Flavonoids are present in foods bound to sugars as Î²-glycosides (with the exception of catechins) that make hard to absorption of flavonoids from the diet. Only aglycones which has not sugar molecule, were considered to be able to pass the gut wall, and no enzymes that can split these predominantly Î² -glycosidic bonds are secreted into the gut or present in the intestinal wall. During degradation of dietary flavonoids hydrolysis starts to occur in the colon by microorganisms. Thus, only a marginal absorption of dietary flavonoids is to be expected. However, research on the mechanisms for aglycone transfer across the gut wall is lacking. According to Hollman et al., (1995) studied onion absorption in humans and found that in a human study with ileostomy, the absorption of orally administered quercetin aglycone was 24% and the absorption of quercetin glycosides from onions was 52%, and 17% for pure quercetin rutinoside, a common glycoside in foods. This research provided information about humans can absorb appreciable amounts of quercetin which is occurring in the small intestine (Crepsy et al., 2001). Thus absorption from small intestine can be important than absorption from colon. The reason supported that flavonols and flavones are absorbed from ingested food, and appear rapidly in the plasma (<7 h). Generally the diffusion of phenolic aglycones occurs passively through biological membranes. However linkage of a phenolic to a sugar or organic acid increases the water solubility and severely limits passive diffusion. According to researches it may be possible for flavonol glucosides to be selectively absorbed in the gut. The reason is that the rate of absorption is affected by attached sugar (Hollman, 1999).
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The initial step in the absorption process for glycosylated flavonoids is deglycosylation which is essential if further metabolism is to occur (Williamson et al., 2000) and it provides conjugation from intestinal enzymes and transport to the serosal or mucosal sides (Williamson, 2004). "Deglycosylation can potentially occur at several sites in the duodenum and jejunum:
(1) within the intestinal lumen;
(2) brush border hydrolases; or
(3) intracellular hydrolases after transport of the flavonoid into the enterocyte (Williamson, 2004)."
There are 2 pathways of absorption for flavonoids. Both way give rise to intracellular aglycone, and in fact transient intracellular free aglycone (Williamson, 2004).
The first step in the absorption process for glycosylated flavonoids is deglycosylation by lactase phlorizin hydrolase (LPH). LPH is an enzyme that is located in the brush border of the small intestine and is responsible for lactose hydrolysis (Williamson, 2004). The deglycosylation reaction produces a free aglycone which can then diffuse into epithelial cells either passively or by facilitated diffusion. The enzyme provides deglycosylation in the lumen such as acting like outside the epithelial without first having to traverse the enterocyte membrane (Williamson, 2004).
The second pathway of absorption involves transport of the flavonoid glycoside into the enterocyte in an intact form via the function of a sugar transporter such as SGLT1. After transporting of flavonoid glycoside into the cell deglycosylation is started by cytosolic Î²-glucosidase (Williamson, 2004). Quercetin-4'-glucoside is a good substrate for the cytosolic-glucosidase and the research on rat everted intestine was shown that the sugar transporter/cytosolic Î² -glucosidase pathway accounted for 20% of the absorbed quercetin while LPH accounted for the remaining 80%. However the LPH pathway for quercetin-3-glucoside accounted for 100% of the absorbed quercetin although it is not a substrate for cytosolic Î²-glucosidase, (Williamson, 2004).
The initial conjugation of flavonoids occurs in the small intestine. The intestine's conjugating capacity has glucuronosyl transferases (UGTs) and glutathione transferases. These enzymes catalyze the conjugation of flavonoids in the human small intestine. Using models of the small intestine studies showed that the transfers of flavonoids from the mucosal (gut) compartment to the serosal (blood) compartment have found quercetin, predominantly in the glucuronidated form (Williamson, 2004). Sometimes glucuronide residues are removed and replaced with a sulphate during glucuronidation reactions then a mixture of glucuronide and sulfated flavonoid conjugates can be present in peripheral blood. The sulfation reaction is thought to occur predominantly in the liver (Williamson, 2004).
The liver receives flavonoids from the blood, including blood from the small intestine. Hepatic cells provide transportation of quercetin glucuronides from the small intestine to liver although quercetin-3-glucuronide and quercetin-7-glucuronide appear to be taken up by a different mechanism. After this transport the glucuronides are deglucuronidated inside the cell by Î²-glucuronidase and then sulfated or methylated. MRP2 export the conjugates of flavonoids into the bile and back to the small intestine. Flavonoids are delivered to tissues throughout the body by the blood. While aglycones could enter peripheral tissues by passive or facilitated diffusion, glucuronide conjugates need to be transported into peripheral tissues, because they are relatively hydrophilic and diffuse through membranes only very slowly (Williamson, 2004). The reason is that not only sulphate conjugates may be relatively hydrophobic. The cells which have Î²-glucuronidase activity are present in the lysosomal fraction and in the lumen of the endoplasmic reticulum; in liver cells. This enzyme provides deconjugation in tissues and it is also active on quercetin glucuronides (Williamson, 2004). According do Williamson the yield of quercetin in the urine less than 1.5% the amount of flavonoids in the urine dependent on the flavonoid and also the urinary content of flavonoids cannot be used as a biomarker of bioavailability or dietary intake. In the small intestine quercetin is much less efficiently absorbed than in the colon probably because quercetin is more readily broken down into low molecular weight phenolics by colonic microflora, and the aglycone of quercetin is unstable (Williamson, 2004).
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Figure 3 Pathway of quercetin metabolism (Williamson et al., 2000)
Materials and Methods
To determine the total quercetin concentrations there have been some numerous analytical methods reported in biological samples, obtained after the enzymatic hydrolysis of conjugated quercetin metabolites, and for the analysis of quercetin metabolites. Analytical methods have included LC/MS, LC/MS/MS, HPLC with UV detection, HPLC with fluorescent detection, HPLC with electrochemical detection and HPLC-radiocounting and tandem mass spectrometry. LC/MS method allows the analysis of low concentrations of parent quercetin in urine with good reproducibility (Ishii et al., 2003).
1.1. Chemical and reagents
Quercetin and rutin were purchased from Sigma Aldrich Inc (St Louis, MO). Stock solutions of quercetin and rutin were prepared by dissolving these compounds in ethanol followed by dilution with water (50% EtOH solution). Taxifolin is dissolved by using pure water. Î²-Glucuronidase were purchased from Sigma Aldrich Inc (St Louis, MO) type IX-A extract from E.Coli. Sulfatase was purchased from Sigma Aldrich Inc (St Louis, MO) type VI extract of Aerobacter aerogenes. All other chemicals and solvent were used without further purification. Methanol, ethanol, acetonitrile and sodium azide were all HPLC grade and purchased from Fisher.
1.2. Preparation of standard solutions for urine samples
A stock solution of 1 mg/ml quercetin was prepared in 50% ethanol solution. Dilution of the stock solution with 20% acetonitrile solution yielded working stock solutions at concentrations of 1, 20, 40, 60, 80, 100Âµg/ml. A stock solution of the internal standard taxifolin was prepared in water at a concentration of 1 mg/ml. A 100Âµl aliquot of quercetin stock solution, 100Âµl taxifolin stock solution and 100Âµl of epicatechin, catechin, caftaric, ferulic acid and caffeic acid were added to 2 ml eppendorf tube and vortexed for 1 min prior and then transfer into vial insert for analysis by LC-MS.
In our study 18 healthy people with no history of major disease recruited. A brief health questionnaire, description of the study, liability waiver, and right to opt out of the study provided to each participant. The study was approved by and performed under the University of Leeds. During the washing period (3 days) detailed dietary questions were requested from participant. Also during the diet volunteers were requested to avoid from certain types of foods containing the quercetin for two days before the consumption and in third day morning before consumption of raisins their baseline urine were collected after that they fed with 1 slice of bread, butter, banana and 100 grams Sun-Maid raisins after consumption they were requested to collect 24h urine. Subjects were coded and data were stored in a form which cannot be traced back to the name of the volunteer and then the addition of raisins; thus, we rely on the research is to determine and quantify which components of foods are absorbed and appear in the urine within a period of 24 hours. This allows us to estimate the bioavailability of quercetin and other compounds in raisins, and to see how much and in what form the body takes up and excretes the compounds. (Appendix form)
1.4. Raisins Extraction
Extraction was done according to Zhao and Halls method. To increase the efficiency of raisins extractions 3 grams well-chopped raisins were extracted with 15 ml solvent (EtOH, MeOH and ACN). The solvents were combined with water to make solvents containing 0, 25, 50, 75 and 95% water. Then raisins extracts vortex for 2 minutes. Following vortex they were homogenized for 5 minutes from low speed to higher speed to reduce the particle size. After that this mixture placed into centrifuge (25 min 20 Â°C 3000rpm) then the pellet re-extracted by adding 15ml of same solvent that re-extract put into centrifuge in the same protocol (A). The solvents from the repeat extractions were combined and stored in the freezer (-20Â°C). Each extraction process was done in triplicate. The aqueous extract filtered through 0.2Âµm polytetrafluoroethylene filter and injected onto HPLC column for the determination of polyphenols that they are investigated. Ethanol had better extraction compare to methanol and acetonitrile. According to Williamson and Carughi effectiveness of extraction is affected by solvent, extraction method, pH, and temperature.
Apart from Zhao and Halls method we used 2 different methods however the results from these extraction were not as good as Zhao and Halls method. One of these methods called protocol 2 was done according to following protocol; 3 grams well-chopped raisins were extracted with 15 ml water after that it was homogenised and sonicated for 10 minutes. From this aliquot 5 ml took out and centrifuge (25 min 20 Â°C 3000rpm) then the pellet called A and supernatant filtered through 0.2Âµm polytetrafluoroethylene filter and injected onto HPLC column. From the left mixture 5 ml took out and re-extracted with 15ml EtOH after that sonicated of mixture for 10 minutes was placed in centrifuge (25 min 20 Â°C 3000rpm) then this pellet called pellet B and supernatant filtered through 0.2Âµm polytetrafluoroethylene filter and injected onto HPLC column. Last 5ml of mixture put into freezer at -20Â°C. The pellets that were obtained from the mixture pellet A and pellet B extracted with 5ml EtOH/H2O (70%/30%) after that they were vortexed and sonicated for 10 minutes. Last mixtures were put into centrifuge (25 min 20 Â°C 3000rpm) then the pellet from pellet A called C and from B called as pellet D. Their supernatants filtered through 0.2Âµm polytetrafluoroethylene filter and injected onto HPLC column. Into pellet C and D 5ml Acetone/H2O (70:30) was added and they were vortexed and sonicated for 10 minutes than were put centrifuge (25 min 20 Â°C 3000rpm) then the pellets put into freezer. Their supernatants filtered through 0.2Âµm polytetrafluoroethylene filter and injected onto HPLC column. Second method called protocol 3 was done by increasing the raisins amount. 10 grams well-chopped raisins were extracted with 10 ml water after that it was homogenised and 10ml MeOH was added into mixture and homogenisation repeated second time. The reason why homogenisation was done for 2 times is to increase the solubility of compounds in solvent. Using measure cylinder 2ml of mixture placed into centrifuge (25 min 20 Â°C 3000rpm). The aqueous extract filtered through 0.2Âµm polytetrafluoroethylene filter and injected onto HPLC column for the determination of polyphenols that they are investigated.
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The HPLC system consisted of an Agilent Eclipse XDB-C18 RRHT threaded column with Merc Hitachi interface D-7000 Lachrom, Merc Hitachi autosampler L-7200, Merc Hitachi column oven L-7300, Merc Hitachi diode array detector L-7450 and Merc Hitachi pump L-7100. Solvent A 95% acetonitrile, 5% distilled water and 0.1% formic acid, solvent B 95% distilled water, 5% acetonitrile and 0.1% formic acid. The elution program at a flow rate of 1.0 ml/min followed a linear gradient from 100% to 10% A and from 0% to 90% B. The pressure limit was between 0 to 400 bar and the temperature was 35 Â°C. Simultaneous detection was at 260, 280, 310 and 370 nm. Total quercetin glycosides were quantitated as rutin and it can be estimated from absorbance measures at 370 nm. All peaks eluted within 26.7 min.
1.5. Urine Sampling and analysis (Analytical Methods of Assay)
We have 4 steps for preparing urine samples which are given below (Laboratory instructions).
2) Preparation for storage
3) Enzymatic hydrolysis
4) Reconstitution and filtration
Twenty-four hour urine samples were collected on day 3 in plastic 3000ml containers containing of 3 g ascorbic acid. Urine samples volume was measured. After that 10X10 portions of urine were measured into 15 mL falcon tubes. These falcon tubes also have 1 ml sodium-azide (0.1% concentration) that used as a biocide and prevents deterioration of samples during storage. They were stored in -20Â°C until analysis. The following flavonoids were quantified in the urine samples by LC-MS quercetin, catechin, epicatechin, ferulic acid, caffeic and caftaric acid. In brief, 100Âµl of 0.01% Taxifolin was added to 1ml of urine sample as internal standard. One control and consumption urines from same volunteer spiked with 100Âµl/ml quercetin and also from the same volunteer 100 Âµl of taxifolin and 100 Âµl of quercetin was added to 1ml of urine sample. The majority of flavonoids in urine will exist as conjugates (sulphates or glucuronides) rather than as aglycones. Therefore enzyme hydrolysis performed to liberate aglycones. To each 1 ml aliquots of urine were hydrolyzed by enzyme-enriched sodium phosphate buffer (0.2M, pH 7). An enzyme solution from E.coli is containing 50 activity units of Î²-glucuronidase and from Aerobacter aerogenes 0.3 activity units of sulfatase in 0.2M sodium phosphate buffer solution (pH 7). The reaction mixture was incubated at 37 Â°C for 2 hours with continuous shaking (100 rpm). After hydrolysis, 275 Âµl of 2% HCL were added to the mixture to stop the enzymatic activity and decrease pH into 3 to provide non-polar analytes removing through ethyl acetate part during washing procedure. Then 3 series of 1500 Âµl ethyl acetate washes provided selectively extraction of non-polar analytes. The resulting supernatant was evaporated by using centrifugal evaporator at 40Â°C for 6-7 hours. After drying up samples they were stored at -20Â°C until reconstitution. Before analysis the residue was dissolved in 50 Âµl ACN and 200 Âµl 0.125% ascorbic acid solution. Until getting the perfect dissolving they were sonicated. After all residue re-dissolved in the tubes, centrifuged at 1700rpm for 10 minutes. Then they placed into vials for LC/MS analysis. Also same protocol applied for urine baseline. Each urine samples was done in duplicate.
1.6. LC/MS analyses
The dried sample was re-constituted in 50Âµl acetonitrile and 200Âµl 0.125% ascorbic acid. After they vortexed they were centrifuging at 1700rpm for 10 minutes in IEC Microcl 17 centrifuge. After they were filtered through 0.2Âµm polytetrafluoroethylene filter, they were placed into LC/MS. Each sample was extracted and analyzed by LC/MS in duplicate. LC/MS Agilent Technologies 6410 Triple Quad LC/MS. Chromatographic separation of the analytes of interest was achieved on a C18 (particle size 3.1micron, 150mmÃ-2.1 mm) column (Phenomenx Kinetix) and the mobile phase consisted of acetonitrile/water with 0.1% formic acid. The injection volume was 5 micro litres. Solvent A has 0.1% formic acid in water and solvent B 0.1% formic acid in acetonitrile. Wang et al. (2005) reported that an acidic mobile phase using formic acid provided optimal separation and quantification of quercetin. The aromatic and phenolic compounds in the samples were identified by comparing retention times and relative retention time.
In this study we investigated the rutin (quercetin glycosides), catechin, epicatechin and caftaric acid content in the raisins than 18 participants was fed with 100 grams Sun Maid raisins. Before consumption of raisins they had 2 days diet (washing period) for the third day they were fed with 100 grams raisins. After consumption they were requested to collect 24 h urine.
Results showed that the yield of raisin extract effected from solvent type and extraction method and each participant has different amount of quercetin absorption depending on their metabolism and diet.
Three different methods were done however Zhao and Halls (2008) method has the best efficiency compare to other two. The 100% and 50% EtOH solvents produced extracts with the lowest rutin content that is close to none. The extracts obtained from 25% ethanol had significantly higher yield than the other treatments that is shown in table 1. Acetonitrile and methanol produced extracts significantly lower levels of rutin than ethanol and MeOH solvent showed better extraction than acetonitrile. The 5 and 100% MeOH solvents produced extracts with none concentration. Furthermore the 5% MeOH had significantly lower rutin compared with concentrations observed for the 5% EtOH. Generally for ACN the main differences for rutin was lower than the comparable alcohol solvents as it seen in table 1. From this information it is clear that extraction method (pH, solvent, temperature (Williamson and Carughi unpublished) effect the apparent composition of polyphenols.
Concentration of solvent (%)
Table 1 The rutin content (Âµg/g) in raisins obtained from ethanol extraction.
Zhao and Halls (2008) reported that the best solvents for total phenolic content is MeOH and EtOH beside from this report it is shown that 60% and 100% EtOH solvent extraction had better extraction in raisins extract and Williamson and Carughi reported that nearly 40mg/100g wet weight quercetin 3-O-rutinoside in raisin extract. According to Williamson and Carughi study they found that 12 mg/100g quercetin 3-O-glycoside and 3 mg/100g quercetin 3-O-rutinoside in raisins so totally 15mg/100g quercetin present. Total losses during extraction are figured out by using taxifolin as an internal standard. Before adding solvent into raisins 100Âµg of taxifolin added that helped to provide information about losses during extraction. Figure 3 showed taxifolin (1mg/ml) and rutin standard (50Âµg/ml) chromatograms besides rutin and taxifolin content from raisin extraction. According to these results lost of taxifolin amount was figured out as 13.4% than compare to this result total amount of rutin content was found 95Âµg/g in raisins. Participants fed with 100 grams raisins in this experiment. Williamson, 2004 reported that the yield of flavonoids in the urine depend on flavonoid type. According to Scalbert and Williamson (2000) excretion of quercetin in urine is less than 1.5% such as for onion 1.39% and for apple 0.44%.
Figure 3 HPLC chromatogram of Sun-Maid Raisins rutin content, internal and rutin standard. (1) Rutin standard (370 nm), (2) taxifolin (310 nm), (3) rutin content in raisin (370 nm), (4) rutin content in 310 nm, (5) taxifolin content in raisin (310 nm).
Phenolic compounds were detected in the participants' 24 h urine samples. The urinary excretion of phenolic compounds increased after feeding with raisins in 24 h urine. The absorption of quercetin was investigated by measuring the total quercetin concentrations present in the urine baseline (before consumption) and 24h urine after consumption. The differences between urine baseline and urine consumption gives the total quercetin concentration in urine. The total amounts of quercetin excreted in urine during the 24 h period were also significantly increased in all participants after feeding. The participants in our study consumed a flavonoid-restricted diet (avoiding fruits, vegetables, and beverages rich in flavonoids) for 2 days before feeding with raisin. According to Hollman et al. (1997) elimination half live of quercetin nearly 24 h therefore 48 h washing period should be enough to clear quercetin that is present in the urine however the amount of quercetin present in urine baseline has shown some variations between 1.5 Âµg/ml and 18.9 Âµg/ml. In figure 4 it is shown that LC/MS chromatograms of extracts of urine consumption (UC) and urine baseline (UB) for one of the volunteer with code 113 and retention time for quercetin is for urine consumption 16.242 while for urine baseline 16.250 so retention time shift left.
Figure 4 LC/MS chromatograms of extracts of urine consumption (UC) and urine baseline (UB) for one of the volunteer with code 113 for quercetin.
Figure 5 LC/MS chromatograms of extracts of urine consumption (UC) and urine baseline (UB) for one of the volunteer with code 113 for taxifolin as internal standard.
Retention time for taxifolin also shifts left as it is shown in figure 5 and the relative retention time confirms that it is quercetin in urine baseline and consumption. The participants in our study consumed a flavonoid-restricted diet (avoiding fruits, vegetables, and beverages rich in flavonoids) for 2 days before feeding with raisin. According to Hollman et al. (1997) elimination half live of quercetin nearly 24 h therefore 48 h washing period should be enough to clear quercetin that is present in the urine however the amount of quercetin present in urine baseline has shown some variations between 1.5 Âµg/ml and 18.9 Âµg/ml. As it is seen in figure 4 urine baseline has some quercetin and the amount of it is 1.5 Âµg/ml. The reason for that might come from different metabolism and some other factors such as not to follow flavonoid-restricted diet. According to raisins extraction each participant fed with 9500Âµg/100g rutin therefore if the excretion of quercetin in urine is accepted less than 1.5% than the amount of total quercetin in urine should be less than 142.5 Âµg. Participants were fed with raisins in the same condition because it was important to provide same conditions for all of them. The reason for that reported by Hollman et al. (1995); absorption of quercetin is effected from diet beside they discovered that quercetin conjugation with sugar provide increase in the absorption of quercetin (Hollman et al., 1995). Firstly all volunteers were fed with bread, butter, banana and water after that they were requested to ate 100 grams raisins. Each participant's 24h urine has different amount of quercetin that is between 238.8 Âµg/ml and 21.8 Âµg/ml. For the final results of total quercetin in urine has shown different values between 229.3 Âµg/ml and 16.2 Âµg/ml so total urinary excretion of quercetin changes between 2.4% to 0.17%. According to Hollman et al. (1995), absorption of quercetin-glucosides from onion was 52% while rutin from onion renders absorption only 15%.
Three different extraction methods were done with different solvents during experiment. The best efficiency was provided in Zhao and Halls (2008) method. According to Zhao and Halls (2008) method three solvents was used for extraction and instead of using raisin extraction well-chopped raisins used. These solvents were ethanol, methanol and acetonitrile. Ethanol extraction solvent had significantly higher yield than the other treatments. Acetonitrile and methanol solvents produced extracts significantly less rutin content than ethanol solvent and MeOH solvent showed better extraction than acetonitrile. AcetonitrileÂ (CH3CN) produced substantially lower yield than yields obtained from the comparable alcohol solvents. Alcohols have an important role in the extraction efficiency (Zhao and Halls, 2008). Other two extractions method had lower yield. Maximum extraction result for protocol 2 (64.4 Âµg/g) and the extracts obtain from protocol 3 was 75.6 Âµg/g. All the three extraction was shown better extraction yield with ethanol. For quercetin and caftaric acid 25% ethanol had better extraction compare to other concentrations and catechin and epicatechin was showed higher yield by using 5% ethanol. A similar trend was reported by Zhao and Halls (2008) and they reported that EtOH and MeOH showed higher extraction than acetone. The presence and identity of quercetin in human urine samples and raisin extraction are verified by two criteria that are a) spiking urine samples and raisin extraction that increase the expected peak heights; b) adding taxifolin as an internal standard into urine samples and raisin extraction to determine relative retention time. Beside losses from the process was identified by calculating the lost taxifolin during process. The retention time for taxifolin was 10.44 and the concentration was 0.01 Âµg/ml and the taxifolin solution was made with 20% ACN. Total loss of taxifolin amount was figured out as 13.4% during process. This loss might come from homogenization process.
Table 2: The amount of total excreted quercetin
The accuracy of measurements was wanted to determine in duplicate by adding 100Âµg of quercetin to 1.0ml aliquots of participant with the code 101. However during re-constitution of this spiked urine some amount of quercetin precipitated. Therefore the accuracy of measurement could not be done by using spiked urine. Total amount of rutin content was found 95 Âµg/g in raisins. Participants fed with 100 grams raisins in this experiment. In that case all participants consumed the amount of 9500 Âµg/100g rutin and Scalbert and Williamson (2000) reported that excretion of quercetin in urine is less than 1.5% such as for onion 1.39% and for apple 0.44%. Hydrolysation of rutin can be done by intestinal microflora with Î±-rhamnosidase and Î²-glucosidase to quercetin and then quercetin is absorbed and the absorbed quercetin excreted into bile and urine (Shimoi, 2003). Scalbert and Williamson (2000) reported that the amount of quercetin present in the urine should lower than 142.5 Âµg/ml. The variation of quercetin excretion is from the concentration 16.2 Âµg/ml to 229.3 Âµg/ml as it is shown in table 2. The participant that has the highest excretion ate chocolate in third day during dinner time. The lowest excretion should base on participant absorption metabolism because 24 h urine sample of this participant (subject code: 116) also has lower amount (21.8 Âµg/ml) although the consumption amount of raisin was same for all participants. During exclusion diet participant with the code 105 consumed dried plums and pineapple therefore baseline urine has high concentration of quercetin. For three days 107 ate mayonnaiseÂ that is a stableÂ emulsionÂ of olive oil, egg yolk and either vinegar or lemon juice with other herbs and spices. Except egg yolk other ingredients of mayonnaiseÂ include polyphenols. Therefore excretion of quercetin is higher for both baseline and consumption urine for this participant. The participants the codes are 111 and 113 have the best exclusion diet compare to other 16 participants. The amount of quercetin in urine baseline is very little. After consumption with raisin as it is seen in table 2 113 has more quercetin compare to 111 in 24 h urine. The reason might be related with different metabolism therefore it can be say 111 has better absorption than 113. Catechin, epicatechin, caffeic acid, caftaric acid and ferulic acid excreted lower than quercetin. Catechin and epicatechin was shown the lower excretion. The reason why quercetin has less absorption compare other polyphenols was reported by several researches. In foods quercetin exists in the form of glycosides and rutin is one of the most commonly occurring glycoside of quercetin (Gross et al., 1996) and first hydrolysed by the microflora before being absorbed (Ishii et al., 2003). Day et al., (2000) reported that naturally flavonols are present as glycosylated forms in foods therefore absorption of quercetin is dependent on the nature of glycoside and metabolism of these compounds hydroxyl groups conjugated with sulphate, glucuronic acid and limited methylation of the catechol functional group (Day et al., 2000). Therefore removal of sugar from flavonols should be providing by enzymes. Williamson (2004) reported that absorption of flavonols is started with deglycosylation by LPH in the lumen. Also quercetin hydrolysis with Î² glucosidase in the small intestine but it cannot hydrolyse in the liver (Scalbert and Williamson, 2000). However flavonols (exp: epicatechin, catechin) can pass through biological membranes and absorbed because there is no needs for hydrolysis or deconjugation (Scalbert and Williamson, 2000). Phenolic acid esters (exp: ferulic acid, caffeic acid) are esterified to organic acids, sugars and lipids however there is no esterases enzymes in humans to metabolise it (Scalbert and Williamson, 2000). According to all these papers it is clear that quercetin cannot metabolise well for some participants and also high amount of present quercetin in urine baseline can be thought there is some interference present in urine which has the same retention time with quercetin. Therefore it might cause increase the amount of present quercetin in urine baseline.
Bioactivities of polyphenols in raisins provide the health benefits. Flavanol, flavonols, and hydroxycinnamics are the major functional components that are responsible for most of the biological activities of raisins. During the recent years the importance of flavonoids which is generally present in fruit and vegetables become from their effect of health. Especially derivatives of quercetin gained importance as dietary constituents that are present high amount in raisins. The amount of quercetin in raisins is applicable to choose this food for feeding volunteers to see the absorption of quercetin. Raisins are a good dietary source of flavonol glycosides and phenolic acids and are considered to be a desirable source of dietary fiber, with polymerized phenolics contributing to that fiber. The antioxidant activity of flavonoids has strong linkage to the antioxidant activity of flavonoids.