ABSTRACT
In the search for compounds with nutritional and functional value, seeds
from several Neltuma species were examined for lipid, protein and
phenolic components. Lipid contents ranged between 9.3 - 12.5 g/100 g
(seed DW), and showed MUFA and PUFA predominating largely. Lipids were
also a good source of tocopherols (170 – 603 mg total tocopherols/kg).
Proteins accounted for 14.7 - 35.5 g/100 g seed. Essential amino acids
(EAA) comprised 26.1 – 40.2 % of the total AA content. Based on the
EAA score, proteins from all the species analysed were deficient in
leucine, isoleucine and valine, but could meet requirements for
histidine, threonine, phenylalanine, tyrosine, and lysine. The
qualitative patterns of phenolic compounds were similar each other
(predominance of flavonoids, particularly apigenin derivatives). Keeping
in mind the examined chemical components, Neltuma seeds have
potential value as a source of healthy macronutrients and bioactive
compounds.
Keywords Amino acids, Lipid components, Phenolic compounds,Neltuma spp., Seeds
Abbreviations AA, amino acid; EAA, essential amino acid; DW,
dry weight; FA, fatty acid; MUFA, monounsaturated fatty acids; PUFA;
polyunsaturated fatty acids; TT, total tocopherol; TPC, total phenol
content.
*Corresponding author: romina.bodoira@mi.unc.edu.ar
Introduction
Cultivated legumes are widely consumed as food worldwide. They provide a
good source of protein and many other important nutritional and
functional components (Grela et al., 2017; Bennetau-Pelissero 2019).
Several wild legume species have also been investigated as a source of
nutrients for humans and livestock (Arinathan et al., 2003; Bhat and
Karim 2009). In many cases, they are present in agroforestry systems
where besides providing invaluable benefits such as ecosystem services,
can contribute sustainable food resources especially in rural
communities.
The species of the genus Neltuma (ex Prosopis , Fabaceae)
are important vegetation elements, particularly in arid and semiarid
regions of the world. Many of them produce abundant pods which, since
ancient times, have provided basic foodstuffs for man as well as forage
for wildlife and domestic herbivores. Most of research on composition
and nutritional value of Neltuma pods has focused on the mesocarp
components (Felker et al., 2013). Studies on seed components are largely
unaddressed. This could be due to the belief that the seeds have little
nutritional benefit. So, in some regions and countries Neltumafoodstuffs are obtained by using only the pods, and seeds are discarded.
Currently, there are few reports on seed chemical components ofNeltuma spp. Fatty acid and sterol compositions have been
investigated in N. alpataco and N. denudans from the
Patagonian region in Argentina (Mazzuca and Balzaretti 2003). By
analysing chemical and nutritional properties of different parts ofN. alba fruits, it has been found that the seeds contribute the
majority of the protein; in addition, they provide significant amounts
of total polyphenols (Sciammaro et al., 2016). Interestingly, protein
concentrates obtained from N. alba seeds have been proposed for
formulation of functional foods because of their good amino acid
profiles and important biological and functional properties (Cattaneo et
al., 2014).
In Argentina, several Neltuma species have become widespread
(Joseau et al., 2013). They can grow and fruit in marginal areas for
conventional crops, under conditions of very low water availability. Our
general interest is to contribute information to nutrient databanks of
underexploited plant resources. In the present study, fourNeltuma species were evaluated for unexplored seed components
with a focus on nutritional and functional compounds.
Materials and methods
Pods from Neltuma species (N. flexuosa , N. nigra ,N. chilensis , N. alba ) were obtained from trees of natural
populations in the north-western region of Córdoba province (Argentina).
The identity of the named species was previously authenticated by using
herbarium specimens from the botanical museum CORD (Argentina). From
each species, three pod samples (about 1 kg each) - each obtained from
at least five individual trees - were used for analytical
determinations. Pods were harvested at full maturity stage and, after
drying in darkness, the seeds were separated by hand and stored at -20
°C until use. Before proceeding to the different analyses, seeds were
ground to a fine powder using a universal cutting mill (Tecno Dalvo,
Argentina). Seed dry matter content was determined by oven drying at 80
ºC (AOCS 2009).
Lipid analyses
Lipid extraction for total lipid content determination was performed
using Soxhlet devices with n-hexane as solvent (AOCS 2009). Fatty acid
(FA) composition was evaluated - as methyl esters (FAME) - by gas
chromatography (GC) according to protocols for lipid sample preparation
and GC conditions previously reported (Cittadini et al., 2021). Identity
of FAME was confirmed by means of GC (Clarus 580, Perkin-Elmer, Shelton,
USA) - mass spectrometry (MS, Clarus SQ8S) analysis. Separations were
performed on a CP Wax 52 CB (Varian, Walnut Creek, USA) fused-silica
capillary column using helium (flow rate 1 mL/min) as carrier gas. The
GC oven temperature was initially maintained at 180 ºC (5 min) and then
increased at 2 ºC/min to 220 ºC. Both injector and detector temperatures
were set at 250 ºC. The FAME components were identified by mass spectra
matching using the Wiley mass spectra search library.
For tocopherol analysis, ground seed samples (20 g each) were extracted
with n-hexane (40 mL x three times) at room temperature (22 - 25º C).
The solvent was removed using a rotary vacuum evaporator at 40 ºC. The
lipid samples obtained were analyzed by high performance liquid
chromatography (HPLC, Perkin-Elmer, Shelton, USA) following analytical
methods used previously (Cittadini et al., 2021). Accurately-weighted
lipid samples (1 g each) were diluted with n-hexane to 10 mL. The
solutions were filtered through 0.45 μm pore filters. Aliquots of 20 mL
of the filtrated solutions were injected into a Supelcosil LC-NH2-NP
column (25 cm x 4.6 mm, Supelco, Bellefonte, USA). The mobile phase was
n-hexane/ethyl acetate (70/30 V/V) with a flow rate of 1 mL/min. UV
detection at 295 nm was performed. Individual tocopherols were
identified by comparing their retention times with those of authentic
standards (α-, γ- and δ-tocopherols, ICN Biomedicals, Costa Mesa, USA),
and were quantified by the external standard method. The linearity of
the response was verified by fitting to line results of each one
tocopherol individuals (eight standard solutions with known
concentrations) covering the concentration range from 2 to 800 ppm, with
linearity regression coefficients (R2) varying between
0.98 and 0.99.
Protein analyses
The Kjeldahl method (total nitrogen x 6.25) was used to quantify the
seed total protein content (AOAC 2009). For amino acid (AA) analysis,
defatted seed samples (500 mg) were hydrolysed following the standard
AOCS (2009) procedure. Hydrolysed samples were filtered using 0.45 μm
membrane filters and then submitted to pre-column derivatization. For
this purpose, accurately-weighted hydrolysed samples were placed in
screw-capped tubes and re-suspended in 500 µL HCl 0.1 M plus 1.8 mL
borate buffer, 0.75 mL methanol and 15 µL diethyl
ethoxymethylenemalonate (Sigma-Aldrich, St. Louis, USA). The tubes were
kept at constant temperature (25 °C) for 24 h. The obtained N-[2,2-
bis (ethoxycarbonyl) vinyl] AA derivatives were analysed by
reversed-phase HPLC (Perkin-Elmer, Shelton, USA) using a C18 column
(Zorbax Eclipse Plus C18, Agilent Technologies, Santa Clara, USA)
thermostated at 40 ºC. Solvents (A, sodium acetate buffer, 25 mM pH 6;
B, methanol/acetonitrile/water, 45/45/10 v/v) were used at a flow rate
of 0.9 mL/min, according to the following gradient: 100 % A for the
first 18 min, 57 % to 100 % B for the next 5 min, 0 % B from 23.1 to
30 min. Detection of AA derivatives was performed at 280 nm.
Identification was carried out by using authentic AA standards
(Sigma-Aldrich, St. Louis, USA), and quantification by means of the
external standard method. The linearity of the response was verified by
fitting to line results of each one amino acid individuals (five
standard solutions with known concentrations). Calibration curves from
the tested amino acid standards showed linearity regression coefficients
(R2) ranging between 0.95 and 0.99. The contents of
the various identified essential AA (EAA) were compared with the
FAO/WHO/UNU (2002) reference pattern. Individual EAA scores were
determined according to FAO/WHO/UNU using the formula: AA score = mg of
AA in 1 g test protein / mg of AA in requirement pattern. Limiting AA
were identified as those having a ratio lesser than 1.
Phenolic compound analyses
Phenolic compounds were obtained from 10 g of ground seed samples
previously defatted at room temperature (n-hexane 50 mL x three times).
Defatted samples (5 g) were extracted thrice with methanol (80 % V/V,
50 mL every time). The extracts were combined and washed twice (50 mL
every time) with n-hexane. The hydro-alcoholic phase was recovered,
filtrated through 0.45 μm pore filters, concentrated under vacuum below
50 ºC to a final volume of 1 mL, and further used for both total phenol
content (TPC) and HPLC-ESI-MS/MS analyses according to procedures
reported elsewhere (Bodoira et al., 2022).
The composition of phenolic extracts was analyzed by means of
HPLC-ESI-MS/MS using an Agilent 1200 HPLC Series system (Agilent
Technologies, Santa Clara, USA). The chromatographic separations were
achieved on a Kromasil (Bohus, Sweden) reversed-phase C18 column (5 µm,
250 mm x 4.60 mm i.d.) under analytical conditions used previously
(Bodoira et al., 2022). The MS detector was programmed to perform a
MS/MS scan of the most abundant ions, using collision energy of 13.0 eV.
The identification of phenolic compounds was based on their retention
times (Rt), elution order, and comparison of UV–Vis spectra and mass
spectrometry data reported in the literature (Picariello et al., 2017;
Singh et al., 2017; Cittadini et al., 2021). The Compass version 3.1
software and DataAnalysis version 4.1 software were used for data
acquisition and processing, respectively.
Statistical analysis
Statistical differences between species in the analysed chemical
parameters were estimated from ANOVA test at the 5 % level (P ≤ 0.05)
of significance. Whenever ANOVA indicated a significant difference, a
pair-wise comparison of means by least significant difference (LSD) was
carried out. Statistical analyses were performed using the InfoStat
program (InfoStat version 2018, National University of Córdoba,
Argentina).
Results and discussion
Lipid content and composition
The seeds of the analysed Neltuma species had similar lipid
content (11.2 % in average) (Table 1). Overall, it results higher than
those found in Prosopis farcta (3.2 %, Lajnet et al., 2015) andN. alpataco (7.0 %, Mazzuca and Balzaretti, 2003), and similar
to those reported in N. caldenia (10.7 %) and N.
argentina (14.7 %) (Lamarque et al., 1994). By way of comparison with
other wild edible legumes, the lipid contents we found compare well with
those from seeds of Canavalia spp. (1.4 – 12.1 %),Cassia spp. (3.8 – 7.0 %) and Mucuna spp. (6.7 – 14.4
%), as well as with data from several tree legume species recently
investigated which showed seed oil yields ranging from 2.1 to 14.9 %
(Grygier et al., 2023). On the other hand, seed lipid contents fromNeltuma result meaningfully higher than those from some
cultivated legumes, such as peas (Pisum sativum ), lentils
(Lens culinaris ) and beans (Phaseolus vulgaris ) which
usually contain about 2 g lipid per 100 g seed (Padhi et al., 2017).
Fatty acid (FA) composition is summarized in Table 1. In accordance with
data from wild and cultivated legumes worldwide, palmitic, oleic and
linoleic acids were present as major FAs (Arinathan et al., 2003; Bhat
and Karim, 2009; Padhi et al., 2017; Grygier et al., 2023). AllNeltuma species evaluated presented the same FA components but
with some differences in concentrations, especially regarding
unsaturated FA levels. Neltuma alba and N. chilensis seeds
had very similar composition; with the exception of palmitoleic and
arachidic acids, no significant differences were found between their FA
profiles. Meanwhile, N. nigra and N. flexuosa seeds showed
significantly higher amounts of oleic and linoleic acids, respectively,
whereas the remaining FA percentages were practically unchanged with
respect to the two species first named. In general, the concentrations
of the main FAs from the Neltuma species we studied are similar
to those found in other species of the genus. As a difference, it could
mention lower linoleic acid percentages in N. alba , N.
chilensis and N. nigra (about 41, 39 and 32 %, respectively)
with respect to those observed in N. argentina (51.1 %) andN. alpataco (56.1 %) seed oils (Lamarque et al., 1994; Mazzuca
and Balzaretti 2003).
Based on the lipid fraction, a comparison of the FA composition between
the Neltuma species studied and some cultivated legumes reveals
that the former have lesser PUFA (linoleic and linolenic acids) contents
than the later (Padhi et al., 2017). However, when FA concentrations are
calculated on the basis of dry seed weight, the PUFA content inNeltuma accounts for 4.0 – 6.3 g/100 g seed, which represent
amounts substantially higher than those present in varieties of peas,
lentils and beans (1.05, 0.86 and 1.34 g PUFA/100 g seed, respectively),
and similar to those found in chickpeas (5.03 g PUFA/100 g seed) (Padhi
et al., 2017).
To the best of our knowledge, there are no published studies on
tocopherol composition of Neltuma spp. Lipids from the studied
species contained 170.9 – 603.5 mg/kg of total tocopherols (TT) which
were composed mostly of α-tocopherol (167.5 – 544.6 mg/kg lipid) and
minor amounts of γ-tocopherol (3.3 – 58.9 mg/kg lipid) (Table 1). On a
DW basis of whole seeds, the range for tocopherol contents from all the
species evaluated (from 2.35 mg TT/100 g in N. chilensis seeds to
7.21 mg TT/100 g in N. flexuosa seeds) is similar to that
observed in edible pulses (2.2 – 5.7 mg TT/100 g) such as beans,
lentils and peas (Padhi et al., 2017).
Protein content and composition
Protein content from Neltuma spp. accounted for 14.7 – 35.5 %
of the seed dry matter content (Table 2); the highest one was observed
in N. flexuosa . The values recorded are in line with those
reported in other species of the genus such as P. argentina (26.9
%) and P. caldenia (32.2 %) (Lamarque et al., 1994). Moreover,
they resemble the values found in some tribal pulses (12.9 – 24.7 %,
Arinathan et al., 2003), and in cultivated peas (Lathyrus spp.)
and lentils (Vicia spp.) (17.7 – 25.6 % and 20.1 – 32.0 %,
respectively) (Pastor-Cavada et al., 2011 a, b).
Amino acid (AA) composition is shown in Table 2. Except for isoleucine -
which was below the detection limit we used - and leucine - not
quantified in N. nigra seeds -, the species analysed showed the
protein AA profiles usually reported in cultivated legume species
(Pastor-Cavada et al., 2011 a, b; Grela et al., 2017; Bennetau-Pelissero
2019). The percentage of essential AA (EAA), which is considered an
indicator of protein nutritional quality, ranged between 26 – 40 % of
the total AA concentration. The values found in N. alba andN. chilensis were similar to those present in whole-wheat flour
(about 27 %), which is a widespread and valuable source of proteins.
Meanwhile, the values from N. nigra and N. flexuosacompared well with those of cultivated beans such as Lathyrus andVicia species (39 – 42 %) (Pastor-Cavada et al., 2011 a, b).
So far, there is very little information on EAA contents inNeltuma seeds. A study on seed protein isolates from N.
alba showed all EAA ( Cattaneo et al., 2014), in quantities above the
established requirements for human adults ( FAO/UNU/WHO 2002). Besides
the absence of isoleucine, proteins from all the species we studied were
deficient in leucine and valine, but could meet requirements for
histidine, threonine, phenylalanine + tyrosine, and lysine. Seed
proteins from N. alba and N. flexuosa could also satisfy
methionine requirements (Table 2). Worthy of mention are the relatively
high amounts of histidine, threonine and lysine, particularly inN. nigra and N. flexuosa (12.7 – 13.2, 6.84 – 9.15, and
10.5 – 11.2 g/100 g protein, respectively). These contents are
substantially higher than those present in several Lathyrus andVicia species which showed histidine, threonine and lysine
contents ranging between 2.2 – 3.3, 4.0 – 6.2 and 6.4 – 8.0 g/100 g
protein, respectively (Pastor-Cavada et al., 2011 a, b). While histidine
has been associated with factors that improve the so-called metabolic
syndrome in humans (Moro et al., 2020), threonine plays a vital role in
the modulation of nutritional metabolism, macromolecular biosynthesis,
and gut homeostasis (Tang et al., 2021). The requirement for lysine is
also a matter of interest as it is limiting in cereals, especially
wheat. So, the high contents of these AA in N. nigra and N.
flexuosa highlight the potential of their seed meals to improve the AA
profile of cereal flour-based foods.
In line with findings reported from several seed proteins - including
those of legumes and cereals - the most abundant non-EAA were those from
the two pairs namely aspartic acid + asparagine and glutamic acid +
glutamine. Interestingly, the aminograms also revealed higher amounts of
arginine than those usually present in edible pulses (Margier et al.,
2018). Arginine is considered a semi-essential AA involved in multiple
metabolic processes and biological functions (Morris 2006).
Phenolic compounds
Firstly, it is worth noting the wide variation between theNeltuma species in the total content of seed phenolic compounds
(Table 3); N. nigra presented the highest concentration whileN. alba showed the lowest abundance. In cultivated legume seeds,
the total phenolic content may also vary widely (Singh et al., 2017). By
way of comparison, total phenolics in N. nigra (about 96 mg/100 g
seed) may account for a similar amount to those found in cowpeas (about
100 mg/100 g) and pigeon pea seeds (78 – 120 mg/100 g), but occur in
lower concentration than reported in chickpea and lentil varieties
(Singh et al., 2017).
A total of 39 phenolic compounds were detected in the examinedNeltuma seeds (Table 3). With the exception of compound 14, which
was assigned tentatively as ferulic acid derivative, the remaining
components were identified as flavonoids and structurally related
substances. Apigenin derivatives were found to show the highest contents
in all Neltuma species we studied. Apigenin 8-C and 6-C
glucosides – commonly named vitexin and isovitexin – were by far the
most abundant compounds reaching concentrations near to 40 mg/100 g (DW)
in N. nigra and N. flexuosa seeds. Such concentrations are
within the ranges found in some cultivated legumes such as the mung bean
(17 – 62.4 and 22.6 – 73.6 mg/100 g, for vitexin and isovitexin,
respectively), which is known as a major source of polyphenols (Hou et
al., 2019). As a matter of interest, the protective effects of these
compounds in several oxidative stress-related diseases are widely
recognised, and have been reviewed in a recent published review (Babaei
et al., 2020).
Another relatively abundant polyphenolic compounds in Neltumaseeds were the apigenin 6-C-glc-8-C-arab and 6-C-arab-8-C-glc isomers,
which are usually known as schaftoside and isoschaftoside, respectively.
They reached concentrations close to 4 mg/100 g in P. nigraseeds. All Neltuma spp. examined also contained minor amounts of
further glycosylated derivatives (hexosides) of both compounds (peaks
number 3 and 4). Schaftoside and isoschaftoside have been found in high
amounts (41 - 61 and 337 - 518 mg/100 g, respectively) in the seed germ
flour from some Neltuma species and in Ceratoniasiliqua seeds (Picariello et al., 2017). Interestingly, all these
compounds have been described as potent α-glucosidase inhibitors (Zhao
et al., 2019) thus suggesting that seeds from all the named plant
materials could be used as a source of bio-actives contributing to
modulate carbohydrate digestion in humans.
Peaks number 6 and 7 were identified as apigenin 6,8-C-di-glucosides.
The occurrence of two peaks with identical [M-H]-signal and major fragment ions suggests the possibility of stereoisomers
already described and sometime named as vicenin (Picariello et al.,
2017). Both peaks were relatively abundant in N. flexuosa but
scarce in the other species. Nevertheless, the amounts found in N.
flexuosa seeds (about 1.2 mg/100 g of both substances) were lesser to
those reported in the seed germ flour (18 - 51 mg/100 g) from otherNeltuma species (Picariello et al., 2017).
Kaempferol aglycone was almost absent in the materials studied but some
derivatives - particularly kaempferol hexoside - were abundant inN. nigra seeds. The presence of these compounds is worthy of
mention due to their wide range of bioactive and pharmacological
properties including antioxidant, anti-cancer, anti-inflammatory, and
cardioprotective effects (Imran et al., 2019). Other common flavonoids -
such as luteolin, quercetin, chrysin, chrysoeriol and isorhamnetin -
were generally present - mostly as glycosylated forms – in the examinedNeltuma seeds, but always in small quantities.
Conclusions
Seeds from the studied Neltuma spp. presented valuable lipid
contents showing a well-balanced fatty acid composition (low percentage
of saturated fatty acids, moderate or high MUFA and PUFA concentrations)
and good amount of tocopherols. Proteins accounted for about 15 - 35 %
of dry seeds, and included 26 – 40 % of essential amino acids. Based
on their AA scores, they were deficient in leucine, isoleucine and
valine. Proteins from N. nigra and N. flexuosa presented
meaningful quantities of the EAA histidine, threonine and lysine. In
general, the qualitative patterns of phenolic compounds were similar
each other (predominance of apigenin derivatives), although they
differed markedly in the amounts of the individual components. Overall,
seeds from the examined Neltuma species represent an undervalued
source of healthy macronutrients and functional components. Moreover, as
a matter of interest, these species can produce fertile hybrids (Joseau
et al., 2013), so they may be a source of great genetic diversity. This
in turn opens up the possibility of searching for interspecific hybrids
with improved composition of target seed metabolites.
Conflict of interest The authors have declared no conflict of
interests.
Compliance with ethics requirements This article does not
contain any studies with human and animal subjects.
Funding This research was financed with funds from SECyT
(Secretaría de Ciencia y Tecnología de la Universidad Nacional de
Córdoba – Argentina).