antioxidants

Review
Anthocyanins from Purple Tomatoes as Novel
Antioxidants to Promote Human Health
Silvia Gonzali and Pierdomenico Perata *
PlantLab, Institute of Life Sciences, Scuola Superiore Sant’Anna, 56127 Pisa, Italy; s.gonzali@santannapisa.it
* Correspondence: p.perata@santannapisa.it; Tel.: +39-05-088-1901




Received: 29 September 2020; Accepted: 16 October 2020; Published: 20 October 2020


Abstract: Anthocyanins are plant secondary metabolites belonging to the class of polyphenols,
whose beneficial roles in the prevention and treatment of several important human diseases have
been demonstrated in many epidemiological studies. Their intake through diet strictly depends on
the eating habits, as anthocyanins are contained in red and purple fruit and vegetables as well as in
some processed foods and beverages, such as red wine. Genetic engineering and breeding programs
have been recently carried out to increase the content of anthocyanins in candidate plant species
which cannot offer satisfactory levels of these precious compounds. Tomato (Solanum lycopersicum) is
a vegetable commodity where these strategies have resulted in success, leading to the production of
new anthocyanin-rich fruit varieties, some of which are already marketed. These varieties produce
purple fruits with a high nutraceutical value, combining the health benefits of the anthocyanins to the
other classical tomato phytochemicals, particularly carotenoids. The antioxidant capacity in tomato
purple fruits is higher than in non-anthocyanin tomatoes and their healthy role has already been
demonstrated in both in vitro and in vivo studies. Recent evidence has indicated a particular capacity
of tomato fruit anthocyanins to act as scavengers of harmful reactive chemical species and inhibitors
of proliferating cancer cells, as well as anti-inflammatory molecules.

Keywords: tomato; Solanum lycopersicum; delphinidin; petunidin; malvidin; antioxidant;

anti-inflammatory; anti-proliferative

1. Anthocyanins as Beneficial Plant Metabolites

Many phytochemicals have long been recognized as health beneficial compounds and have been
used in traditional medicines for thousands of years. Among them, plant-derived polyphenols are
increasingly attracting research interest due to their positive effects in terms of disease prevention and
treatment. Anthocyanins belong to this group of metabolites. They represent a water-soluble subclass
of flavonoids, responsible for red, purple and blue colorations of flowers, fruits and leaves in many
species. Their functions in plants are different—as coloured pigments, they attract pollinators and seed
dispersers in reproductive organs; as antioxidant compounds, they play a pivotal role in counteracting
various types of stress, such as excessive light, in vegetative organs. As a consequence, their content
can be strongly affected by environmental factors, such as light and temperature, as well as by biotic
and abiotic stress conditions [1].
Anthocyanins are the glycosylated derivatives of the anthocyanidin molecules, whose chemical
structure is based on a C15 skeleton formed by two polyhydroxylated or polymethoxylated aromatic
rings (A and B) linked by a C3 benzene ring (C) (Figure 1A). Glucose, galactose, arabinose, rutinose,
rhamnose and xylose are the sugars most commonly attached, as mono-, di- or tri-saccharides, to
the 3-position on the C ring or the 5- or 7-position on the A ring. The sugar moieties can be further
acylated with aliphatic and aromatic acids. Several anthocyanidins can be synthesized, differing in
the substituent groups on the structural rings, but six of them represent 90% of the total, namely

Antioxidants 2020, 9, 1017; doi:10.3390/antiox9101017 www.mdpi.com/journal/antioxidants

Antioxidants 2020, 9, 1017 2 of 17

pelargonidin, cyanidin, delphinidin, peonidin, petunidin and malvidin [2]. Glucose represents the
Antioxidants 2020, 9, x FOR PEER REVIEW 2 of 17
main glycosylating agent, and the 3-O-glucose derivatives are the most common anthocyanins found
in nature
main[3,4] (Figure 1A).
glycosylating agent, and the 3-O-glucose derivatives are the most common anthocyanins found
in nature [3,4] (Figure 1A).

Figure
Figure 1. General
1. General anthocyanin
anthocyanin structureand
structure and list
list of
of the
the most
mostcommon
common anthocyanins
anthocyaninsfound in nature
found in nature
(from [5]) (A). Examples of purple tomato fruits obtained by breeding: Aft/Aft x atv/atv x
(from [5]) (A). Examples of purple tomato fruits obtained by breeding: Aft/Aft × atv/atv × hp2/hp2 hp2/hp2 in
MicroTom background (on the left); Aft/Aft x atv/atv in Ailsa Craig background (B). Structure of
in MicroTom background (on the left); Aft/Aft × atv/atv in Ailsa Craig background (B). Structure
petunidin-3-(trans-p-coumaroyl)-rutinoside-5-glucoside, one of the most abundant anthocyanin
of petunidin-3-(trans-p-coumaroyl)-rutinoside-5-glucoside, one of the most abundant anthocyanin
molecules identified in purple tomato fruits (C). Redrawn from [6].
molecules identified in purple tomato fruits (C). Redrawn from [6].
Antioxidants 2020, 9, 1017 3 of 17

Anthocyanins’ stability can be affected by different factors, including pH, temperature,

concentration, light, oxygen, presence of other flavonoids or metallic ions [7]. In aqueous solution,
anthocyanins can assume four molecular structures according to pH [8]. In acidic solutions (pH 1–3),
they exist primarily as 2-phenylbenzopyrylium (flavylium) cations (red), the most stable forms.
Progressively increasing pH above 4, anthocyanins adopt the form of the carbinol pseudobase
(colourless) and chalcone (yellowish). In basic conditions, the predominant form is the quinoidal base
(blue). Other factors, such as degree of hydroxylation or methylation and glycosylation patterns of the
aromatic rings, can also affect the colour, with more hydroxyl groups giving a bluish shade, and more
methoxyl groups increasing redness [8]. The aglycone forms are highly unstable and rarely found
in nature, whereas glycosylation and esterification with organic and phenolic acids increase stability.
On the contrary, stability is decreased by B-ring hydroxylation [9]. The formation of complexes with
other flavonoids (copigmentation) can further stabilize anthocyanins.
The chemical structure of the different anthocyanin molecules affects their corresponding chemical
activities, particularly in terms of antiradical and antioxidant properties, which represent a key
mechanism through which anthocyanins can be beneficial for human health [10]. Substituents on the
flavylium cation strongly influence the radical scavenging activity [11]; hydroxyl and methoxyl groups
are electron-donating structures, and therefore their type, number and position are very important in
relation to the antioxidant properties [12,13]. On the contrary, glycosylation, by reducing free hydroxyls
and metal chelation sites, reduces the antioxidant power [8].
Anthocyanins provide many health benefits, from anti-inflammatory and anti-carcinogenic to
antimicrobial, neuroprotective and anti-obesity effects [14]. As a consequence, their consumption can
be helpful in reducing the incidence of cardiovascular, metabolic and degenerative or chronic diseases
and of certain types of cancer. The strong antioxidant capacity represents the main mechanism of
action. However, in some body compartments, e.g., in the venous system, their concentrations seem
too low to allow scavenging of reactive oxygen species (ROS), whereas they would be adequate to
influence signal transduction and gene expression pathways [15].
Anthocyanins are usually assumed with the diet. Rich sources are mostly represented by red
and purple fruits or dark vegetables (e.g., berries, cherries, plums, grapes, black beans, red onions,
eggplant, red cabbage, purple sweet potatoes) [16], where they can reach up to 1% of dry weight
(DW) [17]. However, anthocyanins from fruits and vegetables are not the same, as their molecules are
in general more glycosylated and contain more aromatic acyl groups in vegetables than in fruits [18].
Anthocyanins can enter human diet also through processed foods and beverages, such as red wine,
juices and yogurt. Their intake in diet is strongly influenced by eating habits, and their highest
consumption is associated with diets rich in fruit and vegetables and red wines, such as those common
in the Mediterranean countries [19].
The beneficial effects of anthocyanin-enriched foods also depend on the bioavailability of these
compounds, that is, the rate and extent to which they or their active metabolites become available at
the sites of action within the human body. Many studies have been recently carried out highlighting
the crucial role of the digestion process [7]. Anthocyanins can be absorbed from the stomach and
the intestines, either as intact molecules or after degradation. The structure of the molecule, and in
particular the glycone (the sugar moiety) and the presence of acylated groups, affects the rate and extent
of absorption, which is generally lower for the more complex structures [20–22]. Anthocyanins can be
rapidly absorbed from the stomach and can enter the systemic circulation within minutes, where they
reach maximum concentrations after a few hours, then rapidly decline [23]. The gastrointestinal mucosa
thus appears highly permeable to these molecules and the existence of active transporters has been
hypothesized [24–26]. Anthocyanin concentrations are not the same in the different compartments;
they can achieve micromolar values in the intestinal tissues, while maximum plasma levels are in the
nanomolar range [15]. Moreover, it is probable that anthocyanins are extensively metabolized before
entering the systemic circulation since some of their metabolites (e.g., phenolic acids) are generally more
concentrated than the original compounds [27]. In studies with animal models, intact anthocyanins
Antioxidants 2020, 9, 1017 4 of 17

have been found in kidney or liver as well as in the brain [28,29]. They can finally reach the large
intestine and be partially decomposed here by the microbiota, giving rise to products which may
contribute to their health effects [30–32].

2. Purple Tomatoes: Their Origins and Specificities

Tomato is the second most cultivated vegetable worldwide and one of the most consumed, both
fresh and processed [33]. Therefore, tomato fruits represent one of the main sources of carotenoids,
polyphenols and vitamins of the human diet, even if the concentrations of bioactive compounds are
not as high as those of other vegetables [34].
With domestication, several different tomato varieties have been developed all over the world,
differing in fruit size, shape and colour [35]. The nutraceutical value of tomato fruits relies on their
content of carotenoids, polyphenols, soluble sugars, organic acids, minerals and vitamins, particularly
vitamin C and vitamin E [33,36,37], as well as of volatile compounds [38]. Their antioxidant capacity
depends on both lipophilic (carotenoids and vitamin E) and hydrophilic (vitamin C and phenolic
compounds) fractions [39]. Both composition and nutritional value are highly affected by genotype as
well as nutrition and cultivation environment [40,41]. Pre- and post-harvest processing operations [42]
can represent other important factors.
Carotenoids represent the main secondary metabolites in tomatoes, with the red lycopene as the
most abundant one, followed by the colourless phytoene and phytofluene and the orange β-carotene
(Table 1). Lycopene content increases during ripening, as the concentration of chlorophylls decrease,
and it is higher in the pericarp [43,44]. Lycopene is a strong antioxidant compound, with a very
important role in the prevention of several diseases [44,45].

Table 1. Content of the main carotenoids and polyphenols found in tomato ripe fruits from different
cultivated varieties [44]. Values are expressed as mg bioactive compound 100 g−1 fresh weight (FW).

Carotenoid Carotenoid Concentration Polyphenol Polyphenol Concentration

Lycopene 7.8–18.1 Naringenin chalcone 0.9–18.2
Phytoene 1.0–2.9 Rutin (quercetin-3-O-rutinoside) 0.5–4.5
Phytofluene 0.2–1.6 Quercetin 0.7–4.4
β-Carotene 0.1–1.2 Chlorogenic acid 1.4–3.3
γ-Carotene 0.05–0.3 Caffeic acid 0.1–1.3
δ-Carotene 0–0.2 Naringenin 0–1.3
Lutein 0.09 Kaempferol-3-O-rutinoside 0–0.8
Neurosporene 0–0.03 p-Coumaric acid 0–0.6
α-Carotene 0–0.002 Ferulic acid 0.2–0.5
Neoxanthin - Kaempferol 0–0.2
Violaxanthin - Myricetin -
Cyanidin -
Pelargonidin -
Delphinidin -

Polyphenolic compounds include flavonols and their derivatives, hydroxycinnamic acids and
flavanones [41,46] (Table 1). Naringenin chalcone (a flavonoid biosynthetic pathway intermediate) is
the most abundant, followed by rutin, which is responsible for the yellow colour of the peel. Rutin is a
glycosylated derivative of quercetin, which represents the main flavonol of tomatoes. The flavanone
naringenin is present at lower concentrations, as well as other flavonols, such as kaempferol and
myricetin. Hydroxycinnamic acids mainly include chlorogenic and caffeic acids, but also p-coumaric
and ferulic acids, which are quite common in tomato. Remarkably, the flavonoid pathway is active only
in the fruit peel [47,48], where flavonols and their derivatives can be accumulated [49]. Anthocyanins
are not synthesized in tomato fruits, due to mutations in regulatory genes of their specific biosynthetic
pathway [5]. On the contrary, they are produced in tomato vegetative tissues [50], provided that
adequate developmental and environmental stimuli are present.
Antioxidants 2020, 9, 1017 5 of 17

Due to the increasingly acknowledged health beneficial role of anthocyanins, several attempts
to activate their biosynthetic pathway in tomato fruits have been successfully made in recent years,
either with genetic engineering or traditional breeding approaches [51].
After some preliminary attempts [5], the first “purple” engineered tomatoes were produced
in 2008, when two transcription factor-encoding genes from snapdragon, Delila (Del) and
Rosea1 (Ros1), were expressed in the cultivar MicroTom under a fruit-specific promoter [52].
The high anthocyanin concentrations reached in these fruits determined an intense purple
coloration in both peel and flesh, enhancing in a significant way their hydrophilic
antioxidant power. The activation of the biosynthetic pathway led to the synthesis
of different compounds, with delphinidin-3–(trans-p-coumaroyl)-rutinoside-5-glucoside and
petunidin-3-(trans-p-coumaroyl)-rutinoside-5-glucoside as major molecules [53] (Table 2). The same
exogenous gene combination was expressed in other tomato varieties with similar results [52,54].
By using a multi-level genetic engineering approach [55], the content of anthocyanins was then
almost doubled in transgenic Del/Ros1 tomato lines by concomitantly expressing the Arabidopsis thaliana
MYB12 gene, able to activate the upstream reactions from primary metabolism to flavonoid biosynthesis
(Table 2).
An alternative strategy to obtain anthocyanin-rich tomatoes has been the breeding approach,
made possible by the existence of wild Solanum species bearing fruits that synthesize anthocyanins under
suitable conditions [5,51]. Different genetic combinations were obtained by crossing S. lycopersicum
with different interfertile wild species. The most stable anthocyanin-rich fruit genotypes were those
homozygous for both Anthocyanin fruit (Aft) [56] and atroviolacea (atv) [57] alleles (Table 2). Differently
from the engineered tomatoes, the anthocyanin phenotype in these fruits is limited to fruit peel and is
environmentally dependent, being particularly induced under high light and low temperatures [48,58].
As a consequence, the quantity of pigments inversely correlates with fruit size—the maximum amounts
(up to 300 mg 100 g−1 FW) could be reached by small fruits, where the ratio peel/flesh was higher [58].
Carotenoid levels in these fruits were similar to those in non-anthocyanin ones, indicating that the
increased flux of metabolites into the flavonoid pathway was not detrimental to the carotenoid synthesis.
From the Aft/Aft × atv/atv starting breeding material [58], the “Indigo Rose” purple tomato lines were
bred [59].
Antioxidants 2020, 9, 1017 6 of 17

Table 2. Main purple tomato lines obtained through genetic engineering or breeding programs and relative anthocyanin content and composition. Abbreviations: Del,
delphinidin; Pet, petunidin; Mal, malvidin; Cya, cyanidin; Peo, peonidin; rut, rutinoside; glc, glucoside; glyc, glycoside; hex, hexoside; FW, fresh weight.

Tomato Line Origin Main Anthocyanins Detected Anthocyanin Concentration in Fruit Ref.
Pet-3-(trans-p-coumaroyl)-rut-5-glc;
Del-3-(trans-p-coumaroyl)-rut-5-glc;
Del/Ros1 Genetic engineering 2.835 ± 0.456 mg g−1 FW [52]
Pet-3-(feruloyl)-rut-5-glc;
Del-3-(feruloyl)-rut-5-glc
Del-3-(trans-p-coumaroyl)-rut-5-glc; peel: 5.1 ± 0.5 g kg−1 DW
Pet-3-(trans-p-coumaroyl)-rut-5-glc; flesh: 5.8 ± 0.3 g kg−1 DW
Del/Ros1 Genetic engineering [54]
Mal-3-(p-coumaroyl)-rut-5-glc; whole fruit: 5.2 ± 0.5 g Peo-3-glc
Mal-3-(feruloyl)-rut-5-glc equivalent kg−1 DW, or 0.5% of DW
Del-3-(trans-p-coumaroyl)-rut-5-glc; 1.154 ± 0.011 mg g−1 FW
Pet-3-(trans-p-coumaroyl)-rut-5-glc; 2.857 ± 0.218 mg g−1 FW
Del/Ros1 x AtMYB12 Genetic engineering [55]
Pet-3-(feruloyl)-rut-5-glc; 0.922 ± 0.102 mg g−1 FW
Mal-3-(p-coumaroyl)-rut-5-glc 0.598 ± 0.011 mg g−1 FW
Pet-3-(p-coumaroyl)-rut-5-glc;
Aft/Aft × atv/atv Breeding peel: 116.11 mg 100 g−1 FW [58]
Del-3-rut
Pet-3-(trans-p-coumaroyl)-rut-5-glc;
Sun Black (Aft/Aft × atv/atv) Breeding more than 1 mg g−1 DW [6,60]
Mal-3-(trans-p-coumaroyl)-rut-5-glc
Pet-3-(p-coumaryl)-rut-5-glc; 50.18 mg 100 g−1 DW
V118 Breeding Pet-3-caffeoyl-rut-5-glc; 9.04 mg 100 g−1 DW [61]
Mal-3(p-coumaryl)-rut-5-glc 13.09 mg 100 g−1 DW
Pet + p-coumaroyl + rut + glyc;
Blue Japan Indigo tomato peel: 17 mg g−1 DW
Breeding Mal + p-coumaroyl + rut; [62]
(Aft/Aft × atv/atv) pulp: 0.1 mg g−1 DW
Del
Pet-(p-coumaroyl)-rut-hex
Delphinidin-3-(p-coumaroyl)-rut-glyc
Pet-(p-coumaroyl)-rut-hex
Aft/Aft × atv/atv × hp2/hp2 Breeding peel: 90.91 mg 100 g−1 FW [63]
Pet-3-(cafeoyl)-rut-5-glyc
Mal-3-(p-coumaroyl)-rut-5-glyc
Cya-3-O-rut
Antioxidants 2020, 9, 1017 7 of 17

A second independently developed Aft/Aft × atv/atv genotype was named “Sun Black”
(SB) for the light conditional purple fruits [48,51,60] (Figure 1B). In SB, the content
of anthocyanins could reach more than 1 mg g−1 DW and the two main molecules
identified were petunidin-3-(trans-p-coumaroyl)-rutinoside-5-glucoside (56.6% of the total) and
malvidin-3-(trans-p-coumaroyl)-rutinoside-5-glucoside (21.4%) [6] (Table 2). In this line, other
flavonoids increased, compared to the non-anthocyanin background, in proportion to the anthocyanin
concentration, with rutin reaching 0.8 mg g−1 DW and a total phenolic content of 8.6 mg g−1 DW.
Consequently, the antioxidant capacity of the hydrophilic extract was significantly high. Total carotenoid
content, exceeding 200 µg g−1 DW, remained similar to non-anthocyanin varieties, whereas the amount
of vitamin C (37.3 mg 100 g−1 FW) was found higher [6].
The line V118, whose genotype was not made known, was also developed using a breeding
approach [61]. Similarly to the previous ones, carotenoid and polyphenol amounts and compositions
in this accession did not differ significantly from other cultivated varieties, with the exception of the
presence of anthocyanins, which mainly resulted in acylglycosides of petunidin and malvidin (Table 2).
The phenolic compounds represented the major contributors to the antioxidant power measured in V118
purple tomatoes [61]. Successive studies carried out on this line [64] indicated the good bioavailability of
the anthocyanin fraction in a cell-based antioxidant assay. Furthermore, in a simulated gastrointestinal
digestion model, it was found that both the carotenoid and the phenolic profiles significantly changed
during digestion, indicating the occurrence of a strong degradation process. This also affected the
lipophilic and hydrophilic antioxidant activities of V118 fruits. The anthocyanin fraction, however,
reduced in quantity but not in its composition, suggesting that a significant degradation did not occur
during digestion.
A high-anthocyanin breeding line further described was a “blue” tomato, corresponding to a
cultivar of “Indigo Rose” [59] cultivated in Japan [62]. In this cv., several eleven types of anthocyanins
were identified, including five delphinidin-, four petunidin- and four malvidin-derived, mostly
accumulated in the peel (Table 2). The antioxidant activity of the blue tomato peel extract was highly
dependent on the anthocyanin content and similar to that of strawberry fruits, and half if compared
with that of blueberries [62].
In order to maximize the nutraceutical properties of tomato fruits, increasing both anthocyanins
and carotenoids, breeding programs were also carried out by using “high pigment” (hp) mutants to
be crossed with high-anthocyanin accessions [58,65]. Very interesting results were obtained in terms
of anthocyanin accumulation as well as carotenoid and vitamin C contents, with the introgression
in the same background of different mutations increasing the content of specific phytonutrients [66]
(Figure 1B). The combination of the alleles hp2, Aft and atv in a cherry tomato background, in particular,
led to uniformly purple fruits with total enhanced nutrient contents [63] (Table 2). However, these allele
introgressions also led to changes in volatile compounds, particularly enriched by phenolic profiles,
which could modify the typical tomato flavour and are therefore worthy of further studies [63].
To summarize, independently from their genotypes, purple tomato lines obtained by breeding
material maintained the original content of the tomato bioactive compounds and were characterized
by the accumulation of anthocyanidins in the peel, which mainly belong to petunidin, malvidin
and delphinidin classes, the same that can be found in tomato vegetative tissues [50]. One of
the most common and abundant anthocyanin molecules identified in purple tomatoes was
petunidin-3-(trans-p-coumaroyl)-rutinoside-5-glucoside (Figure 1C). The antioxidant power of these
fruits was higher than in non-anthocyanin varieties, because they summed a higher hydrophilic
activity due to the polyphenolic fraction to the lipophilic antioxidant activity of the carotenoids.
The concentration of anthocyanins varied according to fruit size and light exposition and could
reach considerable values (more than 1 mg g−1 DW). The purple tomato lines produced by genetic
engineering further maximized all these aspects, being characterized by a uniform phenotype with
high anthocyanin concentrations in both peel and flesh. However, whereas some purple bred-tomato
Antioxidants 2020, 9, 1017 8 of 17

varieties have been already patented and marketed, none of the GMO genetically engineered lines are
commercially available yet.

3. Anthocyanins from Purple Tomatoes and Human Health

In parallel with the research activities aimed at producing new anthocyanin-rich tomato lines,
several studies were carried out to characterize the pharmacological activities of these fruits by
in vitro and in vivo studies. In many cases, direct comparison between purple tomatoes and
their non-anthocyanin backgrounds allowed the identification of effects specifically exerted by the
anthocyanin fraction, thus demonstrating the added nutraceutical value that these pigments can offer.
As mentioned before, several health beneficial effects are currently accredited to anthocyanins,
and pigments from different sources, in particular berries, have been tested for their pharmacological
effects (recently reviewed in [67,68]). Due to the anthocyanin-rich tomatoes’ recent origin,
epidemiological studies on such tomatoes are still scarce. We review what is currently known
in the following paragraphs.
Antitumor activity. The beneficial role of tomato consumption in terms of reduced cancer incidence,
thanks to their content of carotenoids and polyphenols, is well known [44]. However, anthocyanins
from many different sources have also demonstrated anti-carcinogenic activity and multiple and
overlapping mechanisms of action have been proposed, specifically in the different phases of cancer
development and progression (extensively reviewed in [69]). In the initial stage of tumorigenesis,
anthocyanins, due to their strong antioxidant capacity, may protect cellular genomes from possible
DNA damage and gene mutations induced by oxidative stress or other mutagens, thus preventing the
transformation of normal cells into cancer cells. Moreover, anthocyanins can control the expression
and secretion of inflammatory factors, whose continuous or increased production may induce chronic
inflammation processes, leading in many cases to tumour development (see also next paragraphs).
In the cancer formation stage, anthocyanins can induce terminal differentiation of tumour cells, through
their differentiation towards normal and mature cells, thus blocking tumorigenesis. They can also
inhibit cellular transformation, a mechanism that is often associated with tumour development induced
by inflammation signalling pathways, as well as cellular proliferation, that is, the uncontrolled cell
cycle, characterized by continuous divisions, that is typical of cancer cells. This occurs due to the
ability of anthocyanins to inhibit different kinase signalling pathways, and to arrest the cell cycle
at different division phases. In the cancer developmental stage, anthocyanins can induce apoptosis
of malignant cells through mechanisms involving different pathways (e.g., mitochondrial signalling
pathway, caspase-dependent and -independent signalling pathways, endoplasmic reticulum signalling
pathway). Finally, anthocyanins are potential antiangiogenic agents and thus may prevent the formation
of new blood vessels that, by supplying oxygen to the tumour cells, represent an important factor in
cancer development.
The recent studies carried out on the anthocyanin fractions isolated from purple tomatoes
supported these lines of evidence, in particular the specific anti-proliferative effects against different
types of tumour cell lines both in vitro and in vivo. One of the first indications came from the
Del/Ros1 purple tomatoes that, in a pilot test administered to cancer-susceptible Trp53–/– mice, led to
a significant extension of their life span [52]. Subsequently, extracts from SB tomato were tested
in vitro for proliferation inhibitory effects on human colon (HT-29) and ovarian (HeLa) cancer cells [60].
They showed a significant anti-proliferative action on both kinds of cells in a dose-dependent manner,
and this effect appeared genotype-specific because anthocyanin-rich extracts from eggplant, red grape
and red cabbage did not show the same effectiveness. This was probably due to the differences in the
extracts’ composition; different anthocyanidin structures can indeed show different biological activity
as well as a different susceptibility to cell uptake [70,71].
Photoprotection. The beneficial effect of anthocyanins on vision and visual acuity has been
known for a very long time [72,73]. Recent evidence confirms that intact anthocyanins and their
metabolites can pass through the blood–brain and the blood–retinal barriers in animal models and
Antioxidants 2020, 9, 1017 9 of 17

their concentrations in several ocular tissues can be higher than in plasma, suggesting that they
can concentrate there [74]. Anthocyanins can exert different effects on vision health, as attested by
many recent studies (reviewed in [74]): (i) they showed relaxing activities on ciliary smooth muscle,
which is involved in the control of lens focus, in the production of aqueous humour and in the
maintenance of ocular pressure, thus demonstrating possible therapeutic uses in the treatment of
visual fatigue and important ocular disorders such as myopia and glaucoma; (ii) they stimulated
the regeneration of rhodopsin, after being photo-bleached by light, a mechanism that is necessary to
make this photoreceptor active to carry out again its function in the visual photo transduction process;
(iii) being able to inhibit the elongation of axial and ocular lengths caused by wearing a negative lens
in animal models, they could be useful in the prevention of myopia, a very common cause of impaired
vision in young and adult people, due to a refractive error induced by the elongation or curvature
of the eye shape; (iv) they improved dark adaptation and night vision by the enhanced generation
of retinal pigments; and (v) they increased retinal blood circulation and could therefore be useful in
preventing the early stages of diabetic retinopathy or glaucoma. The antioxidant and scavenging
activities of anthocyanins could also affect the production of ROS inducing the apoptotic degeneration
of the photoreceptor cells, which is responsible for the gradual constriction of the central visual field,
leading to blindness in retinitis pigmentosa [75,76]. In this context, the extract of the Japan blue tomato
was tested in vitro on murine photoreceptor cells and found to significantly reduce cone cell death by
scavenging hydrogen peroxide [62]. This was attributed to the anthocyanin fraction, whereas lycopene
showed no effect. This study indicated that petunidin-derived anthocyanins, the most abundant in
Japan blue tomato extract, could inhibit ROS production due to their scavenging activities, supporting
previous positive results obtained in similar models with other kinds of anthocyanin molecules, such as
malvidin glycosides from Chinese blueberries [77] and delphinidin glycosides from Maqui berries [78].
Anti-inflammatory activities. Inflammation is a complex response of the immune system through
which the animal body may fight infections, injuries, toxins or other stress events. In the acute
inflammation process, cell surface receptors recognize the detrimental stimuli, with the activation
of inflammatory pathways characterized by the release of inflammatory markers from the injured
cells. These markers include inflammatory cytokines, nitric oxide (NO), interleukins, tumour necrosis
factor-alpha (TNF-α), interferon gamma and prostaglandins. Finally, inflammatory cells, such as
leukocytes, are recruited from the venous system to the injured tissue [79]. All these events contribute
to the restoration of tissue homeostasis and the resolution of the acute inflammation. When this
does not occur, a long-term reaction to an inflammatory stimulus accompanied by changes aimed at
wound healing can lead to a chronic inflammation. This in turn can create conditions able, with time,
to trigger a chronic disease such as an autoimmune disease, a metabolic disorder (atherosclerosis and
obesity), fibrosis or certain types of cancer [80]. Fighting the acute inflammation causes and interfering
with the inflammatory cascades may help in preventing such important pathologies. In this context,
several phytochemicals showed anti-inflammatory actions and anthocyanins appeared effective in
the inhibition of signalling cascades involving cytokines, NO production and expression of some
pro-inflammatory genes [81]. Cyanidin glycosides, in particular, were able to inhibit cyclooxygenase
enzyme activities that are necessary to convert arachidonic acid to pro-inflammatory cytokines
(prostaglandins), which play a fundamental role in the inflammation response [82].
The anti-inflammatory potential of purple tomatoes was analysed in several studies. Extracts from
V118 tomatoes, for example, were tested in vivo in the carrageenan-induced paw oedema rat model [64].
This phlogistic model is commonly used to investigate new anti-inflammatory drugs and has been used
to evaluate the anti-inflammatory activity of other tomato phytochemicals. Lycopene, in particular,
was able to significantly inhibit paw oedema formation in both acute and chronic treatments [83].
Carrageenan in rats leads to increases in paw volume through the induction of an inflammatory process
characterized by two distinct phases. In the first two hours, inflammation mediators are released, then
ROS such as hydrogen peroxide and superoxide radical, as well as prostaglandins, are produced [83].
Moreover, inflamed tissue attacked from free radicals accumulate malondialdehyde (MDA), an end
 from V118 tomatoes, for example, were tested in vivo in the carrageenan-induced paw oedema rat
model [64]. This phlogistic model is commonly used to investigate new anti-inflammatory drugs and
has been used to evaluate the anti-inflammatory activity of other tomato phytochemicals. Lycopene,
in particular, was able to significantly inhibit paw oedema formation in both acute and chronic
treatments
Antioxidants [83].
2020, Carrageenan in rats leads to increases in paw volume through the induction 10
9, 1017 of ofan17
inflammatory process characterized by two distinct phases. In the first two hours, inflammation
mediators are released, then ROS such as hydrogen peroxide and superoxide radical, as well as
product of lipid peroxidation,
prostaglandins, are produced [83].and Moreover,
NO, whileinflamed
trying totissue
counteract oxidative
attacked stress
from free by inducing
radicals accumulate the
activities of antioxidant enzymes such as glutathione peroxidase (GPx) and
malondialdehyde (MDA), an end product of lipid peroxidation, and NO, while trying to counteract superoxide dismutase
(SOD) [84].stress
oxidative V118 by purple tomato
inducing theextracts showed
activities significant
of antioxidant and dose-dependent
enzymes anti-inflammatory
such as glutathione peroxidase
effects
(GPx) (Figure 2), in addition
and superoxide to those
dismutase exerted
(SOD) [84]. by thepurple
V118 other tomato
tomato phytochemicals,
extracts showed i.e., carotenoids
significant and
dose-dependent
and other phenolic anti-inflammatory
compounds [64]. effects
These(Figure
effects2),resulted
in addition to those
in both exerted
direct by the other
and indirect tomato
antioxidant
phytochemicals,
activities i.e., carotenoids
in oedematous andscavenging
tissue, with other phenolic compounds
of free oxygen and [64].nitrogen
These effects resulted
radicals, in both
reduction of
directproduction
MDA and indirect andantioxidant
increase ofactivities
GPx andinSOD oedematous
enzymatic tissue, with scavenging of free oxygen and
activities.
nitrogen radicals, reduction of MDA production and increase of GPx and SOD enzymatic activities.

Figure Schematicrepresentation
Figure 2.2. Schematic representation
of of
thethe main
main positive
positive effects
effects exerted
exerted by anthocyanins
by anthocyanins as
as anti-
anti-inflammatory compounds.
inflammatory compounds. Abbreviations:
Abbreviations: ROS,
ROS, reactive
reactive oxygen
oxygen species;RNS,
species; RNS,reactive
reactivenitrogen
nitrogen
species;
species;COX,
COX,cyclooxygenase
cyclooxygenaseenzyme.
enzyme. Adapted
Adapted from [85].

Genetically
Genetically engineered
engineered “Indigo”
“Indigo” tomatoes,
tomatoes, characterized by high
characterized by high anthocyanin
anthocyanin and
and flavonol
flavonol
contents, as well as other polyphenol-enriched
contents, as well as other polyphenol-enriched tomatotomato lines, were tested in a spontaneous ulcerative
tested in a spontaneous ulcerative
colitis
colitismouse
mousemodel
modelfor fortheir
their potential
potential effects
effects on
on the
the host
host gut
gut microbiota, inflammatory
inflammatory responses
responses
and
andthethesymptoms
symptomsof ofinflammatory
inflammatory bowel
bowel diseases
diseases (IBDs), a group
group of common
common chronic
chronic intestinal
intestinal
inflammation syndromes associated with intestinal dysbiosis [86,87]. Two-week diets supplemented
with polyphenol-enriched tomatoes were able to change the composition of the gut microbiota in
healthy mice [86], as well as the dysbiotic intestinal microbiota communities in the colitis mouse
model [87]. Polyphenol-enriched diets thus created unfavourable conditions for distinct bacterial
species. Suppression of the production of pro-inflammatory cytokines also indicated induction of an
anti-inflammatory pathway [86]. Finally, it was demonstrated that the observed effects specifically
related to the particular combination of the different polyphenolic classes present in these tomato fruits,
acting synergistically, rather than to single specific molecules. The positive anti-inflammatory effects of
the anthocyanin fraction were thus maximised here by the food matrix context.
A parallel study focused on the effects of anthocyanins and flavonols on the pro-inflammatory
signalling pathways associated with IBDs, by using a murine colonic epithelial cell-based inflammatory
assay [88]. A dysregulation of the intestinal immune response to resident intestinal microbes, altering
the delicate equilibrium between immunogenicity against pathogens and tolerance of the commensal
microbiota, is among the possible causes of IBDs [89]. Extracts from engineered high-anthocyanin and
high-flavonol tomatoes were able to inhibit (significantly more than non-anthocyanin tomato controls)
the epithelial secretion of a set of pro-inflammatory cytokines and chemokines mediating interaction
between the gut epithelium cells and the underlying mucosal dendritic cells (DCs), which are effectors
of both innate and adaptive immune responses [88]. This had the effect of reducing influx and
accumulation of DCs into the gut mucosa, a process which plays a key role in the pathogenesis of
Antioxidants 2020, 9, 1017 11 of 17

colitis [90]. Moreover, flavonoids from engineered tomatoes significantly reduced the activation of
SAPK/JNK and p38 MAPK pathways [88], both important in the regulation of pro-inflammatory
responses in IBDs [91–93]. In the end, anthocyanins and flavonols modulated epithelial cells to become
hyporesponsive to bacterial stimulation.
Cardiovascular protection. Several recent epidemiological studies have demonstrated that
the consumption of polyphenol-rich foods may induce beneficial effects in pathways related to
cardiovascular health. Anthocyanin intake, in particular, positively correlated with a reduction
in cardiovascular risk [94], both improving vascular function and reducing atherosclerotic plaque
development [95–97]. As is the case for other beneficial effects, the cardiovascular protection exerted by
anthocyanins was firstly ascribed to their antioxidant nature, allowing them to delay the progression of
atherosclerosis due to the scavenging activity against the oxidation of low-density lipoprotein cholesterol
and associated inflammatory processes [98]. However, anthocyanins seem to be involved in more
complex molecular mechanisms. Recent studies have demonstrated their ability to reduce the adhesion
of human circulating monocytes to inflamed endothelial cells, which represents one of the initial steps
of the atherosclerotic process [99]. Moreover, increasing evidence has indicated that anthocyanins
can also modulate the expression of genes encoding enzymes involved in antioxidant defences or the
expression of microRNA acting in the regulation of cellular processes like inflammation and apoptosis.
They were also found to regulate cell-signalling pathways, e.g., based on phosphorylation events [100].
In a study carried out by Blando and colleagues [10], purified anthocyanin samples (PASs),
differing in composition from various plant materials including SB purple tomatoes, were compared in
terms of radical scavenging and anti-inflammatory activities in human endothelial cells. A model of
vascular inflammation, represented by cultured human microvascular endothelial cells-1 treated with
the pro-inflammatory mediator TNF-α, was used in the study. Endothelial cells were pre-exposed to the
different PASs, and the relative anti-inflammatory properties were evaluated by measuring their ability
to inhibit TNF-α-stimulated expression of two endothelial adhesion molecules, namely the vascular
cell adhesion molecule-1 and the intercellular adhesion molecule-1, two crucial vascular inflammatory
antigens [101]. All the different PASs showed anti-inflammatory properties, but the anthocyanin
chemical structures affected the results [10]. Anthocyanins were more effective when non-acylated
(e.g., PASs from mahaleb cherry and blackcurrant) than when acylated (e.g., PASs from black carrot
and SB tomato). These differences could be partially explained considering the relative antioxidant
capacities, since the non-acylated anthocyanins showed the highest activities. However, since the
different PASs were administered at the same concentrations, the lower molar concentrations of the
acylated anthocyanins (characterized by higher molecular weights) could have in part affected their
efficacy. In any case, anthocyanins showed anti-inflammatory and anti-atherosclerotic effects in this
study, supporting the previous evidence on the positive roles they may play in cardiovascular protection.

4. Conclusions
Although the pharmacological effects of the specific classes of anthocyanins accumulated in purple
tomatoes have not been completely characterized and the number of epidemiological studies carried out
with this “new” source of anthocyanins is still low, the first results obtained in both in vitro and in vivo
research studies nevertheless support what was already known from larger and more consolidated
works (Figure 3). Anthocyanin intake from purple tomatoes, particularly from the breeding lines able
to accumulate the pigments only in the peel, cannot reach the amounts characterizing other traditional
sources, such as berries. However, tomato consumption is high and almost daily in many human
diets, thanks to both fresh and processed products. Purple tomato consumption could thus easily
guarantee a habitual anthocyanin supply without changing eating habits and in a rich food matrix
context, which can offer extra values for the synergistic actions of all its phytochemical components,
certainly not obtainable through pharmacological supplements. All the experimental results obtained
so far thus appear very promising for the valorisation of purple tomatoes as a functional food.
Antioxidants
Antioxidants 2020,
2020, 9,
9, x1017
FOR PEER REVIEW 12
12 of
of 17
17

Figure 3. Overview of the positive effects carried out by anthocyanins from purple tomato lines as
health beneficial molecules. Results effects
from incarried
vitro and 1
Figure 3. Overview of the positive outinbyvivo studies published
anthocyanins in recent
from purple years:
tomato lines[60];
as
2 [52]; 3 [62]; 4 [64]; 5 [86]; 6 [87]; 7 [88]; 8 [10].
health beneficial molecules. Results from in vitro and in vivo studies published in recent years: 1 [60];
2 [52]; 3 [62]; 4 [64]; 5 [86]; 6 [87]; 7 [88]; 8 [10].
Future research in the plant biology field will be needed to further increase the amount of
anthocyanins in tomato
Author Contributions: S.G.fruits, inducing,
and P.P. conceived for
theexample, their
review; S.G. biosynthetic
drafted pathway
the paper; and P.P.also in thecritical
provided flesh
(which could allow the use of purple tomatoes not only as fresh but also
feedback. All authors have read and agreed to the published version of the manuscript. as processed products) or
making more uniform their synthesis on the peel [5]. Moreover, it can be envisaged that additional
Funding: This research received no external funding.
studies will be carried out on the nutraceutical value of these vegetables, with new and more in-depth
Acknowledgments: We acknowledge
research on the peculiar mechanisms L.E.ofPereira
actionPeres from
of the Universidade
anthocyanin de Sãothey
fraction Paulo, Brazil, as
contain, forwell
kindly
as
providing us with seeds of the Aft/Aft atv/atv hp2/hp2 line in MT background.
preclinical and clinical trials to better evaluate their health beneficial effects.
Conflicts of Interest: S.G. and P.P. are among the authors of the “Solenero” tomato varieties, derived from the
Author
Sun Contributions:
Black S.G.material.
original breeding and P.P. conceived the review; S.G. drafted the paper; and P.P. provided critical
feedback. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
References
Acknowledgments: We acknowledge L.E. Pereira Peres from Universidade de São Paulo, Brazil, for kindly
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