Characterisation of phenolic antioxidants in fruits and vegetables : bioavailability of raspberry phenolics in humans and rats

摘要

Epidemiological studies have suggested associations between the consumption of plant foods and beverages, especially those rich in phenolic compounds, and the prevention of chronic diseases, and there is a growing evidence indicating that these dietary phytochemicals are involved in enhancing long-term health. The antioxidant capacity of these compounds appears to be, at least in part, responsible the reduction of certain oxidative stress pathologies. Common fruits, vegetables and nuts available in supermarkets in Scotland were screened to identify products that are rich in phenolic antioxidants. The selection of products analyzed was based on their availability in local supermarkets. Two different assays were used to assess antioxidant acitivity, electron-spin resonance spectroscopy (ESR) and Ferric Reducing Antioxidant Potential (FRAP). Total Phenolics content (TPC) was also quantified using the Folin-Ciocalteau assay. Due to interest in the Zutphen study in the early 1990's that showed that dietary intake of flavonols was inversely correlated with the incidence of coronary heart disease, the analysis of quercetin, isorhamnetin and kaempferol was done. There were differences of up to 758-fold in the total FRAP antioxidant capacity (AOC) of the individual products. Those especially rich in antioxidants included peanuts, almonds, broad beans, blueberries, raspberries, strawberries and purple broccoli. The vitamin C and flavonol concentrations were not correlated to the AOC. The results obtained by FRAP and ESR are significantly high correlated. There was no correlation between FRAP and flavenols. The data obtained in this study were used in a separate collaborative investigation that is not included in this thesis to evaluate the overall intake of antioxidants in the UK. Berries were selected for further analysis because they constitute a group of fruits with very high AOC. Raspberries (Rubus idaeus), blueberries (Vaccinium corymbosum), blackcurrents (Ribes nigrum), redcurrants (Ribes rubrum) and cranberries (Vaccinium oxycoccus) were included in the study. A detailed analysis revealed not only the nature and concentration of individual phenolic compounds but also their contribution to the overall antioxidant activity of the berries. The importance of such a detailed quantitative and qualitative analysis of phenolics from any specific source is considerable because their chemical structure has an impact on the absorption and bioavailability although the mechanisms involved are still unclear. Changes in the chemical structure of dietary phenolics following ingestion by humans and animal models cna in part help unravel this puzzle. The phenolic compounds present in major quantities are principally responsible for the antioxidant capacity in blackcurrants and blueberries were the anthocyanins. Blackcurrants contained large amounts of anthocyanins (5446 nmol/g) and vitamin C (2328 nmol/g) and had the highest AOC of the five berries. Blueberries were the second highest with anthocyanins levels of 4908 nmol/g but the sample study contained no vitamin C. Raspberries, redcurrant and cranberries contained anthocyanins but in lower amounts. Ellagitannins such as sanguiin H-6 were responsible for 58 per cent of the AOC of raspberries. Flavonols (16 per cent) and vitaimin C (23 per cent) were important antioxidants in cranberries while in redcurrants a number of unidentified peaks were the major contributors of the AOC (33 per cent) along with 28 per cent from anthocyanins. Raspberries were chosen for an intervention study with human subjects. The bioarailability of anthocyanins, ellagitannins and ellagic acid in raspberries was investigated. Plasma and urine were collected from six healthy human subjects after ingestion of 300 g of raspberries. Three healthy volunteers with an ileostomy were included in the study, providing ileal fluid, plasma and urine at different time points for 24 h after consuming a 300 g raspberry portion. All samples were analyzed using HPLC-PDA-MS[superscript 2]. No anthocyanins, ellagitannins or their metabolites or breakdown products were detected in the plasma of any of the volunteers. Eight of the anthocyanins identified in raspberries were detected and quantified in ileal fluid in their native form. They reached a maximum level of 36.5 per cent of intake in samples collected 0-4 h after supplementation and after 24 h there was an overall anthocyanin recovery of 39.6 per cent. With regard to ellagitannins, 16.3 [.moles] of sanguiin H-6 was detected in ileal fluid after 0-4 h with 26.2 per cent of intake being detected over the 0-24 h collection period. No lambertianin or sanguiin H-10 were found. The levels of ellagic acid in 0-24 h ileal fluid corresponded to 239.4 percent of intake 24 h with 162.9 per cent being collected 0-4 h after ingestion. This coincided with the peak levels of ellagic acid in urine although the levels, 13.1 nmoles and 33.6 nmoles for non ileostomy and ileostomy volunteers respcectively, were low and equivalent to no more than 0.4 per cent of intake. No ellagic acid or metabolites were detected in plasma at any time point. This study, therefore, found a low absorption and excretion of anthocyanins and ellagic acid in human subjects. The ca. 40 per cent recovery of these compounds in ileal fluid indicates that in healthy subjects with a colon substantial quantities pass from the small to the large intestine where they will be cataabolised by the gut microflora. The present study also investigated the distribution of anthocyanins, ellagitannins and their metabolites in the gastrointestinal tract and their presence in other tissues of rats fed 2.77 mL of raspberry juice by gavage. One hour after feeding the ellagitannins, sanguiin H-6 and lambertianin C had disappeared with only traces of ellagic acid being detected in the stomach. Up to 2 h after supplementation there was a very high recovery of unmetabolised anthocyanins, principally cyanidin-3-sophoroside, cyanidin-3-(2[superscript G]-glucosylrutinoside) and cyanidin-3-glucoside, a they passed from the stomach to the duodenim.jejunum and ileum. After 3 h, less than 50 per cent was recovered, after 4 h this declined to 11 per cent of intake and after 6 h only 2 per cent remained. Only trace quantities of anthocyanins were detected in the caecum, colon, and faeces and tehy were absent in extracts of liver, kidneys and brain. These findings imply anthocyanins are poorly absorbed and that which does occur takes place before ileum, in keeping with evidence indicating that the stomach and jejunum are sites of anthocyanin absorption in mice and rats. Because anthocyanins are poorly absorbed substantial amounts pass from the small to the large intestine where their rapid disappearance suggests they are degraded by faecal bacteria.