Nutrition 21 (2005) 920 –924
www.elsevier.com/locate/nut

Applied nutritional investigation

Melatonin in walnuts: Influence on levels of melatonin and total
antioxidant capacity of blood
Russel J. Reiter, Ph.D.*, L. C. Manchester, Ph.D., and Dun-xian Tan, M.D., Ph.D.
Department of Cellular and Structural Biology, The University of Texas Health Science Center, San Antonio, Texas, USA
Manuscript received November 15, 2004; accepted February 4, 2005.

Abstract

Objective: We investigated whether melatonin is present in walnuts (Juglans regia L.) and, if so,
tested whether eating walnuts influences melatonin levels and the total antioxidant status of theblood.
Methods: Melatonin was extracted from walnuts and quantified by high-performance liquid
chromatography. After feeding walnuts to rats, serum melatonin concentrations were measured
using a radioimmunoassay and the “total antioxidant power” of the serum was estimated by using
the trolox equivalent antioxidant capacity and ferric-reducing ability of serum methods.
Results: Mean ⫾ standard error melatonin concentrations were 3.5 ⫾ 1.0 ng/g of walnut. After
food restriction of rats and then feeding them regular chow or walnuts, blood melatonin concentrations in the animals that ate walnuts were increased over those in the rats fed the control diet.
Increases in blood melatonin were also accompanied by increases in trolox equivalent antioxidant
capacity and ferric-reducing ability of serum values.
Conclusions: Melatonin is present in walnuts and, when eaten, increase blood melatonin concentrations. The increase in blood melatonin levels correlates with an increased antioxidative capacityof this fluid as reflected by augmentation of trolox equivalent antioxidant capacity and ferricreducing ability of serum values. © 2005 Elsevier Inc. All rights reserved.

Keywords:

Walnuts; Melatonin; Cardiovascular system; Antioxidant; Trolox equivalent antioxidant capacity; Ferric-reducing ability of serum

Introduction
Walnuts (Juglans regia L.) are receiving increasing interest as a healthy foodstuff because their regular consumption has been reported to decrease the risk of heart disease[1–3]. These findings are sufficiently compelling to thepoint where the U.S. Food and Drug Administration has
affirmed the following health claim for walnuts: “Supportive but not conclusive research shows that eating 1.5 ounces
per day of walnuts as part of a diet low in saturated fat andcholesterol may reduce the risk of heart disease” (July 14,2003). The published basic and clinical research on thebenefits of walnut consumption on cardiovascular physiology was used to support this claim.
This study was supported by a grant from the California WalnutCommission.
* Corresponding author. Tel.: ⫹210-567-3859; fax: ⫹210-567-6948.
E-mail address: reiter@uthscsa.edu (R.J. Reiter).
0899-9007/05/$ – see front matter © 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.nut.2005.02.005

The health benefits of walnuts are usually attributed totheir high content of ␻-3 fatty acids [3–5] and, to a lesser
degree, to the amount of vitamin E they contain [6], in
particular ␥-tocopherol, which promotes the cellular uptakeof ␣-tocopherol. In addition, walnuts have other components that may be beneficial for health including a low
lysine:arginine ratio and high levels of arginine, folate,fiber, tannins, and polyphenols [1,7]. Importantly, walnut
consumption does not cause a net gain in body weight wheneaten as a replacement food [8].
Several reports that have appeared in the past decade havealso identified melatonin, a molecule with beneficial effects on
the cardiovascular system [9,10], in edible foods and seeds[11,12]. Melatonin (N-acetyl-5-methoxytryptamine) is synthesized and widely distributed in the animal kingdom [13,14],and its presence in plants has prompted investigations into thepotential health benefits of consumption of foodstuffs containing this indole. Given the numerous observations regarding thepositive effects of walnut consumption on cardiovascular

R.J. Reiter et al. / Nutrition 21 (2005) 920 –924

pathophysiology [1,2], we anticipated that, in addition to otherheart-healthy constituents [4 –7], walnuts may contain melatonin. Thus, the goals of the present research were to test whether
melatonin is found in walnuts and, if so, to determine whether
consumption of walnuts would alter circulating levels of thisconstituent in rats. Further, because melatonin is an antioxidant, we determined the total antioxidant capacity of blood
after rats had eaten walnuts.

Materials and methods
Materials
Bulk walnuts (mixed variety) were supplied by the California Walnut Commission. Chromatographically pure
melatonin was a gift from the Helsinn Chemical Company(Biasca, Switzerland). Other chemicals and reagents werepurchased from Sigma Chemical Company (St. Louis, MO,
USA).
Melatonin extraction from walnuts and measurement
Five different 1-g samples of raw walnuts were pulverized with a mortar and pestle by using 15 ml of methanol as
the homogenizing solution. The homogenates were centrifuged at 10 000g at 4° C for 30 min. After removing thelipid phase, the supernatant was collected and evaporated
under vacuum (20 psi). Residues were redissolved in 1 ml
(pH 7.4, 20 mM) of phosphate buffered saline. Two milliliters of chloroform were added to this mixture and horizontally shaken for 2 min. After centrifugation at 1000g for
5 min, the water phase was discarded and the organic phase
was evaporated under a vacuum. The residue then was
dissolved in 100 ␮L of high-performance liquid chromatography (HPLC) mobile phase for melatonin analysis using
HPLC. Before analysis, samples were filtered with a
0.22-␮m filter under centrifugation at 15 000g for 1 h.
Thirty microliters of each sample was injected into the
HPLC-ECD system, which consisted of an ESA 580 dual
pump, 504 autosampler, and a Coularray 8-channel coulometric array electrochemical detector (ESA, Chelmsford,
MA, USA). A C18 reverse-phase column was used to separate melatonin. The mobile phase was constituted with 0.1
mM potassium phosphate buffer (pH 4.5) with acetonitrile(20%) at a flow rate of 1 mL/min. Applied potentials were
initiated at 200 mV for channel 1 and was increased by 100
mV for each remaining channel.

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ambient temperature of 22 ⫾ 1°C and 55% humidity. During this interval animals had free access to Purina rodent
chow and fresh tap water. Rats were then assigned to one of
two groups (control fed and walnut fed) of eight rats eachand fasted for 24 h; after the period of fasting (at 9:00 AM),
control rats were given access to rodent chow, whereas theremaining eight animals were given walnuts to eat. After
4 h, all animals were killed by decapitation and trunk blood
was collected. Serum was separated by centrifugation andstored at ⫺80°C until assayed for melatonin.
Serum melatonin was estimated by means of a direct
radioimmunoassay [15,16] using the following parameters.
A 250-␮L serum sample was combined with 100 ␮L ofantiserum (initial dilution 1:9000; Batch G/S/704-8483;
Stockgrand Ltd., Guilford, UK) and 100 ␮L of [3H]melatonin (TRK 798 Amersham, Buckinghamshire, UK;approximately 2000 cpm/tube) in an assay buffer (0.1 M
tricine buffer, pH 8.0, containing 0.1% gelatin, 0.9% NaCland 0.1% NaN3). After an overnight incubation period (18h) at 4°C, 500 ␮L of chilled dextran-coated charcoal solution was added (0.5 g of charcoal and 0.05 g of dextran in
100 mL of assay buffer). After vortexing and incubation for
15 min at 4°C, tubes were centrifuged at 15 000g for 15 min
at 4°C. Thereafter, 750 ␮L of the supernatant was added to
7.5 mL of scintillation fluid (Liquiscini, National Diagnostics, Manville, NJ, USA) and radioactivity in the tubes wascounted for 10 min in a beta-counter. Assay sensitivity wasbetter than 2 pg/mL, with intra- and interassay variations of
7.8% and 12.8%, respectively.
Trolox equivalent antioxidant capacity andferric-reducing ability of serum studies and assays
Two-month-old male Sprague-Dawley rats were purchased from Harlan. Rats initially had free access to foodand water and were maintained under conditions as described above. After a 1-wk acclimation period, animals
were assigned to a control group (n ⫽ 8) and into a walnutfed group (n ⫽ 12) and subsequently deprived of food for
24 h. After the period of fasting, animals were provided
Purina rodent chow (control fed) or walnuts to eat. After 5 h
30 min, all rats were killed by decapitation and trunk blood
was collected. Blood samples were centrifuged and serum
was assayed for trolox equivalent antioxidant capacity(TEAC) and ferric-reducing ability of serum (FRAP) values. The TEAC and FRAP assays were performed precisely
as described by Re et al. [17] and Benzie and Strain [18],respectively.

Serum melatonin study and assay
Statistical analyses
Two-month-old male Sprague-Dawley rats (mean bodyweight 150 –175 g at time of purchase) were purchased from
Harlan (Indianapolis, IN, USA). During a 1-wk period of
acclimation, animals were kept under a 12-h:12-h light:darkcycle (lights off at 7:00 PM and on at 7:00 AM daily) with an

Data are expressed as mean ⫾ standard error. Unpairedt test was used to compare differences between control-fed
and walnut-fed rats. P ⱕ 0.05 was considered statisticallysignificant.

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R.J. Reiter et al. / Nutrition 21 (2005) 920 –924

Fig. 1. Serum melatonin levels in blood from rats that were fasted overnight and then given regular rodent chow or walnuts to eat. As shown inthis report, walnuts contain melatonin and their consumption is associatedwith increased circulating levels of melatonin. **P ⬍ 0.01.

Results

become apparent is that the concentration of this indoleamine varies widely according to the plant species studied[11,12,20]. The highest concentrations of plant melatoninreported to date exist in Chinese herbal products [20 –22].
To our knowledge, the walnut is the first commonly eaten
tree nut in which melatonin has been measured.
The function of melatonin in plants is presumably similar
to that in animals, where, among a variety of functions, it isa free radical scavenger and antioxidant [23–25]. Its presence in the walnut may be to protect the highly oxidizable
lipids from oxidation, thereby preserving the viability of thenut (seed) so it will subsequently successfully germinate.
Manchester et al. [26] proposed this function for melatoninin seeds.
Whether melatonin in plant products is synthesized insitu or whether it is taken up from the soil remains unknown. If the latter is the case, it may be possible to increase
the melatonin concentration in plants by adding it to the soilin which they grow. However, there is a modicum of evidence that suggests that plants have the enzymatic machin-

Using the described method of extraction and after correction of the values for the recovery rate (about 40%), thecalculated concentrations of melatonin in walnuts was 3.5 ⫾1.0 ng/g.
All procedures for the treatment of animals described inthis report were approved by the institutional animal careand utilization committee. After fasting for 24 h, feeding of
the control diet (rodent chow) or walnuts, and then death,the stomach of each animal was inspected. In both experiments and for all rats, the stomach was filled with food atthis time. Judging from the contents of the stomach, it wasestimated that each rat had eaten 3.0 to 3.5 g of food during
the interval from the initiation of food availability to tissue
collection.
After consumption of rodent chow or walnuts, walnutfed animals had significantly higher blood melatonin levels
at the conclusion of the experiment (Fig. 1). The mean valueof melatonin in the rodent chow group was 11.5 ⫾ 1.9
pg/mL and that of the walnut group was 38.0 ⫾ 4.3 pg/mL.
As with melatonin levels, consuming walnuts significantly increased the serum TEAC and FRAP values overthose of the chow-fed rats (Fig. 2A and 2B, respectively),
although the FRAP values appeared to increase more markedly than the TEAC levels after walnut consumption, in
both cases P ⬍ 0.01.

Discussion
Using an HPLC method, melatonin was identified in
extracts of walnuts in the present study. The first reports
describing melatonin in plant material appeared less than adecade ago [11,19]. Since then, only a few plant products
have been analyzed for their melatonin levels, and what has

Fig. 2. (A) TEAC and (B) FRAP values of blood from rats that were fasted
overnight and then given regular rodent chow or walnuts to eat. TEAC and
FRAP values increased in the blood of rats that consumed walnuts. **P ⬍0.01. FRAP, ferric-reducing ability of serum; TEAC, trolox equivalentantioxidant capacity.

R.J. Reiter et al. / Nutrition 21 (2005) 920 –924

ery to synthesize melatonin [27]. Further, one study hasreported that melatonin levels may be synthesized moreabundantly at night [27], in at least one plant species, as inanimals [13].
The present study also shows that when walnuts are
consumed, the levels of this constituent in the blood increase. Certainly, melatonin taken orally in pure form isreadily absorbed by the gastrointestinal tract [28,29]. The
present study in a mammal is consistent with a report inwhich chickens that consumed melatonin-containing foodproducts also had measurably increased melatonin levels intheir blood [11].
Rats were food restricted for 24 h and then given exclusively walnuts to eat for 4 h before blood was collected.
This experimental design was used because, had normalchow also been available, the rats may not have consumed
a sufficient quantity of walnuts to significantly influence
blood melatonin concentrations. The purpose of the present
study was to test whether melatonin from consumed walnuts
is absorbed by the gastrointestinal tract. Blood levels of
melatonin increased roughly three-fold after walnut consumption. The final levels achieved (about 40 pg/mL) weresignificantly lower than those normally present in the bloodof most mammals at night [13].
The present study also documented that increased melatonin levels in the blood after walnut consumption positively correlate with an increase in total antioxidant capacityof the serum as reflected by increases in TEAC and FRAP.
Heretofore it has been shown that in mammals, including
humans, fluctuations in blood melatonin concentrations
strongly correlate with the ability of the blood to detoxify
toxic free radicals and related reactants [30,31]. It should be
noted that in the present study the increases in TEAC and
FRAP levels were not necessarily or exclusively related tothe increase in melatonin. Walnuts contain other importantantioxidants, e.g., vitamin E [6] and polyphenols [7], thatare also absorbed from the gut and influence the total antioxidant capacity of serum.
The repeated demonstration that walnuts, if consumed ona regular basis, decrease cardiovascular risk factors has been
ascribed to ␻-3 fatty acids that walnuts contain [1– 4].
Although this is very likely the case, melatonin may contribute to the beneficial cardiovascular actions of walnuts
because it has been found to have important cardioprotective effects [9]. These beneficial actions of melatonin areapparent at physiologic levels of the indole [10], suggestingthat the increase in blood melatonin levels after walnut
consumption would increase protection of the heart against
oxidative damage, which is the basis of a variety of deteriorative cardiac conditions.
In addition to its direct protective actions against oxidative damage to the cardiovascular system, melatonin has
been shown to synergize with other antioxidants, e.g., vitamin E [32–34], which are found in walnuts. Thus, thecomposite action of a variety of beneficial molecules in923

walnuts, e.g., ␻-3 fatty acids, vitamin E, and melatonin, may
be involved in their antioxidative actions.
In addition to melatonin’s beneficial actions on the heart,
this indole decreases the initiation of cancer by limiting
oxidative damage to DNA [35], and it curtails the growth oftumors once they are established [36]. Melatonin achieves
these actions by a number of means including inhibiting theuptake of growth factors such as ␻-6 fatty acids by cancer
cells [36,37]. Further, melatonin’s effects on cancer inhibition are achieved at physiologic concentrations [38], andphytomelatonin, such as that in walnuts, has been proposed
as a potential means of limiting the growth of establishedtumors.
Of interest is that melatonin [36] and one of the ␻-3 fatty
acids found in walnuts [4], i.e., eicosapentaenoic acid, act
by a similar mechanism to inhibit tumor growth [39]. Thus,
these molecules decrease the uptake and metabolism of ␻-6linoleic acid by acting on membrane receptors, thereby
stimulating an inhibitory G-protein and decreasing intracellular levels of cyclic adenosine monophosphate [39]. The
decrease in cyclic adenosine monophosphate decreases linoleic acid transport into cancer cells. Linoleic acid is a
growth factor for a variety of tumors. Given their common
mechanisms of action on tumor growth, melatonin andeicosapentaenoic acid, both of which are present in walnuts,
may act synergistically to inhibit cancer cell proliferation.
In conclusion, melatonin is present in walnuts, and whenthese nuts are eaten blood levels of the indoleamine increase
to values where they could be protective against cardiovascular damage and cancer initiation and growth.

References[1] Feldman EB. The scientific evidence for a beneficial health relationship between walnuts and coronary heart disease. J Nutr 2002;132:
1062S–101.
[2] Iwamoto M, Imaizumi K, Sato M, Hirooka Y, Sakai K, Takeshita A,
Kono M. Serum lipid profiles in Japanese women and men duringconsumption of walnuts. Eur J Clin Nutr 2002;56:629 –37.
[3] Ros E, Nunez I, Perez-Heras A, Serra M, Gilabert R, Casals E,
Deulofeu R. A walnut diet improves endothelial function in hypercholesterolemic subjects: a randomized crossover trial. Circulation
2004;109:1609 –14.
[4] Kris-Etherton PM, Yu-Poth S, Sabate J, Ratcliffe HE, Zhao G, Etherton TD. Nuts and their bioactive constituents: effects on serum lipids
and other factors that affect disease risk. Am J Clin Nutr 1999;70:
504S–11.
[5] Amaral JS, Casal S, Pereira JA, Seabra RM, Oliveira BP. Determination of sterol and fatty acid composition, oxidative stability andnutritional value of six walnuts (Juglan regia L) cultivars grown in
Portugal. J Agric Food Sci 2003;51:6698 –702.
[6] Maguire LS, O’Sullivan SM, Galvin K, O’Connor TP, O’Brien NM.
Fatty acid profile, tocopherol, squalene and phytosterol content of
walnuts, almonds, peanuts, hazelnuts and the macadamia nut. Int J
Food Sci Nutr 2004;55:171– 8.
[7] Anderson KJ, Teuber SS, Gobeille A, Cremin P, Waterhouse AL,
Steinberg FM. Walnut polyphenolics inhibit in vitro human plasma
and LDL oxidation. J Nutr 2001;131:2837– 42.

924

R.J. Reiter et al. / Nutrition 21 (2005) 920 –924

[8] Sabate J. Nut consumption and body weight. Am J Clin Nutr 2003;
78:647S–50S.
[9] Reiter RJ, Tan DX. Melatonin: a novel protective agent against
oxidative injury of ischemic/reperfused heart. Cardiovasc Res 2003;
58:10 –9.
[10] Sahna E, Parlakspinar H, Ozer MK, Ozturk F, Ozugurlu F, Acet A.
Melatonin protects against myocardial doxorubicin toxicity in rats:role of physiological concentrations. J Pineal Res 2003;35:257– 61.
[11] Hattori A, Migitaka H, Iigo M, Itoh M, Yamamoto K, Ohtani-Kandro
R, et al. Identification of melatonin in plants and its effect on plasma
melatonin levels and binding to melatonin receptors in vertebrates.
Biochem Mol Biol Int 1995;35:627–34.
[12] Reiter RJ, Tan DX, Burkhardt S, Manchester LC. Melatonin in plants.
Nutr Rev 2001;59:286 –90.
[13] Reiter RJ. Pineal melatonin: cell biology of its synthesis and of its
physiological interactions. Endocr Rev 1991;12:151– 80.
[14] Hardeland R, Poeggeler B. Non-vertebrate melatonin. J Pineal Res
2003;34:233– 41.
[15] Fraser S, Cowen P, Franklin M, Franey C, Arendt J. Direct radioimmunoassay of melatonin in plasma. Clin Chem 1983;29:1703– 4.
[16] Webley GE, Mehl H, Willey KP. Validation of a sensitive direct
assay for melatonin for investigation of circadian rhythms in different
species. J Endocrinol 1985;106:387–94.
[17] Re R, Pellegrini N, Proteggente A, Panala A, Yang M, Rice-Evans C.
Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med 1999;26:1231–7.
[18] Benzie IFF, Strain JJ. The ferric reducing ability of plasma (FRAP) asa measure of “antioxidant power”: the FRAP assay. Anal Biochem
1996;239;70 – 60.
[19] Dubbels R, Reiter RJ, Klenke E, Goebel A, Schnakenberg E, Ehlers
C, et al. Melatonin in edible plants identified by radioimmunoassay
and by high performance liquid chromatography-mass spectrometry.
J Pineal Res 1995;18:28 –31.
[20] Murch SJ, Simmons CB, Saxena PK. Melatonin in feverfew and othermedicinal plants. Lancet 1997;350:1598 –9.
[21] Badria FA. Melatonin, serotonin, and tryptamine in some Egyptian
food and medicinal plants. J Med Food 2002;5:153–7.
[22] Chen G, Huo Y, Tan DX, Liang Z, Zhang W, Zhang Y. Melatonin in
Chinese herbal medicines. Life Sci 2003;73:19 –26.
[23] Tan DX, Chen LD, Poeggeler B, Manchester LC, Reiter RJ. Melatonin: a potent, endogenous hydroxyl radical scavenger. Endocr J
1993;1:57– 60.
[24] Allegra M, Reiter RJ, Tan DX, Gentile C, Tesoriere L, Livrea MA.
The chemistry of melatonin’s interaction with reactive species. J
Pineal Res 2003;34:1–10.
[25] Tan DX, Manchester LC, Hardeland R, Lopez-Barillo S, Mayo JC,
Sainz RM, Reiter RJ. Melatonin: a hormone, a tissue factor, an
autocoid, a paracoid, and an antioxidant vitamin. J Pineal Res 2003;
34:75–78.
[26] Manchester LC, Tan DX, Reiter RJ, Park W, Monis K, Qi W. Highlevels of melatonin in the seeds of edible plants: possible function in
germ cell protection. Life Sci 2000;67:3023–9.

[27] Kolar J, Johnson CH, Machackova I. Presence and possible role ofmelatonin in a short-day flowering plant, Chenopodium rubrum. Adv
Exp Med Biol 1999;460:391–3.
[28] Rosales-Corral S, Tan DX, Reiter RJ, Valdivia-Velazquez M,
Martinez-Barboza C, Acosta-Martinez JP, Ortiz GG. Orally administered melatonin reduces oxidative stress and proinflammatory cytokines induced by amyloid-beta peptide in rat brain: a comparative in
vivo study versus vitamin C and E. J Pineal Res 2003;35:80 – 4.
[29] Sharman EH, Sharman KG, Ge YW, Lahiri K, Bondy SC. Agerelated changes in murine CNS mRNA gene expression are modulated by dietary melatonin. J Pineal Res 2004;36:165–70.
[30] Benot S, Molinero P, Soutto M, Goberna R, Guerrero JM. Circadianvariations in the rat total antioxidant status: correlation with melatonin levels. J Pineal Res 1998;25:1– 4.
[31] Benot S, Goberna R, Reiter RJ, Garcia-Maurino S, Osuna C, Guerrero
JM. Physiological levels of melatonin contribute to the antioxidant
capacity of human serum. J Pineal Res 1999;27:59 – 64.
[32] Morreale M, Livrea MA. Synergistic effect of glycolic acid on theantioxidant activity of alpha-tocopherol and melatonin in lipid bilayers and in human skin homogenates. Biochem Mol Biol Int 1997;42:1093–102.
[33] Gitto E, Tan DX, Reiter RJ, Karbownik M, Manchester LC, Cuzzocrea S, et al. Individual and synergistic actions of melatonin: studies
with vitamin E, vitamin C, glutathione and desferrioxamine (deferoxamine) in rat liver homogenates. J Pharm Pharmacol 2001;53:
1393– 401.
[34] Lopez-Burillo S, Tan DX, Mayo JC, Sainz RM, Manchester LC,
Reiter RJ. Melatonin, xanthurenic acid, resveratrol, EGCG, vitamin C
and alpha-lipoic acid differentially reduce oxidative DNA damage
induced by Fenton reagents: a study of their individual and synergistic effects. J Pineal Res 2003;34:269 –77.
[35] Qi W, Reiter RJ, Tan DX, Garcia JJ, Manchester LC, Karbownik M,
Calvo JR. Chromium (III) induced 8-hydroxydeoxyguanosine in
DNA and its reduction by antioxidants: comparative effects of melatonin, ascorbate and vitamin E. Environ Health Perspect 2000;108:399 – 402.
[36] Blask DE, Sauer LA, Dauchy RT. Melatonin as a chronobiotic/
anticancer agent: cellular, biochemical, and molecular mechanisms ofaction and their implications for circadian-based cancer therapy. Curr
Topics Med Chem 2002;2:113–32.
[37] Reiter RJ. Mechanism of cancer inhibition by melatonin. J Pineal Res
2004;37:213– 4.
[38] Blask DE, Dauchy RT, Sauer LA, Krause JA. Melatonin uptake andgrowth prevention in rat hepatoma 7288CTC in response to dietary
melatonin: melatonin receptor-mediated inhibition of tumor linoleic
acid metabolism to the growth signaling molecule 13-hydroxyoctadecadienoic acid and the potential role of phytomelatonin. Carcinogenesis 2004;25:951– 60.
[39] Sauer LA, Dauchy RT, Blask DE. Polyunsaturated fatty acids,
melatonin, and cancer prevention. Biochem Pharmacol 2001;61:
1455– 62.