except brain. The kinds and levels of metabolites were different in different tissues. These new findings were good for studying toxicity and biological effects of resveratrol.
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