Journal of Food Composition and Analysis 105 (2022) 104180

Contents lists available at ScienceDirect

Journal of Food Composition and Analysis

journal homepage: www.elsevier.com/locate/jfca

Original Research Article

Melatonin content in walnuts and other commercial nuts. Influence of

cultivar, ripening and processing (roasting)
Antía Verde a, Jesús M. Míguez b, Jose Manuel Leao-Martins c, Ana Gago-Martínez c,
Mercedes Gallardo a, *
a
Universidade de Vigo, Departamento de Biología Funcional y C.C. del Suelo, 36310 Vigo, Spain
b
Universidade de Vigo, Departamento de Biología Funcional y C.C. de la Salud, 36310 Vigo, Spain
c
Universidade de Vigo, Laboratorio de Química Analítica y Alimentaria, 36310 Vigo, Spain

A R T I C L E I N F O A B S T R A C T

Keywords: Nuts are important components of a healthy diet since they provide nutritional value and bioactive components.
Melatonin Melatonin is a well-known molecule in plants, and its relevance in foodstuffs is increasing. This study investi­
Walnuts gated the presence of melatonin in nuts using chromatographic techniques and optimized extraction procedures
Commercial nuts
according to the high oil content of nuts. Melatonin was detected in four walnut cultivars with levels similar to
Ripening
Processing
those previously reported. Moreover, the melatonin content in walnut seeds decreased sharply during the
ripening process from the ripe green stage to the mature dehydrated fruit and increased after harvesting when
the fruits were edible. A number of other commercial nuts were also measured, with melatonin contents varying
markedly, and generally being lower than in walnuts. The presence of melatonin was lower in commercial
roasted nuts than in raw nuts, with the exception of peanuts, where melatonin content increased with roasting. It
seems that this industrial processing negatively alters the structure of this molecule and its availability, which
should be taken into account when estimating its levels during nut consumption. Therefore, this study reveals
new data on the presence of melatonin in walnut seeds in a natural format and its evolution with maturation as
well as in other commercial nuts. We also highlight the importance that processing has on melatonin and other
antioxidants in the nuts that reach the consumer.

1. Introduction into the diet as a supplement to or instead of other foodstuffs. In recent

Nuts are so named because they share the characteristic that in their
natural composition (without human manipulation), they have less than
50 % water. Therefore, in nuts, the usable part as food is not the fruit
itself but the seed. The most popular are tree nuts such as almonds,
hazelnuts, walnuts and pistachios, in which the seeds are enclosed in
hardened or woody shells that are in turn covered with a fleshy or
fibrous outer layer that falls off before harvesting. Other edible seed nuts
include cashews, pecans, pine nuts and chestnuts. Additionally, peanuts,
which are botanically legumes, are widely identified as part of the nut
food group by consumers. All these nuts are nutrient-rich foodstuffs that
have been regular constituents of the human diet for hundreds of years
(Eaton and Konner, 1985). They are typically used as snack foods in seed
format (raw or roasted) or as components of butters, oils, sauces, or
ingredients of other foods, such as cookies, chocolates, and cakes. Other
seeds, such as those of pumpkin and sesame, have been incorporated
decades, the consumption of nuts has increased exponentially because
they are highly recommended by nutritionists and dietitians due to their
multiple health-promoting properties. Nuts are highly caloric foods,
although their consumption does not seem to be related to the risk of
weight gain (Ros, 2010; Tindall et al., 2018). Meanwhile, clinical and
epidemiological studies indicate that regular consumption of nuts,
particularly walnuts, is linked with a lower incidence of coronary heart
disease, with the associated reduction of cholesterol levels in the blood,
as well as diabetes, cancer and inflammatory diseases (Feldman, 2002;
Grosso et al., 2015).
Nuts are rich in vegetable proteins and fats, mostly unsaturated fatty
acids (Ros, 2010), and are a good source of dietary fibre. In addition,
nuts provide a wide range of nutrients, such as vitamins (folic acid,
niacin, tocopherols, vitamin B6, etc.), minerals (calcium, magnesium,
potassium, etc.) and many other bioactive components, such as phy­
tosterols (Phillips et al., 2005; Segura et al., 2006) and phenolic

* Corresponding author.
E-mail address: medina@uvigo.es (M. Gallardo).

https://doi.org/10.1016/j.jfca.2021.104180
Received 7 April 2021; Received in revised form 15 September 2021; Accepted 16 September 2021
Available online 28 October 2021
0889-1575/© 2021 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
A. Verde et al.
compounds, which act as powerful antioxidants in the body (Chang
et al., 2016). In most nuts, these antioxidants are located in the skin, as
occurs in almonds (Garrido et al., 2008; Chen et al., 2019), hazelnuts
and pistachios (Arcan and Yemenicioğlu, 2009), walnuts (Jahanba­
n-Esfahlan et al., 2019) and peanuts (Lou et al., 2004). This means that
after peeling, a large part of the antioxidant capacity of the fruits is lost.
Something similar happens with industrial bleaching, which is applied
for peeling purposes and causes antioxidant activity to be reduced, as
has been shown for almonds (Garrido et al., 2008; Oliveira et al., 2020)
and peanuts (Yu et al., 2006). However, roasting in general increases the
antioxidant activity in nuts (Chang et al., 2016).
Many studies have tried to characterize the functional bioactive
compounds in vegetables and derived foodstuffs, particularly those that
increasing their antioxidant activity, such as melatonin. This molecule is
widespread among living organisms and is involved in many physio­
logical processes, including circadian rhythms, hormonal regulation,
temperature, mood and immune system responses (Cipolla-Neto and
Amaral, 2018). Due to its pharmacological properties and its use as a
chronobiotic, melatonin has been proposed to alleviate the disruption of
circadian rhythms, as occurs in jet lag and insomnia (Zisapel, 2018).
Moreover, melatonin is an amphipathic molecule that is able to cross cell
membranes and the blood–brain barrier and possesses intrinsic and
powerful antioxidant activity that is effective in scavenging free radical
oxygen and nitrogen species (Tan et al., 2007). Consequently, melatonin
exhibits anticancer (Di Bella et al., 2013), anti-inflammatory (Hardeland
et al., 2015) and neuroprotective effects (Pandi-Perumal et al., 2013).
Additionally, it has also been proposed to control chronic diseases such
as diabetes, cardiovascular diseases and obesity (Karamitri and Jockers,
2019).
Melatonin is a ubiquitous molecule in the vegetal kingdom (Dubbels
et al., 1995; Van Tassel et al., 2001; Hardeland, 2016). In edible plants, it
is present in most of their organs, with the highest contents reported in
seeds, roots, leaves, flowers and in some fruits (Van Tassel et al., 2001;
Iriti et al., 2010; Nawaz et al., 2016; Arnao and Hernández-Ruiz, 2020).
Many of these are common constituents of a healthy diet, suggesting that
plant melatonin contributes to the benefits of consuming plants (Gar­
cia-Parrilla et al., 2009; Meng et al., 2017). An outstanding example of
this is the presence of melatonin in the Mediterranean diet, which is rich
in fruits, vegetables and seeds, which provide bioactive phytochemicals,
i.e., phenolic antioxidants, and contributes to reducing the risk of cancer
and cardiovascular and neurodegenerative diseases (Iriti et al., 2010;
Arnao and Hernández-Ruiz, 2018). Nuts are part of the Mediterranean
diet, and melatonin has been reported in several nuts, including popular
nuts such as almonds, pistachios (Paroni et al., 2019) and walnuts (Meng
et al., 2017). However, as in other plants, even for the same species, the
melatonin content detected in some nuts differs widely between labo­
ratories (Reiter et al., 2005; Tapia et al., 2013; Kocadağlı et al., 2014;
Paroni et al., 2019). Plant-related factors, such as the variety, cultivar,
harvesting season and fruit maturity (Kocadağlı et al., 2014), as well as
the analytical complexity of the plant tissues, which also vary between
species and within each fruit, advise increasing the number of studies on
the presence of melatonin in nuts, as well as the methodologies for its
analytical determination. Furthermore, following harvest, most nuts are
dried and stored until they are used in consumption or are processed, i.
e., roasted, salted, before commercial, which affects antioxidant
composition (Chang et al., 2016). However, the impact of processing on
melatonin has been studied little (Paroni et al., 2019).
In this study, an optimized solid-phase extraction method combined
with liquid chromatography techniques was used to identify and
quantify melatonin in walnuts and then in a number of commercial nuts.
The importance of the maturation stage and the commercial processing
(roasted) of nuts were also investigated.
2
Journal of Food Composition and Analysis 105 (2022) 104180
2. Materials and methods
2.1. Chemicals
The organic solvents methanol, acetonitrile, chloroform, hexane and
isopropanol were of HPLC grade and were provided by Merck (Darm­
stadt, Germany). Melatonin (pure reagent) was obtained from the same
supplier. Ethanol and benzene were purchased from VWR-Prolabo
(Fontenay sous Bois, France).
2.2. Plant material and processing
This study included several nuts common in the human diet, as listed:
walnuts (Juglans regia L.) of several varieties and origins, as follows:
“Pizarro” (Spain), “Franquette” (France), “Hartley” (California), and
unrated walnut of local origin (A Estrada, Galicia, Spain), hazelnuts
(Corylus avellana L), pistachios (Pistacea vera L.), almonds (Prunus dulcis
L.), peanuts (Arachis hypogaea L.), chestnuts (Castanea sativa Miller),
pine nuts (Pinus pinea L.), pumpkin seed (Cucurbita pepo L.), sunflower
seeds (Helianthus annuus L.), cashew nuts (Anacardium occidentale L.),
macadamia nuts (Macadamia integrifolia Maiden & Betche) and Brazil
nuts (Bertholletia excelsa Humb. & Bonpl.). All these nuts were obtained
from marketplaces and supermarkets in Galician (Spain), either as nat­
ural (raw) or processed (toasted) products.
Once in the laboratory, the nuts were opened manually, the shell was
carefully removed, and the seeds were immediately weighed and ho­
mogenized with the help of a mortar and pestle, protecting them from
light at all times.
Melatonin during fruit ripening in walnuts
The evaluation of melatonin content during different stages of fruit
ripening was carried out on locally sourced walnuts harvested in 2018.
The study involved five stages of maturation: i) ripe green, the fruit has
completed its growth and is ready to begin ripening; ii) ripe pre-
abscission, the fruit has reached maturity on the tree; iii) post-
abscission ripe the ripe fruit abscises and falls to the ground; iv) matu­
ration of the seed, dehydration of the seed (edible part of the nut) begins;
and v) desiccation of the seed, the seed completes maturation and shows
a dry stage (edible nuts). Except in the ripe green stage, in which the
melatonin contents were determined in both the pericarp and the seed,
the assays were carried out on the seed that constitutes the edible part of
the nut.
2.2.1. Melatonin extraction in walnuts
Walnut kernels are very high in oils (~65 to 70 %) (Martínez et al.,
2010). Because melatonin exhibits amphipathic characteristics, inade­
quate choice in the extraction method could lead to poor melatonin
recovery due to the molecule’s solubility in lipids. Therefore, optimi­
zation of the melatonin extraction was tried and included several assays
with solvents of different polarities, such as methanol, ethanol, ethyl
acetate and isopropanol, with the best results obtained with methanol.
However, methanol extraction in nut homogenates was not sufficient
since interference of lipids with melatonin reduced its recovery.
Therefore, mixtures of methanol and low-polarity solvents such as
benzene and hexane were assayed. In terms of melatonin extraction, the
best results were obtained with the hexane:methanol:water system
(3:3:1), which was developed as a modification of chloroform-based
methods (Bligh and Dyer, 1959; Kates, 1986) and according to Luzia
and Jorge (2013). This solution allows the lipids to be extracted in a
biphasic system, forming an upper layer, whereas the lower layer con­
tains the hydroalcoholic solution with a residual amount of lipids and
most of the extracted melatonin.
The procedure in walnut was carried out as follows: 1 g samples of
raw nuts were homogenized in a mortar with 7 mL of hexane:methanol:
water (3:3:1 v/v) in the absence of light. The resulting homogenate was
mixed for 10 min and then centrifuged at 4000 x g at 4 ◦ C for 10 min.
The upper phase (lipids in hexane) was discarded, and 1 mL of the
A. Verde et al.
hydroalcoholic phase was collected and evaporated to dryness under
vacuum at 30 ◦ C using a Speed-Vac (Eppendorf concentrator plus). The
residue was resuspended in 0.3 mL of 5% acetonitrile acidified with 0.1
% formic acid (pH 2.5) and used for a twofold solid-phase extraction as
described by Muñoz et al. (2009), with modifications. Briefly, the res­
idue was mixed (1:4 v:v) with chloroform, and the mixture was vortexed
for two min and centrifuged (4000 x g, 10 min). The aqueous layer was
aspirated and discarded. The organic layer was cleaned once with 0.5
mL of 0.2 N NaOH. After stirring and posterior centrifugation, the upper
aqueous phase was eliminated, and the organic solvent was evaporated
under vacuum at 30 ◦ C using a SpeedVac. The final residue was dis­
solved in 0.1 mL of 5% acidified acetonitrile and filtered through a
0.45-μm filter. An aliquot (20 μL) of filtrate was used for injection into
the HPLC system.
The efficiency of the extraction method and linearity to different
melatonin concentrations were calculated after spiking pure melatonin
(0.35–8.75 ng/ml) into homogenates of six nut samples and assaying in
triplicate. Intra- and interassay variation coefficients were determined
after pooling homogenates of six samples containing added melatonin
(range from 0.35 to 8.75 ng/mL) that were extracted and analysed on
the same day or on three different days, respectively.
A similar extraction protocol was used for the other nuts (1 g fresh
weight) included in this study. Indeed, the content of oily material was
lower than that in walnuts, resulting in a clearer final extract. In all
cases, seeds were obtained carefully, avoiding contamination with other
tissues.
Influence of temperature on melatonin extraction yield
To assess whether temperature could influence the melatonin
extraction yield, three temperature conditions were tested: cold, 2–4 ◦ C;
standard, 20–25 ◦ C; and moderately high, 40–45 ◦ C, which were applied
during all the homogenate preparation and extraction procedures.
2.2.2. Determination of melatonin by HPLC-FD
Many analytical techniques have been applied to determine mela­
tonin in plants and plant foods (see Discussion). In our study, an HPLC
method with fluorescence detection (FLD) was validated to analyse the
melatonin content in nuts. The system consisted of a Hewlett Packard
1100 HPLC system equipped with a quaternary pump (HP 1311A
module), an online degasser (HP G1322A) and a fluorescence detector
(HP G131A module) set at 285/345 excitation/emission wavelengths.
Separation was performed on a Kinetex C-18 column (2.6 μm particles,
150 mm × 4.6 mm, Phenomenex, USA) kept at 25 ◦ C (Jasco column
oven) with a flow rate of 1.0 mL/min. A mobile phase binary gradient
was performed with two solutions: (A) 60 % acetonitrile containing 0.1
% (v:v) formic acid and (B) 0.1 % formic acid. The elution profile was
15%–60% A from 0 to 15 min, 60%–90% A from 15 to 20 min and 90 %
to 15 % A from 20 to 25 min to recover the initial conditions. HP 1100
ChemStation software was used for acquisition and integration of the
chromatograms. Quantification was performed by comparing peak areas
with those of melatonin standards dissolved in 5% acetonitrile con­
taining 0.1 % formic acid.
2.2.3. Confirmation of melatonin by liquid chromatography/mass
spectrometry
The LC–MS system consisted of an HP 1100 series LC compact system
equipped with a binary pump coupled to a mass spectrometer (Agilent
Tech 1100 series MSD) that included an electrospray ionization module
and a single quadrupole mass analyser. Chromatographic separation
was performed with a reverse stationary phase column (Vydac 201TP52,
C18, 25 cm x 2.1 mm with a 5-μm pore), and the mobile phase consisted
of a linear gradient system (5%–95% in 15 min) from the mixture of two
mobile phases: 100 % acetonitrile (LCMS grade) with 0.1 % (v/v) formic
acid and water (LCMS grade) with 0.1 % (v/v) formic acid. LC separa­
tion was carried out at room temperature and a flow rate of 0.4 mL/min.
The ion source (ESI interface) of the mass spectrometer was operated
with the following parameters: nitrogen (N2) as the drying gas at a flow
3
Journal of Food Composition and Analysis 105 (2022) 104180
rate of 7 L⋅min-1 and temperature of 300 ◦ C, nebulizer pressure of 50 psi,
positive capillary voltage of 4000 V and fragmentor energy of 30 V. MS
acquisition data were in SIM (single ion monitoring) mode at transitions
m/z 233.1 for [M+H]+, 255.1 for [M+Na]+, and 174.1 and 159.1 for
selective ions that resulted from melatonin molecule fragmentation.
2.3. Statistical analysis
Data are shown as the means ± standard errors of the mean (S.E.M.).
Melatonin levels in the different groups were analysed by one-way
ANOVA and the Student-Newman–Keuls multiple comparison test. Dif­
ferences were significant when the p value was less than 5% (p < 0.05).
3. Results
3.1. Melatonin extraction and quantification
Melatonin was routinely measured in nuts by HPLC-FLD with a
previous extraction in organic solvents. The method was optimized in
walnut since it showed the highest oil content and the greatest chro­
matographic complexity of all nuts. Assays with different organic sol­
vents were carried out in walnuts to estimate the best efficiency in terms
of cleaning properties and recovering melatonin from the samples
(Table 1). Methanol showed greater efficiency in the extraction of
melatonin from walnut homogenates than other solvents, such as
ethanol, ethyl acetate and isopropyl alcohol; however, we visually
detected oily material dissolved in methanol with the additional risk of
interference in the subsequent extraction step. This was improved by the
use of a hydroalcoholic solution (methanol:water 3:1 v:v), although
melatonin recovery was lowered. As an alternative, the combination of
methanol with less polar solvents such as hexane and benzene in the
presence of different proportions of water was assessed. The best results
were obtained with the hexane-methanol-water system (3:3:1, v:v:v), in
which partitioning of lipids and melatonin occurs largely in two phases
that can be easily separated.
The performance and reproducibility of the extraction and chro­
matographic procedures were evaluated by adding serial concentrations
of melatonin (from 0.35 to 8.75 ng/mL) to homogenates (pools of six
walnuts) assayed in triplicate. As shown in Table 2, the percentage of
melatonin recovery oscillated between 81 % and 92 %, depending on the
spiked concentration. The intra- and interassay variation coefficients
ranged from 1.35 % to 3.38 % and from 1.70 % to 5%, respectively,
depending on the melatonin concentration, which met the demands in
terms of method precision. The detection limits (LOD: 38.55 pg/mL;
LOQ, 128.50 pg/mL) were adequate for routine analysis of a broad
spectrum of nut samples.
Finally, the influence of temperature on the extraction yield and the
subsequent quantification of melatonin was evaluated by processing the
walnut samples under three temperature ranges: 2–4 ◦ C, 20–25 ◦ C, and
40–45 ◦ C (Table 3). Melatonin levels were much higher when assayed at
moderately high temperatures (40–45 ◦ C) than when assayed at cold
and room temperatures. Moreover, samples assessed under a cold
environment showed significantly lower levels than those tested at room
temperature, although the latter was chosen for routine analysis.
Table 1
Melatonin levels in walnut samples extracted with different solvents.
Extraction solvent Means (pg/g) Concentrations (pg/g)
Methanol 100 % 906.5 804− 1009
Ethanol 100 % 75.5 59− 150
Hexane:Methanol:Water (3:3:1) 4015.0 910− 6210
Hexane:Isopropanol (3:2) 212.0 110− 314
Benzene:Methanol (1:4) 260.0 108− 412
Data are the mean values and the range of concentrations resulting from the
independent analysis of six samples for each solvent assessed.
A. Verde et al. Journal of Food Composition and Analysis 105 (2022) 104180

Table 2
Performance and reproducibility of the extraction method.
Melatonin added (pg/ Mean values (pg/ Recovery Repetitivity (% RSD, intra- Reproducibility (% RSD, inter- Linearity LOD (pg/ LOQ (pg/
mL) mL) (%) assay) assay) mL) mL)

350 325.05 92.87 3.38 3.95

875 749.46 85.65 2.81 2.26 y = 0.9043x −
0.099
1750 1424.24 81.38 2.96 5.23 38.55 128.50
3500 2887.45 82.50 1.35 4.43 r2 = 0.9985
8750 7887.08 90.14 1.92 1.70

Different concentrations of melatonin were added at the beginning of the processing of the walnut samples. The yield values express the percentage of the amount of
melatonin measured in the sample with respect to that added. Variability (% RSD) is calculated as the percentage of the standard deviation in relation to the mean.
Intra-assay and inter-assay data were obtained from pools (6 pieces) of walnuts processed in triplicate and analysed on the same day or on successive days.

255.1, 233.1, 174.1 and 159.1 specific ions obtained by melatonin

Table 3
fragmentation in mass spectrometry. Melatonin peak identification in
Effect of temperature on the melatonin extraction yielding in walnuts.
walnuts was confirmed by LC–MS data obtained for sample extracts and
Temperature from specific HPLC-FLD fractions collected at melatonin retention time,
Low (0− 4 ◦ C) Standard (20− 25 High (40− 45 ◦ C) which were compared against scan data obtained for melatonin standard
◦
C) solution (Fig. 1B). We also identified a chromatographic signal (peak 1)
Melatonin (pg/g 329.39 ± 16.70 1120.26 ± 236.86 b 2167.56 ± 318.97 with a longer retention time than melatonin but that produces the same
FW) a c
ion fragment profile. This means that peak 1 is related to the presence of
Results are means ± SEM of six walnuts per group. Different letters indicate a melatonin analogue, but no further specific studies were performed.
statistical (p < 0.05) differences.
3.3. Melatonin content in walnut varieties
3.2. Confirmation of melatonin by LC–MS
The melatonin contents in the different types of walnut studied are
The sample extracts analysed by HPLC-FLD were also processed by shown in Fig. 2. Regarding the commercial walnut varieties, the highest
LC–MS for confirmatory purposes. Fig. 1A shows an example of a levels corresponded to Hartley (3301 ± 92 pg/g FW), which were three
chromatogram that contains the superposition of signals obtained at m/z times higher than the levels in Franquette (1600 ± 74 pg/g) and Pizarro
(1191 ± 57 pg/g). The walnuts obtained from local producers showed
intermediate melatonin levels (2070 ± 60 pg/g).

3.4. Melatonin content during nut ripening

Using the local walnut variety as a model, the melatonin content was
studied in the different parts of the fruit (pericarp and seed) as well as in
different physiological stages of fruit ripening, in this case only in the
seed (Table 4). In the mature green stage, the pericarp is the tissue that
shows the highest concentration of melatonin in the walnut (8044 pg/g),
which is almost five times higher than in the seed of the same stage.
Meanwhile, the content of melatonin in the seed varied sharply from the
initial stage of ripe green (approximately 1534 pg/g) to a long period of
low levels (approximately 120 pg/g) that coincided with the pre- and
post-abscission times. Finally, melatonin levels rose when the fruit was
completely dehydrated, reaching close to 2.5 ng/g.

3.5. Melatonin contents in raw and processed nuts

The melatonin contents in different nuts of commercial origin are

depicted in Fig. 3. Melatonin was measured in all the studied nuts, both
in raw and processed formats when available, showing values that range

Fig. 1. (A) LC–MS total ion count chromatogram obtained for the analysis of a
walnut extract. MEL peak corresponds to the retention time of a melatonin
standard. Unknown Peak 1 was also run for mass spectrum analysis as
explained in the M&M section. (B) SCAN MS data with ion fragment structures
for MEL and peak 1 in walnut extract ranged from 100 to 300 m/z (Fig. 1B).
Fig. 2. Melatonin content in different walnut varieties. Data are the means ±
SEM of ten individual samples into each group. Different letters indicate sta­
tistical (p < 0.05) differences.

4
A. Verde et al.
Table 4
Melatonin content during maturation stages in walnuts.
Tissue Maturation stage Number of samples Melatonin (pg/g)
a were assessed, including SPE and chloroform, although their perfor­
Pericarp Ripe green 6 8044.77 ± 1222.60
Seed Ripe green 6 1534.36 ± 693.14b
Seed Pre-abscission green 6 164.39 ± 65.33c
Seed Post-abscission ripe 6 124.21 ± 58.22c
Seed Dehydrated fruit 6 96.63 ± 17.65c
Seed Dry fruit 6 2531.13 ± 983.21b
Results are means ± SEM. In the mature green stage, the pericarp and the seed
were analyzed separately. In the other stages, only the seed was included in the
analysis. Different letters indicate significant differences (p < 0.05).
Fig. 3. Melatonin content in different types of nuts both in raw and processed
format. Data are the means ± SEM of ten individual samples into each group.
Where applicable, asterisks indicate statistical (p < 0.05) differences between
formats for each type of nut.
from 83 pg per gram in the case of peanuts up to 1417 pg per g in the
case of chestnuts, both of them in raw format. In general, raw nuts
showed significantly higher amounts of melatonin than those that were
subjected to processing (roasted fruit). As an exception, the melatonin
level in peanuts was much higher when roasted (589 pg/g) than when
raw (83 pg/g).
4. Discussion
Melatonin is present in a wide variety of plant species and in different
organs of a given plant (Reiter et al., 2007; Hernández-Ruiz and Arnao,
2008; Arnao and Hernández-Ruiz, 2020). Different techniques have
been used for its quantification, such as radioimmunoassay (RIA),
enzyme-linked immunosorbent assay (ELISA) and chromatography
(liquid or gas). The first two are techniques based on the use of anti­
bodies that are more unspecific for plant material due to cross-reactivity
with other metabolites of plant extracts (Kólar et al., 1995; Nawaz et al.,
2016). HPLC techniques coupled to either electrochemical or fluori­
metric detectors display high sensitivity and accuracy, as well as suffi­
cient versatility to be applied in a wide spectrum of biological matrices;
therefore, they have often been used to measure melatonin in plants and
plant products (Reiter et al., 2007; Nawaz et al., 2016). Mass spec­
trometry is an option that is gaining acceptance today because it ensures
the identification of melatonin and allows its measurement in a wide
range of plants, although even in this case, matrix problems may seri­
ously affect the quantification limits (Cao et al., 2006).
To quantify the melatonin present in nuts, we adapted an HPLC-FLD
method that has been previously validated in animals (Muñoz et al.,
2009). This analytical method fit well to plant sample demand, showing
great sensitivity and good linearity in the response to a wide range of
melatonin concentrations, as well as good reproducibility, as shown by
the intra- and interassay coefficients. The versatility of the method was
verified when applied to different types of nuts, although in our case, the
walnut was the model chosen for fine-tuning the trials. The composition
5
Journal of Food Composition and Analysis 105 (2022) 104180
of walnut with a high oil percentage adds difficulty for sample pro­
cessing and the melatonin extraction itself due to the molecule’s
amphipathic properties. A number of extraction methods for melatonin
mance was rather low, and they poorly purified the extracted material.
The capacity of several other organic solvents, such as ethanol, meth­
anol, isopropanol and benzene:methanol, was also evaluated. From
them, methanol turned out to be a very effective solvent in recovering
melatonin from walnuts, although when applied as a pure reagent, it
also carries oily components, which makes further processing difficult.
Hexane, a nonpolar solvent that mixes well with more polar solvents
such as isopropanol and methanol, was also useful to recover melatonin,
but as a pure reagent, it is highly volatile, which poses greater risks for
the manipulator. Based on a lipid extraction method (Luzia and Jorge,
2013), the hexane:methanol:water solution (ratio 3:3:1 by volume) was
revealed as the most suitable for our purposes. With this mixture, two
well-separated phases are obtained so that hexane extracts almost all
lipids, while melatonin remains dissolved in the lower hydroalcoholic
phase. A subsequent step with a chloroform:water mixture allowed
increasing the concentration of melatonin in residuals to be injected into
the chromatographic system.
Moreover, we observed that the temperature at which the nuts are
processed may be a relevant factor when estimating melatonin content
in samples. Moderately high temperature (40–45 ◦ C) yields twice the
melatonin recovery rate compared with room temperature, and this
performance is seriously compromised when cold temperature (0–4 ◦ C)
is applied. Pranil et al. (2020) reported that temperature is a relevant
factor when storing melatonin water solutions and fruit juices since high
temperatures (60–90 ◦ C) negatively affected melatonin stability,
whereas cold conditions (4 ◦ C) and dark and acidic solvents improved
storage. Our data in walnut seeds suggest that the increase in melatonin
extraction yield at temperatures of 45–50 ◦ C is mainly due to the
decrease in viscosity of organic solvents so that the solvent penetrates
better into the matrix; therefore, an improvement in extraction yield is
observed (Dean and Saim, 1998). However, the most relevant of these
results is the low extraction capacity of solvents in a cold environment,
something that is common in research to preserve the molecules under
study.
The LC–MS technique was used for confirmatory purposes of mela­
tonin detection in walnut sample extracts. According to the mass spec­
trum of purified melatonin, ion fragments with m/z 159.1, 174.1, 233.1
and 255.1 were used for LC/MS identification of melatonin in sample
extracts and in HPLC-FLD fractions eluting at specific peak retention
times. Moreover, an unknown peak (peak 1, Fig. 1A) with a relatively
longer retention time but having the same molecular fragmentation
profile of melatonin was found. This peak could be related to the pres­
ence of a melatonin analogue, which is consistent with the previously
reported detection of a melatonin isomer in walnuts (Kocadağlı et al.,
2014). Further tandem mass spectrometry (MS/MS) and HRMS studies
should be carried out to establish the exact mass of this compound and
its relative abundance in relation to melatonin.
The melatonin levels measured in walnuts were in a similar range as
those reported by other studies in different walnut cultivars. Specif­
ically, the melatonin content in Hartley (California) was similar to that
reported by Reiter et al. (2005) in the same cultivar, while Pizarro and
Franquette were in the range of values observed by Tapia et al. (2013)
for four varieties of walnuts: Serr, Hartley, Chandler and Howard.
Likewise, our data show that melatonin levels varied conspicuously
according to the walnut variety studied. Among the most common
commercial walnuts, Hartley exhibited the highest levels of melatonin,
followed by a non-classified local walnut that indeed had higher values
than all those included in the study of Tapia et al. (2013). These dif­
ferences between types of walnuts may be due to the constitution of the
plant itself, the harvesting time or the analytical methods, but other
factors, such as the time passed since harvest or the storage conditions,
could also be involved.
A. Verde et al.
Melatonin presence in fruits has been reported to be affected by
ripening degree (Wang et al., 2020), but it has not been investigated in
nuts. In this study, we explored the changes in melatonin content during
different stages of ripening in walnuts. A considerable amount of
melatonin was found in the seed at the physiological stage called ripe
green, in which the fruit has already reached its final size and ripening
begins. These levels clearly decreased in the pre- and post-abscission
stages of the fruit and even at the dehydration process that occurs
when seed maturity begins, ultimately increasing when the seed is in a
dry state. In the mature green stage, the melatonin levels were also
measured in the fruit pericarp (including the epicarp and mesocarp) and
were surprisingly high. We were not able to find data in the literature on
the impact that ripening has on the levels of melatonin in nuts. In other
fruits, such as grapes (Xu et al., 2018), tomatoes (Okazaki and Ezura,
2009), cherries (Tijero et al., 2019) or mulberry fruits (Wang et al.,
2016), the content of melatonin varied during fruit development, nor­
mally showing high levels during growth that decrease with the ripening
process, which would be in line with our results.
The evolution of melatonin levels observed in walnuts suggests that
this molecule may play a relevant role in the process of maturity. Hence,
melatonin could interact with the hormones that regulate the process, as
has been noted in other fruits (Sun et al., 2015; Wang et al., 2020),
and/or the seed itself by acting at the cellular level as an antioxidant
(Manchester et al., 2000). Studies on the presence of melatonin in seeds
reported high levels of the molecule that could protect the germ tissue
during oxidative stress caused by abiotic factors (Balabusta et al., 2016).
This is consistent with the higher levels of melatonin found in this study
in walnut kernels in the dry state compared to previous stages. On the
other hand, Manchester et al. (2000) showed that melatonin content is
especially high in seeds rich in fatty acids. Given that walnut contains
high levels of long-chain polyunsaturated fatty acids, which are easily
oxidized (Tapia et al., 2013), melatonin could contribute to the pro­
tection of these cellular constituents. In this role, melatonin could add to
other bioactive components, such as phenols, which also have high
antioxidant activity (Salcedo et al., 2010; Tapia et al., 2013). Further­
more, Pycia et al. (2019) found that the content of phenolic compounds
in walnut (seed) depended on the degree of maturation in such a way
that it had decreased significantly from those collected in July to
September. This trend is similar to that found for melatonin in the
present study. In turn, the high levels of melatonin present in the peri­
carp of the green fruit could indicate that not only the seeds but also the
additional cover of the fruit (including the skin, epicarp and mesocarp)
are melatonin-rich tissues. The outermost covers are also rich in anti­
oxidant polyphenols that subsequently decrease during fruit ripening
(Castrejón et al., 2008; Oliveira et al., 2008; Pycia et al., 2019). The
pericarp opens in the mature fruit before falling to the ground (Oliveira
et al., 2008); this phase coincides with the lowest level of melatonin in
the walnut seeds. In postharvested dehydrated kernels, melatonin con­
centration increased dramatically, which seems to respond to tissue
quality deterioration, which occurs together with the loss of phenolic
antioxidants (Ortiz et al., 2019). Therefore, our data suggest that
melatonin both at the level of the green pericarp and in the kernel itself
may be a relevant antioxidant constituent during walnut ripening, but
this molecule could also have a role in maintenance of the seed during
the postharvest period.
In this study, the content of melatonin was also determined in a wide
variety of nuts, such as hazelnut, pine nut, chestnut, pistachio, and
peanut, all of which contained lower concentrations of melatonin than
walnuts. The amounts present in these fruits were in accordance with
the literature (Meng et al., 2017), with the exception of pistachio, whose
content was much lower than that described by Oladi et al. (2014) in
several cultivars of pistachio (more than 230 μg/g) using spectro­
fluorimetric and GC/MS methods. In contrast, Paroni et al. (2019), using
LC–MS/MS analysis, found melatonin levels in pistachios to be
approximately 600–2000 pg/g, which is similar to the range reported
herein. The reasons for these discrepancies may come from differences
6
Journal of Food Composition and Analysis 105 (2022) 104180
in the selectivity of the extraction methods and the chromatographic
techniques applied, which usually entail differences in quantification of
the compounds of interest.
All nuts included in our study are common in the diet, as their con­
sumption is highly recommended due to their nutritional properties.
Nuts stand out for their contributions of protein, fibre, minerals, vita­
mins and the presence of unsaturated and monounsaturated fatty acids,
which provide them with health-beneficial properties. In some cases, as
in walnut, daily consumption is recommended, as the nuts contain
linoleic and linolenic acids (60 % of the total fatty acid content), which
also help reduce cardiovascular risk (Feldman, 2002; Grosso et al.,
2015). In addition, the high content of trace elements (especially mag­
nesium) diminishes cellular susceptibility to peroxidation. Conse­
quently, studies in rats fed meals supplemented with walnuts for several
weeks showed a higher antioxidant capacity in blood plasma than in
animals fed standard diets, which occurred in parallel with the increase
(up to 3 times) in the serum content of melatonin (Reiter et al., 2005).
Usually, nuts are available for consumers in a raw state (without pro­
cessing) or after processing that can be more or less complex. Nuts are
often found in roasted and salted formats to adapt their aroma, texture
and colour to the consumer’s tastes. Even in its raw form, nuts generally
undergo processes of high temperature and humidity to remove the shell
and/or to clean the shell and the skin of the kernel, i.e., bleaching.
Although manufacturers try to preserve the nutritional quality of the
product as much as possible, it is likely that the processing of nuts affects
the level of its bioactive compounds, including fatty acids and phenols
(Chang et al., 2016; Oliveira et al., 2020). In this study, the levels of
melatonin in several commercially available roasted nuts, such as al­
monds, hazelnuts, pumpkin seeds and pistachios, were measured and
compared with similar products in raw format. Of all of them, only the
roasted pistachios showed significantly lower values than the raw pis­
tachios, although there was also a trend (though not significant) in
hazelnut. Meanwhile, roasting clearly reduced the melatonin content in
almonds and pumpkin seeds. To the best of our knowledge, only the
study of Paroni et al. (2019) reported melatonin levels in nuts with
different processing formats. In this study, the melatonin levels in
non-roasted, non-salted pistachios were higher than those detected in
the processed pistachios, although with great differences depending on
the origin of the product. Our data also suggest that roasted nuts have
lost part of their melatonin content during processing, which may
contribute to a decrease in their antioxidant capacity, as reported by
others (Chang et al., 2016). However, drawing conclusions from our
study could be risky given the different origins of the commercial nuts
that were used, and which were probably subjected to different pro­
cessing depending on the marketing company.
In summary, this study demonstrates the presence of high amounts of
melatonin in walnuts and, to a lesser extent, in other commercial nuts.
Our data corroborate the importance of characterizing the presence of
melatonin in nuts, including those that have been processed to improve
their attractiveness for consumption. The lower levels of melatonin that
were measured in some roasted nuts compared to those in raw nuts
highlight the need for further studies that focus not only on the nutri­
tional value of processed foods but also on their content of bioactive
components, including melatonin.
5. Conclusions
Our results demonstrate the presence of high amounts of melatonin
in walnut seeds, which vary with ripening and even after harvest when
the fruits are edible. A possible modulatory and antioxidant role of
melatonin in ripening can be suggested, which could also lead to better
seed maintenance during the post-harvest period. Melatonin is also
present in other commercial nuts, with variable levels among them,
although they were generally lower than those present in walnuts. On
the other hand, melatonin levels measured in some roasted nuts were
lower than those found in raw nuts. This highlights the need for further
A. Verde et al.
studies that focus not only on the nutritional value of processed foods,
but also on their content of bioactive components, including melatonin.
Author statement
Antía Verde: Investigation, data curation, writing- original draft
preparation and editing; J.M. Míguez: Supervision, conceptualization,
methodology, writing-review and editing; J.M. Leao: Methodology, re­
sources, validation; Ana Gago: Methodology, validation, resources.
Mercedes Gallardo: Supervision, conceptualization, methodology, re­
sources, writing-review and editing. All authors reviewed the results and
approved the final version of the manuscript.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgements
The authors thank L. Souto and E. Rodríguez for support in walnut
analyses. Antía Verde Rodríguez is a fellowship recipient from Xunta de
Galicia in the pre-doctoral formation programme (Reference ED481A-
2017/387). Open access charge is funding by Universidade de Vigo/
CISUG.
References
Arcan, I., Yemenicioğlu, A., 2009. Antioxidant activity and phenolic content of fresh and
dry nuts with or without the seed coat. J. Food Compos. Anal. 22 (3), 184–188.
https://doi.org/10.1016/j.jfca.2008.10.016.
Arnao, M.B., Hernández-Ruiz, J., 2018. The potential of phytomelatonin as a
nutraceutical. Molecules 23 (1), 238. https://doi.org/10.3390/molecules23010238.
Arnao, M.B., Hernández-Ruiz, J., 2020. Melatonin in flowering, fruit set and fruit
ripening. Plant Reprod. 33 (2), 77–87. https://doi.org/10.1007/s00497-020-00388-
8.
Balabusta, M., Szafrańska, K., Posmyk, M.M., 2016. Exogenous melatonin improves
antioxidant defense in cucumber seeds (Cucumis sativus L.) germinated under chilling
stress. Front. Plant Sci. 7, 575. https://doi.org/10.3389/fpls.2016.00575.
Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification.
Can. J. Biochem. Physiol. 37 (8), 911–917. https://doi.org/10.1139/o59-099.
Cao, J., Murch, S.J., O’Brien, R., Saxena, P.K., 2006. Rapid method for accurate analysis
of melatonin, serotonin and auxin in plant samples using liquid chromatography-
tandem mass spectrometry. J. Chromatogr. A 1134, 333–337. https://doi.org/
10.1016/j.chroma.2006.09.079.
Castrejón, A.D.R., Eichholz, I., Rohn, S., Kroh, L.W., Huyskens-Keil, S., 2008. Phenolic
profile and antioxidant activity of highbush blueberry (Vaccinium corymbosum L.)
during fruit maturation and ripening. Food Chem. 109 (3), 564–572. https://doi.
org/10.1016/j.foodchem.2008.01.007.
Chang, S.K., Alasalvar, C., Bolling, B.W., Shahidi, F., 2016. Nuts and their co-products:
the impact of processing (roasting) on phenolics, bioavailability, and health
benefits–a comprehensive review. J. Funct. Foods 26, 88–122. https://doi.org/
10.1016/j.jff.2016.06.029.
Chen, C.Y.O., Milbury, P.E., Blumberg, J.B., 2019. Polyphenols in almond skins after
blanching modulate plasma biomarkers of oxidative stress in healthy humans.
Antioxidants 8 (4), 95. https://doi.org/10.3390/antiox8040095.
Cipolla-Neto, J., Amaral, F.G.D., 2018. Melatonin as a hormone: new physiological and
clinical insights. Endocr. Rev. 39 (6), 990–1028. https://doi.org/10.1210/er.2018-
00084.
Dean, J.R., Saim, N., 1998. Modern alternatives to supercritical fluid extraction. In:
Ramsey, E.D. (Ed.), Analitical Supercritical Fluid Extraction Techniques. Kluwer
Academic Publishers, London, pp. 392–417. https://doi.org/10.1007/978-94-011-
4948-8_10.
Di Bella, G., Mascia, F., Gualano, L., Di Bella, L., 2013. Melatonin anticancer effects. Int.
J. Mol. Sci. 14 (2), 2410–2430. https://doi.org/10.3390/ijms14022410.
Dubbels, R., Reiter, R.J., Klenke, E., Goebel, A., Schnakenberg, E., Ehlers, C.,
Schiwara, H.W., Schloot, W., 1995. Melatonin in edible plants identified by
radioimmunoassay and by high performance liquid chromatography-mass
spectrometry. J. Pineal Res. 18 (1), 28–31. https://doi.org/10.1111/j.1600-
079X.1995.tb00136.x.
Eaton, S.B., Konner, M., 1985. Paleolithic nutrition: a consideration of its nature and
current implications. N. Engl. J. Med. 312 (5), 283–289. https://doi.org/10.1056/
NEJM198501313120505.
Feldman, E.B., 2002. The scientific evidence for a beneficial health relationship between
walnuts and coronary heart disease. J. Nutr. 132 (5), 1062S–1101S. https://doi.org/
10.1093/jn/132.5.1062S.
Garcia-Parrilla, M.C., Cantos, E., Troncoso, A.M., 2009. Analysis of melatonin in foods.
J. Food Compos. Anal. 22 (3), 177–183. https://doi.org/10.1016/j.
jfca.2008.09.009.
7
Journal of Food Composition and Analysis 105 (2022) 104180
Garrido, I., Monagas, M., Gómez-Cordovés, C., Bartolomé, B., 2008. Polyphenols and
antioxidant properties of almond skins: influence of industrial processing. J. Food
Sci. 73 (2), C106–C115. https://doi.org/10.1111/j.1750-3841.2007.00637.x.
Grosso, G., Yang, J., Marventano, S., Micek, A., Galvano, F., Kales, S.N., 2015. Nut
consumption on all-cause, cardiovascular, and cancer mortality risk: a systematic
review and meta-analysis of epidemiologic studies. Am. J. Clin. Nutr. 101 (4),
783–793. https://doi.org/10.3945/ajcn.114.099515.
Hardeland, R., 2016. Melatonin in plants–diversity of levels and multiplicity of functions.
Front. Plant Sci. 7, 198. https://doi.org/10.3389/fpls.2016.00198.
Hardeland, R., Cardinali, D.P., Brown, G.M., Pandi-Perumal, S.R., 2015. Melatonin and
brain inflammaging. Prog. Neurobiol. 127, 46–63. https://doi.org/10.1016/j.
pneurobio.2015.02.001.
Hernández-Ruiz, J., Arnao, M.B., 2008. 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. 56 (22), 10567–10573. https://doi.org/10.1021/jf8022063.
Iriti, M., Varoni, E.M., Vitalini, S., 2010. Melatonin in traditional Mediterranean diets.
J. Pineal Res. 49 (2), 101–105. https://doi.org/10.1111/j.1600-079X.2010.00777.x.
Jahanban-Esfahlan, A., Ostadrahimi, A., Tabibiazar, M., Amarowicz, R., 2019.
A comparative review on the extraction, antioxidant content and antioxidant
potential of different parts of walnut (Juglans regia L.) fruit and tree. Molecules 24
(11), 2133. https://doi.org/10.3390/molecules24112133.
Karamitri, A., Jockers, R., 2019. Melatonin in type 2 diabetes mellitus and obesity. Nat.
Rev. Endocrinol. 15 (2), 105–125. https://doi.org/10.1038/s41574-018-0130-1.
Kates, M., 1986. Techniques of lipidology: isolation, analysis and identification of lipids.
Lab. Techniq. Biochem. Molec. Biol. 3 (2). Amsterdam (Netherlands): Elsevier.
Kocadağlı, T., Yılmaz, C., Gökmen, V., 2014. Determination of melatonin and its isomer
in foods by liquid chromatography tandem mass spectrometry. Food Chem. 153,
151–156. https://doi.org/10.1016/j.foodchem.2013.12.036.
Kólar, J., Machackova, I., Illnerova, H., Prinsen, E., Van Dongen, W., Van Onckelen, H.A.,
1995. Melatonin in higher plant determined by radioimmunoassay and liquid
chromatography-mass spectrometry. Biol. Rhythm Res. 26, 406–409.
Lou, H., Yuan, H., Ma, B., Ren, D., Ji, M., Oka, S., 2004. Polyphenols from peanut skins
and their free radical-scavenging effects. Phytochemistry 65 (16), 2391–2399.
https://doi.org/10.1016/j.phytochem.2004.06.026.
Luzia, D.M., Jorge, N., 2013. Bioactive substance contents and antioxidant capacity of
the lipid fraction of Annona crassiflora Mart. seeds. Ind. Crops Prod. 42, 231–235.
https://doi.org/10.1016/j.indcrop.2012.05.027.
Manchester, L.C., Tan, D.X., Reiter, R.J., Park, W., Monis, K., Qi, W., 2000. High levels of
melatonin in the seeds of edible plants: possible function in germ tissue protection.
Life Sci. 67 (25), 3023–3029. https://doi.org/10.1016/S0024-3205(00)00896-1.
Martínez, M.L., Labuckas, D.O., Lamarque, A.L., Maestri, D.M., 2010. Walnut (Junglans
regia L.): genetic resources, chemistry, by-products. J. Sci. Food Agric. 90,
1959–1967. https://doi.org/10.1002/jsfa.4059.
Meng, X., Li, Y., Li, S., Zhou, Y., Gan, R.Y., Xu, D.P., Li, H.B., 2017. Dietary sources and
bioactivities of melatonin. Nutrients 9 (4), 367. https://doi.org/10.3390/
nu9040367.
Muñoz, J.L., Ceinos, R.M., Soengas, J.L., Míguez, J.M., 2009. A simple and sensitive
method for determination of melatonin in plasma, bile and intestinal tissues by high
performance liquid chromatography with fluorescence detection. J. Chromatogr. B
877 (22), 2173–2177. https://doi.org/10.1016/j.jchromb.2009.06.001.
Nawaz, M.A., Huang, Y., Bie, Z., Ahmed, W., Reiter, R.J., Niu, M., Hameed, S., 2016.
Melatonin: current status and future perspectives in plant science. Front. Plant Sci. 6
https://doi.org/10.3389/fpls.2015.01230. Article 1230.
Okazaki, M., Ezura, H., 2009. Profiling of melatonin in the model tomato (Solanum
lycopersicum L.) cultivar Micro-Tom. J. Pineal Res. 46 (3), 338–343. https://doi.org/
10.1111/j.1600-079X.2009.00668.x.
Oladi, E., Mohamadi, M., Shamspur, T., Mostafavi, A., 2014. Spectrofluorimetric
determination of melatonin in kernels of four different Pistacia varieties after
ultrasound-assisted solid–liquid extraction. Spectrochim. Acta A. Mol. Biomol.
Spectrosc. 132, 326–329. https://doi.org/10.1016/j.saa.2014.05.010.
Oliveira, I., Sousa, A., Ferreira, I.C., Bento, A., Estevinho, L., Pereira, J.A., 2008. Total
phenols, antioxidant potential and antimicrobial activity of walnut (Juglans regia L.)
green husks. Food Chem. Toxicol. 46 (7), 2326–2331. https://doi.org/10.1016/j.
fct.2008.03.017.
Oliveira, I., Meyer, A.S., Afonso, S., Sequeira, A., Vilela, A., Goufo, P., Trindade, H.,
Gonçalves, B., 2020. Effects of different processing treatments on almond (Prunus
dulcis) bioactive compounds, antioxidant activities, fatty acids, and sensorial
characteristics. Plants 9 (11), 1627. https://doi.org/10.3390/plants9111627.
Ortiz, C.M., Vicente, A.R., Fields, R.P., Grillo, F., Labavitch, J.M., Donis-Gonzalez, I.,
Crisosto, C.H., 2019. Walnut (Juglans regia L.) kernel postharvest deterioration as
affected by pellicle integrity, cultivar and oxygen concentration. Postharvest Biol.
Technol. 156, 110948 https://doi.org/10.1016/j.postharvbio.2019.110948.
Pandi-Perumal, S.R., BaHammam, A.S., Brown, G.M., Spence, D.W., Bharti, V.K.,
Kaur, C., Hardeland, R., Cardinali, D.P., 2013. Melatonin antioxidative defense:
therapeutical implications for aging and neurodegenerative processes. Neurotox.
Res. 23 (3), 267–300. https://doi.org/10.1007/s12640-012-9337-4.
Paroni, R., Dei Cas, M., Rizzo, J., Ghidoni, R., Montagna, M.T., Rubino, F.M., Iriti, M.,
2019. Bioactive phytochemicals of tree nuts. Determination of the melatonin and
sphingolipid content in almonds and pistachios. J. Food Compos. Anal. 82, 103227
https://doi.org/10.1016/j.jfca.2019.05.010.
Phillips, K.M., Ruggio, D.M., Ashraf-Khorassani, M., 2005. Phytosterol composition of
nuts and seeds commonly consumed in the United States. J. Agric. Food Chem. 53
(24), 9436–9445. https://doi.org/10.1021/jf051505h.
Pranil, T., Moongngarm, A., Loypimai, P., 2020. Influence of pH, temperature, and light
on the stability of melatonin in aqueous solutions and fruit juices. Heliyon 6 (3),
e03648. https://doi.org/10.1016/j.heliyon.2020.e03648.
A. Verde et al.
Pycia, K., Kapusta, I., Jaworska, G., 2019. Impact of the degree of maturity of walnuts
(Juglans regia L.) and their variety on the antioxidant potential and the content of
tocopherols and polyphenols. Molecules 24 (16), 2936. https://doi.org/10.3390/
molecules24162936.
Reiter, R.J., Manchester, L.C., Tan, D.X., 2005. Melatonin in walnuts: influence on levels
of melatonin and total antioxidant capacity of blood. Nutrition 21 (9), 920–924.
https://doi.org/10.1016/j.nut.2005.02.005.
Reiter, R.J., Tan, D.X., Manchester, L.C., Simopoulos, A.P., Maldonado, M.D., Flores, L.J.,
Terron, M.P., 2007. Melatonin in edible plants (phytomelatonin): identification,
concentrations, bioavailability and proposed functions. World Rev. Nutr. Diet. 97,
211–230. https://doi.org/10.1159/000097917.
Ros, E., 2010. Health benefits of nut consumption. Nutrients 2 (7), 652–682. https://doi.
org/10.3390/nu2070652.
Salcedo, C.L., de Mishima, B.A.L., Nazareno, M.A., 2010. Walnuts and almonds as model
systems of foods constituted by oxidisable, pro-oxidant and antioxidant factors. Food
Res. Int. 43 (4), 1187–1197. https://doi.org/10.1016/j.foodres.2010.02.016.
Segura, R., Javierre, C., Lizarraga, M.A., Ros, E., 2006. Other relevant components of
nuts: phytosterols, folate and minerals. Br. J. Nutr. 96 (S2), S36–S44. https://doi.
org/10.1017/BJN20061862.
Sun, Q., Zhang, N., Wang, J., Zhang, H., Li, D., Shi, J., Li, R., Weeda, S., Zhao, B., Ren, S.,
Guo, Y.D., 2015. Melatonin promotes ripening and improves quality of tomato fruit
during postharvest life. J. Exp. Bot. 66 (3), 657–668. https://doi.org/10.1093/jxb/
eru332.
Tan, D.X., Manchester, L.C., Terron, M.P., Flores, L.J., Reiter, R.J., 2007. One molecule,
many derivatives: a never-ending interaction of melatonin with reactive oxygen and
nitrogen species? J. Pineal Res. 42 (1), 28–42. https://doi.org/10.1111/j.1600-
079X.2006.00407.x.
Tapia, M.I., Sánchez-Morgado, J.R., García-Parra, J., Ramírez, R., Hernández, T.,
González-Gómez, D., 2013. Comparative study of the nutritional and bioactive
Journal of Food Composition and Analysis 105 (2022) 104180
compounds content of four walnut (Juglans regia L.) cultivars. J. Food Compos. Anal.
31 (2), 232–237. https://doi.org/10.1016/j.jfca.2013.06.004.
Tijero, V., Muñoz, P., Munné-Bosch, S., 2019. Melatonin as an inhibitor of sweet cherries
ripening in orchard trees. Plant Physiol. Biochem. 140, 88–95. https://doi.org/
10.1016/j.plaphy.2019.05.007.
Tindall, A.M., Petersen, K.S., Lamendella, R., Shearer, G.C., Murray-Kolb, L.E.,
Proctor, D.N., Kris-Etherton, P.M., 2018. Tree nut consumption and adipose tissue
mass: mechanisms of action. Curr. Dev. Nutr. 2 (11), nzy069 https://doi.org/
10.1093/cdn/nzy069.
Van Tassel, D.L., Roberts, N., Lewy, A., O’Neill, S.D., 2001. Melatonin in plant organs.
J. Pineal Res. 31 (1), 8–15. https://doi.org/10.1034/j.1600-079X.2001.310102.x.
Wang, C., Yin, L.Y., Shi, X.Y., Xiao, H., Kang, K., Liu, X.Y., Zhan, J.C., Huang, W.D., 2016.
Effect of cultivar, temperature, and environmental conditions on the dynamic
change of melatonin in mulberry fruit development and wine fermentation. J. Food
Sci. 81 (4), M958–M967. https://doi.org/10.1111/1750-3841.13263.
Wang, S.Y., Shi, X.C., Wang, R., Wang, H.L., Liu, F., Laborda, P., 2020. Melatonin in fruit
production and postharvest preservation: a review. Food Chem., 126642 https://doi.
org/10.1016/j.foodchem.2020.126642.
Xu, L., Yue, Q., Xiang, G., Yao, Y., 2018. Melatonin promotes ripening of grape berry via
increasing the levels of ABA, H2O2, and particularly ethylene. Hortic. Res. 5 (1),
1–11. https://doi.org/10.1038/s41438-018-0045-y.
Yu, J., Ahmedna, M., Goktepe, I., Dai, J., 2006. Peanut skin procyanidins: composition
and antioxidant activities as affected by processing. J. Food Compos. Anal. 19 (4),
364–371. https://doi.org/10.1016/j.jfca.2005.08.003.
Zisapel, N., 2018. New perspectives on the role of melatonin in human sleep, circadian
rhythms and their regulation. Br. J. Pharmacol. 175 (16), 3190–3199. https://doi.
org/10.1111/bph.14116.