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CHAPTER: 3.3
Saponins
*Corresponding author: Jacopo TROISI [email protected]
Authors:
J. TROISIa*, R. DI FIOREa, C. PULVENTOb, R. D’ANDRIAb, ANTONIO VEGA-GÁLVEZc, MARGARITA
MIRANDAc, ENRIQUE A. MARTÍNEZd , A. LAVINI b
Laboratorio Chimico Merceologico, Az. Spec. CCIAA, Corso Meridionale 58, I-80134 Napoli, Italy.
CNR – Institute for Agricultural and Forest Mediterranean System (ISAFoM)), Ercolano (NA), Italy
c
Universidad de La Serena, Facultad de Ingeniería, Av. Raúl Bitrán s/n, Box 599, La Serena, Chile.
d
Centro de Estudios Avanzados en Zonas Áridas, CEAZA, Avda. Raúl Bitrán s/n, La Serena, Chile.
a
b
Abstract
The term saponin comes from the Latin word sapo,
meaning “soap”, reflecting a readiness to form stable
soap-like foams in aqueous solutions. The biological
role of saponins is not completely understood, but
they are generally considered to be part of a plant’s
defence system against pathogens and herbivores,
particularly because of their bitter flavour. Saponins
comprise aglycones and sugar, each representing
about 50% of the total weight of the molecule. In
quinoa, saponins are a complex mixture of triterpene glycosides that derive from seven aglycones:
oleanolic acid, hederagenin, phytolaccagenic acid,
serjanic acid, 3β-hydroxy-23-oxo-olean-12-en-28oic acid, 3β-hydroxy-27-oxo-olean-12-en-28-oic
acid and 3β,23α,30β-trihydroxy-olean-12-en-28-oic
acid, while the most common sugars are arabinose,
glucose and galactose. Saponins are traditionally
considered very antinutritional because of their
haemolytic activity, and there is therefore a longstanding controversy about their functions in food.
It is believed that saponins can form complexes
with membrane sterols of the erythrocyte, causing
an increase in permeability and a subsequent loss
of haemoglobin. However, recent extensive studies
of the biological activity of saponins in vitro and in
vivo have identified associations with several health
benefits, including anti-inflammatory, anticarcinogenic, antibacterial, antifungal and antiviral effects.
Saponins are also of interest as valuable adjuvants
and the first saponin-based vaccines have been introduced commercially. Traditionally, quinoa seeds
are either abraded mechanically to remove the
bran – which is where the saponins are predominantly located – or washed with water to remove
bitterness prior to use. During washing, valuable
nutrients are lost and the chemical composition and
amino acid profiles of quinoa seeds can be altered.
Following treatment, the level of saponin content
in to-be-consumed quinoa seeds remains a major
concern in terms of bitterness and possible negative biological effects. A mathematical model based
on Fick’s second law has been created to optimize
the leaching process of saponins from quinoa seeds
during washing with water.
Many studies have focused on the effects of agronomic variables (e.g. irrigation and salinity) on the
saponin profiles of quinoa. It has been observed
that saponins decrease in samples that have been
exposed to drought and saline regimes – suggesting
that irrigation and salinity may regulate the saponin
content in quinoa and affect its nutritional and industrial values.
Studies are underway to evaluate and compare
the saponin content in seven varieties of quinoa
grown in Italy and six varieties grown in Chile under rainfed or low irrigation conditions. Seeds from
the more arid or stressing Chilean localities have a
higher saponin content.
CHAPTER: 3.3 saponis
268
1. Introduction
1.1 Saponin chemistry
Saponins are compounds found in many plants
(Sparg et al., 2004) and they have the distinctive feature of forming foam. The name probably
comes from the plant Saponaria whose roots were
historically used to make soap (Latin sapo = soap)
(Augustin et al., 2011). Chemically, they are glycosides with a polycyclic aglycone (glycoside-free portion), which may occur in the form of a steroid or
a triterpenoid choline bound via the C3 carbon by
means of an ethereal bond to a side sugar chain.
The aglycone is commonly referred to as sapogenin,
while the subset of steroidal saponins is commonly
referred to as sarapogenin. Saponins are amphipathic because of their fat-soluble aglycone function
and their water-soluble saccharide chain. This char-
acteristic is the basis of the ability to form foam.
Saponins are perceived as bitter, and this reduces
the organoleptic characteristics and the palatability
of any products rich in them. Only a few (usually
those with a triterpenoic aglycone) have a nice flavour, reminiscent of liquorice root.
1.2 Saponin Biosynthesis
Evidence that the overexpression of squalene synthase may induce an up-regulation of saponins and
phytosterols (Lee et al., 2004) suggests that this
enzyme is involved in the branching of biosynthetic
pathways leading to the synthesis of phytosterols
and saponins. This observation led to the theory
(now consolidated) that saponins derive from the
same anabolic process that leads to the formation
of phytosterols. All terpenoids derive from condensation of 5-carbon building blocks designated
IPP (3-isopentenyl pyrophosphate) and DMAPP
(dimethylallyl pyrophosphate). In plants, IPP and
DMAPP drift from condensation of acetyl-CoA in
the mevalonate pathway or from pyruvate and
phosphoglyceraldehyde. Terpenoid biosynthesis in
plants is extensively compartmentalized: steroids,
triterpenes and saponins are mainly synthesized in
the cytosol utilizing IPP from the mevalonate pathway.
Flores-Sanchez et al. (2002) conducted experiments
in which the activity of HMG-CoA reductase – a key
enzyme in mevalonate and squalene synthesis –
was inhibited, and this led to a reduction of phytosterols and of ursolic/oleanolic acid biosynthesis,
confirming the hypothesis that the biosynthetic
pathway of saponins is linked to that of plant sterols
by means of squalene synthesis.
Figure 1: Summarizes the seven aglycones identified so
far in the different parts of quinoa (flowers, fruits, seedcoats and seeds) (Kuljanabhagavad et al., 2008). These
structures have been obtained by means of extensive
characterizations in NMR (nuclear magnetic resonance)
and mass spectrometry. Most of the variability is generated by the saccharide side chains – indeed, the seven
aglycones give birth to more than 20 saponins (Table 1).
IPP and DMAPP undergo condensation to the
10-carbon intermediate GPP (geranyl pyrophosphate), and the addition of a second IPP unit leads
to FPP (farnesyl pyrophosphate, C15), the common
precursor of the vast array of sesquiterpenes produced by plants. Linkage of two FPP units leads to
formation of squalene (C30). This is then epoxygenated to 2,3-oxidosqualene (C30), considered the
last common precursor of triterpenoid saponins,
phytosterols and steroidal saponins. The steps at
which steroidal saponin and phytosterol biosynthesis diverge have not been elucidated, although Kalinoswska et al. (2005) suggest that cholesterol is a
precursor of steroidal saponins.
CHAPTER: 3.3 saponis
269
Table 1: Saponins derived from the 7 aglycones found in quinoa.
Compound
Sugar side chain
Aglycone
1
I
2
II
3
III
4
β-D-Glc(1→3)-α-L-Ara
IV
5
V
6
VI
7
VII
8
III
9
α-L-Ara
V
10
VI
11
III
12
β-D-GlcA
IV
13
VI
14
III
15
β-D-Glc(1→2)-β-D-Glc(1→3)-α-L-Ara
16
17
18
19
20
IV
V
β-D-Xyl(1→3)-β-D-GlcA
β-D-Glc(1→3)-β-D-Gal
β-D-Glc(1→4)-β-D-Glc(1→4)-β-D-Glc
The first committed step in the biosynthesis of triterpenoid saponins and phytosterols is the cyclization
of 2,3-oxidosqualene. During this process, internal
bonds are introduced into the oxidosqualene backbone, resulting in the formation of predominantly
polycyclic molecules containing varying numbers of
5- and 6-membered rings. The high number of possibilities for establishing different internal linkages
during cyclization gives rise to a vast array of diverse
structures, and over 100 different triterpene skeletons have been found in nature. However, from
this vast range, only a limited number of possible
cyclization products appear to be utilized in saponin
biosynthesis.
Following the formation of basal sapogenin backbone structures, these common precursors usually
undergo various modifications prior to glycosylation. The most common sapogenin modifications
IV
V
VI
V
are small functional groups, such as hydroxyl-, keto,
aldehyde - and carboxyl-moieties at various positions of the backbone.
Glycosylation patterns of saponins are often considered crucial for their biological activities. Typical
triterpenoid saponin glycosylation patterns consist
of oligomeric sugar chains of 2–5 monosaccharide
units, most often linked at positions C3 and/or C28.
Less often, 1–2 monosaccharide units have been
reported to occur at positions C4, C16, C20, C21,
C22 and/or C23. Glucose, galactose, glucoronic
acid, rhamnose, xylose and arabinose are the most
abundant hexoses and pentoses in the saccharide
chains. Saponin glycosylation presumably involves
sequential activity of different enzymes belonging
to the multigene family of uridin diphosphate glycosyltransferases (UGTs).
CHAPTER: 3.3 saponis
270
1.3
Biological role
Saponins have different biochemical activities.
Francis et al. (2002) reported, among others, strong
haemolytic, antimicrobial, fungicidal, allelopathic,
insecticidal and molluscicidal activity, while VegaGálvez et al. (2010) reported their effects as a vaccine coadjuvant. Therefore, although the true biological significance of saponins in quinoa still needs
to be fully determined, the current line of thought
is that they are part of the plant’s apparatus to defend off predators.
1.3.1 Haemolytic activity
One of the systems used to probe the presence of
saponins in a plant extract or in a drug is based on
incubation of the extract with blood red cells and
verification of the degree of haemolysis of the sample. The ability of saponins to break the membrane
of the erythrocytes is linked to their ability to bind
membrane sterols (Khalil et al., 1994). When the
membrane bursts, there is an increase in permeability and a loss of haemoglobin. Baumann et al.
(2000) have investigated the effect of saponins on
the membrane structure through haemolysis of human erythrocytes. The findings show that saponinlysed erythrocytes do not reseal, indicating that
saponin-induced damage to the lipid bilayer is irreversible. The level of haemolytic activity has been
attributed to the type of aglycone and to the presence of the sugar side chains (Wang et al., 2007).
1.3.2 Anti-inflammatory activity
In the carrageenan-induced oedema assay, many
saponins isolated from plant sources produce an
inhibition of inflammation. Kim et al. (1999) suggested that the anti-inflammatory activity of these
saponins is related to anticomplementary action through the classical inflammation pathway.
Oleanolic acid and ginsenoside Ro show the highest
anticomplementary activity.
1.3.3 Antifungal/antiyeast activity
Triterpenoid saponins from the seeds of Chenopodium quinoa Willd. (Chenopodiaceae) have been
reported to have antifungal activity (Woldemichael
and Wink, 2001). A study by Bader et al. (2000)
revealed that the antifungal activity of saponins
against different Candida albicans strains can be
influenced by variation of the etherglycosidically
bonded carbohydrate units and the acylglycosidically bonded oligosaccharide at C-28 of the aglycone. However, only crude saponin mixture inhibits
the growth of Candida albicans. Pure compounds
show little or no activity, which suggests a possible
synergistic effect between these saponins.
1.3.4 Antibacterial/antimicrobial activity
Saponins have also been reported to have antimicrobial activity (Killeen et al., 1998). Alcohol soluble
saponins have antimicrobial activity towards both
prokaryotic and eukaryotic organisms, but only at
low cell densities, and they do not inhibit microbial
growth of dense populations.
1.3.5 Cytotoxicity and antitumour activity
Numerous reports highlight the highly cytotoxic
properties of many saponins (Musende et al., 2009;
Man et al., 2010). In particular, oleananes show
an antitumour effect in various pathways, including anticancer, antimetastasis, immunostimulation
and chemoprevention. The detailed mechanisms
are complex but involve dephosphorylate Stat3 in a
variety of human tumour cell lines and lead to a decrease in the transcriptional activity of Stat3, which
regulates proteins such as c-myc, cyclin D1, Bcl2,
survivin and VEGF. Moreover, several immunostimulating activities, such as induced growth of human
T lymphocytes, promoting apoptosis and triggering
autophagic cell death have been reported. They
decrease respiratory activity and induced ATP efflux after inhibition of the voltage-dependent anion
channel in the outer mitochondrial membrane.
2.
Saponin removal
Saponins are generally bitter, so before consumption they must to be eliminated from quinoa. Traditionally, quinoa seeds are either mechanically
abraded to remove the bran, where the saponins
are predominantly located, or washed with water to
remove bitterness prior to use. Wright et al. (2002)
report that during this washing process, valuable
nutrients are also lost and the chemical composition and amino acid profiles in quinoa seeds may be
altered. The final level of saponin content in to-beconsumed quinoa seeds remains a major concern
in terms of its bitterness and possible negative biological effects.
CHAPTER: 3.3 saponis
2.1
2.2
Kinetic
The removal of saponins from quinoa seeds during
washing can be described according to the rules
governing solid–liquid extraction and by applying
mathematical models generally used to evaluate
process kinetics.
The total saponin concentration inside quinoa
seeds rapidly tends towards an asymptotic value
following an initial leaching. Fuentes et al. (2013)
show that this asymptomatic value decreases as the
washing temperature increases.
Saponin ratio (SR) – defined according to equation
1 – is the most commonly used parameter for modelling the saponin leaching kinetics of quinoa seeds.
SR represents a dimensionless concentration used
to study the leaching kinetics, supposing a mechanism of diffusion inside the solid and negligible external mass transfer under conditions of intensive
stirring.
SR=
Xst-Xse
Xs0-Xse
Eq.1
where Xst is the saponin content in real time
(g/100gdm), and Xs0 and Xse are the initial and residual saponin contents.
Table 2 represents the most important model
adopted for modelling SR in saponin removal.
271
Uses of Saponins
Saponins are used in industry as additives in foods
and cosmetics. They can also be used in other industrial applications (Yang et al., 2010; Chen et al.,
2010; Price et al., 1987; Hostettmann and Marston,
1995) as, for example, preservatives, flavour modifiers, detergents (due to their chemical properties
and abilities as foaming agents) and agents for cholesterol removal from dairy products.
Notably, saponins can also activate the mammalian immune system, arousing significant interest
in their potential as vaccine adjuvants (Sun et al.,
2009). Their unique capacity to stimulate both the
Th1 immune response and the production of cytotoxic T-lymphocytes (CTLs) against exogenous antigens makes them ideal for use in subunit vaccines
and vaccines directed against intracellular pathogens, as well as in therapeutic cancer vaccines.
3.
Quinoa saponin content
3.1
Analytical methods
Several analytical methods have been developed for
the determination of saponins from various matrices, including quinoa seeds. The simplest methods
are used to detect typical saponin features, such as
their ability to form foam or their haemolytic ability. The most commonly used methods, however,
are chromatographic. Both liquid chromatography
(with detection by mass spectrometry, DAD and
Table 2: Mathematical models selected to describe saponin leaching kinetics
Model
Equation
Reference
Midilli–Kuçuk
Vega-Gàlvez et al.
(2011)
Weibull
Corzo et al. (2008)
Logarithmic
Akpinar (2006)
Henderson–Pabis
(modified)
Sacilik & Elicin (2006)
Two terms
Page (modified)
Lahsasni et al. (2004)
Tog˘ rul& Pehlivan
(2003)
CHAPTER: 3.3 saponis
272
ELSD), and gas chromatography (with detection by
mass spectrometry and FID) have been employed.
Gas chromatography has been widely used, although providing for a longer extraction protocol
and a delicate silanization reaction. The first studies
to include determination by gas chromatography
were those by Ridout et al. (1991) and Price et al.
(1986). In gas chromatography, saponins are generally extracted after acid hydrolysis of the degrased
sample with a polar solvent; the extract after silanization is analysed with non-polar or slightly polar
columns and eluted at high temperatures. The analysis in HPLC, on the other hand, entails a simpler
preparation consisting of extraction with alcohols
and purification with a C18 SPE. Separation is usually achieved with C18 stationary phases and elutions
in water-acetonitrile gradient, both for photometric
detection (DAD, ELSD) and in mass spectrometry.
3.2 Saponin evaluation in Chilean quinoa ecotypes
3.2.1. Ecotypes present in Chilean quinoa agro-
ecological regions
Five quinoa ecotypes are described for the Andean
region. They come from the Inter-Andean valleys
of Colombia, Ecuador and Peru, the Altiplano of
Peru and Bolivia, Yunga in the Bolivian subtropical
forest, Salare (salt flats) in Bolivia, Chile and Argentina, and the Coastal (lowlands) or sea level areas of
Chile. Their origins and possible expansion routes
have been reviewed by Fuentes et al. (2012). In
Chile, just two of the five ecotypes have been found
(Salare and Coastal). However, within these two
ecotypes many landraces or local farmers’ varieties
exist in the country. In the Altiplano (highlands) at
4 000 m asl (19°S), farmers hold at least 12 of these
landraces (Alfonso, 2008; Alfonso and Bazile, 2009),
known by the local Aymara people as, for example,
‘Pandela’ (red seeds), ‘Jankú’ (white seeds), ‘Churi’
(yellow seeds), ‘Chullpe’ (brown seeds), ‘Khánchi’
(dark pink seeds) and ‘Chále’ (mixed colours). In
central (34°S) and southern (39°S) Chile, the landraces appear less abundant because there is less
diversity of seed colour, as most are whitish, yellowish, beige and grey, the latter being more abundant
at southern latitudes (39°S), as is also observed in
seed bank collections used for testing comparative
yields (Martínez et al., 2007).
Of these three regions, the climatic conditions are
more stressful in the high Andes of northern Chile
where annual rainfall is 100–200 mm (Lanino,
2006), while in central and southern Chile, it is over
400 mm (Miranda et al. 2013).
3.2.2 Saponin content
The total saponin content evaluated in whole seeds
of Chilean landraces and in one hybrid variety (‘Regalona’) is over 1%. They are, therefore, all bitter
(i.e. saponins > 0.11%) but with significant variation among them. Unexpectedly, high Andes Salare landraces do not always contain higher values
of saponins (2%). Those from central Chile have the
highest values, reaching as much as 4% (Miranda et
al., 2012). When seeds are sown in a different locality, particularly cultivated under the drier conditions of arid Chile (at 30°S with no rainfall between
October and May), harvested seeds increased their
saponin content, at least for the ‘Regalona’ hybrid,
from 2.2% to 3.2%. This phenomenon, however, is
not observed for another landrace from Villarrica in
southern Chile. The latter maintains a saponin content of 2.11–2.38% when cultivated in arid northern Chile (Miranda et al., 2013). The higher saponin
content in landraces from central Chile might be due
to the particular stressing conditions of high salinity in some coastal soils. These soils are sometimes
naturally irrigated in the winter with brackish waters
from the neighbouring rivers influenced by the high
tides of the Pacific Ocean (Orsini et al., 2011).
3.2.3 Conclusions
1. Saponin content has to date been studied in
seeds from Chilean landraces of quinoa belonging to the Salare and Coastal Andean ecotypes.
Their saponin content is high (> 2%), compared
with some sweet quinoas of the Altiplano (<
0.11%).
2. Unexpectedly, saponin content is higher in
coastal landraces from central Chile
3. The saponin content of some quinoa seeds
changes when grown under different conditions, normally increasing in a more stressing
climate (drought).
3.3 Italian research activity
From 2006, different field trials have been performed at ISAFoM-CNR to test quinoa. The strategic
objectives of these studies have been: to evaluate
CHAPTER: 3.3 saponis
Table 3: Saponin content (mg/100 gdm) in the two accessions
Accession
Total saponin
Oleanolic ac. Hederagenin Phytolaccagenin
% of total saponin
mg 100 g of DW
238.9 ± 10.87
78.2
16.7
5.1
213.8 ± 7.52
76.3
18.9
4.8
329.0 ± 6.78
85.3
10
4.7
-1
KVapril
KVmay
RB
the quantitative and qualitative responses of quinoa accessions under combined abiotic stresses
(salt and drought stress) and their adaptability in
the Mediterranean environment of southern Italy
(see Chapter 6.3); to improve food production by
introducing quinoa as a possible alternative crop for
this area (potentially high value food cash crops);
and to verify the opportunities for use of quinoa
seeds, flours and derivatives in product lines for
children and for people with coeliac disease, with
potentially interesting growth prospects in specialized sectors.
At the experimental station of the National Research Council (CNR), Institute for Agricultural and
Forest Mediterranean Systems (ISAFoM) in Vitulazio
(CE) (14°50’E, 40°07’N, 25 m asl), a 2-year (2006–
07) field trial was carried out to compare two quinoa genotypes: ‘Titicaca’ (‘KVLQ52’) and ‘Regalona
Baer’ (‘RB’) under rainfed conditions (Pulvento et
al., 2010). Comparison was also made between two
sowing dates (April and May) for ‘KVLQ52’ (‘KV’april
and ‘KV’may). In this period, quinoa was studied within the project “CO.Al.Ta. II” (Alternative Crops to Tobacco), set up by the European Community (CE), to
explore the possibilities of diversification of Italy’s
traditional tobacco-growing areas and to evaluate
seed quality, and in particular saponin content, in
collaboration with the Department of Food Technology (DISTAAM) of the University of Molise.
Results show that April is the best sowing time for
quinoa in the Mediterranean region (Table 2). Of
the two genotypes, ‘RB’ records better growth and
productivity, apparently being more tolerant to abiotic stress (high temperatures associated with water stress).
The study includes quantitative/qualitative assessment of saponins. Gas chromatography analysis
shows that the two varieties of quinoa are in an
intermediate position between “sweet” and “bitter” genotypes. In particular, the total saponin content of 238.9 and 213.8 mg/100 gdm for genotype
‘KV’april (sown in April) and ‘KV’may (sown in May),
respectively, was obtained. For genotype ‘RB’, the
saponin content is 328 mg/100 gdm. From a qualitative point of view, confirmed by bibliographic
data (Ridout et al., 1991), oleanolic acid is the main
saponin component (76–85%), followed by hederagenin (10–18%) and phytolaccagenin (4–5%).
Since saponins are mainly located in the outer layers of the seed, these components were removed
through the process of pearling. The process was
performed using a laboratory perlator model (TM05-Takayama, testing Mill) with an abrasive roller
(40P). A 50% reduction in total saponins % compared with the initial value for the product with a
pearling degree of 20% was observed by gas chromatographic analysis. However, the final product
still had a saponin content which could be detected
at sensory level. Application of pearling at 30% reduced the saponin content by about 80%. In fact,
saponin values dropped from 238.9 mg/100 gdm to
33.47 mg/100 gdm in the pearled product (Table 3).
Ash, protein and lipid content in ‘Titicaca’ is higher
after abrasion of the pericarp. In particular, the linoleic omega fatty acid is very high in ‘Titicaca’ seed
and flour.
Seed abrasion tends also to increase oleic, linoleic
and palmitic fatty acid in ‘Titicaca’.
From 2008 to 2013, ISAFoM-CNR participated as a
partner in the UE project “Sustainable water use securing food production in dry areas of the Mediterranean region” (SWUP-MED).
Quinoa genotype ‘Q52’ (‘Titicaca’) was grown in
an open field trial in 2009 and 2010 to investigate
273
CHAPTER: 3.3 saponis
274
the effects of salt and water stress on quantitative
and qualitative aspects of the yield. Treatments irrigated with well water (‘Q100’, ‘Q50’ and ‘Q25’)
and corresponding treatments irrigated with saline
water (‘Q100S’, ‘Q50S’ and ‘Q25S’) with an electrical conductivity (ECw) of 22 dS/m were compared.
Sample
Soxhlet Extraction
Defatted sample
Saline and water stress in both years do not cause
significant yield reduction, and quinoa may be defined as tolerant to salinity and drought (Pulvento
et al., 2012).
Chemical composition of quinoa seeds confirms a
higher protein and fibre content compared with
common cereals, while the highest level of saline
water determines higher mean seed weight and, as a
consequence, higher fibre and total saponin content
in quinoa seeds. It has been observed that irrigation
with 25% full water restitution, with and without the
addition of salt, is associated with an increase in free
phenolic compounds of 23.16% and 26.27%, respectively. In contrast, bound phenolic compounds are
not affected by environmental stresses.
The effects of the different agronomic variables,
such as irrigation and salinity, on the saponin profiles of quinoa were analysed.
Saponins were evaluated in terms of sapogenins
(Gomez-Caravaca et al., 2012; Lavini et al., 2011)
(Figure 2).
Sapogenin
GC analisis
Quantitative and qualitative
evaluation of sapogenis
Figure 2: Schematic diagram for the extraction of
saponins
A gas chromatographic procedure was applied for
the evaluation of saponin aglycones (sapogenins)
derived from the acid hydrolysis of samples (Ridout
et al., 1991; Woldemichael and Wink, 2001). Three
major quinoa saponin aglycones were identified:
oleanolic acid (36–50% total), hederagenin (27–
28%) and phytolaccagenic acid (21–36%) (Figure 3).
62
62
61
61
1
60
60
3
59
59
2
58
58
57
57
56
56
55
55
54
54
53
53
52
52
51
51
50
50
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
Figure 3: GC chromatogram of Titicaca saponin (1 Oleanolic acid, 2 Hederagenin, 3 Phytolaccagenic acid)
30
CHAPTER: 3.3 saponis
When considering the total amount of saponins
(Table 5) it was observed that ‘Titicaca’ is a bitter
variety. In fact, quinoa seeds with a saponin concentration > 0.11% are usually considered to be bitter genotypes (Vega-Gálvez et al., 2010).
The highest saponin values were observed in samples obtained without deficit irrigation treatments
(1 633.3 mg/100 g for ‘Q100S’ and 1 140.1 mg/100
gdm for ‘Q100’, respectively). The samples treated
with a water deficit (‘Q25’ and ‘Q50’) showed a decrease in saponin content compared with ‘Q100’.
The ‘Q50’ samples, compared with ‘Q100’, showed
a decrease in saponins of 32%; while the samples grown with a higher irrigation deficit (‘Q25’)
showed a 45% decrease in saponins. These results
are in agreement with the study of Soliz-Guerrero et
al. (2002), who reported that saponin content is affected by a soil-water deficit, to the extent that high
water deficits promote low saponin contents. Samples treated with saline water also show significant
differences at different irrigation levels (‘Q100S’,
‘Q50S’ and ‘Q25S’); the decrease in saponin content
in the ‘Q50S’ and ‘Q25S’ samples is very high compared with ‘Q100S’ (40% and 42% for ‘Q25S’ and
‘Q50S’, respectively).
From 2011 to 2013, field trials were performed in
Vitulazio on quinoa, and others are ongoing at ISAFoM within the “CISIA” project, funded by the National Research Council, and the “Quinoa Felix” project – Introduction of quinoa (Chenopodium quinoa
Willd.) – in the Campania region for high nutritional
and functional value food production, in collaboration with the University of Molise and CNR-Institute
of Food Science (ISA) of Avellino. The aim of these
activities is to evaluate yield and seed quality of
Chenopodium quinoa varieties grown under rainfed
conditions in southern Italy, and to assess milling
performance and protein, ash, lipid and saponin
content of the seed.
All analyses are performed on whole seeds and on
“pearling” grain, after removal of the pericarp, to
define the potential nutritional characteristics of
each quinoa variety. Since there is no genetic resource of quinoa as a domesticated variety in Italy,
the studies are conducted using seeds received from
foreign institutions and of different origins. Testing
is being done on the Danish quinoa cultivars ‘Puno’
and ‘Titicaca’ selected from material originating in
southern Chile and provided by the University of
Copenhagen; four Bolivian cultivars ‘Kurmi’, ‘Janca
grano’ ‘Blanquita’ and ‘Real’; the Peruvian ‘Amarilla
de Marangani’; and ‘Jujuy rosada’ originating in Argentina. The Danish cultivars ‘Titicaca’ and ‘Puno’
give the higher yield, while ‘Janca grano’, ‘Real’ and
‘Kurmi’ give the lowest yields; ‘Blanquita’ does not
produce under Mediterranean conditions.
All seven aglycones have been assayed. The variety
‘Jujuy Rosada’ is richest in saponins (4.99%), while
‘Real’ is the poorest (0.1%). Although the concentration profiles of the seven aglycones vary greatly
among the varieties – in particular, in ‘Jujuy rosada’,
72.5% of saponins contain 3β-hydroxy-23-oxo-olean12-en-28-oic acid as aglycone, while in ‘Real’, oleanolic acid is the most represented aglycone (despite only
24.80%) – there is a more homogeneous distribution
of all seven aglycones. However, 3β,23,30-trihydroxy
olean-12-en-28-oic acid is the least represented aglycone in all the varieties studied.
4. Conclusion and perspective
Saponins present both an obstacle and an opportunity. The deployment as food of many pseudocereals, especially quinoa, is hindered by the presence
of these antinutritional elements, both because of
reduced palatability due to their bitter taste, and
because of the serious effects they can have on human health. On the other hand, these molecules
are proving to be extremely interesting in several
fields: from pharmaceutical (as the basis for the development of new cancer drugs, new antifungals or
adjuvants in vaccines), to chemical, but especially in
the field of agronomy, where they are proving to be
excellent and versatile insecticides. Saponin insecticidal activity is based on three different mechanisms (Chaieb, 2010): interference with feeding, entomotoxicity (various forms of chronic toxicity, such
as female fertility reduction and decreased rate of
blossoming eggs, are observed in many insect species) and growth regulation (research shows that
saponins are able to regulate the growth of many
insect species). The effects of saponins are generally
associated with disturbance of the developmental
stages and moulting failure. Nevertheless, there is
still massive scope for understanding and improving
this use of saponins, regarding in particular: stability (because the bulk of insecticide activity is due to
the sugar side chains and these are very susceptible
to pH values and enzymatic activity), application,
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CHAPTER: 3.3 saponis
276
action of residual saponins and their antinutritional
properties, and, finally, their difficult synthesis. The
latter could be solved by means of extraction protocols from varieties that produce large amounts of
saponins or are grown under conditions that generate larger quantities (good water supply and high
salinity of the soil), while knowledge of the pedoclimatic effects on saponin content may allow the
development of varieties requiring sustainable agronomic treatments to eliminate these dangerous
antinutritional agents.
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