- Email: [email protected]
Bioavailability of Ferulic Acid
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
253, 222–227 (1998)
RC989681
Bioavailability of Ferulic Acid Louise C. Bourne and Catherine Rice-Evans1 International Antioxidant Research Centre, Guy’s King’s College, and St. Thomas’s School of Biomedical Sciences, King’s College, Guy’s Campus, St. Thomas’s Street, London SE1 9RT, United Kingdom
Received October 13, 1998
There is a wealth of evidence for the powerful antioxidant properties in vitro of flavonoid components of the diet. However, few studies have been undertaken concerning the hydroxycinnamates, major constituents of fruit, some vegetables, beverages, and grains, particularly the extent to which they are absorbed in vivo from the diet. The study described here has investigated the bioavailability of ferulic acid in humans, from tomato consumption, through the monitoring of the pharmacokinetics of excretion in relation to intake. The results show that the peak time for maximal urinary excretion is approximately 7 h and the recovery of ferulic acid in the urine, on the basis of total free ferulic acid and feruloyl glucuronide excreted, is 11– 25% of that ingested. © 1998 Academic Press Key Words: ferulic acid; urinary excretion; hydroxycinnamate; bioavailability; tomato; antioxidant; free radical; oxidation.
Hydroxycinnamic acids are among the most widely distributed phenylpropanoids in plant tissues [1]. Ferulic acid (4-hydroxy-3-methoxycinnamic acid) and its dimers are ubiquitous components of the primary cell walls of plants [2]. The monomer is conjugated covalently with mono- and disaccharides, plant cell wall polysaccharides, glycoproteins, polyamines, lignin and hydroxy fatty acids in suberin and cutin. Ferulic acid and its precursors, p-coumaric acid (p-hydroxycinnamic acid) and caffeic acid (3,4-dihydroxycinnamic acid), are synthesized in plants from the shikimate pathway from phenylalanine or L-tyrosine. Several roles for ferulic acid in plants have been proposed, especially following dimerization. It cross-links vicinal pentosan chains of arabinoxylans and hemicellulose in cell walls [3–5] associated with the cessation of cell wall expansion. Such cross-linking is also essential in 1
To whom correspondence should be addressed at International Antioxidant Research Centre, Guy’s, King’s College and St. Thomas’s School of Biomedical Sciences, King’s College, Guy’s Campus, St. Thomas’s Street, London SE1 9RT, UK. Fax: -44-(0)171 955 4983. E-mail: [email protected]. 0006-291X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
the formation of barriers to invading pathogens [6]. Ferulate has also been used as a model compound in studying the formation of dehydrogenation polymers in vitro to understand the nature of these reactions in the wall [7, 8]. The antioxidant properties of the hydroxycinnamates have been demonstrated in vitro against peroxidizing polyunsaturated fatty acids in lipid systems and in low-density lipoproteins [9–12]. Investigations on the partitioning of ferulic acid in plasma have elucidated its association with the aqueous phase and its greater efficacy as an antioxidant against LDL oxidation than the hydrophilic ascorbic acid [13]. Simple phenolics such as hydroxycinnamates are also effective scavengers of reactive nitrogen species, protecting against the nitration of tyrosine induced by peroxynitrite [14]. There is considerable interest in the role of the major phenolic phytochemical components of fruit, vegetables, beverages and grains as dietary antioxidants. However, until recently, there has been a paucity of information on the absorption of flavonoids in humans. A number of studies are beginning to detect flavonoids and/or their metabolites in plasma and urine of humans consuming specific diets or supplements. In spite of the high levels in fruit and some vegetables, very few studies have been undertaken concerning the uptake of the hydroxycinnamate antioxidants from the diet. The purpose of this study was to investigate the bioavailability of ferulic acid from tomatoes in humans through the monitoring of the pharmacokinetics of their excretion in relation to intake. MATERIALS AND METHODS Study protocol. Ethical permission was obtained from The Guy’s Ethical Committee. Five healthy volunteers (one male and four female), mean age 27.2 6 7 years, BMI 22.8 6 5.6 kg/m2, consumed a single bolus of 360 –728 g tomatoes (equivalent to 8 g/kg body wt), providing approximately 21– 44 mg ferulic acid (Table 1). Volunteers fasted for 12 h prior to the study and refrained from taking antioxidant supplements for 1 week prior to the study and from specific phenolic-rich dietary agents for 48 h. These include bran cereals; whole grain products; vegetables such as broad beans, broccoli, brussel sprouts, cabbage, celery, chives, endive, aubergine, French beans, garlic, kale, leeks, lettuce, onions, parsley, radish, spinach, sweet
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Characteristics of Volunteers and Their Supplementation Subject
BMI (kg/m2)
Age (years)
Gender
Tomatoes ingested (g)
Tomatoes ingested (g)/body wt (kg)
Ferulic acid ingested (mg)
T101 T102 T103 T104 T105
18.26 18.82 19.15 28.4 29.4
25 28 22 22 39
F F F F M
360 400 368 728 640
8 8 8 8 8
21.71 24.12 22.19 43.89 38.6
Mean
22.8 6 5.6
27.2 6 7
30.1
peppers and tomatoes; fruits such as apples, apricot, blackberries, cranberries, grapes, grapefruit, pears, plums; strawberries; and beverages such as beer, coffee, fruit juices, tea and wine. Volunteers consumed a single bolus of fresh whole tomatoes of relevant weight, individual urine samples were collected in sterile tubes immediately prior to and for 24 hr post tomato consumption and stored at 270°C until analysis. Chemicals. Methanol and acetonitrile, all HPLC grade were obtained from Rathburn Chemicals (Walkerburn, Scotland). Ferulic acid and all other hydroxy-cinnamates were obtained from Extrasynthese (ZI Lyon Nord, BP 62, 69730 Genay, France). Tomatoes (Flavia variety) were from a major local United Kingdom supermarket (Sainsbury). Elgastat UHP double-distilled water (18 V grade) is used in all experiments. b-Glucosidase was obtained from ICN Biomedicals Inc. (Ohio 44202) and salicylic acid and b-glucuronidase (Type IX-A from E coli) is from Sigma (UK). Standards. Stock solutions of the standards were prepared by dissolving 1–2 mg of sample into either methanol or mobile phase (20% methanol, 0.1% HCl). Urine standards were prepared for analysis by the addition of ferulic acid stock solution to blank pooled human urine. The standards ranged from 0 to 300 ng of ferulic acid. Salicylic acid was used as internal standard for the HPLC analysis. HPLC analysis of the phenolic composition of tomatoes. The fruit was chopped into small pieces and lyophilized under liquid nitrogen, after recording the wet weight. For aqueous extraction, water (15 ml), methanol (15 ml) and salicylic acid (300 ml of stock solution, concentration 2 mg/ml) were added to 0.5–1 g of freeze dried material. The contents were refluxed for 30 min on a heating mantle (40°C). After cooling the mixture was filtered using a Buchner flask and Whatman No. 4 filter paper. The extract was then subjected to rotary evaporation under vacuum at 40 –50°C and the methanol removed. After transferring to an amber HPLC vial, the sample was ready for analysis. For enzymatic hydrolysis, 1 ml of the aqueous extract was incubated with 4084 units of b-glucoside in a stoppered culture tube for 1 h at 37°C. The extract was then diluted 1:1 (v/v) with mobile phase into an amber HPLC vial, ready for injection. Urine sample preparation. Urine samples were thawed and mixed well. A 1-ml sample was diluted into a 5-ml disposable culture tube containing 5ml salicylic acid as internal standard (stock solution 2 mg/ml). To this, 2.4 ml of methanol was added and 100 ml of HCl (5 M) the sample then stoppered and mixed for 30 s. Samples were centrifuged at 800g for 10 min at 4°C, the supernatant collected, and the methanol removed by rotary evaporation under vacuum at 40°C. The resultant aqueous fraction was filtered using a Flowpore 0.22-mm sterile nonpyrogenic membrane filter (Whatman, UK) directly into a HPLC vial. To cleave the glucuronidated conjugates, samples were hydrolyzed with b-glucuronidase. For enzymatic hydrolysis 1 ml of urine containing 5 ml salicylic acid (as internal standard) was incubated with 500 units/ml (final concentration) b-glucuronidase in a stoppered culture tube for 24 h at 37°C. Following the incubation, 100 ml of HCl
(5 M) and 2.4 ml methanol were added. The samples were stoppered and mixed for 30 s, as described above. Analysis of phenolics by gradient HPLC. HPLC analysis was conducted according to the method of Paganga and Rice-Evans [15]. The HPLC system consisted of an autosampler with a Peltier temperature controller, a 626 pump with a 600S controller, a photodiode array detector, and a software system that controlled all the equipment and carried out data processing. A Nova-Pak C18 column (4.6 3 250 mm) with a 4-mm particle size was used, and the temperature maintained by the column oven set at 30°C. The injection was by means of an autosampler, with a fixed loop, and the volume injected was 30 ml. Elution (0.8 ml/min) was performed using a solvent system composed of solvent A (20% methanol in 0.1% hydrochloric acid) and acetonitrile (solvent B) mixed using a linear gradient held at 95% solvent A for 10 min and then decreasing to 50% solvent A at 50 min, back to 95% solvent A at 55 min, and held under these conditions for a further 5 min. There was a 10-min delay before the next injection to ensure re-equilibration of the column. The chromatograms were obtained with photodiode array detection at 320 nm. All injections were performed in duplicate. Sample identification was established by comparing retention times and absorption spectra to reference standards chromatographed under identical conditions and spiking of samples with suspected compounds for confirmation. Quantification was carried out using calibration of the ferulic acid standard. Four calibration runs with ferulic acid were executed routinely. For the urine assay, calibration was performed by following the procedures for the standard solutions as described for urine samples. A linear regression calculation was performed on the resulting plot of peak area versus amount of ferulic acid, and the regression line used to calculate the amount of ferulic acid present.
RESULTS HPLC analysis of the phenolic composition of tomato extract, before (Fig. 1A) and after (Fig. 1B) glucosidase treatment, is illustrated in Fig. 1. Identification of the peaks for the glucosidase-treated tomato extract are 5.74, 7.80, 13.14, and 15.50 min for the hydroxycinnamates, chlorogenic, caffeic, p-coumaric and ferulic acid, respectively (Fig. 1B). These correspond in the non-enzyme treated extract (Fig. 1A) to 5.74 min chlorogenic acid, 5.91 min p-coumaroyl glycoside, with peaks at 5.11, 6.41, and 7.78 representing conjugates of hydroxycinnamates. The spectra of the peaks at 28.8 and 34.2 min suggest other conjugates of hydroxycinnamic acids, but clearly not glucosides as they are resistant to glucosidase hydrolysis over a range of en-
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FIG. 1. HPLC analysis with photodiode array detection at 320 nm of the phenolic composition of aqueous methanolic extracts of tomato fruit before (A) and after (B) glucosidase treatment to cleave the glucosides to the aglycone for spectral identification. Conditions: reverse phase C18 column, gradient elution with methanol/HCl/acetonitrile. (A) Major peaks eluting at 5.74, 5.91, 25.18, and 35.58 min correspond to chlorogenic acid, p-coumaroyl glycoside, rutin, and naringenin, respectively; (B) peaks at eluting 5.74, 7.8, 13.14, 15.5, 25.24, and 35.64 min are identified as chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, rutin, and naringenin, respectively. 224
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creases to a range of approximately 2.7–5.5 mg (range of volumes of 24-h urines being 1.2–3.0 liters). The individual concentrations excreted at specific time points for volunteer T103 are also demonstrated (Fig. 3). On the concentration versus time plot, the time at which maximal excretion occurs is ca. 7 h. DISCUSSION
FIG. 2. The cumulative urinary excretion of ferulic acid in mg over 24 h postintake for all subjects: (A) Free ferulic acid; (B) total ferulic acid, free and conjugated.
zyme concentrations. Rutin (quercetin-3-rutinoside) known to be resistant to glucosidase treatment, and naringenin, previously suggested to be present as the aglycone in tomatoes [16], are identified with retention times at around 25 and 35.6 min, respectively, in both enzyme- and non-enzyme-treated extracts, with the internal standard, salicylic acid, eluting at 26.6 min. Estimation of levels of ferulic acid derived from ferulic acid conjugates extracted from the fresh tomatoes were 6 mg ferulic acid/100 g fresh wt. The cumulative excretion of free ferulic acid after ingestion of tomatoes (Fig. 2A) increases with time post-supplementation, giving a similar profile for all the volunteers. Excretion continues progressively up to 7–9 h, after which it reaches a plateau, showing no further excretion. The concentration of free ferulic acid excreted over 24 h was in the range of 0.9 –3.0 mg for all subjects, approximately 4 –5% of the ferulic acid ingested. Quantification of the excretion of conjugated ferulic acid, as the glucuronide, was determined from the increase in the appearance of ferulic acid detected by HPLC after treatment of the urine samples with b-glucuronidase. The results (Fig. 2B) show that a considerable proportion of ferulic acid is excreted as the glucuronide in all subjects, the relative proportions of the free to conjugated forms varying from subject to subject. The total amount of ferulic acid excreted in-
Early studies suggested that the metabolic fate of ferulic acid was qualitatively identical to that of caffeic acid, for which a large number of metabolites have been reported (Fig. 4) [17, 18]. The range of metabolites and their relative proportions will depend on many factors, including dose, route of administration and animal species. A variety of pieces of experimental data have substantiated the notion of very similar metabolic routes for these two hydroxycinnamates.. For example, following ingestion of caffeic acid or ferulic acid, the same 3-hydroxyphenyl- and 3-methoxy-4-hydroxyphenyl-derivatives of phenyl-propionic acid, hydracrylic acid and glycine conjugates were excreted in human urine (Fig. 4) [19 –21, 17]. Feeding studies in rats with ferulic acid revealed metabolism to a dehydroxylated compound and the same hydroxymethoxy derivatives, as in the human studies, with ferulic acid itself being partly excreted as the glucuronide [20]. Studies of others [22] support the suggestion that 3-hydroxyphenylpropionic acid is a major urinary metabolite of ferulic acid in rats after intra-peritoneal administration. The study described here provides evidence for the absorption and bioavailability of the hydroxycinnamate ferulic acid from tomato consumption. The peak time for maximal excretion is approximately 7 h and the recovery of ferulic acid in the urine, on the basis of the total amount of free ferulic acid and feruloyl glucuronide excreted, is 11–25% of that ingested. To function physiologically dietary phenolics should
FIG. 3. The concentration (mM) of ferulic acid excretion for volunteer 103 at individual time points: Upper curve, total ferulic acid concentration profile, free and conjugated; lower curve, free ferulic acid concentration.
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FIG. 4. The metabolic fate of ferulic acid and caffeic acid [17]: 1, substituted cinnamic acid derivatives; 2, phenylproprionic acid metabolites; 3, phenylhydracrylic acid metabolites; 4, substituted cinammoyl- and benzoyl-glycine conjugates; 5, benzoic acid metabolites.
not only be absorbed intact, but also have an appropriate lifetime in the general circulation and these studies show that dietary ferulic acid has the appropriate pharmacokinetic properties. Much research has focused on lycopene as the active constituent of tomatoes with a role in disease prevention [23–27]. Our studies suggest that the phenolic components may also play a contributory role to the health benefits of fruit, vegetables and grains and this study provides evidence for the bioavailability of ferulic acid. ACKNOWLEDGMENT We thank the Ministry of Agriculture, Fisheries, and Food for financial support for this research.
REFERENCES 1. Macheix, J-J., Fleuriet, A., and Billot, J. (1990) Fruit Phenolics, CRC Press, Boca Raton, FL. 2. Graf, E. (1992) Free Radical Biol. Med. 13, 435– 448. 3. Fry, S. C. (1986) Annu. Rev. Plant Physiol. 37, 165–186.
4. Fry. S. C. (1995) Annu. Rev. Plant Physiol. Mol. Biol. 46, 497– 520. 5. Iiyama, K., Lam, T. B. T., and Stone, B. A. (1994) Plant Physiol. 104, 315–320. 6. Bolwell, G. P. (1993) Int. Rev. Cytol. 146, 261–324. 7. Ralph, J., Quideau, J. H., Grabber, J. K., and Hatfield, D. (1994) J. Chem. Soc. Perkins Trans. I, 3485–3498. 8. Zimmerlin, A., Wojtaszk, P., and Bolwell, G. P. (1994) Biochem. J. 299, 747–753. 9. Chimi, H., Cillard, J., Cillard, P., and Rahmani, M. (1991) J. Am. Oil Chem. Soc. 68, 307–312. 10. Castelluccio, C., Paganga, G., Melikian, N., Bolwell, G. P., Pridham, J., Sampson, J., and Rice-Evans, C. (1995) FEBS Lett. 368, 188 –192. 11. Laranjinha, J., Almeida, L., and Maderia, V. (1995) Free Radical Biol. Med. 19, 329 –337. 12. Bourne, L., and Rice-Evans, C. (1997) Free Radical Res. 27, 337–344. 13. Castelluccio, C., Bolwell, G. P., Gerrish, C., and Rice-Evans, C. (1996) Biochem. J. 316, 691– 694. 14. Pannala, A., Razaq, R., Halliwell, B., Singh, S., and Rice-Evans, C. (1998) Free Radical Biol. Med. 24, 594 – 606. 15. Paganga, G., and Rice-Evans, C. (1997) FEBS Lett. 401, 78 – 82.
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16. Wardale, D. A. (1980) Phytochemistry 12, 1523–1528. 17. Scheline, R. R. (1991) CRC Handbook of Mammalian Metabolism of Plant Compounds, CRC Press, Boca Raton, FL. 18. DeEds, F., Booth, A. N., and Jones, F. T. (1955) Fed. Proc. Fed. Am. Soc. Exp. Biol. 14, 332. 19. Shaw, K. N. F., and Trevarthen, J. (1958) Nature 182, 792. 20. Scheline, R. R. (1968) Acta Pharmacol. Toxicol. 26, 189 –193. 21. Booth, A. N., Emmerson, O. H., Jones, F. T., and DeEds, F. (1957) J. Biol. Chem. 229, 51.
22. Teuchy, H., and Van Sumere, C. F. (1971) Arch. Int. Physiol. Biochim. 79, 589 –597. 23. Beecher, G. R. (1998) Proc. Soc. Exp. Biol. Med. 218, 98 –100. 24. Clinton, S. K. (1998) Nutr. Rev. 56, 35–51. 25. La Vecchia, C. (1998) Proc. Soc. Exp. Biol. Med. 218, 125– 128. 26. Gartner, C., Stahl, W., and Sies, H. (1997) Am. J. Clin. Nutr. 66, 116 –122. 27. Gerster, H. (1997) J. Am. Coll. Nutr. 16, 109 –126.
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RC989681
Bioavailability of Ferulic Acid Louise C. Bourne and Catherine Rice-Evans1 International Antioxidant Research Centre, Guy’s King’s College, and St. Thomas’s School of Biomedical Sciences, King’s College, Guy’s Campus, St. Thomas’s Street, London SE1 9RT, United Kingdom
Received October 13, 1998
There is a wealth of evidence for the powerful antioxidant properties in vitro of flavonoid components of the diet. However, few studies have been undertaken concerning the hydroxycinnamates, major constituents of fruit, some vegetables, beverages, and grains, particularly the extent to which they are absorbed in vivo from the diet. The study described here has investigated the bioavailability of ferulic acid in humans, from tomato consumption, through the monitoring of the pharmacokinetics of excretion in relation to intake. The results show that the peak time for maximal urinary excretion is approximately 7 h and the recovery of ferulic acid in the urine, on the basis of total free ferulic acid and feruloyl glucuronide excreted, is 11– 25% of that ingested. © 1998 Academic Press Key Words: ferulic acid; urinary excretion; hydroxycinnamate; bioavailability; tomato; antioxidant; free radical; oxidation.
Hydroxycinnamic acids are among the most widely distributed phenylpropanoids in plant tissues [1]. Ferulic acid (4-hydroxy-3-methoxycinnamic acid) and its dimers are ubiquitous components of the primary cell walls of plants [2]. The monomer is conjugated covalently with mono- and disaccharides, plant cell wall polysaccharides, glycoproteins, polyamines, lignin and hydroxy fatty acids in suberin and cutin. Ferulic acid and its precursors, p-coumaric acid (p-hydroxycinnamic acid) and caffeic acid (3,4-dihydroxycinnamic acid), are synthesized in plants from the shikimate pathway from phenylalanine or L-tyrosine. Several roles for ferulic acid in plants have been proposed, especially following dimerization. It cross-links vicinal pentosan chains of arabinoxylans and hemicellulose in cell walls [3–5] associated with the cessation of cell wall expansion. Such cross-linking is also essential in 1
To whom correspondence should be addressed at International Antioxidant Research Centre, Guy’s, King’s College and St. Thomas’s School of Biomedical Sciences, King’s College, Guy’s Campus, St. Thomas’s Street, London SE1 9RT, UK. Fax: -44-(0)171 955 4983. E-mail: [email protected]. 0006-291X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
the formation of barriers to invading pathogens [6]. Ferulate has also been used as a model compound in studying the formation of dehydrogenation polymers in vitro to understand the nature of these reactions in the wall [7, 8]. The antioxidant properties of the hydroxycinnamates have been demonstrated in vitro against peroxidizing polyunsaturated fatty acids in lipid systems and in low-density lipoproteins [9–12]. Investigations on the partitioning of ferulic acid in plasma have elucidated its association with the aqueous phase and its greater efficacy as an antioxidant against LDL oxidation than the hydrophilic ascorbic acid [13]. Simple phenolics such as hydroxycinnamates are also effective scavengers of reactive nitrogen species, protecting against the nitration of tyrosine induced by peroxynitrite [14]. There is considerable interest in the role of the major phenolic phytochemical components of fruit, vegetables, beverages and grains as dietary antioxidants. However, until recently, there has been a paucity of information on the absorption of flavonoids in humans. A number of studies are beginning to detect flavonoids and/or their metabolites in plasma and urine of humans consuming specific diets or supplements. In spite of the high levels in fruit and some vegetables, very few studies have been undertaken concerning the uptake of the hydroxycinnamate antioxidants from the diet. The purpose of this study was to investigate the bioavailability of ferulic acid from tomatoes in humans through the monitoring of the pharmacokinetics of their excretion in relation to intake. MATERIALS AND METHODS Study protocol. Ethical permission was obtained from The Guy’s Ethical Committee. Five healthy volunteers (one male and four female), mean age 27.2 6 7 years, BMI 22.8 6 5.6 kg/m2, consumed a single bolus of 360 –728 g tomatoes (equivalent to 8 g/kg body wt), providing approximately 21– 44 mg ferulic acid (Table 1). Volunteers fasted for 12 h prior to the study and refrained from taking antioxidant supplements for 1 week prior to the study and from specific phenolic-rich dietary agents for 48 h. These include bran cereals; whole grain products; vegetables such as broad beans, broccoli, brussel sprouts, cabbage, celery, chives, endive, aubergine, French beans, garlic, kale, leeks, lettuce, onions, parsley, radish, spinach, sweet
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Characteristics of Volunteers and Their Supplementation Subject
BMI (kg/m2)
Age (years)
Gender
Tomatoes ingested (g)
Tomatoes ingested (g)/body wt (kg)
Ferulic acid ingested (mg)
T101 T102 T103 T104 T105
18.26 18.82 19.15 28.4 29.4
25 28 22 22 39
F F F F M
360 400 368 728 640
8 8 8 8 8
21.71 24.12 22.19 43.89 38.6
Mean
22.8 6 5.6
27.2 6 7
30.1
peppers and tomatoes; fruits such as apples, apricot, blackberries, cranberries, grapes, grapefruit, pears, plums; strawberries; and beverages such as beer, coffee, fruit juices, tea and wine. Volunteers consumed a single bolus of fresh whole tomatoes of relevant weight, individual urine samples were collected in sterile tubes immediately prior to and for 24 hr post tomato consumption and stored at 270°C until analysis. Chemicals. Methanol and acetonitrile, all HPLC grade were obtained from Rathburn Chemicals (Walkerburn, Scotland). Ferulic acid and all other hydroxy-cinnamates were obtained from Extrasynthese (ZI Lyon Nord, BP 62, 69730 Genay, France). Tomatoes (Flavia variety) were from a major local United Kingdom supermarket (Sainsbury). Elgastat UHP double-distilled water (18 V grade) is used in all experiments. b-Glucosidase was obtained from ICN Biomedicals Inc. (Ohio 44202) and salicylic acid and b-glucuronidase (Type IX-A from E coli) is from Sigma (UK). Standards. Stock solutions of the standards were prepared by dissolving 1–2 mg of sample into either methanol or mobile phase (20% methanol, 0.1% HCl). Urine standards were prepared for analysis by the addition of ferulic acid stock solution to blank pooled human urine. The standards ranged from 0 to 300 ng of ferulic acid. Salicylic acid was used as internal standard for the HPLC analysis. HPLC analysis of the phenolic composition of tomatoes. The fruit was chopped into small pieces and lyophilized under liquid nitrogen, after recording the wet weight. For aqueous extraction, water (15 ml), methanol (15 ml) and salicylic acid (300 ml of stock solution, concentration 2 mg/ml) were added to 0.5–1 g of freeze dried material. The contents were refluxed for 30 min on a heating mantle (40°C). After cooling the mixture was filtered using a Buchner flask and Whatman No. 4 filter paper. The extract was then subjected to rotary evaporation under vacuum at 40 –50°C and the methanol removed. After transferring to an amber HPLC vial, the sample was ready for analysis. For enzymatic hydrolysis, 1 ml of the aqueous extract was incubated with 4084 units of b-glucoside in a stoppered culture tube for 1 h at 37°C. The extract was then diluted 1:1 (v/v) with mobile phase into an amber HPLC vial, ready for injection. Urine sample preparation. Urine samples were thawed and mixed well. A 1-ml sample was diluted into a 5-ml disposable culture tube containing 5ml salicylic acid as internal standard (stock solution 2 mg/ml). To this, 2.4 ml of methanol was added and 100 ml of HCl (5 M) the sample then stoppered and mixed for 30 s. Samples were centrifuged at 800g for 10 min at 4°C, the supernatant collected, and the methanol removed by rotary evaporation under vacuum at 40°C. The resultant aqueous fraction was filtered using a Flowpore 0.22-mm sterile nonpyrogenic membrane filter (Whatman, UK) directly into a HPLC vial. To cleave the glucuronidated conjugates, samples were hydrolyzed with b-glucuronidase. For enzymatic hydrolysis 1 ml of urine containing 5 ml salicylic acid (as internal standard) was incubated with 500 units/ml (final concentration) b-glucuronidase in a stoppered culture tube for 24 h at 37°C. Following the incubation, 100 ml of HCl
(5 M) and 2.4 ml methanol were added. The samples were stoppered and mixed for 30 s, as described above. Analysis of phenolics by gradient HPLC. HPLC analysis was conducted according to the method of Paganga and Rice-Evans [15]. The HPLC system consisted of an autosampler with a Peltier temperature controller, a 626 pump with a 600S controller, a photodiode array detector, and a software system that controlled all the equipment and carried out data processing. A Nova-Pak C18 column (4.6 3 250 mm) with a 4-mm particle size was used, and the temperature maintained by the column oven set at 30°C. The injection was by means of an autosampler, with a fixed loop, and the volume injected was 30 ml. Elution (0.8 ml/min) was performed using a solvent system composed of solvent A (20% methanol in 0.1% hydrochloric acid) and acetonitrile (solvent B) mixed using a linear gradient held at 95% solvent A for 10 min and then decreasing to 50% solvent A at 50 min, back to 95% solvent A at 55 min, and held under these conditions for a further 5 min. There was a 10-min delay before the next injection to ensure re-equilibration of the column. The chromatograms were obtained with photodiode array detection at 320 nm. All injections were performed in duplicate. Sample identification was established by comparing retention times and absorption spectra to reference standards chromatographed under identical conditions and spiking of samples with suspected compounds for confirmation. Quantification was carried out using calibration of the ferulic acid standard. Four calibration runs with ferulic acid were executed routinely. For the urine assay, calibration was performed by following the procedures for the standard solutions as described for urine samples. A linear regression calculation was performed on the resulting plot of peak area versus amount of ferulic acid, and the regression line used to calculate the amount of ferulic acid present.
RESULTS HPLC analysis of the phenolic composition of tomato extract, before (Fig. 1A) and after (Fig. 1B) glucosidase treatment, is illustrated in Fig. 1. Identification of the peaks for the glucosidase-treated tomato extract are 5.74, 7.80, 13.14, and 15.50 min for the hydroxycinnamates, chlorogenic, caffeic, p-coumaric and ferulic acid, respectively (Fig. 1B). These correspond in the non-enzyme treated extract (Fig. 1A) to 5.74 min chlorogenic acid, 5.91 min p-coumaroyl glycoside, with peaks at 5.11, 6.41, and 7.78 representing conjugates of hydroxycinnamates. The spectra of the peaks at 28.8 and 34.2 min suggest other conjugates of hydroxycinnamic acids, but clearly not glucosides as they are resistant to glucosidase hydrolysis over a range of en-
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FIG. 1. HPLC analysis with photodiode array detection at 320 nm of the phenolic composition of aqueous methanolic extracts of tomato fruit before (A) and after (B) glucosidase treatment to cleave the glucosides to the aglycone for spectral identification. Conditions: reverse phase C18 column, gradient elution with methanol/HCl/acetonitrile. (A) Major peaks eluting at 5.74, 5.91, 25.18, and 35.58 min correspond to chlorogenic acid, p-coumaroyl glycoside, rutin, and naringenin, respectively; (B) peaks at eluting 5.74, 7.8, 13.14, 15.5, 25.24, and 35.64 min are identified as chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, rutin, and naringenin, respectively. 224
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creases to a range of approximately 2.7–5.5 mg (range of volumes of 24-h urines being 1.2–3.0 liters). The individual concentrations excreted at specific time points for volunteer T103 are also demonstrated (Fig. 3). On the concentration versus time plot, the time at which maximal excretion occurs is ca. 7 h. DISCUSSION
FIG. 2. The cumulative urinary excretion of ferulic acid in mg over 24 h postintake for all subjects: (A) Free ferulic acid; (B) total ferulic acid, free and conjugated.
zyme concentrations. Rutin (quercetin-3-rutinoside) known to be resistant to glucosidase treatment, and naringenin, previously suggested to be present as the aglycone in tomatoes [16], are identified with retention times at around 25 and 35.6 min, respectively, in both enzyme- and non-enzyme-treated extracts, with the internal standard, salicylic acid, eluting at 26.6 min. Estimation of levels of ferulic acid derived from ferulic acid conjugates extracted from the fresh tomatoes were 6 mg ferulic acid/100 g fresh wt. The cumulative excretion of free ferulic acid after ingestion of tomatoes (Fig. 2A) increases with time post-supplementation, giving a similar profile for all the volunteers. Excretion continues progressively up to 7–9 h, after which it reaches a plateau, showing no further excretion. The concentration of free ferulic acid excreted over 24 h was in the range of 0.9 –3.0 mg for all subjects, approximately 4 –5% of the ferulic acid ingested. Quantification of the excretion of conjugated ferulic acid, as the glucuronide, was determined from the increase in the appearance of ferulic acid detected by HPLC after treatment of the urine samples with b-glucuronidase. The results (Fig. 2B) show that a considerable proportion of ferulic acid is excreted as the glucuronide in all subjects, the relative proportions of the free to conjugated forms varying from subject to subject. The total amount of ferulic acid excreted in-
Early studies suggested that the metabolic fate of ferulic acid was qualitatively identical to that of caffeic acid, for which a large number of metabolites have been reported (Fig. 4) [17, 18]. The range of metabolites and their relative proportions will depend on many factors, including dose, route of administration and animal species. A variety of pieces of experimental data have substantiated the notion of very similar metabolic routes for these two hydroxycinnamates.. For example, following ingestion of caffeic acid or ferulic acid, the same 3-hydroxyphenyl- and 3-methoxy-4-hydroxyphenyl-derivatives of phenyl-propionic acid, hydracrylic acid and glycine conjugates were excreted in human urine (Fig. 4) [19 –21, 17]. Feeding studies in rats with ferulic acid revealed metabolism to a dehydroxylated compound and the same hydroxymethoxy derivatives, as in the human studies, with ferulic acid itself being partly excreted as the glucuronide [20]. Studies of others [22] support the suggestion that 3-hydroxyphenylpropionic acid is a major urinary metabolite of ferulic acid in rats after intra-peritoneal administration. The study described here provides evidence for the absorption and bioavailability of the hydroxycinnamate ferulic acid from tomato consumption. The peak time for maximal excretion is approximately 7 h and the recovery of ferulic acid in the urine, on the basis of the total amount of free ferulic acid and feruloyl glucuronide excreted, is 11–25% of that ingested. To function physiologically dietary phenolics should
FIG. 3. The concentration (mM) of ferulic acid excretion for volunteer 103 at individual time points: Upper curve, total ferulic acid concentration profile, free and conjugated; lower curve, free ferulic acid concentration.
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FIG. 4. The metabolic fate of ferulic acid and caffeic acid [17]: 1, substituted cinnamic acid derivatives; 2, phenylproprionic acid metabolites; 3, phenylhydracrylic acid metabolites; 4, substituted cinammoyl- and benzoyl-glycine conjugates; 5, benzoic acid metabolites.
not only be absorbed intact, but also have an appropriate lifetime in the general circulation and these studies show that dietary ferulic acid has the appropriate pharmacokinetic properties. Much research has focused on lycopene as the active constituent of tomatoes with a role in disease prevention [23–27]. Our studies suggest that the phenolic components may also play a contributory role to the health benefits of fruit, vegetables and grains and this study provides evidence for the bioavailability of ferulic acid. ACKNOWLEDGMENT We thank the Ministry of Agriculture, Fisheries, and Food for financial support for this research.
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