Bioorganic & Medicinal Chemistry Letters 21 (2011) 5784–5786

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Bioorganic & Medicinal Chemistry Letters

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Microbial metabolism. Part 13.1 Metabolites of hesperetin

Wimal Herath a, Ikhlas Ahmad Khan a,b,⇑a National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, University, MS 38677, USAb Department of Pharmacognosy, Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, University, MS 38677, USA

a r t i c l e i n f o

Article history:Received 2 June 2011Revised 29 July 2011Accepted 1 August 2011Available online 9 August 2011

Keywords:HesperitinMicrobial metabolismMucor ramannianus

0960-894X/$ - see front matter � 2011 Elsevier Ltd.doi:10.1016/j.bmcl.2011.08.004

⇑ Corresponding author. Tel.: +1 662 915 7821; faxE-mail address: ikhan@olemiss.edu (I.A. Khan).

a b s t r a c t

The fungal culture, Mucor ramannianus (ATCC 2628) transformed hesperitin (1) to four metabolites: 40-methoxy-5,7,8,30-tetrahydroxyflavanone (8-hydroxyhesperetin) (2), 5,7,30,40-tetrahydroxyflavanone(eriodictyol) (3), 40-methoxy-5,30-dihydroxyflavanone 7-sulfate (hesperetin 7-sulfate) (4) and 5,7,30-tri-hydroxyflavanone 40-O-a-quinovopyranoside (eriodictyol 40-O-a-quinovopyranoside) (5). The structureswere established by spectroscopic methods.

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The flavonoid, hesperetin is the aglycone of hesperidin found insweet oranges, other citrus fruits and some herbs.2–4 It is the mostconsumed flavonoid amounting to about 30% of the total intake.4

Both compounds are associated with beneficial health effects.2 Bio-logical activities of hesperetin include antioxidant, bone-sparingand lipid-lowering effects.5 The antioxident property together withthe ability to penetrate the blood–brain barrier is suggested to helpin neuroprotection against oxidative damage.6 Hesperetin alsoplays a significant role in inflammation and cancer inhibition.2 Itis known that the Nuclear Factor-kappa B (NF-jB) found in thecytoplasm promotes inflammation-associated cancer. NF-jB, inaddition, activates the genes responsible for inflammation, amongothers and prevents cancer cell destruction by inactivating tumorsuppressor proteins.7,8 It also plays a major role in the aging pro-cess.9 Animal experiments show the importance of antioxidantsas inhibitors of NF-jB.10 Thus, phytochemicals like flavanoids withsuch properties may hold promise for cancer prevention,7 providedthat they block specific signal partways which depend on NF-jbactivation to prevent adverse side effects.10 One such compoundis hesperitin. In vitro biological activities of hesperitin are well doc-umented but little is known about its in vivo efficacy. Despitemany investigations carried out to date, its bioavailability is poorlyunderstood.3,4,11–16 Using an experiment where hesperetin itself isadministered orally to humans,11 some conclusions would havebeen drawn as to the fate of hisperetin in vivo, if the chemicalstructures of the blood plasma metabolites were elucidated.12 Itis vital to learn the bioavailability and selectivity of hesperetinand its metabolites on choosing NF-jB as the target to prevent can-

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: +1 662 915 1006.

cer.10 Since, microorganisms can be used as predictive models formammalian drug metabolism we investigated retrospectively themicrobial transformation of hesperetin (1)17 to isolate and charac-terize metabolites which may help to predict its fate in mamma-lian systems.18,19

Forty microorganisms were screened to evaluate the ability totransform hesperitin (1) to its metabolites.20 Fermentations werecarried out according to a two-stage procedure21 in medium a.22

Suitable controls were used to ensure that the metabolites werea result of enzyme activity and not a consequence of non-meta-bolic transformation.22 Mucor ramannianus (ATCC 2628) was se-lected for the preparative stage due to its higher transformationefficiency compared to several other organisms which showedconversion capability.

M. ramannianus transformed hesperitin (1) (Fig. 1) to 40-meth-oxy-5,7,8,30-tetrahydroxyflavanone (8-hydroxyhesperetin) (2),5,7,30,40-tetrahydroxyflavanone (eriodictyol) (3), 40-methoxy-5,30-dihydroxyflavanone 7-sulfate (hesperetin 7-sulfate) (4) and5,7,30-trihydroxyflavanone 40-O-a-quinovopyranoside (eriodictyol40-O-a-quinovopyranoside) (5).

The molecular formulae of all the metabolites were determinedby HR-ESI-MS. Structures were elucidated by spectroscopicmethods.23

Metabolite 224 (0.9 mg, 0.03%) was isolated as a white solidwith a molecular formula, C16H14O7. It was more polar than hes-peretin (1). The NMR data were similar to those of 1 except forthe presence of an additional hydroxyl group in ring A. NMR corre-lations along with the published data were used to characterizethe compound as 40-methoxy-5,7,8,30-tetrahydroxyflavanone(8-hydroxyhesperitin).25

The white solid 326 (10 mg, 0.27%) with a molecular ion peak atm/z 287.0450 [M�H]+ in its HR-ESI-MS corresponded to the

O

O

R2

R5

1

35

7

1'4'

R3

R4

R1 3'

8A C

B

R1 R2 R3 R4 R5

1 H OH OH OH OMe2 OH OH OH OH OMe3 H OH OH OH OH4 H OSO3H OH OH OMe5 H OH OH OH quinovose

quinovose =

O

H

HO

H

HO

H

OHOHH

H1''3''5''

6''

4'' 2''

Figure 1. Structures of hesperetin (1) and its metabolites obtained from M.ramannianus.

W. Herath, I. A. Khan / Bioorg. Med. Chem. Lett. 21 (2011) 5784–5786 5785

elemental formula, C15H12O6. The 1H NMR data showed closeresemblance to those of 1 except for the absence of a three protonsinglet due to methoxy group.

It was identified as 5,7,30,40-tetrahydroxyflavanone (eriodictyol)(3) by correlation spectra together with published data.27

Compound 428 (3.7 mg, 0.13%) was isolated as an off-white solidwith a molecular formula C16H14O9S. Comparison of this com-pound with 1 revealed significant similarity except for the pres-ence of a sulfate moiety, the position of which was determinedas C-7 by correlation NMR spectra. It was further supported bythe shielding of the signal due to the ipso carbon at C-7 positionby 5.3 ppm and the deshielding of C-6 and C-8 carbons ortho toand C-10 carbon para to the sulfation site by values of 3.3, 3.1and 2.1 ppm respectively, in the 13C NMR spectrum when com-pared with that of 1.29 The loss of 80 mass units together with1270, 1025 and 825 absorptions observed in the MS and IR spectra,respectively, gave further evidence to the presence of a sulfategroup in the molecule. The forgoing data suggested 40-methoxy-5,30-dihydroxyflavanone 7-sulfate (hesperetin 7-sulfate) as thestructure of compound 4.

Metabolite 530 (05 mg, 0.13%), was assigned the molecular for-mula C21H22O10 based on m/z 435.1378 [M+H]+. The 1H NMR spec-tral data of the compound was similar to those of 1, except for thepresence of a glycosyl unit. It showed resonances between d 3.54–5.21 in the 1H NMR and five carbon resonances between d 67.8–100.5 in the 13C NMR spectrum. The small coupling constant ofthe anomeric proton d 5.21 (1H, d, J = 3.6 Hz, H-100) indicated ana-configuration of the glycosyl moiety. The occurrence of O-glyco-sylation at C-40 was suggested by the presence of three-bond cor-relation of the anomeric proton with the quaternary carbon at d145.7 (C-40) which in turn is in three-bond correlations with H-20

(d 6.92) and H-60 (d 6.84). The large coupling constants betweenH-200/300, H-300/400, H-400/500 protons indicated trans-diaxial relation-ships. The NMR data are in agreement with those published forquinovopyranose.31–33 Thus, the structure of metabolite 5 wasdetermined as 5,7,30-trihydroxyflavanone 40-O-a-quinovopyrano-side (eriodictyol 40-O-a-quinovopyranoside).

Hesperetin (1), found in sweet orange and lemon as a rhamnog-lucoside (hesperidin) is shown to have important biological activ-ities in vitro.3 However, knowledge about absorption, distribution,metabolism and excretion along with physiological behavior arerequired to evaluate its benefits and risks following oral intake.34

Hesperedin, after oral ingestion undergoes hydrolysis by colonicmicroflora to yield the aglycon, hesperetin which is absorbed bythe intestine and metabolized by UDP-glucuronosyltransferaces(UGTSs) and sulfotransferaces (SULTs) to glucuronides and sulfo-nates in the intestinal cells or during further first-pass metabolismleading to differences in regioselectivity and kinetics for conju-gates.35 This was demonstrated using different UGT and UGTS en-zymes along with selected tissues of humans and rats.35

Experiments revealed that during bio-transformation, hesperetingets conjugated at 7 and/or 30 positions.35 In vitro metabolismstudies using Caco-2 cell monolayers as a small intestine modelgave Hesperetin 7-O-glucuronide and hesperetin 7-O-sulfate.12 Invivo studies too showed the formation of glucuronide and sulfatemetabolites.4,36–38 In the present investigation we isolated hes-peretin 7-sulfate (4) and eriodictyol 40-O-a-qinovopyranoside (5)as prominent metabolites showing sulfation and glycosylation asthe pathway of metabolism of hesperetin in fungi as well, indicat-ing the ability of microbes to mimic mammalian metabolism.39 It isknown that microbes unlike mammals use different sugars instateof glucuronides to conjugate polar groups.39 Formation of 5 is sig-nificant as homoeriodictyol conjugates have been detected in ratplasma,36 indicating the possible conversion of hesperetin to eri-odictyol (3) in mammalian and fungal systems. Eriodictyol and8-hydroxyhesperitin (2) detected in the present study show strongantioxidant properties.25,40,41 2 is a microbial transformation prod-uct of 1, isolated from Aspergillus saitoi culture extract.25 The pres-ence of a free C-7 OH group together with O-dihydroxyl groups in3, contribute to its strong antioxidant activity.41 Similarly, com-pound 2 shows distinct antioxidant activity comparable to thatof a-tocopherol due to the presence of adjacent hydroxyl groupsat C-7 and C-8.25 Isolation of these microbial products with antiox-idant capacity much higher than that of hesperitin (1) may be ofhelp to evaluate the activity of 1 in terms of these metabolites hith-erto not detected in vivo experiments.

Acknowledgments

The authors thank Mr. Frank Wiggers for assistance in obtaining2D NMR spectra and Dr. Bharathi Avula for conducting HRESIMSanalysis. This work was supported, in part, by the United StatesDepartment of Agriculture, Agricultural Research Specific Coopera-tive Agreement No. 58-6408-2-00009.

References and notes

1. Mikell, J. R.; Herath, W.; Khan, I. A. Chem. Pharm. Bull. 2011, 59, 692.2. Tomás-Barberán, F. A.; Clifford, M. N. J. Sci. Food Agric. 2000, 80, 1073.3. Garg, A.; Garg, S.; Zaneveld, L. J. D.; Singla, A. K. Phytother. Res. 2001, 15, 655.4. Manach, C.; Morand, C.; Gil-Izquierdo, A.; Bouteloup-Demange, C.; Rémésy, C.

Eur. J. Clin. Nutr. 2003, 57, 235.5. Horcajada, M. N.; Habauzit, V.; Trzeciakiewicz, A.; Morand, C.; Gil-Izquierdo, A.;

Mardon, J.; Lebecque, P.; Davicco, M. J.; Chee, W. S. S.; Coxam, V.; Offord, E. J.Appl. Physiol. 2008, 104, 648.

6. Hwang, S.; Yen, G. J. Agric. Food Chem. 2008, 56, 859.7. Prasad, S.; Phromnol, K.; Yadav, V. R.; Chaturvedi, M. M.; Aggarwal, B. B. Planta

Med. 2010, 76, 1044.8. Pikarsky, E.; Porat, R. M.; Stein, I.; Abramovitch, R.; Amit, S.; Kasem, S.;

Gutkovich-Pyrest, E.; Urieli-Shoval, S.; Galun, E.; Ben-Neriah, Y. Nature 2004,431, 461.

9. Kim, J. Y.; Jung, K. J.; Choi, J. S.; Chung, H. Y. Aging Cell 2006, 5, 401.10. D’Acquisto, F.; May, M. J.; Ghosh, S. Mol. Interv. 2002, 2, 22.11. Kanaze, F. I.; Bounartzi, M. I.; Georgarakis, M.; Niopas, I. Eur. J. Clin. Nutr. 2007,

61, 472.12. Brand, W.; van der Wel, P. A. I.; Rein, M. J.; Barron, D.; Williamson, G.; van

Bladeren, P. J.; Rietjens, I. M. C. M. Drug Metab. Dispos. 2008, 36, 1794.

5786 W. Herath, I. A. Khan / Bioorg. Med. Chem. Lett. 21 (2011) 5784–5786

13. Karoon, P. A.; Clifford, M. N.; Crozier, A.; Day, A. J.; Donovan, J. L.; Manach, C.;Williamson, G. Am. J. Clin. Nutr. 2004, 80, 15.

14. Spencer, J. P. E.; Chowrimootoo, G.; Choudhury, R.; Debnam, E. S.; Srai, S. K.;Rice-Evans, C. FEBS Lett. 1999, 458, 224.

15. Nielsen, I. L. F.; Chee, W. S. S.; Poulsen, L.; Offord-Cavin, E.; Rasmussen, H.;Enslen, M.; Barron, D.; Horcajada, M.; Williamson, G. J. Nutr. 2006, 136,404.

16. Erlund, I.; Meririnne, E.; Alfthan, G.; Aro, A. J. Nutr. 2001, 131, 235.17. Hesperetin (1) was purchased from Sigma–Aldrich Chemical Co. (Milwaukee,

Wisconsin) and its authenticity was confirmed by NMR data.18. Rosazza, P. N.; Duffel, M. W. In Alkaloid Chemistry and Pharmacology; Brossi, A.,

Ed.; Academic Press: New York, 1986; Vol. 27, pp 391–392. Chapter 4.19. Clark, A. M.; McChesney, J. D.; Hufford, C. D. Med. Res. Rev. 1985, 5, 231.20. Forty microorganisms from The National Center for Natural Products Research

of The University of Mississippi were used to select organisms capable oftransforming hesperetin to its metabolites. M. ramannianus was selected forpreparative scale fermentation.21 Six 1 L flasks, each containing 100 mg ofsubstrate dispersed in 500 mL of medium-a were used.22 The culture mediawere extracted with EtOAc. The residues of the combined extracts werecolumn chromatographed (Silica Gel 60 F254) to isolate the metabolites.Repeated column and preparative thin layer (Silica Gel 60 F254)chromatography were performed to purify the metabolites. Substrate andculture controls were run along with the above experiments.22

Microbial transformation of hesperitin (1) by M. ramannianus (ATCC 20129):Column chromatographic separation (Si Gel 230–400 mesh: E. Merck, 30 g,column diameter: 20 mm) was carried out using CH2Cl2 enriched with MeOHas the eluent. Repeated column and preparative layer chromatography(CH2Cl2–MeOH, 24:1) were used to further purify the column fractions. Fourmetabolites, 2–5 were isolated. They were identified by spectroscopic methodsalong with published data.17–26

21. Abourashed, E. A.; Khan, I. A. Chem. Pharm. Bull. 2000, 48, 1996.22. Herath, W. H. M. W.; Ferreira, D.; Khan, I. A. Nat. Prod. Res. 2003, 17, 269.23. Unless otherwise stated the 1H and 13C NMR were obtained in CDCl3 and

DMSO-d6 on a Bruker DRX-500 spectrometer. UV spectra were obtained using aHewlett Packard 8452A diode array spectrometer. IR spectra were measured inCHCl3 on an ATI Mattson Genesis series FTIR spectrophotometer. HR-ESI-MSdata were obtained using a Bruker GioApex 3.0.

24. 40-Methoxy-5,7,8,30-tetrahydroxyflavanone (8-hydroxyhesperitin) (2) wasobtained as a yellow amorphous solid (0.9 mg, 0.03%). Rf 0.36 [hexane–MeOH–CH2Cl2 (3:1:13)]; ½a�27

D +8.22 (c 0.07, MeOH); UV kmax (MeOH) nm(log e): 336.9 (3.40), 289.0 (3.99), 231.5 sh (4.01), 207.0 (4.21); IR mmax(CHCl3)cm�1: 3358, 2925, 2854, 1639, 1616, 1459, 1344, 1160, 1086, 1005, 874; HR-ESI-MS [M�H]+: (m/z) 317.0556 (calcd for C16H14O7�H+: 317.06601). It wasidentified by comparison with literature data.25

25. Miyake, Y.; Minato, K.; Fukumoto, S.; Yamamoto, K.; Oya-Ito, T.; Kawakishi, S.;Osawa, T. Biosci. Biotechnol. Biochem. 2003, 67, 1443.

26. 5,7,30 ,40-Tetrahydroxyflavanone (eriodictyol) (3) (10 mg, 0.27%), was isolatedas an amorphous yellow solid with a Rf 0.40 [MeOH–CH2Cl2 (2:11.5)]; ½a�27

D+4.14 (c 0.19, MeOH); UV kmax (MeOH) nm (log e): 403.5 (3.17), 335.1 sh (3.54),287.9 (4.16) 222.0 (4.23); IR mmax (CHCl3) cm�1: 3187, 2925, 2856, 1639, 1630,1459, 1277, 1162, 1080, 824; HR-ESI-MS [M�H]+: (m/z) 287.0450 (calcd forC15H12O6�H+: 287.05545). The metabolite was identified by comparison withpublished spectroscopic data.27

27. Moretti, C.; Sauvain, M.; Lavaud, C.; Massiot, G.; Bravo, J. A.; Muñoz, V. J. Nat.Prod. 1998, 61, 1390.

28. 40-Methoxy-5,30-dihydroxyflavanone 7-sulfate (hesperetin 7-sulfate) (4)(3.7 mg, 0.13%), was a light yellow solid with a Rf 0.28 [hexane–MeOH–CH2Cl2 (20:7:33)]; ½a�27

D +4.8 (c 0.33, MeOH); UV kmax (MeOH) nm (log e): 332.1(3.49), 286.1 (4.11), 2.23.1 (4.25); IR mmax (CHCl3) cm�1: 3170, 2926, 2856,1638, 1606, 1514, 1457, 1270, 1135, 1025, 825; 1H NMR 400 MHz (DMSO-d6)d: 2.77 (1H, dd, J = 17 Hz, 3 Hz, H-3eq), 3.25 (1H, dd, J = 17 Hz, 12.5 Hz, H-3ax),3.78 (s, OCH3), 5.43 (1H, dd, J = 12.5 Hz, 2.5 Hz, H-2), 6.33 (1H, d, J = 2 Hz, H-8),6.36 (1H, d, J = 2 Hz, H-6), 6.89 (1H, dd, J = 17 Hz, 3 Hz, H-60), 6.95 (1H, d, J = 8.5,H-50), 6.96 (1H, br s, H-20). 13C NMR 150 MHz (DMSO-d6) d 42.8 (C-3), 60.3(OCH3), 78.7 (C-2), 98.7 (C-8), 99.7 (C-6), 104.1 (C-10), 114.5 (C-20), 131.5 (C-10), 147.0 (C-30), 148.4 (C-40), 162.5 (C-5), 162.5 (C-7), 162.8 (C-9). HR-ESI-MS[M�H]+: (m/z) 381.0575 (calcd for C16H14O9S�H+: 281.02795). Spectroscopicanalysis confirmed the structure of 4.

29. Agrawal, P. K.; Bansal, M. C. In Carbon-13 NMR of Flavonoids; Agrawal, P. K., Ed.;Elsevier: Amsterdam, 1989; pp 168–171. pp. 286–293, Chapter 6.

30. 5,7,30-Trihydroxyflavanone 40-O-a-quinovopyranoside (eriodictyol 40-O-a-quinovopyranoside) (5) (05 mg, 0.13%), was a white amorphous solid with aRf 0.14[MeOH–CH2Cl2 (2:23)]; ½a�27

D �128.5 (c 0.05, MeOH); UV kmax (MeOH)nm (log e): 330.0 (3.93), 286.1 (4.40), 222.0 (4.52); IR mmax (CHCl3) cm�1: 3353,2925, 2857, 1639, 1630, 1459, 1281, 1162, 1087, 842; 1H NMR 400 MHz(DMSO-d6) d: 1.06 (1H, d, J = 6.6 Hz, H-600), 2.67 (1H, dd, J = 17.4 Hz, 3 Hz, H-3eq), 3.16 (1H, dd, J = 17.4 Hz, 12.6 Hz, H-3ax), 3.54 (1H, br m, H-400), 3.66 (1H,dd, J = 10.2, 3.6 Hz, H-200), 3.81 (1H, dd, J = 10.2, Hz, H-300), 3.92 (1H, br m,J = 6.6 Hz, H-500), 5.21 d (1H, d, J = 3.6 Hz, H-100), 5.39 (1H, dd, J = 12.6, 3 Hz, H-2), 5.83 (1H, d, J = 2.4 Hz, H-8), 5.84 (1H, d, J = 2.4 Hz, H-6), 6.84 (1H, d,J = 8.4 Hz, H-60), 6.92 (1H, br s, H-20), 7.06 (1H, d, J = 8.4 Hz, H-50). 13C-NMR150 MHz (DMSO-d6) d 16.9 (C-600), 42.5 (C-3), 67.8 (C-500), 68.6 (C-200), 69.9 (C-300), 71.8 (C-400), 78.6 (C-2), 95.4 (C-8), 95.6 (C-6), 100.5 (C-100), 102.0 (C-10),114.8 (C-20), 117.1 (C-50), 118.2 (C-60), 133.6 (C-10), 145.7 (C-40), 163.2 (C-5),163.9 (C-9), 196. 4 (C-4). HR-ESI-MS [M+H]+: (m/z) 435.1378 (calcd forC21H22O10+H+: 435.12924). Spectroscopic data confirmed the structure of 5.

31. Findlay, J. A.; Jaseja, M.; Burnell, D. J.; Brison, J.-R. Can. J. Chem. 1987, 65, 1384.32. León-Rivera, I.; Mirón-López, G.; Estrada-Soto, S.; Aguirre-Crespo, F.; Gutiérrez,

M. C.; Molina-Salinas, G. M.; Hurtado, G.; Navarrete-Vázquez, G.; Montiel, E.Bioorg. Med. Chem. Lett. 2009, 19, 4652.

33. Enriquez, R. G.; Leon, I.; Perez, F.; Walls, F.; Carpenter, K. A.; Puzzuoli, F. V.;Reynolds, W. F. Can. J. Chem. 1992, 70, 1000.

34. Prasain, J. K.; Barnes, S. Mol. Pharm. 2007, 4, 846.35. Brand, W.; Boersma, M. G.; Bik, H.; Hoek-van den Hil, E. F.; Vervoort, J.; Barron,

D.; Meinl, W.; Glatt, H.; Williamson, G.; van Bladeren, P. J.; Rietjens, I. M. C. M.Drug Metab. Dispos. 2010, 38, 617.

36. Matsumoto, H.; Ikoma, Y.; Sugiura, M.; Yano, M.; Hasegawa, Y. J. Agric. FoodChem. 2004, 52, 6653.

37. Brett, G. M.; Hollands, W.; Needs, P. W.; Teucher, B.; Dainty, J. R.; Davis, B. D.;Brodbelt, J. S.; Kroon, P. A. B. J. Nutr. 2009, 101, 664.

38. Gardana, C.; Guarnieri, S.; Riso, P.; Simonetti, P.; Porrini, M. B. J. Nutr. 2007, 98,165.

39. Davis, P. J. Microbial Transformations: Models of Mammalian Metabolism andCatalysts in Synthetic Medicinal Chemistry. In Antibiotics and MicrobialTransformations; Lamba, S. S., Walker, C. A., Eds.; CRC Press: Boca Raton,Florida, 1987; pp 47–70. Chapter 3.

40. Miyake, Y.; Minato, K.; Fukumoto, S.; Shimomura, Y.; Osawa, T. Food Sci.Technol. Res. 2004, 10, 70.

41. Takamatsu, S.; Galal, A. M.; Ross, S. A.; Ferreira, D.; ElSohly, M. A.; Ibrahim, A.S.; El-Feraly, F. S. Phytother. Res. 2003, 17, 963.