The presence of melatonin in grapevine (Vitis vinifera L.) berrytissues

Introduction

The presence of melatonin (N-acetyl-5-methoxytryptamine)

in food plants has been known formore than a decade, and inboth mono- and dicotyledonous families including fruits,vegetables, cereals, edible seeds, and medicinal herbs [1–11].

As a result, it has been postulated that melatonin may be abioactive component of certain dietary styles, such as theMediterranean diet [12]. Interestingly, in animals includingthe human, the intake of foodstuffs containing melatonin

may contribute to increase the serum levels of this compoundas well as the urinary concentrations of its metabolite 6-sulfatoxymelatonin [13–15]. Among plant foods typical of

theMediterranean diet, melatonin has been detected in grape(Vitis vinifera L.) and olive oil [16–18].

In grape products, melatonin was first found in berry

exocarp (skin) of Italian and French varieties grown inNorthwest Italy, with levels ranging from 0.005 to 0.96ng/g [16]. Similar results (from 0.6 to 1.2 ng/g) werereported by Stege and colleagues [19] for berry skin of

some varieties cultivated in Argentina. In Merlot varietygrown in Canada, the melatonin concentrations measured

by Murch and coworkers in the whole berry varied

approximately between 100 and 150 lg/g, according tothe phenological stage [20]. On the contrary, in a recentreport, melatonin was not detected in berry tissues of

different varieties [21].Other studies ascertained the presence of melatonin in

wine. Mercolini and colleagues [22] found it, at 0.5 and0.4 ng/mL, in Sangiovese red and Trebbiano white wine,

respectively. Stege and coworkers [19] reported melatoninin Chardonnay, Malbec and Cabernet Sauvignon wines inthe concentration range from 0.16 to 0.32 ng/mL. More

recently, our results showed that the levels of melatoninin Groppello and Merlot wines varied between 5.2 and8.1 ng/mL, depending on agrochemicals treatments [23],

whereas Rodriguez-Naranjo and colleagues measuredmuch higher melatonin concentrations, up to 400 ng/mL,in racked wines [21, 24]. Noticeably, many endogenous and

external factors may influence melatonin and polyphenoliclevels in grapevine and their products, such as the genetictraits of the cultivar, the berry tissue/grapevine organanalyzed, the phenological stage, pathogen infections and

phytosanitary treatments, agro-meteorological conditions

Abstract: Melatonin has been reported in a variety of food plants and,

consequently, in a number of plant-derived foodstuffs. In grapevine (Vitis

vinifera L.) products, it was found in berry exocarp (skin) of different

cultivars and monovarietal wines. Herein, we assessed, by means of mass

spectrometry, the occurrence of melatonin in all berry tissues (skin, flesh, and

seed) at two different phenological stages, pre-veraison and veraison. We

detected the highest melatonin content in skin, at pre-veraison, whereas, at

veraison, the highest levels were reported in the seed. Furthermore, during

ripening, melatonin decreased in skin, while increasing in both seed and flesh.

The relative concentrations of melatonin in diverse berry tissues were

somewhat different from those of total polyphenols (TP), the latter measured

by the Folin-Ciocalteau assay, and more abundant in seed at pre-veraison

and in exocarp at veraison. The highest antiradical activity, determined by

both DPPH (2,2-diphenyl-1-pycryl hydrazyl) and ABTS [(2,2¢-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] radical-scavenging assay, was

reported at pre-veraison in seed. To the best of our knowledge, we reported,

for the first time, the occurrence of melatonin in grape seeds.

Sara Vitalini1,2, Claudio Gardana3,Alessandro Zanzotto4, PaoloSimonetti3, Franco Faoro1,5,Gelsomina Fico2,6 and MarcelloIriti1

1Dipartimento di Produzione Vegetale,

Universita degli Studi di Milano, Milano, Italy;2Orto Botanico �G. E. Ghirardi�, Dipartimento di

Biologia, Universita degli Studi di Milano,

Toscolano Maderno, Brescia, Italy; 3DiSTAM –

Sezione Nutrizione, Universita degli Studi di

Milano, Milano, Italy; 4CRA, Centro di Ricerca

per la Viticoltura, Conegliano Veneto, Treviso,

Italy; 5CNR, Dipartimento Agroalimentare,

Istituto di Virologia Vegetale, Sezione di

Milano, Milano, Italy; 6Dipartimento di Biologia,

Universita degli Studi di Milano, Milano, Italy

Key words: antioxidant power, bioactive

phytochemicals, grape seeds, Mediterranean

diet, polyphenols

Address reprint requests to Marcello Iriti,

Dipartimento di Produzione Vegetale,

Universita degli Studi di Milano, via Celoria 2,

20133 Milano, Italy.

E-mail: marcello.iriti@unimi.it

Received March 1, 2011;

Accepted April 8, 2011.

J. Pineal Res. 2011; 51:331–337Doi:10.1111/j.1600-079X.2011.00893.x

� 2011 John Wiley & Sons A/S

Journal of Pineal Research

331

Mo

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lar,

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and environmental stresses, the vintage and wine-makingprocedures [20, 21, 23, 25, 26, 27].Because, to date, melatonin was detected only in grape-

vine berry exocarp and wine, the present work tested for thepresence and the amount of this indoleamine in all berrytissues (skin, flesh, and seed) at two different phenologicalstages, pre-veraison and veraison. Furthermore, we com-

pared the levels of melatonin with those of polyphenols andstimulated the antiradical power of the different tissuefractions.

Materials and methods

Plant material

The study was carried out during 2009 in an experimentalvineyard of Merlot grape (Vitis vinifera L. cv. Merlot)

located at Conegliano Veneto (CRA, Centro di Ricerca perla Viticoltura, Treviso, Italy) and managed according to theGood Agricultural Practices (http://www.fao.org/prods/

gap/). Phytosanitary conditions of grapevines were con-trolled by conventional phytoiatric treatments. Samplingwas scheduled at two phenological phases: pre-veraison

(�pea-size� stage, before the end of the berry�s growth cycle)and 100% veraison (the stage when berry begin to turn colorand soften); bunches were randomly collected from plants

during the morning and stored at )20�C until analyses.

Reagents and working solutions

Melatonin was purchased from Sigma-Aldrich (St. Louis,MO, USA). Methanol, acetonitrile, and trifluoroacetic acid(TFA) were from Merck (Darmstadt, Germany). Water

was obtained from a MilliQ apparatus (Millipore, Milford,MA, USA). The stock solutions of melatonin (0.1 mg/mL)were prepared in methanol and diluted to give working

solutions in the range of 0.5–50 ng/mL. Stock and workingsolutions were stored at )80�C and )20�C, respectively.

Determination of polyphenol compounds andantiradical activity

The extraction procedure and all the subsequent analyses

were carried out in dim light conditions, to preventmelatonin degradation. Different tissues (skin, flesh andseeds) of frozen berries, randomly selected from bunches,

were carefully separated with a chilled scalpel and homog-enized in liquid nitrogen with a chilled mortar and pestle.After weighing, a MeOH aliquot (3 mL/g) was added and

the mixtures were sonicated for 10 min and then stirred for20 min. Each sample was decanted and the supernatantcentrifuged at 10,000 g for 10 min. All phases wereconducted at 4�C. The resulting extract was filtered through

a 0.20-m filter and combined with that of a secondextraction, finally concentrated under nitrogen gas.

Total polyphenols

Total polyphenols (TP) content was measured by the Folin-

Ciocalteu colorimetric assay, using gallic acid as referencestandard [28]. Briefly, an aliquot (2.5 lL) of sample was

added to 50 lL of Folin-Ciocalteau reagent. The solutionswere mixed and allowed to stand for 3 min. Next, 100 lL ofa saturated sodium carbonate solution and distilled water to

final volume of 2.5 mLwere added. After 1 hr of incubation,in the dark, at room temperature, the absorbance was read at725 nm. Polyphenol quantification was based on a standardcurve (15–500 mg/L, y = 0.0228x ) 0.0069, R2 = 0.9986)

of gallic acid, and results were expressed as gallic acidequivalents.

2,2-Diphenyl-picryl hydrazyl (DPPH·)radical-scavenging assay

DPPH· radical-scavenging activity was performed as pre-viously reported [28]. Briefly, aliquots of each sample, atfive different concentrations (from 1 to 100 lm), were addedto a 15 lm EtOH solution of DPPH· free radical. After a

reaction time of 15 min in the dark, the absorbance at517 nm was determined by spectrophotometer (Jenway6310). The IC50 was calculated with Prism� 4 (GraphPad

Software Inc., La Jolla, CA, USA).

2,2¢-Azino-bis(3-ethylbenzothiazoline-6-sulfonicacid) (ABTS·+) assay

ABTS·+ radical cation-scavenging capacity was determined

according to Iriti et al. [29] with some modifications. TheABTS·+ radical cation was produced by reacting 7 mm

ABTS with 2.45 mm potassium persulfate (final concentra-tion) and maintaining the mixture in the dark at room

temperature for, at least, 6 hr before use. The ABTS·+

solution was diluted with ethanol to an absorbance of 0.7(± 0.02) AU at 734 nm and equilibrated at 30�C. Ten

microlitres of each diluted sample, ethanol (negativecontrol), and standard solution of the synthetic antioxidant6-hydroxy-2,5,7,8-tetramethychroman-2-carboxylic acid

(Trolox, positive control) were mixed for 30 s with 1 mLof diluted ABTS·+ solution. Their absorbance was read at734 nm, at room temperature, 50 s after the initial mixing.The results are expressed as Trolox equivalent antioxidant

capacity (TEAC, mmol of Trolox equivalents/kg of grapes).

UPLC-MS/MS analysis of melatonin in grape berrytissues

All analyses on berry tissues were carried out in dim light

conditions, to avoid photooxidation of plant metabolites.The chromatographic system consisted of an UPLC (ultra-performance liquid chromatograph) mod. Acquity (Waters,

Milford, MA, USA) coupled to a triple quadrupole massspectrometer mod. Quattromicro (Waters). A 1.8-m C18

HSS column (100 · 2.1 mm; Waters) was used for theseparation at a flow rate of 0.5 mL/min. The column was

maintained at 60�C, and the separation was performed bymeans of a linear gradient elution (eluent A, 0.05% TFA;eluent B, 0.05% TFA in acetonitrile). The gradient was as

follows: 5–50% B in 1.4 min, 50–70% B in 5 s, and then70% B for 0.5 min. The capillary voltage was set to 3 kV,the cone voltage and the collision energy was 16 and 14 eV,

respectively. The source temperature was 130�C, thedesolvating temperature was 350�C and argon was used

Vitalini et al.

332

at 2.0 · 10)3 mbar to improve fragmentation in thecollision cell. Masslynx 4.0 software acquired data withQuan-Optimize option for fragmentation study. The frag-

mentation transition for the multiple reaction monitoring(MRM) was (m/z)+ 233 fi 174, with a dwell time of 0.2 s.

Statistical analysis

For each experiment, results are expressed as mean ±standard deviation (S.D.) of data from three determina-

tions recorded for at least two independent extractions.Data were subjected to one-way analysis of variance(ANOVA) and comparison among means was determined

according to Fisher�s least significant difference (LSD) test.Significant differences were accepted at P < 0.05 andrepresented by different letters.

Results

To quantify melatonin in the different grapevine berry

tissues, the MRM mode was employed, and ion transitionsof the melatonin standard were recorded by UPLC-MS/MSanalysis. The calibration curve was generated with a 233/

174 transition area (tR = 1.75 min) for each of the sevenmelatonin concentrations (from 0.5 to 50 ng/mL), andgood linearity was obtained (r = 0.9995). Fig. 1 shows the

chromatographic profile of a tissue extract relative to themelatonin peak.

Two different trends have been observed in berry tissues,with melatonin decreasing in skin, from pre-veraison to

veraison, while increasing both in seed and flesh (Fig. 2).The highest melatonin concentration was reported in skinat pre-veraison (17.5 ± 0.28 ng/g grapes), decreasing by

47% at veraison (9.3 ± 0.14 ng/g grapes, P < 0.05)(Fig. 2). Furthermore, at pre-veraison, significantly lowermelatonin levels were detected in seed and flesh (3.6 ± 0.07

and 0.2 ± 0.03 ng/g grapes, respectively, P < 0.05)(Fig. 2). The transition from pre-veraison to veraison raisedthe melatonin content by 63%, in seed (from 3.6 ± 0.07 to10.04 ± 0.49 ng/g grapes, P < 0.05), and by 95%, in flesh

(from 0.2 ± 0.03 to 3.9 ± 0.06 ng/g grapes, P < 0.05)

(Fig. 2). At veraison, the melatonin concentration of theseed was significantly higher than that measured in skin andflesh (10.04 ± 0.49 versus 9.3 ± 0.14 and 3.9 ± 0.06 ng/g

grapes, respectively, P < 0.05) (Fig. 2). Differently frommelatonin, TP significantly increased, from pre-veraison toveraison, by 11.5% in skin (1589 ± 27.3 versus1783 ± 57.9 mg/kg grapes, P < 0.05), while decreasing

by 57% (from 2914 ± 75.6 to 1244 ± 10.0 mg/kg grapes,P < 0.05) and 72% (from 225 ± 9.1 to 63 ± 2.4 mg/kggrapes, P < 0.05) in seed and flesh, respectively (Fig. 3).

DPPH and ABTS radicals are among the most usedchromogen compounds to measure the antiradical power ofbiological samples. The scavenging activity of the different

berry tissues against the DPPH radical, expressed as IC50, isreported in Fig. 4. In particular, at pre-veraison, the DPPHradical-scavenging capacity increased in the order:flesh (31.6 ± 1.1 lm) < skin (19.6 ± 0.71 lm) < seed

(2.01 ± 0.07 lm) (Fig. 4). Similarly, at veraison, the sameactivity increased in the order: flesh (74.7 ± 2.4 lm) < skin(17.2 ± 1.34 lm) < seed (7.3 ± 0.12 lm) (Fig. 4). All dif-

ferences were statistically significant (P < 0.05). Interest-ingly, at pre-veraison, the sample with the lowest IC50 (i.e. themaximum antiradical power), the seed, showed the highest

TP content, whereas, at veraison, the sample with themaximum DPPH radical-scavenging activity, the seed, wasthat with the highest levels of melatonin (Fig. 4).

ABTS radical-scavenging assay showed that, as forresults obtained by the DPPH test, the highest TEACvalues were measured in seed (18.8 ± 0.37 mmol eqTrolox/kg grapes) at pre-veraison (Fig. 5). At veraison, the

maximum TEAC was detected in skin (5.6 ± 0.14 mmol eqTrolox/kg grapes), though, in this tissue, no significantdifference in TEAC values was reported in the two

phenological stages (5.47 ± 0.39 mmol eq Trolox/kggrapes at pre-veraison, P > 0.05) (Fig. 5).For grapevine berry tissues, a correlation analysis based

on simple linear regression was performed on the assayedvariables at the 95% confidence level (Figs 6 and 7).Melatonin concentration was highly correlated with both

DPPH and ABTS radical-scavenging activity only atveraison (R2 = 0.999 and R2 = 0.831, respectively)

1.35–10

90

%

1.40 1.45 1.50 1.55Time

1.60 1.65 1.70 1.75

Fig. 1. Typical UPLC-MS/MS chro-matogram of a grapevine (Vitis vinifera L.cv. Merlot) berry tissue extract. The frag-mentation transitions for the melatoninwas (m/z) + 233*174; the first 1.2 min ofthe analysis was wasted to keep the massinterface clear.

Melatonin in grape berry tissues

333

(Figs 6C and 7C). A high linear correlation coefficient wasalso reported for polyphenols and DPPH (R2 = 0.986 atpre-veraison and R2 = 0.812 at veraison) (Fig. 6B,D).

Similarly, TP content and ABTS were highly correlatedboth at pre-veraison (R2 = 0.926) (Fig. 7B) and at veraison(R2 = 0.999) (Fig. 7D). From these results, it seems that

melatonin and polyphenols play main roles in determiningthe antiradical power of grapevine berry tissues, at least inour experimental conditions.

Discussion

The presence of melatonin in grapevine berry tissues has

been reported in this work. The grapevine berry consists ofthree major types of tissues: skin, flesh, and seed. Forwinemaking, skins have greater importance, because the

polyphenols are more easily extracted from the skins thanfrom the seeds. Even though the latter represent only 1–6%of berry weight, they are an abundant source of polyphe-

nols (mainly proanthocyanidins) [30, 31]. Furthermore, wehave shown that melatonin levels in berry tissues changedepending on the phenology. The development of wine

grape berry is characterized by a double-sigmoidal pattern,i.e., two successive sigmoid curves separated by a plateau(the lag phase) and corresponding to three stages differingconsiderably in the biochemical activity of berry and its

subsequent composition. During stage I (first phase ofberry growth), the berry grows initially by cell division andlater by cell expansion; also during this stage, all seed

structures are differentiated and the seed approaches its fullsize. During phase II (lag phase), berry growth slowsmarkedly and little change occurs in berry size, whereas

organic acid concentration reaches its highest level; the seedaccumulates reserves and the embryo develops with aconcomitant hardening of the seed coat. Stage III (secondperiod of berry growth) is the ripening phase, when sugars

and aroma components rapidly accumulate, whereasorganic acids decline. The beginning of this phase ischaracterized by berry softening and change in skin color

(in French, veraison) because of the accumulation of redpigments (anthocyanins, a class of flavonoidic polyphe-nols). By veraison, which represents the onset of ripening,

the seed is almost entirely developed and its color begins to

20

15

10

ng/g

gra

pes

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0

Skin Seed Flesh

e

f

d

c

b

aPre-véraison

Véraison

Fig. 2. Melatonin concentration (ng/g grapes) evaluated byUPLC-MS/MS in skin, seed and flesh of grapevine (Vitis viniferaL. cv. Merlot) berry at pre-veraison and veraison (see �Materialsand methods� for details). Results are expressed as mean ± S.D. ofdata from three determinations recorded for at least two indepen-dent extractions. Bars carrying different letters indicate meanssignificantly different at P < 0.05 (Fisher�s least significant differ-ence test).

3000

2500

2000

1500

1000

500

0

Skin Seed Flesh

e f

d

c

ba

Pre-véraison

Véraison

mg/

kg g

rape

s

Fig. 3. Total polyphenol content (mg/kg grapes) measured by theFolin-Ciocalteau colorimetric assay in skin, seed and flesh ofgrapevine (Vitis vinifera L. cv. Merlot) berry at pre-veraison andveraison (see �Materials and methods� for details). Results areexpressed as mean ± S.D. of data from three determinationsrecorded for at least two independent extractions. Bars carryingdifferent letters indicate means significantly different at P < 0.05(Fisher�s least significant difference test).

80

70

60

50

40

30

20a

b c

de

f

g

10

0

Skin

Asc

orbi

cac

id Seed Flesh

Pre-véraison

Véraison

IC50

(µM

)

Fig. 4. DPPH (2,2-diphenyl-picrylhydrazyl) radical-scavengingactivity (IC50) measured in skin, seed and flesh of grapevine (Vitisvinifera L. cv. Merlot) berry at pre-veraison and veraison (see�Materials and methods� for details). Results are expressed asmean ± S.D. of data from three determinations recorded for atleast two independent extractions. Bars carrying different lettersindicate means significantly different at P < 0.05 (Fisher�s leastsignificant difference test).

Pre-véraison

mm

ol e

q T

rolo

x/kg

gra

pes

20

15

10

5

0

Skin Seed Flesh

d e

c

b

aa

Véraison

Fig. 5. ABTS [2,2¢-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)]radical-scavenging activity (TEAC, mmol eq. Trolox/kg grapes)measured in skin, seed and flesh of grapevine (Vitis vinifera L. cv.Merlot) berry at pre-veraison and veraison (see �Materials andmethods� for details). Results are expressed as mean ± S.D. ofdata from three determinations recorded for at least two indepen-dent extractions. Bars carrying different letters indicate meanssignificantly different at P < 0.05 (Fisher�s least significant differ-ence test).

Vitalini et al.

334

change from an initial green to a dark brown at harvest,because of polyphenol oxidation in the seed coat [31–34].

In previous studies, melatonin was detected in berry skin,at levels near or below 1 ng/g [16, 19, 35], though Murch

and colleagues measured much higher concentrations,

between 100 and 150 lg/g, in the whole berry (i.e. skin,flesh, and seeds analyzed together) [20]. In skin, Boccalan-dro and coworkers (H.E. Boccalandro, C.V. Gonzales,D.A. Wunderlin, et al., submitted) reported a higher

melatonin content at dawn (159 ng/g) than during the

(A) 35

30

25

20

15

10

5

0

80

70

60

50

40

30

20

10

0

0 2 4 6 8 10

y = –0.1311x + 18.667R2 = 0.0065

y = –0.011x + 35.065R2 = 0.9863

y = –0.0373x + 71.475R2 = 0.8125

y = –10.897x + 117.34R2 = 0.999

Melatonin (ng/g grapes)

IC50

(µM

)IC

50 (

µM)

80

70

60

50

40

30

20

10

0

IC50

(µM

)

35

30

25

20

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10

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IC50

(µM

)

12

0 2 4 6 8 10Melatonin (ng/g grapes) Total polyphenols (mg/kg grapes)

Total polyphenols (mg/kg grapes)

12 0 200 400 600 800 1000 1200 1400 1600 1800 2000

14 16 18 20 0 500 1000 1500 2000 2500 3000 3500

(B)

(D)(C)

Fig. 6. Correlation analysis based on simple linear regression at the 95% confidence level between melatonin concentration (ng/g grapes)and DPPH radical-scavenging activity (IC50) at (A) preveraison and (C) veraison, and between polyphenol content (mg/kg grapes) andDPPH at (B) preveraison and (D) veraison.

(A) (B)

(D)(C)

00

6

5

4

3

2

1

0

2

4

6

8

10

12

14

16

18

20

2 4 6 8 10Melatonin (ng/g grapes)

12

0 2 4 6 8 10Melatonin (ng/g grapes)

12

14 16 18 20

Total polyphenols (mg/kg grapes)0 200 400 600 800 1000 1200 1400 1600 1800 2000

Total polyphenols (mg/kg grapes)

0 500 1000 1500 2000 2500 3000 3500

mm

ol e

q T

rolo

x/kg

gra

pes

mm

ol e

q T

rolo

x/kg

gra

pes

6

–5

0

5

10

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20

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4

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0

mm

ol e

q T

rolo

x/kg

gra

pes

mm

ol e

q T

rolo

x/kg

gra

pes

y = –0.0786x + 8.8929R2 = 0.0058

y = 0.0068x – 2.3143R2 = 0.9266

y = 0.0032x – 0.1084R2 = 0.9999

y = 0.767x – 2.7515R2 = 0.831

Fig. 7. Correlation analysis based on simple linear regression at the 95% confidence level between melatonin concentration (ng/g grapes)and ABTS radical-scavenging activity (TEAC, mmol eq Trolox/kg grapes) at (A) pre-veraison and (C) veraison, and between polyphenolcontent (mg/kg grapes) and ABTS at (B) pre-veraison and (D) veraison.

Melatonin in grape berry tissues

335

night (9 ng/g), and the latter value is comparable to thelevels found in our samples (17.5 and 9.3 ng/g at pre-veraison and veraison, respectively, in skin).

It was also shown that, in the whole berry, melatonindecreases with ripening (i.e. from pre-veraison to veraison)[20]. Our results have confirmed this trend only in skinsamples, whereas themelatonin content increased at veraison

both in seed and in flesh. However, in a recent study,melatonin was not detected in grapes [21], in contrast to ourand previous data [16, 19, 20, 35]. Many factors may have

caused these differences, including the day-night fluctuationsof melatonin in grape tissues (H.E. Boccalandro, C.V.Gonzales, D.A. Wunderlin, et al., submitted) [36].

To the best of our knowledge, this work is the firstreporting melatonin in grapevine berry flesh and seed. Thepresence of melatonin in seed suggests a primary role of thiscompound as part of the antioxidant defense system, able

to protect the germ tissues from external stresses, whichcould compromise the germination potential of the seed.Therefore, the functional role of melatonin in seed may be

explained by the necessity, for the parental generation, toprotect the germ tissues (the next generation) particularlyvulnerable to the oxidative damage [4]. Owing to its

amphipathic nature and antioxidant activity [37–39], mel-atonin may exert such a function, being able to permeate allseed tissues, particularly rich in storage lipids, and mem-

branes, by virtue of its both lipophilic and hydrophilicproperties, thus reaching subcellular compartments wherereactive oxygen species are endogenously produced [40].Furthermore, as previously mentioned, seeds reach matu-

rity at veraison, thus becoming more susceptible to damagecaused by external detrimental conditions, and when themelatonin concentration is higher than in pre-veraison. In

skin, an opposite trend has been observed, with melatonindecreasing from pre-veraison to veraison, while polyphenolcontent increases sustaining (with the anthocyanic fraction)

the berry color change. In plants, melatonin and polyphe-nols arise from tryptophan and phenylalanine, respectively,through two distinct secondary metabolic pathways; in

turn, both aromatic amino acids derive from the shikimatebiosynthetic route [16]. Therefore, it is possible that, atveraison, the carbon flux is mainly directed toward phen-ylalanine, to guarantee the berry coloring, a primary need

for the ecology of the plant, thus adding resources andprecursors to the melatonin biosynthesis. Additionally, atthis stage, the synthesis of tryptophan derivatives (including

melatonin) might be subjected to a down-regulation, atgene and/or enzyme level or to a hormonal control.From a physiological point of view, grape berry ripening

is a complex process regulated at the hormonal level. Thefirst step of berry growth (pre-veraison) is under the controlof developmental hormones (auxins, cytokinins, and gibb-erellins), mostly produced by seed tissues and promoting

cell division and expansion [34]. Among these, auxins (theplant �growth hormones�) are indolic derivatives arisingfrom tryptophan and sharing structural similarities with

melatonin, for which an auxin-like activity has beensuggested [41, 42]. At veraison, when auxin biosynthesisdecreases, a higher availability of precursors might generate

higher levels of melatonin, as observed at least in seed(where it might be produced). Moreover, at this phase,

hormones involved in ripening (berry softening and color-ing), such as ethylene and abscisic acid, positively influencethe accumulation of polyphenols in skin [34]. Consequently,

it may be hypothesized that one or some of these hormonesmay negatively regulate the increase in melatonin, at leastin skin, where, at this stage, cell metabolism is deeplyinvolved in the coloring phase.

In conclusion, apart from the implications for plantphysiology and food technology, some mention should bemade related to the pharmaconutritional aspects of grape

seeds. Many health-promoting effects have been attributedto the grape seed extract, mainly because of proanthocy-anidins, a class of polyphenols considered the active

ingredients [43, 44]. Therefore, the discovery of melatoninin seed may add a new element in this field, by virtue of itsneuroprotective and antitumoral properties [45–48], possi-bly helping to explain the potential of grape seed extract for

human health [43, 44].

Acknowledgement

The authors are grateful to Mr Daniele Contino for theexcellent technical assistance in laboratory analyses.

References

1. Dubbels R, Reiter RJ, Klenke E et al. Melatonin in edible

plants identified by radioimmunoassay and by high perfor-

mance liquid chromatography-mass spectrometry. J Pineal Res

1995; 18:28–31.

2. Hattori A, Migitaka H, Iigo M et al. Identification of

melatonin in plants and its effects on plasma melatonin levels

and binding to melatonin receptors in vertebrates. Biochem

Mol Biol Int 1995; 35:627–634.

3. Murch SJ, Simmons CB, Saxena PK. Melatonin in feverfew

and other medicinal plants. Lancet 1997; 350:1598–1599.

4. Manchester LC, Tan DX, Reiter RJ et al. High levels of

melatonin in the seeds of edible plants: possible function in

germ tissue protection. Life Sci 2000; 67:3023–3029.

5. Badria FA. Melatonin, serotonin, and tryptamine in some

Egyptian food and medicinal plants. J Med Food 2002; 5:153–

157.

6. Chen G, Huo Y, Tan DX et al. Melatonin in Chinese

medicinal herbs. Life Sci 2003; 73:19–26.

7. Paredes SD, Korkmaz A, Manchester LC et al. Phytome-

latonin: a review. J Exp Bot 2009; 60:57–69.

8. Posmyk MM, Janas KM. Melatonin in plants. Acta Physiol

Plant 2009; 31:1–11.

9. Arnao MB, Hernandez-Ruiz J. Chemical stress by different

agents affects the melatonin content of barley roots. J Pineal

Res 2009; 46:295–299.

10. Okazaki M, Ezura H. Profiling melatonin in the model to-

mato (Solanum lycopersicum L.) cultivar Micro-Tom. J Pineal

Res 2009; 46:338–343.

11. Murch SJ, Alan AR, Cao J et al. Melatonin and serotonin

flowers and fruits of Datura metel L. J Pineal Res 2009; 47:277–

283.

12. Iriti M, Varoni EM, Vitalini S. Melatonin in Mediterranean

diets. J Pineal Res 2010; 49:101–105.

13. Reiter RJ, manchester LC, Tan DX. Melatonin in walnuts:

influence on levels of melatonin and total antioxidant capacity

of blood. Nutrition 2005; 21:920–924.

Vitalini et al.

336

14. Maldonado MD, Moreno H, Calvo JR. Melatonin present

in beer contributes to increase the levels of melatonin and

antioxidant capacity of the human serum. Clin Nutr 2009;

28:188–191.

15. Garrido M, Parede SD, Cubero J et al. Jerte Valley cherry-

enriched diets improve nocturnal rest and increase 6-sulfat-

oxymelatonin and total antioxidant capacity in the urine of

middle-aged and elderly humans. J Gerontol A Biol Sci Med

Sci 2010; 65A:909–914.

16. Iriti M, Rossoni M, Faoro F. Melatonin content in grape:

myth or panacea? J Sci Food Agric 2006; 86:1432–1438.

17. Iriti M. Melatonin in grape, not just a myth, maybe a pana-

cea. J Pineal Res 2009; 46:353.

18. de la Puerta C, Carrascosa-Salmoral MP, Garcıa-Luna

PP et al. Melatonin is a phytochemical in olive oil. Food Chem

2007; 104:609–612.

19. Stege PW, Sombra LL, Messina G et al. Determination of

melatonin in wine and plant extracts by capillary electrochro-

matography with immobilized carboxylic multi-walled carbon

nanotubes as stationary phase. Electrophoresis 2010; 31:2242–

2248.

20. Murch SJ, Hall BA, Le CH et al. Changes in the levels of

indoleamine phytochemicals during veraison and ripening of

wine grapes. J Pineal Res 2010; 49:95–100.

21. Rodriguez-Naranjo MI, Gil-Izquierdo A, Troncoso AM

et al. Melatonin is synthesized by yeast during alcoholic fer-

mentation in wines. Food Chem 2011; 4:1608–1613.

22. Mercolini L, Saracino MA, Bugamelli F et al. HPLC-F

analysis of melatonin and resveratrol isomers in wine using an

SPE procedure. J Sep Sci 2008; 31:1007–1114.

23. Iriti M, Vitalini S, Di Tommaso G et al. A new chitosan

formulation induces grapevine resistance against powdery

mildew and improves grape quality traits. Aust J Grape Wine

Res 2011; doi:10.1111/j.1755-0238.2011.00149.x

24. Rodriguez-Naranjo MI, Gil-Izquierdo A, Troncoso AM

et al. Melatonin: a new bioactive compound in wine. J Food

Compost Anal 2011; doi:10.1016/j.jfca.2010.12.009

25. Burkhardt S, Tan DX, Manchester LC et al. Detection

and quantification of the antioxidant melatonin in Montmo-

rency and Balaton tart cherries (Prunus cerasus). J Agric Food

Chem 2001; 49:4898–4902.

26. Lachman J, Sulc M, Faitova K et al. Major factors influ-

encing antioxidant contents and antioxidant activity in grapes

and vines. Int J Wine Res 2009; 1:101–121.

27. Cohen SD, Kennedy JA. Plant metabolism and the envi-

ronment: implications for managing phenolics. Crit Rev Food

Sci Nutr 2010; 50:620–643.

28. Vitalini S, Grande S, Visioli F et al. Antioxidant activity of

wild plants collected in Valsesia, an alpine region of northern

Italy. Phytother Res 2006; 20:576–580.

29. Iriti M, Di Maro A, Bernasconi S et al. Nutritional traits of

bean (Phaseolus vulgaris) seeds from plants chronically ex-

posed to ozone pollution. J Agric Food Chem 2009; 57:201–

208.

30. Gonzalez-Manzano S, Rivas-Gonzalo JC, Santos-Buelga

C. Extraction of flavan-3-ols from grape seed and skin into

wine using simulated maceration. Anal Chim Acta 2004;

513:283–289.

31. Cadot Y, Minana-Castello MT, Chevalier M. Anatomi-

cal, histological, and histochemical changes in grape seeds

from Vitis vinifera L. cv. Cabernet franc during fruit devel-

opment. J Agric Food Chem 2006; 54:9206–9215.

32. Kennedy JA, Matthews MA, Waterhouse AL. Changes in

grape seed polyphenols during fruit ripening. Phytochemistry

2000; 55:77–85.

33. Kennedy JA, Troup GJ, Pilbrow JR et al. Development of

seed polyphenols in berries from Vitis vinifera L. cv. Shiraz.

Aust J Grape Wine Res 2000; 6:244–254.

34. Conde C, Silva P, Fontes N et al. Biochemical changes

throughout grape berry development and fruit and wine

quality. Food 2007; 1:1–22.

35. Rayne S. Concentrations and profiles of melatonin and sero-

tonin in fruits and vegetables during ripening: a mini-review.

Nat Prec 2010; doi:10.1038/npre.2010.4722.1

36. Hernandez-Ruiz J, Arnao MB. Distribution of melatonin in

different zones of lupin and barley plants at different ages in

the presence and absence of light. J Agric Food Chem 2008;

56:10567–10573.

37. Tan DX, Chen LD, Poeggeler B et al. Melatonin, a potent,

endogenous hydroxyl radical scavenger. Endocr J 1993; 1:57–60.

38. Romero A, Egea J, Garcıa AG et al. Synergistic neuropro-

tective effect of combined low concentrations of galantamine

and melatonin against oxidative stress in SH-SY5Y neuro-

blastoma cells. J Pineal Res 2010; 49:141–148.

39. Xu Sc, He MD, Zhong M et al. Melatonin protects against

Nickel-induced neurotoxicity in vitro by reducing oxidative

stress and maintaining mitochondrial function. J Pineal Res

2010; 49:86–94.

40. Reiter RJ, Paredes SD, Manchester LC et al. Reducing

oxidative/nitrosative stress: a newly-discovered genre for mel-

atonin. Crit Rev Biochem Mol Biol 2009; 44:125–200.

41. Hernandez-Ruiz J, Cano A, Arnao MB. Melatonin: a

growth-stimulating compound present in lupin tissues. Planta

2004; 220:140–144.

42. Hernandez-Ruiz J, Cano A, Arnao MB. Melatonin acts as a

growth-stimulating compound in some monocot species.

J Pineal Res 2005; 39:137–142.

43. Kaur M, Agarwal C, Agarwal R. Anticancer and cancer

chemopreventive potential of grape seed extract and other

grape-based products. J Nutr 2009; 139:1806S–1812S.

44. Pasinetti GM, Ho L. Role of grape seed polyphenols in

Alzheimer�s disease neuropathology. Nutr Diet Suppl 2010;

2:97–103.

45. Borah A, Mohanakumar KP. Melatonin inhibits 6-hy-

droxydopamine production in the brain to protect against

experimental parkinsonism. J Pineal Res 2009; 47:293–300.

46. Olcese JM, Cao C, Mori T et al. Protection against cognitive

deficits and markers of neurodegeneration by long-term oral

administration of melatonin in a transgenic model of Alzhei-

mer�s disease. J Pineal Res 2009; 47:82–96.

47. paradies G, Petrosillo G, Paradies V et al. Melatonin,

cardiolipin and mitochondrial bioenergetics in health and

disease. J Pineal Res 2010; 48:297–310.

48. Bonnefont-Rousselot D, Collin F. Melatonin: action as

antioxidant and potential applications in human disease and

aging. Toxicology 2010; 278:55–67.

Melatonin in grape berry tissues

337