Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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COMPOSITIONS AND METHODS TO ADD VALUE TO PLANT PRODUCTS, INCREASING
THE COMMERCIAL QUALITY, RESISTANCE TO EXTERNAL FACTORS AND POLYPHENOL
CONTENT THEREOF
Field of the Invention
The invention is related to compositions and methods that protect against
ultraviolet radiation,
thus giving protection against sunburn to plants, plant parts, fruits and/or
flowers during their
development. The invention is also related to compositions and methods to
naturally improve the
color of plants, plant parts, fruits and/or flowers by inducing the natural
synthesis of flavonoids
and anthocyanins present in plants. Likewise, the present invention is
directed to improving the
nutritional value of plants, plant parts, fruits and/or flowers by increasing
the normal levels of
polyphenolic compounds, especially flavonoids, present therein. Additionally,
the present
invention is related to compositions and methods that give more resistance to
plants, plant parts,
fruits and/or flowers against pathogens as bacteria and fungi. Finally, the
present invention is
related to plants, plant parts, fruits, flowers and/or propagating material
treated with the
compositions described in the present document.
Background of the Invention
A suitable color development, which normally occurs simultaneously with
ripening, is one of the
most important parameters that affect the commercial value of fruits. In fact,
a large percentage of
produced fruit loses its commercial value due to the lack of quality standards
caused by sunburn
and/or lack of good color. Sunburn is generated while fruit is still on the
tree and is exposed to
particularly high amounts of solar radiation and high temperatures. Various
degrees of sunburn
can be distinguished on fruit, ranging from a slight discoloration of the
natural fruit pigment to a
severe burn that completely destroys (ulcerates) the plant tissue, in the
worst case. When
sunburn is present in produced fruit, either in a slight or maximum degree,
the producer finds the
sell price of its production dropping dramatically in all markets, being
impossible to export said
fruit due to consumer market norms and therefore the producer cannot
participate of the market
that yields the largest returns. Sunburn is not the only problem that affects
fruit production, as
crops are constantly at risk due to pathogens as fungi and bacteria. To fight
this problem,
producers need to use diverse agrochemical products in order to guarantee a
good development
of their future crops. Nevertheless, now the world market tendency is to be
increasingly more
cautious to accept the use of pesticides or fungicides on crops.
Simultaneously, each year the
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world demand for healthy, pesticide-free and chemical-free food, as fruits and
vegetables, is
growing. Likewise, the interest for functional food, i.e. food having
beneficial properties for human
health, is also growing. Therefore, it would be very advantageous to have a
product that naturally
protects crops against these pathogens, simultaneously adding nutritional
value to the food that
consumers are increasingly eager to incorporate to their diet.
Part of human diet includes fruits and vegetables that in the whole contribute
with vitamins, fiber,
sugars, lipids, antioxidants and the like. One of the antioxidants produced by
plants in large
amounts to protect themselves against oxidative damage caused by UV radiation
or other
environmental aggressions is the group of molecules known as flavonoids.
Anthocyanins are one
subtype of flavonoids. They are molecules with red, blue or violet color that
are present in plant
tissues and are responsible for the characteristic colors of many ripe fruits.
A flavonoid-rich diet
helps removing free radicals from the body, retarding natural aging processes
and fighting cancer
development in the body, among many other health benefits. As many flavonoids
are unstable
molecules that do not resist cooking, it is recommended to consume them in the
diet as fresh
fruits and vegetables. Given the abovementioned facts, it would very
interesting to have a way to
improve the quality and amount of flavonoids available in plant food consumed
by humans.
The present invention is directed to compositions and methods to decrease the
harmful effects of
solar radiation on plants, plant parts, flowers and fruits, also improving the
color thereof and,
additionally, increasing the nutritional value of plants by increasing the
natural synthesis of
antioxidants. This could imply a direct increase of the value and quality of
fruit and the net income
for the producers. The present invention proposes, definitely, the use of
novel compositions for an
original purpose.
Sunburn is caused by a combination of excessive heat and a high dose of UV
radiation. In order
to prevent or decrease sunburn or sun damage, plants, plant parts, fruits
and/or flowers are
sought to be protected against the harmful effects of heat and excessive UV
radiation. If harmful
UV effects are to be avoided, excess radiation can be reflected or screened by
using clays or, in
the other hand, said radiation excess can be absorbed by using chemical
filters as waxes or
some of the compounds of the present invention. Additionally, if the desired
effect is a decrease
in temperature, it can be achieved by shadowing the trees or by spraying water
to cool the
orchard by evaporation, as explained hereinafter. At present, some methods are
known to protect
plants (fruits) against sunburn, which are based in different indirect
methodologies. Next, known
commercial products of the present atate of the art that offer protection
against sunburn are
mentioned:
"Raynox ~- : Their manufacturers define it as a product that is applied over
the tree (fruits), which is
based on UV-absorbing plant waxes (carnauba wax). This wax deteriorates with
solar radiation
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as time goes by, and due to this reason it is necessary to apply the product
many times to
achieve a notorious positive result.
"Surround .- : Their manufacturers define it as a reflecting product that is
applied over the tree
(fruits), where it screens UV and general solar radiation. This product is
made from kaolin
(mineral clay) as a suspension of finely divided white clay and it does not
only reflect incident
radiation on the fruits but also radiation that reaches the tree leaves. This
obviously decreases
the efficiency of the photosynthetic processes of the tree, which affects
their own growth and the
growth of its fruits, and also the general health of the tree. This clay
suspension can also limit tree
respiration because of stoma blockage. All this effects cause a higher general
weakness of the
plant and/or a decrease of fruit quality or amount, both in their color as in
their size. In the other
hand, it is possible that fruit growth causes the clay layer to break, thus
losing a part of its
protective ability, also requiring many applications during fruit growth and
ripening.
"Sunshield~- : Their manufacturers define it as a product that is applied over
the tree (fruits),
where it filters UV radiation. This product is described as a biodegradable
propolymer protein
micro-layer. Sunshield's principle of action is similar to that of Raynox.
"Kool-Kore ": Their manufacturers define it as a product that is applied over
the tree (fruits),
based on silica and surfactants. This product has a principle of action
similar to Surround's.
Another product is defined as secret mixtures of calcium carbonate, slime and
clay. These
mixtures are reflecting clays, and accordingly they operate using the same
principle of action as
Surround.
Another way to avoid harmful effects of solar radiation is by using protective
meshes that filter
solar light, thus shadowing the trees. Meshes are put over the trees in such a
way that they
shadow the fruits, by which it is possible to achieve a temperature reduction
in the orchard,
simultaneously reducing incident radiation over the trees. These meshes are
expensive and the
lower amount of light also affects negatively photosynthesis and fruit
coloration processes.
Another technique used to reduce sunburn in fruit is water aspersion over the
canopy of trees or
between them during the hours of higher solar irradiation, in order to
decrease the temperature of
the fruit. Nevertheless, this option requires an expensive installation,
constant water spraying
removes agrochemical products previously applied over the trees and the
increase in
environmental humidity favors the development of plagues and weeds, affecting
as a whole the
phytosanitary status of the orchard.
As set forth hereinabove, sunburn is caused by a combination of excessive heat
and a high dose
of UV radiation. In order to prevent or decrease sunburn or sun damage,
plants, plant parts, fruits
and/or flowers are sought to be protected against the harmful effects of heat
and excessive UV
radiation. Therefore, as exposed when previous art was discussed, both
products and methods to
reflect or screen said radiation excess, or products that can absorb said
radiation excess have
been proposed.
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It is well known that the ability of a compound to absorb UV radiation is
directly related to its
molecular structure. Thus, UV absorbing capacity is given by the presence of
conjugated double
bonds, e.g. in an aromatic structure. As the number of unsaturations in an
aromatic molecule
increases, the maximum wavelength said molecule can absorb decreases. UV
radiation is
invisible to the human eye and has a short wavelength comprised between 200
and 380 nm. The
compositions disclosed in the present invention include compound derived from
phenolic acids
and preferably derived from the cinnamic acid skeleton. These compounds have a
molecular
structure that is able to absorb UV radiation by itself. Even when the ability
of these molecules to
absorb UV radiation is limited, these molecules play also an important
protective role in living
tissues as scavengers of free radical produced by diverse oxidative stress
processes. Once one
or more molecules derived from cinnamic acids has captured a harmful free
radical, far from
being inactivated, it can bond to one or more molecules of its own kind, i.e.
another cinnamic acid
derivative, to form new dimeric, trimeric or oligomeric structures. These new
dimeric, trimeric or
oligomeric structures have an even higher ability to absorb UV radiation and
play even more
important roles in the structure of plant cells, mainly as part of the
hemicellulose and lignin
structure of plant cell walls. The cinnamic acid derivatives that mainly play
this structural and UV
absorbing functions as part of lignin in plants are ferulic acid derivatives,
caffeic acid derivatives
and sinapic acid derivatives.
Additionally, obtaining fruit with a good coloration is as important for the
producer as obtaining
fruit with no sunburn damage. For some fruit, apples for example, color is the
main feature that
determines their market price, provided that there is no sunburn damage,
surface bruises or other
physiological disorders. Some of the techniques used at the present time to
manage color in fruits
include increasing the dose of potassium, magnesium or other oligoelements in
fertilizers applied
in the last stage of fruit development and/or stressing the tree by decreasing
water supply, which
affects the final color of the fruit, but limits its growth (size) and makes
the treatment not always
satisfactory.
In the past, Alar (daminozide) was used to increase color in fruit. Daminozide
was used in some
crops, mainly apples and ornamental plants, to improve the balance between
vegetative growth
and fruit production, improve fruit quality and synchronize fruit ripening. In
1989, a by-product of
daminozide, called UDMH (unsymmetrical dimethylhydrazine), was found to be
carcinogenic and
therefore daminozide was classified as a moderate carcinogen and its use was
forbidden for fruits
or food products in many countries, allowing it to be sold only for use in
ornamental plants.
At the present time, there is another phytostimulant alternative product
(ethephon, 2-
chloroethylphosphonic acid) that presents as a side effect the promotion of a
better color in fruit,
but this feature is associated to a higher ripening of said fruit. As
aforementioned, fruit having
better color reaches higher market values, but over-ripened fruit has a
shorter conservation time
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before having to be discarded. Likewise, in Patent Application ES 2,137,893, a
composition able
to stimulate the development of color in skin and flesh of fruit and
vegetables is disclosed. Said
composition comprises 1 to 90% of a compound that contribute phosphite anion,
1 to 10% of
methionine or a salt thereof, and 1 to 10% of glycine or a salt thereof.
Nevertheless, the extra
contribution of phosphite anion has some drawbacks such as the contribution of
a
disproportionate phosphorus amount to the plant in forms other than phosphate
and possible
environmental problems due to accumulation of non-assimilable and non-
degradable phosphorus
in soil.
As no available product is able to substantially improve color quality in
fruits, there is also no
marketed product able to improve color both in fruits and flowers. The
possibility of having a
product able to increase or improve flower coloration is therefore very
interesting. A good color in
flowers, such as roses or tulips, but not limited to those, is fundamental to
achieve a good
exporting market price. The present invention also offers an alternative able
to improve color in
flowers without stressing or genetically modifying the plant. For this, it is
necessary to understand
the mechanisms by which plants develop their colors. In plants there are 3
families of pigments
that give rise to all colors found in the plant kingdom. Green, yellow and
brown colors are given
by chlorophyll-like molecules; the major part of yellow, orange and some red
colors are produced
by pigments known as carotenes; and blue, violet and most red colors are the
result of a family of
pigments known as anthocyanins.
The compositions of the present invention include phenolic acids and, more
specifically, cinnamic
acid derivatives, among which are p-coumaric acid, ferulic acid, caffeic acid
and sinapic acid.
Coumaric and ferulic acids constitute the major part of phenolic acids that
are present in plant cell
walls (Jung [1989], "Forage Lignins and their effect on fiber digestibility".
Agron J 81: 33-38).
Phenolic acids exist in plant cell walls as monomers joined by ester and ether
bonds, as sterified
dimers and as crosslinked ester and ethers between polysaccharides and lignin
(D. Deetz [1993],
"Impact of Methyl -0-(E)-Feruloyl-(x-L-Arabinofuranoside on In-vitro
Degradation of Cellulose and
xylan". J Sci Food Agric 61:423-427), playing structural roles by
strengthening the plant cell wall
and making the plant more resistant to pathogen attack. As the crosslinking
between phenolic
acids belonging to different polysaccharide chains (normally hemicellulose)
increases, the
mechanical strength of the plant cell wall increases accordingly, as it
becomes more tough and,
being more crosslinked and structurally complex, it acquires more resistance
against pathogen
attack. Some phenolic acids and/or their derivatives are not digestible or
even toxic for many soil
or rumen microorganisms (C. Faulds [1991], "The purification and
characterization of 4-hydroxy-
3-methoxycinnamic (ferulic) acid esterase from Streptomyces olivochromogenes".
Journal of
General Microbiology 137: 2339-2345), and therefore their antibiotic action is
due both to the
physical strength increase that they cause in the plant cell wall and to the
toxic effect they have to
certain bacteria. Amongst phenolic acid derivatives, cinnamates or cinnamic
acid derivatives are
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mostly interesting, as they are precursors in hemicellulose chain
lignification pathways during
plant growth and ripening. It has been reported that cinnamic acid
derivatives, particularly
cinnamic acid esters, have antifungic properties ("Synthesis and antifungal
activity of cinnamic
acid esters". Biosci Biotechnol Biochem, 1996, 60(5): 909-10). Cinnamates,
particularly ferulic
acid, can be used as food preservatives because of their ability to inhibit
the oxidation of fatty
acids and other molecules, aside from their function as antibiotics and/or
antifungics, and they
has been used as the active ingredient of some tanning lotions. Additionally,
ferulic acid has been
described to decrease the side effects of chemotherapy and radiotherapy, and
further shows
strong anti-inflammatory properties (C. Faulds [1997], "Novel
biotransformation of agro-industrial
cereal waste by ferulic acid esterases". Industrial Crops and Products 6: 367-
374).
As previously discussed, it is generally well known that UV radiation induces
molecular level
damage inside cells, mainly through two different and interdependent
mechanisms. The first
mechanism is direct damage caused to biomolecules through covalent bond
breakage, which
alters or destroys the biological function thereof. The second damage
mechanism is produced
through the interaction of UV radiation and electrons of organic molecules.
This interaction breaks
molecular bonds and generates free radicals, which auto-propagate in a chain
reaction wherein
each new step gives rise to new free radicals from intact molecules, thus
causing a massive
destruction of the organic molecules of the cell.
It is well known that in plants there is a large variety of secondary
metabolites that play a
protective role against external aggressions. These external aggressions may
be caused by UV
radiation, free radical generation, hydric stress, attack of pathogenic
agents, herbivorous animals
attack, etc. One of the more abundant groups of secondary metabolites having a
protective role in
the cell is the group of flavonoids. Among them, aside from this general role
in the entire plant
kingdom, some sub-families of these compounds play specific non-protective
roles (for example,
pigments), and some flavonoid molecules have different functions inside each
particular plant. In
their chemical structure, biosynthetic pathways and functional role,
flavonoids themselves are
part of and are tightly related to other more general group of compounds,
called in general
phenolic compounds, also abundant in plants, either as primary metabolites
(for example,
tyrosine and phenylalanine) or secondary metabolites. The most relevant
phenolic compounds in
plants are shown in Table 1, along with their basic carbon skeleton (Harborne,
J. B., T. J. Marby,
H. Marby: "The flavonoids". London: Chapman and Hall, 1975).
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Table 1: Most relevant phenolic compound classes in plants
Number of carbon atoms Basic skeleton Class
6 C6 simple phenols, benzoquinones
7 C6 - C, phenolic acids
8 C6 - C2 acetophenones, phenylacetic
acids
9 C6 - C3 hydroxicinnamic acids,
polypropenes, coumarins,
isocoumarins
C6 - C4 naphtoquinones
13 C6 - C, - C6 xanthones
14 C6 - C2 - C6 stilbenes, anthraquinones
C6 - C3 - C6 flavonoids, isoflavonoids
18 (C6 - C3)2 lignans, neolignans
30 (C6 - C3 - C6)2 biflavonoids
n (C6 - C3)n lignins, catecholmelanines
(C6)n (condensed tannins)
(C6 - C3 - C6)n
The starting product for the biosynthesis of most part of the phenolic
compounds, including
flavonoids, is shikimate. Phenols are acid owing to the dissociation of their -
OH group. They are
fairly reactive compounds and, as no steric hindrance due to side chains is
present, they can form
hydrogen bonds. In this way, many flavonoids have intramolecular bonds.
Another relevant
feature is their ability to form chelate complexes with metals. They are also
easily oxidizable and
when subjected to oxidation they generate polymers (dark aggregates). The
browning of cuts or
dead parts in plants is due to this reaction. Flavonoids have generally an
inhibitory effect over
plant growth. Among low molecular weight phenylpropanoid derivatives there is
a variety of
essences as coumarins, cinnamic acid, sinapinic (sinapic) acid, coniferyl
alcohol and others.
These substances and their derivatives are at the same time intermediates of
lignin biosynthesis,
where they are especially useful for their ability to polymerize and thus
covalently crosslinking
hemicellulose fibers.
Often, phenolic compounds, including flavonoids, are not free in plant
tissues. In their major part,
they are coupled to other molecules, most frequently with carbohydrate
moieties (glycosylated),
but they are also found coupled to sulfate or acetyl moieties. It is thought
that one of the
fundamental reasons for that is their toxicity in free state, as they are
detoxified, at least in part,
when coupled. Many low molecular weight compounds (e.g. thymol) are used in
medicine as
antiseptics because of their toxicity. The different types of bonds between a
flavonoid molecule
(for example, an anthocyanidin) and a glycosidic residue, lead to different
derivatives that
increase the color spectrum of flowers (and also their tonalities). Flavonoid
glycosylation has an
additional effect, a not less important function from an ecological point of
view: it has been put
into evidence their connection with protection against pests and other
animals. Based on their
biological functions, phenolic compounds can be classified as shown in Table 2
(Harborne, J. B.,
T. J. Marby, H. Marby: "The flavonoids". London: Chapman and Hall, 1975).
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Table 2: Ecological significance of some phenolic compounds in plants
Function Group Examples of plant species where
the effect has been studied
flower pigments anthocyans Cyanidin-3,5-diglucoside in Rosa
chalcones Coreopsin in Coreopsis tinctoria
aurones Aureusin in Anthirrhinum majus
yellow flavonoids Gossypetin-7-glucoside in
Gossypium
flavones Apigenin-7-glucoside in Bellis
perennis
fruit pigments anthocyans Petunidin glucoside in Atropa
belladonna
isoflavones Osajin in Maclura pomifera
chalcones Okanin in Kyllinagi brevifolia
allelopathic quinones Juglone in Juglans regia
substances phenols Hydroquinone in Arctostaphylos
phenolcarboxylic Sialic acid in Quercus falcata
acids
hydroxicinnamic acids Ferulic acid in Adenostoma
pest protection quinones Juglone in Carya ovata
tannins Gallotannin in Quercus robur
flavonols Quercetin glycosides in Gossypium
fungicides isoflavones Luteone in Lupinus
phenolcarboxylic Protocatechuic acid in Allium
acids
dihydrochalcones Phloridzin in Malus pumila
phytoalexins stilbenes Resveratrol in Arachis hypogaea
phenylanthrenes Orchinol in Orchis militaris
isoflavans Vestitiol in Lotus corniculatus
pterocarpans Pisatin in Pisum sativum
phenylpropanoids Coniferyl alcohol in Linum
utilissimum
furocoumarins Psoralen in Petroselinum crispum
The basic structure of flavonoids is derived from the C15 body of flavone.
They differ from other
phenolic substances in the oxidation degree of the central piran ring and,
more fundamentally,
also in their biological functions. While some flavonoid classes are colorless
(flavanones, for
instance), other classes' members (anthocyans, for example) are always colored
and are known
as pigments of flowers and other plant parts. Anthocyans are normally red or
yellow, their color
depending on pH. Blue pigments are obtained through the formation of chelates
with some
metallic ions (Fe3+ or AI3+, for instance).
Flavonoids in general are divided in subfamilies of compounds. The most
important flavonoid
molecule classes are shown in Table 3, altogether with their biological
significance (Harborne, J.
B., T. J. Marby, H. Marby: "The flavonoids". London: Chapman and Hall, 1975).
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Table 3: Most important flavonoid classes and their biological function in
plants
Class Number of Biological significance (as
representative presently known)
members
Anthocyanin(s) 250 red and blue pigments
Chalcones 60 yellow pigments
Aurones 20 yellow pigments
Flavones 350 cream colored pigments of
flowers
Flavonols 350 feed deterrents for
herbivores in leaves and UV
filters
Dihydrochalcones 10 some of them taste bitter
Proanthocyanidins 50 astringent substances
Catechins 40 some of them have
properties similar to tannins
Biflavonoids 65 protective and pathogen
defensive function
Isoflavonoids 15 estrogenic effect, toxic for
fungi
The variability of flavonoids is based mainly in the hydroxilation and/or
methylation pattern of the
three ring system. A correlation between two flavonoids often points out to a
relationship between
the plant species that produce them. For this reason, they have proven to be
suitable characters
for the study of phylogenetic relationships between superior plants.
The flavonoid biosynthetic pathway is one of the most studied metabolic
pathways in the plant
kingdom, its study being started in 19t" century with the isolation of the
first anthocyanins and
flavonols. From then on, the pathway has been generally characterized for many
plants.
Nevertheless, even in our days this biosynthetic pathway has not been
characterized in its
entirety for any species, as each particular species produces different
molecules depending on its
own genetic information and its particular set of enzymes. A schematic
representation of the
general biosynthesis pathway of the most relevant and/or best characterized
flavonoids as far as
it is known in the present art, is presented in Scheme 1 (extracted from KEGG,
Kyoto
Encyclopedia of Genes and Genomes).
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Scheme 1
OH OH
Metabollsmo Biosintesisdelignina
detenilalanina HO~I CoAS
'Ir pcumaroil-CoA
O O
Acido pcumarico IB
Isotlavo as "~ OH
OH OH HO O
HO O HO OH
OH
OH
OH OH 0 Naringeninachalcona(4)
I Atzelechina (7)
Apigeninidina
OH c
OH OH OH OH
HO O ,aa D HO O aaE HO O .a D HO O aHO
~ K ~ ~OH -- ~OH F - OH
OH OH OH 0 OH 0 OH OH OH
Apitorol(6) Naringenina (8) Dihidrokaempterol(5) cis-3,4-Leucopelargonidina
Pelargonidina(1)
(2)
OCH3 G OH OH
O
HO O J H HO O I HO OH -cr I -- Rutina
OH OH (3)
OH O H I OH C OH O
Acacetina(9) puercetina(3)
OH OH OH OH
HO0 HO_ O` aOH OH F HO OH
H OH
OH O H OH O `L O H
Apigenina(9) Dihidroquercetina(5) (2) y "~OH Cianidina(1)
O ` O OH E ~OH
H OH OH
H HO O OH Catequina(7) HO O OCH3
OH OH OH OH
OH O OH
HO O OH HO Ola OH
J ~ I.õ Miricetina(3) Peonidina(1)
OH O ~TOJH~~O/
OH OH OH
Luteolina(9) Eriodictiol(e) OH OH OH F
1 H K D HO O~OH ~- O o~OH - OH
OH IOH ~OH OH OH
HO O HO Oõaa OH O OH OH OH
OCH3 OH Dihidromiricetina(5) Leucodeltinidina(2) Deltinidina(1)
OH O OH OH L
3'-O-Metil-luteolina(9) Luteotorol(6) OH OCH3 OCH3
OH OH OH OH
HO O o OH HO OCH3 HO OH
HO OH H E
OH OH
OH
H OH OH OH
OH Galocatequina(7) Malvidina(1) Petunidina(1)
Luteolinidina
OH OH
OH O
HO O OH OH
OH
OH O OH O
Tricetina (9) Pentahidroxitlavanona (8)
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In Scheme 1, different representative classes of flavonoids are shown with the
following number
key, which is printed after the particular name of each molecule: (1)
anthocyanidins, (2)
leucoanthocyanidins, (3) flavonols, (4) chalcones, (5) dihydroflavonols, (6)
flavan-4-ols, (7) flavan-
3-ols (catechins), (8) flavanones, (9) flavones, and other compounds.
It is necessary to note that, according to the present knowledge, the entire
flavonoid biosynthetic
pathway naturally begins with p-coumaric acid and Frcoumaroyl-CoA. This
compound is directly
derived from phenylalanine and tyrosine metabolism, and it is also the
entrance point for the plant
lignin biosynthesis pathway. In this biosynthetic pathway, p-coumarate is
transformed also in
other particular derivatives of cinnamic acid, especially cinnamic acid
itself, ferulic acid, caffeic
acid, 5-hydroxiferulic acid and sinapic acid, and their corresponding
aldehydes and alcohols.
Nevertheless, the present state of the art considers that these and other acid
residues do not
participate as major intermediates in flavonoid biosynthesis.
As it can be observed in Scheme 1, the abovementioned large variability of
molecular structure of
flavonoids in the plant kingdom is due to the action of a relatively small
group of enzymes over
the substrates available in the cell along the flavonoid biosynthetic pathway.
Furthermore, it is
evident that there is an interaction between groups of reactions, mediated by
enzymes that
simultaneously participate in two or more reactions. In Scheme 1, the 12 major
known enzymes
that participate in the flavonoid biosynthesis pathway are represented as
follows:
A : EC 6.2.1.12, 4-coumarate-CoA ligase (4CL)
B : EC 2.3.1.74, chalcone synthase (CHS)
C : EC 5.5.1.6, chalcone isomerase (CHI)
D : EC 1.1.1.219, dihydroflavonol reductase (DFR)
E : EC 1.14.11.9, flavanone 3-dioxygenase (FHT)
F : EC 1.14.11.19, leucocyanidin oxygenase (ANS)
G : EC 1.14.11.23, flavonol synthase (FLS)
H : EC 1.14.13.88, flavonoid 3',5'-hydroxilase
I : EC 1.14.13.21, flavonoid 3'-monooxygenase
J : EC 1.14.11.22, flavone synthase
K : EC 1.1.1.234, flavanone 4-reductase
L : EC 1.17.1.3, leucoanthocyanidin reductase
Furthermore, in Scheme 1 it is possible to observe the participation of many
of these enzymes in
different reactions, where substrates differ in the hydroxilation or
methylation degree of the ring
system. Bold arrows indicate reactions catalyzed by enzymes that accept
substrates with different
substituents in the phenyl ring, especially with no substituents (cinnamate
derivatives) or 4-
hydroxi substituted (coumarate derivatives), 4-hydroxi-3-methoxi substituted
(ferulate derivatives),
3,4-dihydroxi substituted (caffeate derivatives), 4,5-dihydroxi-3-methoxi
substituted (5-
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12
hydroxiferulate derivatives), 3,4,5-trihydroxi substituted (5-hydroxicaffeate
derivatives) and 3,5-
dimethoxi-4-hydroxi substituted (sinapate derivatives).
The specificity of each particular enzyme is given by its own molecular
structure and this one
depends in its turn on the species that produces it. In this way, each species
"selects" its
enzymes having varied specificities, which produce a particular and unique
flavonoid set in a
characteristic proportion between their components that is specific for each
species.
Many experiments have been made by altering the expression of these enzymes in
plants at the
level of their genetic sequence. Specifically, gene knock-out tests and over-
expression
experiments has been made with some of these enzymes, normally observing a
final effect in the
concentration and proportion of the different flavonoid molecules produced by
the plant tissue.
Especially, many experiments have been made that report changes in the color
of plant tissues,
both in color tonality and shade, when the function of one of the enzymes of
the metabolic
pathway for anthocyanin biosynthesis is genetically altered. See as an
example, the works of
Saito and Yamazaki (2002), New Phytologist 155:9-23; Gollop, Even, Colova-
Tsolova and Perl
(2002), Journal of Experimental Botany 53(373):1397-1409; Bruce, Folkerts,
Garnaat, Crasta,
Roth and Bowen (2000), Plant Cell 12:65-79; Winkel-Shirley (2001), Plant
Physiology 126:485-
493; Winkel-Shirley (2001), Plant Physiology 127:1399-1404; Muir, Collins,
Robinson, Hughes,
Bovy, De Vos, van Tunen and Verhoeyen (2001), Nature Biotechnology 19:470-474;
DellaPenna
(2001), Plant Physiology 125:160-163; Zhang, Franco, Curtin and Conn (2004),
Journal of
Biomedicine and Biotechnology 5:264-271; and Rosati, Cadic, Duron, Renou and
Simoneau
(1997), Plant Molecular Biology 35(3):303-311. Furthermore, part of the
anthocyanin biosynthetic
pathway has been cloned in Escherichia coli for the production of anthocyanins
and flavonoids in
microorganisms (Yan, Chemler, Huang, Martens and Koffas (2005), Applied
Environmental
Microbiology 71(7) :3617-3623).
In this invention, we have surprisingly found that it is possible to modulate
the flavonoid
biosynthetic pathway by applying in situ phenolic compounds to living plant
tissues, without
genetically altering the expression of enzymes that participate in the
corresponding pathways and
without altering the genetic sequence of the producing plant. More
surprisingly, it has been found
that the external application to said tissues of some compounds that are not
part of the flavonoid
biosynthesis pathway, e.g. belonging to the lignin biosynthesis metabolic
pathway, have a
modulatory effect over metabolic routes that lead to flavonoid synthesis. In
this way, phenolic
compounds that can be used for the purposes of this invention can be part of
the flavonoid
biosynthesis pathway or not, and said phenolic compounds can even be molecules
that are
completely different to those molecules naturally found in the treated plant.
Without losing
generality, it is believed that these different compounds could be
incorporated in the flavonoid
biosynthesis reactions, as the enzymes that participate in said reactions
could be able to accept
substrates (or inhibitors) other than their natural substrates, with a
variable degree of specificity.
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13
In this way, the plant would be able to generate flavonoids in larger amounts
and in different
ratios. It is believed that the plant could also synthesize flavonoid
molecules that are not naturally
present in the plant, by starting from the applied compounds.
Surprisingly, the compounds used in the formulations and applied according to
the methods of
the invention have demonstrated to be able to cross the waxy layer that
protects the plant,
especially over fruit skins, and have also demonstrated to be able to cross
the cell membrane.
Without losing generality, it is believed that, in this way, the applied
compositions can modulate
biosynthetic pathways inside the cells, especially the flavonoid biosynthesis
pathway, and could
participate in the reactions of said metabolic routes and modulate the
function of the different
enzymes that participate in said reactions.
Brief Description of the Invention
The present invention is directed to compositions and protection methods
against sun produced
damage, improving and increasing color in plants and increasing the
nutritional value of plants,
plant parts, flowers and/or fruits through a change or increase of the content
of polyphenolic
compounds thereof, especially flavonoid content.
Said compositions have also antibiotic and antifungic properties associated to
some of their
components and induce antibiotic and antifungic properties that increase the
resistance of treated
plants against external pathogen and pest aggressions. These compositions are
also able to
increase the content of antioxidant compounds, especially polyphenols such as
flavonoids, that
can increase the nutritional value of the plants. The compositions of the
present invention are
similar to other agrochemical compositions and do not represent any risk
during their handling if
normal precautions for agrochemical products are minimally taken.
The present invention is also directed to protection methods against sunburn
(to decrease its
incidence) and to improve the color in plants, through the application of an
effective amount of the
compositions of the invention. Furthermore, the present invention is directed
to methods to
increase the content of polyphenolic compounds in plants, plant parts, flowers
and/or fruits,
through the application of an effective amount of the compositions of the
invention.
Furthermore, the present invention is directed to the plants, plant parts,
flowers and/or fruits
treated with the compositions of the invention.
Detailed Description of the Invention
In this invention, we have found that it is possible to modulate the flavonoid
biosynthetic pathway
by applying in situ compositions that comprise phenolic compounds to living
plant tissues, without
genetically altering the expression of enzymes that participate in the
corresponding pathways and
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14
without altering the genetic sequence of the producing plant. More
surprisingly, it has been found
that the external application to said tissues of some compounds that are not
part of the flavonoid
biosynthesis pathway, e.g. belonging to the lignin biosynthesis metabolic
pathway, have a
modulatory effect over metabolic routes that lead to flavonoid synthesis. In
this way, phenolic
compounds that can be used for the purposes of this invention can be part of
the flavonoid
biosynthesis pathway or not, and even said phenolic compounds can be molecules
that are
completely different to those molecules naturally found in the treated plant.
Without losing
generality, it is believed that these different compounds could be
incorporated in the flavonoid
biosynthesis reactions, as the enzymes that participate in said reactions
could be able to accept
substrates (or inhibitors) other than their natural substrates, with a
variable degree of specificity.
In this way, the plant would be able to generate flavonoids in larger amounts
and in different
ratios. It is believed that the plant could also synthesize flavonoid
molecules that are not naturally
present in the plant. by starting from the applied compounds.
Surprisingly, the compositions applied according to the methods of the
invention have
demonstrated to be able to cross the waxy layer that protects the plant,
especially over fruit skins,
and have also demonstrated to be able to cross the cell membrane. Without
losing generality, it is
believed that, in this way, the applied compositions can modulate biosynthetic
pathways inside
the cells, especially the flavonoid biosynthesis pathway, and could
participate in the reactions of
said metabolic routes and modulate the function of the different enzymes that
participate in said
reactions.
In this invention, it has been found that the effects of the application of
these compositions and
methods of the invention affect the ability of plant tissues to resist
external aggressions.
Specifically, it has been found that fruit treated with these compounds are
more resistant to
damage produced by sunburn, which is caused, at least in part, by incident UV
radiation.
Furthermore, the fruit treated with the compositions and methods of the
invention develops better
color when compared with untreated fruit. Finally, the absence of sunburn
damage and the better
natural color development affect the aesthetic appearance of the fruit and
increase its commercial
value in a very dramatic way.
At last, without being limited, both the resistance against UV radiation and
the better color
development could be explained through an increase in the amount of flavonoids
and
anthocyanins produced by the treatments of the invention, aside from the
natural protective effect
of the compounds of the invention. Without being limited by this, it is
believed that the effect is
principally due to an increase of the concentration of flavonols (especially
quercetin in apples),
dihydrochalcones (especially phloridzin and phloretin in apples) and
anthocyanins (especially
cyanidin and paeonidin in apples). Therefore, the compounds of the invention
thus demonstrate
their modulating ability over flavonoid biosynthesis in vivo.
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An extra aspect of the treatments of the present invention is the increased
amount of flavonoid
molecules present in the plant tissues treated with the formulations and
methods of the invention.
This makes the treated plant, when included in human diet, a better source of
flavonoids, which
increases the nutritional value of the plant product, thus putting said
product into the category of
functional foods. Given the major contribution to human health represented by
a daily intake of
enough amounts of flavonoid compounds, plant products treated according to the
invention would
represent a substantial contribution in daily human diet, in comparison to an
untreated plant
product, thus improving the health state of the consumer. Furthermore, with
this, the treatment of
the invention allows increasing the commercial value of treated plant products
and makes it
possible to open new markets for the commercialization of said products.
Specifically, the compositions of the invention comprise at least one compound
of Formula I:
R4
R5 R3
x 2
R
Z R
Y
A
Formula I
and the salts, esters, ethers, solvates or isomers thereof, or their
corresponding 0- and/or N-
glycosides; wherein R' to R4 are independently selected from the group
consisting of
hydrogen, halogen, -OH, -OR6, -NH2, -NHR6, -NR6R7, -N=R8, -C(=O)OH, -C(=O)OR6,
-
OS(=O)nOH, -OS(=O)nORs, -S(=O)nOH, -S(=O)nORs, -SH, -SR6, -C(=O)H, -C(=O)R6, -
OP(=O)(OH)2, -OP(=O)(OH)(OR6), -OP(=O)(OR6)(OR'), -PH2, -PHR6, -PR6R', -P023 -
PO3
-P=R8, -NO23 -NO, -NH-NH2, -NH-NHR6, -NH-NR6R', -NHOH, -NR6OH, -NHOR6, -
NR6OR', -N2+, -N=NH, -N=NR6, -N=NOH, -N=NOR6, -N33 -CN, -CNS, -SCN, an alkyl
group, an alkenyl group, an alkynyl group, a cycloalkyl group, a cycloalkenyl
group, a
cycloalkynyl group, a heterocycloalkyl group, a heterocycloalkenyl group, a
heterocycloalkynyl
group, an aryl group, a heteroaryl group, an organometallic group, an 0-
glycosyl group or a N-
glycosyl group. Any of these aforementioned alkyl, alkenyl, alkynyl,
cycloalkyl, cycloalkenyl,
cycloalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, aryl
and heteroaryl
groups can be optionally and independently substituted with one or more
substituents selected
from the group consisting of halogen, -R6, -OH3 -OR6, -NH2, -NHR6, -NR6R', -
N=RB, -
6
C(=O)OH, -C(=O)OR, -OS(=O)nOH, -OS(=O)nORs, -S(=O)nOH, -S(=O)nORs, -SH, -SR6, -
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16
C(=O)H, -C(=O)R6, -OP(=O)(OH)2, -OP(=O)(OH)(OR6), -OP(=O)(OR6)(OR'), -PH2, -
PHR6,
-PR6R', -P02, -PO, -P=R8, -NO2, -NO, -NH-NH2, -NH-NHR6, -NH-NR6R', -NHOH, -
NR6OH, -NHOR6, -NR6OR', -N2+, -N=NH, -N=NR6, -N=NOH, -N=NOR6, -N3i -CN, -CNS
or -SCN;
R5 can be either the same as described for R' to R4 or it can be absent and Z
could be directly
bound to the carbon atom indicated in Formula I with an asterisk, thus forming
a 5- to 7-
membered ring, which could also be substituted with one or more independently
selected R9
groups and/or could be part of a fused ring system, optionally substituted
with one or more
independently selected R9 groups;
R6 and R' are independently selected from the group consisting of an alkyl,
alkenyl, alkynyl,
cycloalkyl, cycloalkenyl, cycloalkynyl, heterocycloalkyl, heterocycloalkenyl,
heterocycloalkynyl,
aryl, heteroaryl, organometallic, O-glycosyl or N-glycosyl group, which can be
also substituted
with one or more independently selected R9 groups;
R 8 is =0, =S, =NH, =NR6, =CH2i =CHR6, =CR6R';
R9 is selected from the group consisting of hydrogen, halogen, -OH, -OR'o, -
NH2, -NHR'o -
NR'oR", -N=R12, -C(=O)OH3 -C(=O)OR'o, -OS(=O)nOH, -OS(=O)nOR'o, -S(=O)nOH, -
S(=O),OR'o, -SH, -SR'o, -C(=O)H, -C(=O)R'o, -OP(=O)(OH)2, -OP(=O)(OH)(OR'o), -
OP(=O)(OR'o)(OR"), -PH2, -PHR10, -PR'oR", -P02, -PO, -P=R12, -NO23 -NO, -NH-
NH2, -
NH-NHR10, -NH-NR'oR" -NHOH, -NR10OH, -NHOR10, -NR'oOR", -N2+, -N=NH, -
N=NR10, -N=NOH, -N=NOR'o, -N3i -CN, -CNS, -SCN, an alkyl group, an alkenyl
group, an
alkynyl group, a cycloalkyl group, a cycloalkenyl group, a cycloalkynyl group,
a
heterocycloalkyl group, a heterocycloalkenyl group, a heterocycloalkynyl
group, an aryl group,
a heteroaryl group, an organometallic group, an O-glycosyl group or a N-
glycosyl group. Any
of these aforementioned alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl,
heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, aryl and heteroaryl
groups can be
optionally and independently substituted with one or more substituents
selected from the
group consisting of halogen, -R10, -OH3 -OR10, -NH2, -NHR'o -NR,oRõ -N-R12 -
C(=O)OH, -C(=O)OR'o, -OS(=O)nOH, -OS(=O)nOR'o, -S(=O)nOH, -S(=O)nOR'o, -SH, -
SR'o, -C(=O)H, -C(=O)R'o, -OP(=O)(OH)2, -OP(=O)(OH)(OR'o), -OP(=O)(OR'o)(OR") -
PH2, -PHR'o, -PR'oR", -P023 -PO, -P=R12, -NO23 -NO, -NH-NH2, -NH-NHR'o, -NH-
NR'oR" -NHOH, -NR10OH, -NHOR10, -NR'oOR", -N2+, -N=NH, -N=NR'o, -N=NOH3
-
N=NOR'o, -N33 -CN, -CNS or -SCN;
R10 and R" are independently selected from the group consisting of an alkyl
group, an alkenyl
group, an alkynyl group, a cycloalkyl group, a cycloalkenyl group, a
cycloalkynyl group, a
heterocycloalkyl group, a heterocycloalkenyl group, a heterocycloalkynyl
group, an aryl group,
a heteroaryl group, an organometallic group, an O-glycosyl group or an N-
glycosyl group.
R12 is =0, =S, =NH, =NR10, =CH2i =CHR'o -CR10R1 1 ;
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X, Y and Z are independently selected from the group consisting of hydrogen,
an alkyl group, an
alkenyl group, an alkynyl group, a cycloalkyl group, a cycloalkenyl group, a
cycloalkynyl group,
a heterocycloalkyl group, a heterocycloalkenyl group, a heterocycloalkynyl
group, an aryl
group, a heteroaryl group, halogen, -OH, -OR6, -NH2i -NHR6, -NR6R', -N=R8, -
C(=O)OH, -
C(=O)OR6, -OS(=O)nOH, -OS(=O)nOR6, -S(=O)nOH, -S(=O)nOR6, -SH, -SR6, -C(=O)H, -
C(=O)R6, -OP(=O)(OH)2, -OP(=O)(OH)(OR6), -OP(=O)(OR6)(OR'), -PH2, -PHR6, -
PR6R', -
P02, -PO, -P=R8, -NO23 -NO, -NH-NH2, -NH-NHR6, -NH-NR6R', -NHOH, -NR6OH, -
66OR', -N2+, -N=NH, -N=NR6, -N=NOH, -N=NOR6
NHOR , -NR , -N33 -CN, -CNS, -SCN, an
organometallic group, an O-glycosyl group or a N-glycosyl group. Any of these
aforementioned alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, heterocycloalkyl,
heterocycloalkenyl, heterocycloalkynyl, aryl and heteroaryl groups can be
optionally and
independently substituted with one or more substituents selected from the
group consisting of
halogen, -R6, -OH3 -OR6, -NH2, -NHR6, -NR6R', -N=RB, -C(=O)OH, -C(=O)OR6, -
OS(=O)nOH, -OS(=O)nORs, -S(=O)nOH, -S(=O)nORs, -SH, -SR6, -C(=O)H, -C(=O)R6, -
OP(=O)(OH)2, -OP(=O)(OH)(OR6), -OP(=O)(OR6)(OR'), -PH2, -PHR6, -PR6R', -P023 -
PO3
-P=RB, -NO23 -NO, -NH-NH2, -NH-NHR6, -NH-NR6R', -NHOH, -NR6OH, -NHOR6, -
NR6OR', -N2+, -N=NH, -N=NR6, -N=NOH, -N=NOR6, -N33 -CN, -CNS or -SCN; wherein
X
and Z could be bound forming a 5- to 7-membered ring, which could also be
substituted with
one or more independently selected R6 groups and/or could be part of a fused
ring system,
optionally substituted with one or more independently selected R9 groups; or
R5 can be absent
and Z could be directly bound to the carbon atom indicated in Formula I with
an asterisk, thus
forming a 5- to 7-membered ring, which could also be substituted with one or
more
independently selected R9 groups and/or could be part of a fused ring system,
optionally
substituted with one or more independently selected R9 groups;
A is hydrogen, =0, =S, =NH, =NR6, =CH2i =CHR6, =CR6R', halogen, -OH, -OR6, -
NH2, -NHR6,
-NR6R', -N=R8, -C(=O)OH, -C(=O)OR6, -OS(=O)nOH, -OS(=O)nOR6, -S(=O)nOH, -
S(=O)nOR6, -SH, -SR6, -C(=O)H, -C(=O)R6, -OP(=O)(OH)2, -OP(=O)(OH)(OR6), -
OP(=O)(OR6)(OR'), -PH2, -PHR6, -PR6R', -P023 -PO, -P=R8, -NO23 -NO, -NH-NH2, -
NH-
NHR6, -N-NR6R', -NHOH, -NR6OH, -NHOR6, -NR6OR', -N2+, -N=NH, -N=NR6, -N=NOH3
-N=NOR6, -N33 -CN, -CNS, -SCN, an organometallic group, an O-glycosyl group or
a N-
glycosyl group;
n is 0, 1 or 2;
a discontinuous line parallel to a continuous line represents an optional
double bond;
with the proviso that all R' to R5 groups were simultaneously hydrogen, or at
least one group R'
to R5 must be -OH, -OCH3i -NH2, -NHR6, -NR6R', O-glycosyl or N-glycosyl,
and at least one agrochemically acceptable vehicle.
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More specifically, the compositions of the present invention include
preferably at least one
compound of Formula II:
R4
R5 R3
x 2
R
Z R
Y
A
Formula II
and the salts, esters, ethers, solvates or isomers thereof, or their
corresponding 0- and/or N-
glycosides; wherein R' to R4 are independently selected from the group
consisting of
hydrogen, halogen, -OH, -OR6, -NH2, -NHR6, -NR6R', -N=R8, -C(=O)OH, -C(=O)OR6,
-
S(=O)20H, -S(=O)20R6, -SH3 -SR6, -C(=O)H3 -C(=O)R6, -OP(=O)(OH)2, -
OP(=O)(OH)(OR6), -OP(=O)(OR6)(OR'), -PH2, -PHR6, -PR6R', -NO23 -NO, -NH-NH2, -
NH-NHR6, -NH-NR6R', -NHOH, -NR6OH, -NHOR6, -NR6OR', -N=NH, -N=NR6, -N=NOH3
-N=NOR6, -N33 -CN, -CNS, -SCN, a C1-C7 alkyl group, a C2-C7 alkenyl group, a
C2-C7
alkynyl group, a C3-Cõ cycloalkyl group, a C4-Cõ cycloalkenyl group, a C4-Cõ
cycloalkynyl
group, a 4- to 11 -memebered heterocycloalkyl group, a 4- to 11 -membered
heterocycloalkenyl
group, a 4- to 11-membered heterocycloalkynyl group, an aryl group, a
heteroaryl group, an
organometallic group, an 0-glycosyl group or a N-glycosyl group. Any of these
aforementioned alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, heterocycloalkyl,
heterocycloalkenyl, heterocycloalkynyl, aryl and heteroaryl groups can be
optionally and
independently substituted with one or more substituents selected from the
group consisting of
halogen, -R6, -OH3 -OR6, -NH2, -NHR6, -NR6R', -N=R8, -C(=O)OH3 -C(=O)OR6,-
S(=O)20H, -S(=O)20R6, -SH3 -SR6, -C(=O)H3 -C(=O)R6, -OP(=O)(OH)23 -
OP(=O)(OH)(OR6), -OP(=O)(OR6)(OR'), -PH2, -PHR6, -PR6R', -NO23 -NO, -NH-NH2, -
NH-NHR6, -NH-NR6R', -NHOH, -NR6OH, -NHOR6, -NR6OR', -N=NH, -N=NR6, -N=NOH3
-N=NOR6, -N33 -CN, -CNS or -SCN;
R5 can be either the same as described for R' to R4 or it can be absent and Z
could be directly
bound to the carbon atom indicated in Formula I with an asterisk, thus forming
a 5- to 7-
membered ring, which could also be substituted with one or more independently
selected R9
groups and/or could be part of a fused ring system, optionally substituted
with one or more
independently selected R9 groups;
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R6 and R' are independently selected from the group consisting of a C1-C4
alkyl, C2-C4 alkenyl,
C2-C4 alkynyl, C3-C6 cycloalkyl, C4-C6 cycloalkenyl, C4-C6 cycloalkynyl, 3- to
6-membered
heterocycloalkyl, 4- to 6-membered heterocycloalkenyl, 4- to 6-membered
heterocycloalkynyl,
aryl, heteroaryl, organometallic, O-glycosyl or N-glycosyl group, which can be
also substituted
with one or more independently selected R9 groups;
R 8 is =0, =S, =NH, =NR6, =CH2, =CHR6, =CR6R';
R9 is selected from the group consisting of hydrogen, halogen, -OH, -OR10, -
NH2, -NHR'o -
NR'oR" -N=R12, -C(=O)OH, -C(=O)OR'o, -S(=O)20H, -S(=O)20R'o, -SH, -SR10, -
C(=O)H,
-C(=O)R'o, -OP(=O)(OH)2, -OP(=O)(OH)(OR'o), -OP(=O)(OR10)(OR1), -PH2, -PHR'o -
PR'oR" -NO2, -NO, -NH-NH2, -NH-NHR'o, -NH-NR'oR" -NHOH, -NR10OH, -NHOR'o -
NR'oOR", -N=NH, -N=NR'o, -N=NOH, -N=NOR'o, -N3, -CN, -CNS, -SCN, a C1-C4 alkyl
group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C3-C6 cycloalkyl group,
a C4-C6
cycloalkenyl group, a C4-C6 cycloalkynyl group, a 3- to 6-membered
heterocycloalkyl group, a
4- to 6-membered heterocycloalkenyl group, a 4- to 6-membered
heterocycloalkynyl group, an
aryl group, a heteroaryl group, an organometallic group, an O-glycosyl group
or a N-glycosyl
group. Any of these aforementioned alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl,
cycloalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, aryl
and heteroaryl
groups can be optionally and independently substituted with one or more
substituents selected
from the group consisting of halogen, -R10, -OH3 -OR10, -NH2, -NHR'o -NR,oRõ -
N=R12 -
C(=O)OH, -C(=O)OR'o, -S(=O)20H, -S(=O)20R'o, -SH, -SR'o, -C(=O)H, -C(=O)R'o -
OP(=O)(OH)2, -OP(=O)(OH)(OR'o), -OP(=O)(OR'o)(OR"), -PH2, -PHR10, -PR'oR" -
NO2, -
NO, -NH-NH2, -NH-NHR'o, -NH-NR'oR" -NHOH, -NR10OH, -NHOR10 -NR10OR11-
N=NH, -N=NR'o, -N=NOH, -N=NOR'o, -N3, -CN, -CNS or -SCN;
R10 and R" are independently selected from the group consisting of a C1-C4
alkyl, C2-C4 alkenyl,
C2-C4 alkynyl, C3-C6 cycloalkyl, C4-C6 cycloalkenyl, C4-C6 cycloalkynyl, 3- to
6-membered
heterocycloalkyl, 4- to 6-membered heterocycloalkenyl, 4- to 6-membered
heterocycloalkynyl,
aryl, heteroaryl, organometallic, O-glycosyl or N-glycosyl group;
X, Y and Z are independently selected from the group consisting of hydrogen, a
C1-C7 alkyl
group, a C2-C7 alkenyl group, a C2-C7 alkynyl group, a C3-Cõ cycloalkyl group,
a C4-Cõ
cycloalkenyl group, a C4-Cõ cycloalkynyl group, a 3- to 11 -membered
heterocycloalkyl group,
a 4- to 11-membered heterocycloalkenyl group, a 4- to 11-membered
heterocycloalkynyl
group, an aryl group, a heteroaryl group, halogen, -OH, -OR6, -NH2, -NHR6, -
NR6R', -N=R8,
-C(=O)OH, -C(=O)OR6, -S(=O)20H, -S(=O)20R6, -SH, -SR6, -C(=O)H, -C(=O)R6, -
OP(=O)(OH)2, -OP(=O)(OH)(OR6), -OP(=O)(OR6)(OR'), -PH2, -PHR6, -PR6R', -NO23 -
NO3
-NH-NH2, -NH-NHR6, -NH-NR6R', -NHOH, -NR6OH, -NHOR6, -NR6OR', -N=NH3 -
N=NR6, -N=NOH, -N=NOR6, -N33 -CN, -CNS, -SCN, an organometallic group, an 0-
glycosyl group or a N-glycosyl group. Any of these aforementioned alkyl,
alkenyl, alkynyl,
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cycloalkyl, cycloalkenyl, cycloalkynyl, heterocycloalkyl, heterocycloalkenyl,
heterocycloalkynyl,
aryl and heteroaryl groups can be optionally and independently substituted
with one or more
substituents selected from the group consisting of halogen, -R6, -OH, -OR6, -
NH2, -NHR6, -
NR6R', -N=R8, -C(=O)OH, -C(=O)OR6,-S(=O)20H, -S(=O)20R6, -SH, -SR6, -C(=O)H, -
C(=O)R6, -OP(=O)(OH)2, -OP(=O)(OH)(OR6), -OP(=O)(OR6)(OR'), -PH2, -PHR6, -
PR6R', -
NO2, -NO, -NH-NH2, -NH-NHR6, -NH-NR6R7, -NHOH, -NR6OH, -NHOR6, -NR6OR7,
-
N=NH, -N=NR6, -N=NOH, -N=NOR6, -N33 -CN, -CNS or -SCN; wherein X and Z could
be
bound forming a 5- to 7-membered ring, which could also be substituted with
one or more
independently selected R9 groups and/or could be part of a fused ring system,
optionally
substituted with one or more independently selected R9 groups; or R5 can be
absent and Z
could be directly bound to the carbon atom indicated in Formula I with an
asterisk, thus
forming a 5- to 7-membered ring, which could also be substituted with one or
more
independently selected R9 groups and/or could be part of a fused ring system,
optionally
substituted with one or more independently selected R9 groups;
A is hydrogen, =0, =S, =NH, =NR6, =CH2i =CHR6, =CR6R', halogen, -OH, -OR6, -
NH2, -NHR6,
-NR6R', -N=R8, -C(=O)OH, -C(=O)OR6, -S(=O)20H, -S(=O)20R6, -SH, -SR6, -C(=O)H3
-
C(=O)R6, -OP(=O)(OH)2, -OP(=O)(OH)(OR6), -OP(=O)(OR6)(OR'), -PH2, -PHR6, -
PR6R', -
NO23 -NO, -NH-NH2, -NH-NHR6, -N-NR6R', -NHOH, -NR6OH, -NHOR6, -NR6OR', -N
=NH, -N=NR6, -N=NOH, -N=NOR6, -N33 -CN, -CNS, -SCN, an organometallic group,
an 0-
glycosyl group or a N-glycosyl group;
a discontinuous line parallel to a continuous line represents an optional
double bond;
with the proviso that all R' to R5 groups were simultaneously hydrogen, or at
least one group R'
to R5 must be -OH, -OCH3i -NH2, -NHR6, -NR6R', 0-glycosyl or N-glycosyl,
and at least one agrochemically acceptable vehicle.
Some phenolic compounds specifically preferred are compounds of Formula III:
R4
R5 R3
1
R2
Z R
A
Formula III
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21
and the salts, esters, ethers, solvates or isomers thereof, or their
corresponding 0- and/or N-
glycosides; wherein R' to R5 are independently selected from the group
consisting of
hydrogen, -OH, -OR6, -NH2, -NHR6, -NR6R', an 0-glycosyl group or a N-glycosyl
group;
R6 and R' are independently selected from the group consisting of a C1-C3
alkyl, C2 alkenyl, C2
alkynyl, 0-glycosyl or N-glycosyl group;
Z is selected from the group consisting of hydrogen, -OH, -OR6, -NH2, -NHR6, -
NR6R', a phenyl
group independently substituted with one or more groups -OH, -OR6, -NH2, -
NHR6, -NR6R7,
an 0-glycosyl group or a N-glycosyl group;
A is hydrogen, =0, =S, =NH.
The phenolic compounds of Formula III especially preferred are: cinnamic acid,
o-, m- and p-
coumaric acids, caffeic acid, ferulic acid, sinapic acid, 5-hydroxicaffeic
acid, 5-hydroxiferulic acid,
3,4,5-trimethoxicinnamic acid, o-, m- and p-coumaric alcohols, o-, m- and p-
coumaric aldehydes,
cinnamic alcohol, cinnamic aldehyde, caffeic alcohol, caffeic aldehyde,
ferulic alcohol, ferulic
aldehyde, coniferyl alcohol, sinapic aldehyde, 5-hydroxiferulic alcohol, 5-
hydroxiferulic aldehyde,
5-hydroxicaffeic alcohol, 5-hydroxicaffeic aldehyde, 3,4,5-trimethoxicinnamic
alcohol, 3,4,5-
trimethoxicinnamic aldehyde, chalcone, naringenin-chalcone, eriodictyol-
chalcone,
pentahydroxiflavanone-chalcone, the glycosylated derivatives, dimers, trimers,
and oligomers of
the former compounds, and the like.
Other phenolic compounds specifically preferred are compounds of Formula IV:
R1 X
R 2 I ~ ~ Y
R3 ~ Z A
R4
Formula IV
and the salts, esters, ethers, solvates or isomers thereof, or their
corresponding 0- and/or N-
glycosides; wherein R' to R5 are independently selected from the group
consisting of
hydrogen, -OH, -OR6, -NH2, -NHR6, -NR6R', an 0-glycosyl group or a N-glycosyl
group;
R6 and R' are independently selected from the group consisting of a C1-C3
alkyl, C2 alkenyl, C2
alkynyl, 0-glycosyl or N-glycosyl group;
X and Y are independently selected from the group consisting of hydrogen, -OH,
-OR6, -NH2, -
NHR6, -NR6R', an 0-glycosyl group or an N-glycosyl group;
Z is selected from -CH2-, -CHR6-, -CR6R'-, -0-, -NH-, -NR6-, -S-;
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22
A is hydrogen, =0, =S, =NH.
The phenolic compounds of Formula IV especially preferred are: coumarin,
umbelliferone, 6,7-
dihydroxicoumarin, 7,8-dihydroxicoumarin, 4,5,7-trihydroxicoumarin, 7-
methoxicoumarin (0-
methylumbelliferone), 6,7-dimethoxicoumarin, the glycosylated derivatives,
dimers, trimers, and
oligomers thereof, and the like.
Other phenolic compounds specifically preferred are compounds of Formula V:
R4
R5 R3
R11
R12 , X R ~ R,
R13 Y
14 A
Formula V
and the salts, esters, ethers, solvates or isomers thereof, or their
corresponding 0- and/or N-
glycosides; wherein R' to R5 are independently selected from the group
consisting of
hydrogen, -OH, -OR6, -NH2, -NHR6, -NR6R', an 0-glycosyl group or a N-glycosyl
group;
R6 and R' are independently selected from the group consisting of a C1-C3
alkyl, C2 alkenyl, C2
alkynyl, 0-glycosyl or N-glycosyl group;
R" to R14 are independently selected from the group consisting of hydrogen, -
OH, -OR6, -NH2, -
NHR6, -NR6R', an 0-glycosyl group or an N-glycosyl group;
X is selected from -CH2-, -CHR6-, -CR6R'-, -0-, -NH-, -NR6-, -S-;
Y is selected from the group consisting of hydrogen, -OH, -OR6, -NH2, -NHR6, -
NR6R', an 0-
glycosyl group or an N-glycosyl group;
A is hydrogen, =0, =S, =NH.
The phenolic compounds of Formula V especially preferred are: naringenin,
afzelechin,
apigeninidin, apiforol, dihydrokaempferol, leucopelargonidin, kaempferol,
quercetin, acacetin,
apigenin, dihydroquercetin, leucocyanidin, catechin, miricetin, luteolin,
eriodictyol,
leucopaeonidin, 3'-O-methyl-luteolin, luteoforol, luteolinidin, gallocatechin,
leucodelphinidin,
leucopetunidin, leucomalvidin, tricetin, pentahydroxiflavanone, the
glycosylated derivatives,
dimers, trimers, and oligomers thereof, and the like.
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23
The term "alkyl", unless specified otherwise, when used herein, alone or as
part of another group,
preferably includes straight or branched chain hydrocarbons containing 1 to 20
carbons,
preferably 1 to 10 carbons, more preferably 1 to 8 carbons in the normal
chain, such as methyl,
ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl,
heptyl, 4,4-dimethylpentyl,
octyl, 2,2,4-trimethyl-pentyl, nonyl, decyl, undecyl, dodecyl, the diverse
branched chain isomers
thereof, and the like, and also said groups including 1 to 4 substituents such
as halogen, e.g. F,
Br, Cl or I, or -CF3i alkyl, alkoxi, aryl, aryloxi, aryl(aryl) or diaryl,
arylalkyl, arylalkyloxi, alkenyl,
cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxi, amino, hydroxi, hydroxialkyl,
acyl, heteroaryl,
heteroaryloxi, heteroarylalkyl, heteroarylalkoxi, aryloxialkyl, alkylthio,
arylalkylthio, aryloxiaryl,
alkylamido, alkanoylamino, arylcarbonylamino, nitro, ciano, thiol, haloalkyl,
trihaloalkyl and/or
alkylthio.
The term "cycloalkyl", unless specified otherwise, when used herein, alone or
as part of another
group, includes cyclic saturated or partially unsaturated hydrocarbon groups
having 1 to 3 rings,
including monocyclic alkyl, bicyclic alkyl (or bicycloalkyl) and tricyclic
alkyl (tricycloalkyl),
containing a total of 3 to 20 carbon ring atoms, preferably 3 to 10 carbon
ring atoms, and that
may be fused to 1 or 2 aromatic rings, such as those described for aryl,
including cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl and
cyclododecyl,
cyclohexenyl, adamantyl, bornyl,
C~)
, . .
and the like.
Any of these groups may be optionally substituted with 1 to 4 substituents
such as halogen, alkyl,
alkoxi, hydroxi, aryl, aryloxi, arylalkyl, cycloalkyl, hydroxialkyl,
alkylamido, alcanoylamino, oxo,
acyl, arylcarbonylamino, amino, nitro, ciano, thiol and/or alkylthio, and/or
any of the substituents
specified for "alkyl".
The term "cycloalkenyl" as used herein, alone or as part of another group,
refers to cyclic partially
unsaturated hydrocarbons having 3 to 12 carbon atoms, preferably 5 to 10
carbon atoms, and 1
or 2 double bonds per ring. Exemplary cycloalkenyl groups include
cyclopentenyl, cyclohexenyl,
cycloheptenyl, cyclooctenyl, cyclohexadienyl, and cycloheptadienyl, which may
be optionally
substituted as specified for cycloalkyl.
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24
The term "cycloalkynyl" as used herein, alone or as part of another group,
refers to cyclic partially
unsaturated hydrocarbons having 3 to 12 carbon atoms, preferably 5 to 10
carbon atoms, and 1
or 2 triple bonds. Exemplary cycloalkynyl groups include cyclopentynyl,
cyclohexynyl,
cycloheptynyl, cyclooctynyl, which may be optionally substituted as specified
for cycloalkyl.
The term "alkenyl", unless specified otherwise, as used herein, alone or as
part of other group,
refers to straight or branched chain hydrocarbons having 2 to 20 carbon atoms,
preferably 2 to 12
carbon atoms, and more preferably 1 to 8 carbon atoms in the normal chain, and
having one to
six double bonds in the normal chain, such as vinyl, 2-propenyl, 3-butenyl, 2-
butenyl, 4-pentenyl,
3-pentenyl, 2-hexenyl, 3-hexenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 3-
octenyl, 3-nonenyl, 4-
decenyl, 3-undecenyl, 4-dodecenyl, 4,8,12-tetradecatrienyl, and the like,
which may be optionally
substituted with 1 to 4 substituents, mainly halogen, haloalkyl, alkyl,
alkoxi, alkenyl, alkynyl, aryl,
arylalkyl, cycloalkyl, amino, hydroxi, heteroaryl, cycloheteroalkyl,
alkanoylamino, alkylamido,
arylcarbonyl-amino, nitro, ciano, thiol, alkylthio and/or any of the alkyl
substituents herein
described.
The term "alkynyl", unless specified otherwise, as used herein, alone or as
part of other group,
refers to straight or branched chain hydrocarbons having 2 to 20 carbon atoms,
preferably 2 to 12
carbon atoms, and more preferably 2 to 8 carbon atoms in the normal chain, and
having a triple
bond in the normal chain, such as 2-propinyl, 3-butinyl, 2-butinyl, 4-
pentinyl, 3-pentinyl, 2-hexinyl,
3-hexinyl, 2-heptinyl, 3-heptinyl, 4-heptinyl, 3-octinyl, 3-noninyl, 4-
decinyl, 3-undecinyl, 4-
dodecinyl, and the like, which may be optionally substituted with 1 to 4
substituents, mainly
halogen, haloalkyl, alkyl, alkoxi, alkenyl, alkynyl, aryl, arylalkyl,
cycloalkyl, amino, hydroxi,
heteroaryl, cycloheteroalkyl, alkanoylamino, alkylamido, arylcarbonyl-amino,
nitro, ciano, thiol,
alkylthio and/or any of the alkyl substituents herein described.
The term "halogen" as used herein, alone or as part of other group, refers to
chloro, bromo,
fluoro, and iodo, and also -CF3i preferably chloro and fluoro.
The term "aryl", unless specified otherwise, alone or as part of other group,
refers to monocyclic
or bicyclic aromatic groups having 6 to 10 carbon ring atoms (such as phenyl
or naphtyl, including
1-naphtyl and 2-naphtyl) and they can optionally include one to three
additional rings fused to a
carbocyclic or a heterocyclic ring (such as aryl, cycloalkyl, heteroaryl or
cycloheteroalkyl rings),
for example:
~.. ~
i f
I i
14.
~ ~ , `~r
CA 02647216 2008-09-23
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and that may optionally be substituted at available carbon atoms with 1, 2 or
3 groups selected
from hydrogen, halogen, haloalkyl, alkyl, haloalkyl, alkoxi, haloalkoxi,
alkenyl, trifluoromethyl,
trifluoromethoxi, alkynyl, cycloalkylalkyl, cycloheteroalkyl,
cycloheteroalkylalkyl, aryl, heteroaryl,
arylalkyl, aryloxi, aryloxialkyl, arylalkoxi, arylthio, arylazo,
heteroarylalkyl, heteroarylalkenyl,
heteroarylheteroaryl, heteroaryloxi, hydroxi, nitro, ciano, amino, substituted
amino wherein the
amino include 1 or 2 substituents (which are alkyl, aryl or any of the other
hydrocarbon
compounds mentioned in the definitions), thiol, alkylthio, arylthio,
heteroarylthio, arylthioalkyl,
alkoxiarylthio, alkylcarbonyl, arylcarbonyl, alkylaminocarbonyl,
arylaminocarbonyl, alkoxicarbonyl,
aminocarbonyl, alkylcarbonyloxi, arylcarbonyloxi, alkylcarbonylamino,
arylcarbonylamino,
arylsulfinyl, arylsulfinylalkyl, arylsulfonylamino or arylsulfon-aminocarbonyl
and/or any of the alkyl
substituents herein mentioned.
The term "heterocycloalkyl", unless specified otherwise, as used herein, alone
or as part of other
group, refers to a cycloalkyl having at least one of the carbon atoms of the
ring replaced by an
atom other than carbon, such as nitrogen, oxygen or sulfur, such as e.g.,
morpholinyl,
imidazolidinyl, pyrrolidinyl, pyrazolidinyl, piperidyl, piperazinyl,
isochromanyl, chromanyl, indolinyl,
isoindolinyl, quinuclidinyl, and the like.
The term "heterocycloalkenyl", unless specified otherwise, as used herein,
alone or as part of
other group, refers to a cycloalkenyl having at least one of the carbon atoms
of the ring replaced
by an atom other than carbon, such as nitrogen, oxygen or sulfur, such as
e.g., imidazolinyl,
pyrrolinyl, pyrazolinyl, and the like.
The term "heterocycloalkynyl", unless specified otherwise, as used herein,
alone or as part of
other group, refers to a cycloalkynyl having at least one of the carbon atoms
of the ring replaced
by an atom other than carbon, such as nitrogen, oxygen or sulfur.
The term "heteroaryl", unless specified otherwise, as used herein, alone or as
part of other group,
refers to a monocyclic or bicyclic aromatic nucleus having 5 to 10 elements,
which includes 1, 2,
3 or 4 heteroatoms such as nitrogen, oxygen or sulfur, including possible N-
oxides. The
heteroaryl group may optionally include 1 to 4 substituents such as any of the
substituents herein
described for alkyl. The examples of heteroaryl groups include the following:
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26
N g Q Q
r)
' /N
~,,.!
0
H ~~N N ~
N N
N-N N-N N-N '--~ N_N
C/.S~ Q~ .>
~i o N
thiazolyl, isothiazolyl, indolyl, isoindolyl, indazolyl, purinyl, quinolyl,
isoquinolyl, ftalazinyl,
naftiridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, carbazolyl,
carbolinyl, phenantridinyl,
acridinyl, perimidinyl, phenantrolinyl, phenazinyl, phenothiazinyl,
isoxazolyl, oxazolyl, furazanyl,
phenoxazinyl, imidazolyl, pyranyl, pyrazolyl, benzothienylthianthrenyl,
benzofuranyl,
isobenzofuranyl, piridazinyl, indolizinyl and the like.
The compositions of the invention comprise compounds of Formula I in any
common suitable
agrochemical composition that allows dissolution, emulsion or suspension
thereof. In this type of
composition, compounds of Formula I can be found as a single compound, in
mixtures of different
compounds of Formula I, or in mixtures comprising at least one compound of
Formula I and any
other agrochemical product, and at least one agrochemically acceptable
vehicle.
Said other agrochemical product may be, for example, a pesticide, a fungicide
or an herbicide.
For example, the pesticide may be a pyrethroid-based pesticide such as
allethrin, tetramethrin,
resmethrin, phenothrin, furamethrin, permethrin, cypermethrin, deltamethrin,
cyhalothrin,
cyfluthrin, fenpropathrin, tralomethrin, cycloprothrin, flucythrinate,
fluvalinate, acrinathrin,
tefluthrin, bifenthrin, empenthrin, beta-cyfluthrin, fenvalerate,
esfenvalerate, flubrocythrinate,
metofluthrin, profluthrin, dimefluthrin, silafluofen, pyrethrum extract,
etofenprox, halfenprox and
the like; an organophosphate-based pesticide such as DDVP, cyanophos,
fenthion, fenitrothion,
tetrachlorvinphos, dimethylvinphos, propaphos, methyl parathion, temephos,
phoxim, acephate,
isofenphos, salithion, DEP, EPN, ethion, mecarbam, pyridafenthion, diazinon,
pirimiphos-methyl,
etrimfos, isoxathion, quinalphos, chlorpyrifos-methyl, chlorpyrifos,
phosalone, phosmet,
methidathion, oxydeprofos, vamidothion, malathion, phenthoate, dimethoate,
formothion,
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27
thiometon, ethylthiometon, phorate, terbufos, profenofos, prothiofos,
sulprofos, pyraclofos,
monocrotophos, naled, fosthiazate, trichlorfon, ethoprophos, cadusafos,
chlorfenvinphos,
dichlofenthion, ethylthiometon, methamidophos, dichlorvos, tebupirimfos,
omethoate, triazophos,
oxydemeton-methyl, azinphos-methyl, chlorethoxyphos, dicrotophos, disulfoton,
fenamiphos,
phosphamidon, chlormephos, demeton-S-methyl, mevinphos, parathion and the
like; a
carbamate-based pesticide such as NAC, MTMC, MIPC, BPMC, XMC, PHC, MPMC,
ethiofencarb, bendiocarb, pirimicarb, carbosulfan, benfuracarb, methomyl,
oxamyl, aldicarb,
thiodicarb, alanycarb, carbofuran, methiocarb, fenothiocarb, formetanate,
xylylmethylcarbamate,
propoxur, isoprocarb and the like; a neonicotinoid-based pesticide such as
imidacloprid,
nitenpyram, acetamiprid, dinotefuran, thiamethoxam, thiacloprid, clothianidin
and the like; an
organochlorine-based pesticide such as bromopropylate, dicofol, endosulfan,
lindane and the like;
an insect growth regulator such as diflubenzuron, chlorfluazuron,
teflubenzuron, triflumuron,
flufenoxuron, flucycloxuron, hexaflumuron, fluazuron, diafenthiuron,
novaluron, noviflumuron,
bistrifluron, chromafenozide, halofenozide, methoxyfenozide, lufenuron,
cyromazine, triazamate
and the like; a natural product-based pesticide such as nicotine sulphate,
polynactin complex,
abamectin, milbemectin, lepimectin, BT (Bacillus thuringiensis) agent,
spinosad, rotenone and the
like; cartap, thiocyclam, bensultap, pymetrozine, fipronil, buprofezin,
fenoxycarb,
pyriproxyfen, methoprene, hydroprene, kinoprene, endosulfan, triazuron,
tebufenozide,
benzoepin, emamectin, emamectin benzoate, flupyrazophos, fluacrypyrim,
flufenzin,
indoxacarb, tolfenpyrad, gamma-cyhalothrin, ethiprole, acetoprole,
amidoflumet,
chlorfenapyr, flonicamid, flufenerim, pyridalyl, sodium oleate, potassium
oleate,
azadirachtin, carbam, sodium carbam, propargite, azocyclotin, benzoximate,
metaldehyde, protrifenbute, benclothiaz, flubendiamide, metaflumizole; an
acaricide such
as chlorobenzilate, fenisobromolate, tetradifon, CPCBS (chlorfenson), BPPS,
chinomethionat,
amitraz, benzomate, hexythiazox, fenbutatin oxide, cyhexatin, dienochlor,
clofentezine,
pyridaben, fenpyroximate, fenazaquin, tebufenpyrad, pyrimidifen, acequinocyl,
bifenazate,
etoxazol, spirodiclofen, spiromesifen, amidoflumet and diflovidazin, and other
pesticides with
similar action used in agricultural, horticultural, fruticultural or
floricultural applications.
For example, the fungicide may be an azole-based fungicide such as
triadimefon, hexaconazole,
propiconazole, ipconazole, prochloraz, triflumizole, tebuconazole,
epoxiconazole, difenoconazole,
flusilazole, triadimenol, cyproconazole, metconazole, fluquinconazole,
bitertanol, tetraconazole,
triticonazole, flutriafol, penconazole, diniconazole, fenbuconazole,
bromuconazole,
imibenconazole, simeconazole, myclobutanil, hymexazole, imazalil, furametpyr,
thifluzamide,
etridiazole, oxpoconazole, oxpoconazole fumarate, pefurazoate, prothioconazole
and the like; a
pyrimidine-based fungicide such as pyrifenox, fenarimol, nuarimol, bupirimate
and the like; an
anilinopyrimidine-based fungicide such as mepanipyrim, cyprodinil,
pyrimethanil, diflumetorim and
the like; an acylalanine-based fungicide such as metalaxyl, metalaxyl-M,
oxadixyl, benalaxyl and
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28
the like; a benzimidazole-based fungicide such as thiophanate-methyl, benomyl,
carbendazim,
fuberidazole, thiabendazole and the like; an organosulfur fungicide such as
mancozeb, propineb,
zineb, metiram, maneb, ziram, thiuram, amobam, polycarbamate, thiadiazine and
the like; an
organochlorine fungicide such as tetrachloroisophthalonitrile and the like; a
carboxam-based
fungicide such as ethaboxam, oxycarboxin, carboxin, flutolanil, silthiofam,
mepronil, boscalid and
the like; a morpholine-based fungicide such as dimethomorph, fenpropidin,
fenpropimorph,
spiroxamine, tridemorph, dodemorph, flumorph and the like; a strobilurin-based
fungicide such as
azoxystrobin, kresoxim-methyl, metominostrobin, oryzastrobin, fluoxastrobin,
trifloxystrobin,
dimoxystrobin, pyraclostrobin, picoxystrobin and the like; a dicarboximide-
based fungicide such
as iprodione, procymidone, vinclozolin, chlozolinate and the like; a soil
fungicide such as
flusulfamide, dazomet, methyl isothiocyanate, chloropicrin, methasulfocarb,
hydroxyisoxazole,
potassium hydroxyisoxazole, echlomezol, dichloropropene, carbam, methyl iodide
and the like; a
copper-based fungicide such as basic copper chloride, basic copper sulfate,
copper
nonylphenolsulfonate, oxine copper, DBEDC, anhydrous copper sulfate, copper
sulfate
pentahydrate, copper hydroxide and the like; an inorganic fungicide such as
inorganic sulfur,
wettable sulfur powder, lime sulfur, zinc sulfate, fentin, sodium hydrogen
carbonate, potassium
hydrogen carbonate, hypochlorite salts, metallic silver and the like; an
organophosphate-based
fungicide such as edifenphos, tolclofos-methyl, fosetyl, iprobenfos, dinocap,
pyrazophos and the
like; a melanin biosynthesis inhibitor-based fungicide such as carpropamid,
fthalide, tricyclazole,
pyroquilon, diclocymet, fenoxanil and the like; an antibiotic fungicide such
as kasugamycin,
validamycin, polyoxin derivative, blasticidin S, tecloftalam, oxytetracycline,
mildiomycin,
streptomycin and the like; a natural product-based fungicide such as rape seed
oil, machine oil
and the like; a carbamate-based fungicide such as benthiavalicarb-isopropyl,
iprovalicarb,
propamocarb, diethofencarb and the like; a pyrrole-based fungicide such as
fluoroimide,
fludioxonil, fenpiclonil and the like; a plant activator for leading
resistance to plant diseases such
as probenazole, acibenzolar-S-methyl, tiadinil and the like; a quinoline-based
fungicide such as
quinoxyfen, oxolinic acid and the like; cyflufenamid, fenhexamid, metrafenone,
picobenzamid,
proquinazid, famoxadone, cyazofamid, fenamidone, zoxamide, chlorothalonil,
cymoxanil, captan,
dithianon, fluazinam, folpet, dichlofluanid, triforine, isoprothiolane,
ferimzone, diclomezine,
pencycuron, chinomethionat, iminoctadine acetate, iminoctadine albesilate,
guazatine, chloroneb,
organonickel, dodine, quintozene, tolylfluanid, anilazine, nitrothal-
isopropyl, fenitropan, dicloran,
DPC, dimethirimol, benthiazole, flumetover, mandipropamid and other fungicides
with similar
action used in agricultural, horticultural, fruticultural or floricultural
applications.
Additionally, the compositions of the present invention may optionally include
adjuvants for the
dissolution, emulsion or suspension of the compounds of Formula I. Likewise,
this type of
composition may also comprise other components such as wetting, solvent,
humectant,
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29
dispersing, emulsifier, thickening, and chelating agents, other active
principles with a similar or
different effect than the compositions of the present invention, buffers,
salts, sunscreens, waxes,
penetration agents, covering agents and/or clays.
The compositions of the present invention may be solid or liquid compositions.
The compositions
may be powders, emulsifiable concentrates, concentrates for dilution, wettable
powders,
granules, suspension concentrates, diluted solutions, diluted dispersions and
diluted
suspensions, or combinations thereof. For the purpose of the invention, and
without wishing to be
limited, it is especially preferred to apply the compositions as a liquid, as
this allows a higher
penetration of the active compounds into the plant tissue, mostly in the case
where the active
compound is solubilized or is emulsified in a liquid carrier, preferably
solubilized. Nevertheless,
the liquid composition may be prepared from solid ingredients or mixtures, or
from concentrate
solutions just before application.
Surprisingly, the authors of the compositions of the present invention have
found that only under
certain circumstances the compounds of Formula I used in diluted formulations
and applied
according to the methods of the invention are able to cross the waxy layer
that protects the plant,
especially over fruit skins, and are also able to cross the cell membrane.
Without losing
generality, it is thought that this may be caused by the high concentration
achieved for the active
compounds solubilized in the compositions of the invention, and by the
adjuvants included in the
compositions, which keep active compounds in solution, in an optimal
ionization state, and allow
to achieve a good coverage of the plant parts when sprayed with the
compositions of the
invention. Especially, it has been proven that the use of a volatile pH buffer
in order to keep a
controlled pH in the composition (without losing generality, optimum pH for
acid active
compounds is around 6.0 in the diluted composition just before application)
and the use of a
wetting-humectant agent are relevant to achieve the effects of the
compositions of the present
invention.
The concentration ranges used for each of the compounds of Formula I in the
diluted
compositions of the invention that are directly applied to plants, vary from
0.01% to 20% by
weight, especially from 1% to 15% by weight, preferably from 2% to 10% by
weight, and most
preferably from 2% to 5% by weight, based on the weight of the final diluted
composition.
In molar concentrations, the ranges used for each of the compounds of Formula
I in the diluted
compositions of the invention that are directly applied to plants, vary from 1
to 200 mM, especially
from 5 to 100 mM, preferably from 10 to 80 mM, and most preferably from 10 to
50 mM. As
mentioned before, the diluted composition may comprise either mixtures of one
ore more
compounds of Formula I in concentrations varying from 1 to 100 mM, from 5 to
80 mM, and from
to 60 mM each, and preferably in a concentration from 10 to 40 mM each, or
mixtures with
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other agrochemical products wherein at least one compound of Formula I is
present at said
concentrations. These mixtures having the former concentrations may have been
prepared either
much before application or just before application, and the preparation
procedure comprises
mixing the active agent and at least one agrochemically acceptable vehicle.
Advantageously, the compositions of the present invention may be prepared as
concentrated
liquid solutions to be subsequently diluted, generally in water, just before
application over plants.
This concentrated composition must be suitable to allow subsequent
dissolution, emulsion or
suspension of the compound of Formula I, in such a way to obtain a diluted
liquid with a suitable
composition. The concentration of each compound of Formula I used in these
concentrated
compositions of the invention varies from 0.5% to 50%, especially from 1% to
30%, preferably
from 5% to 30%, and more preferably from 10% to 30% based on the weight of the
concentrated
composition. In molar concentrations, the concentration ranges of each
compound of Formula I
used in these concentrated compositions of the invention varies from 0.05 to
5.00 mM, especially
from 0.1 to 3.0 mM, preferably from 0.5 to 3 mM, and more preferably from 1 to
3 mM. The
concentrated compositions may comprise all the components of the final diluted
formulation, or
they may comprise only some of the components, or even only the active
compound of the
invention, while the remaining components are mixed with the concentrated
composition when
preparing the diluted composition.
The solvent used for the dilution of the concentrated compositions may be the
same solvent used
in the preparation of said concentrated composition, or any other suitable
solvent. The solvent for
the liquid composition to be applied over plants is preferably water,
optionally containing small
amounts of other organic solvents to help solubilize, emulsify or suspend the
compounds of
Formula I. The solvents that can be used for the concentrated formulation of
the invention may be
any organic or inorganic solvent in which the compound of the invention is
soluble in higher
concentrations than the concentration of the solution to be applied over the
plant, in such a way
that the concentrated solution could be diluted before its application.
Specifically, the solvent may
be pure ethanol or water-ethanol mixtures or other pure or mixed organic
solvent. The solvent
may be any solvent used in the agrochemical industry to solubilize active
compounds to be
applied over a plant after dilution. For example, the solvent may be toluene,
xylenes,
dimethylsulfoxide, dimethylformamide, ethanol, methanol, acetone, ether,
ethoxiethanol,
methoxiethanol, or any other suitable solvent, alone or in mixture with other
organic or inorganic
solvent.
The concentrated compositions may also include other components other than the
solvent and
the compound of the invention. For example, the concentrated compositions may
contain co-
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31
solvents, wetting, humectant, surfactant, dispersant, emulsifier, covering,
thickening, and
chelating agents, other active principles with the same effect of the
compounds of the present
invention or other effects, pH buffers, salts, sunscreens, waxes, penetration
agents, clays,
preserving and antioxidant agents, and the like.
The solvent used for dilution, preferably water, may also contain compounds
that contribute
additional effects to the diluted composition, as aforementioned, such as co-
solvents, wetting,
humectant, surfactant, dispersant, emulsifier, covering, thickening, and
chelating agents, other
active principles with the same effect of the compounds of the present
invention or other effects,
pH buffers, salts, sunscreens, waxes, penetration agents, clays, preserving
and antioxidant
agents, and the like.
For example, it has been found that a concentrated composition can be
advantageously prepared
by dissolving the active compounds of Formula I in an organic solvent, such as
ethanol,
dimethylformamide or dimethylsulfoxide, dissolving in water the remaining
components of the
diluted composition at their final concentration, and adding to this solution
the concentrated
composition with the compound of Formula I and the organic solvent just before
application. In
this way, an easy, fast and complete dissolution of the compound of Formula I
is achieved, thus
obtaining a diluted composition that allows the active compound to penetrate
the wax layer that
protects the plant and to achieve a complete effect after application.
In the other hand, the compositions of the invention may be applied by
spraying, irrigation,
fumigation, coverage, immersion or injection. The amount of composition
required for an acre of
land varies according to the selected application method. Nevertheless, it is
preferable to use a
direct application over the fruit to avoid unnecessary losses of the
agrochemical preparation over
the leaves and branches of the trees or into the soil, which is wetted when
using, for example, a
spraying machine.
The most suitable time to apply the compositions is during flower development
or fruit
development and growth, in one, two or more applications before harvest, and
optionally in one,
two or more additional applications after harvest.
It is preferable that applications could be temporally equally-spaced from the
formation of fruit or
flowers up to harvest and optionally leaving a last period before harvest with
no application of the
compositions.
In particular, it is preferable to carry out one, two or more applications,
the first application
between 6-8 weeks before harvest, and the second application between 3-4 weeks
before
harvest.
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As previously mentioned, the compositions of the invention are useful for
decreasing the
incidence of damage caused by sun on plant tissues, especially damage caused
by solar
radiation and temperature, especially damage caused by UV radiation. Without
losing generality,
the compositions of the present invention cause a decrease of the damage
caused by UV and
temperature owing to an increase of the content of molecular species or
compounds able to
absorb UV radiation, visible radiation or both, and/or able to scavenge and/or
stabilize free
radicals in the surface of the plant, in the intercellular space (liquid) or
in the intracellular space.
This effect is due to the increase of concentration of polyphenolic compounds
in the plant, such
as phenols, acetophenones, phenolcarboxylic acids, phenylacetic acids,
cinnamic acids,
hydroxicinnamic acids, polypropenes, coumarins, isocoumarins, flavonoids,
isoflavonoids,
biflavonoids, quinones, tannins, lignans, neolignans, lignins,
catecholmelanines,
phenylpropanoids, stilbenes, phenylanthrenes, pterocarpanes and furocoumarins
and the
glycosylated and polymeric derivatives thereof. The preferred flavonoids are:
anthocyanins,
leucoanthocyanins, chalcones, aurones, flavones, isoflavones, flavans,
isoflavans, flavonols,
flavanols, isoflavonols, isoflavanols, dihydroflavonols, dihydroflavanols,
flavanones,
isoflavanones, dihydrochalcones, proanthocyanidins, catechins, biflavonoids
and isoflavonoids.
Likewise, the compositions of the present invention are useful to alter the
natural color of plant
tissues, especially to achieve an increase of color; specifically red, violet,
purple, blue, yellow,
orange, and red-orange colors, and more specifically changes of color toward
red, violet, purple
or blue. Specifically, and without losing generality, the changes in color are
related to an
alteration of the contents or the proportion of flavonoid compounds in the
plant, such as
anthocyanins, leucoanthocyanins, chalcones, aurones, flavones, isoflavones,
flavans, isoflavans,
flavonoles, flavanols, isoflavonols, isoflavanols, dihydroflavonols,
dihydroflavanols, flavanones,
isoflavanones, dihydrochalcones, proanthocyanidins, catechins, biflavonoids
and isoflavonoids,
and the glycosylated derivatives thereof. More specifically, without losing
generality, a color
change is expected in flowers and/or fruits (inflorescences and/or
infrutescences), especially in
the pericarp and mesocarp.
Additionally, the compositions of the present invention alter the content of
antioxidant species in
plant tissues. Especially, an increase in the content of antioxidant species
is expected, i.e.
molecules able to scavenge and stabilize free radicals, turning them into
unreactive species and
thus blocking the generation chain of more free radicals. More specifically,
an alteration or
increase in the content of polyphenolic compounds is expected, especially
flavonoids such as
flavone, flavonol, 3'-hydroxiflavone, hispidol, chrisin, primetin, 7,4'-
dihydroxiflavone, butein,
sulfuretin, frutinone A, baicalein, 5-deoxikaempferol, galagin, norwogonin,
tectochrisin,
aurantinidin, aureusidin, maritimetin, 4,5-methylenedioxi-6-hydroxiaurone,
phloridzin, phloretin,
okanin, chrisin 5,7-dimethylether, datiscetin, fisetin, geraldone, wogonin,
graveolin, 3-
methylgalangin, 2'-hydroxipseudobaptigenin, 6-hydroxicyanidin, leptosidin,
robinetin, japonin,
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33
baicalein 5,6,7-trimethylether, galangin 3,5,7-trimethylether, lunamarin,
kinobscurinone, stealthin
C, tetrangulol, phenanthroviridone aglycone, isobavachalcone, 8-O-
methyltetrangulol, 19-
hydroxitetrangulol, efloxate, glepidotin A, hypoxylone, 6-(3,3.dimethylallyl)-
galangin, 8-(1,1-
dimethylallyl)-galangin, 19-hydroxi-8-O-methyltetrangulol, dimefline
hydrochloride, chlorflavonin,
ficin, isoficin, 8-(3,3-dimethylallyl)-3-methylgalagin, 8-(1,1-dimethylallyl)-
3-methylgalagin,
skullcapflavone II, PD116740, diospirin, flavoxate, abisinone VI, 6,8-di-(3,3-
dimethylallyl)-chrisin,
6-geranilchrisin, 8-geranilchrisin, tetracenomycins, carthamone, baicalin,
knipholone, raloxifen,
rottlerin, isobutrin, jadomycin B, actinorhodin, malvin, salvianin, 4"'-
demalonylsalvianin,
monardaein, conocurvone, anthocyanidins, anthocyanins, isoliquiritigenin,
liquiritigenin, daidzein,
2'-hydroxidaidzein, formononetin, 2'-hydroxiformononetin, vestitone,
medicarpin, maackiain, 4-
hydroxihomopterocarpin, phaseolin, trifolirhizin, ferreirin, homoferreirin,
toxicarol, sumatrol,
dalpanin, pachirrhizone, millettone, deguelin, tephrosin, rotenone, 12a-
hydroxirotenone,
biochanin A, 2'-hydroxibiochanin A, genistein, 2'-hydroxigenistein,
isoformononetin, calycosin,
orobol, prunetin, pseudobaptigenin, texasin, afrormosin, bowdichione, cajanin,
irilone, pratensein,
sayanedin, tectorigenin, dehydroferreirin, irisolidone, wighteone,
licoisoflavone A, luteone, 7-0-
methyl-luteone, daidzin, osajin, puerarin, genistin, ononin, pomiferin,
rotenonone, tectoridin, iridin,
paniculatin, aurone, aureusidin, riccionidin A, bracteatin, isoetin, betulin,
phloridzin, phloretin,
naringenin-chalcone (isosalipurpol), eriodictyol-chalcone, homoeriodictyol-
chalcone,
pentahydroxiflavanone-chalcone, coreopsin, okanin, sigmoidin B, 5'-prenyl-
homoeriodictyol,
naringenin, eriodictyol, homoeriodictyol, pentahydroxiflavanone, hesperidin,
hesperetin, apigenin,
apiin, malonylapiin, acacetin, luteolin, 3'-O-methyl-luteolin, tricetin,
apiforol, apigeninidin,
luteoforol, luteolinidin, dihydrokaempferol (aromadendrin), dihydroquercetin
(taxifolin),
dihydromyricetin (ampelopsin), kaempferol, quercetin, myricetin, rutin,
diosmin, leucocyanidin,
leucopelargonidin, leucodelphinidin, leucopaeonidin, leucomalvidin,
leucopetunidin, cyanidin,
pelargonidin, delphinidin, paeonidin, malvidin, petunidin, kuromanin,
callistephin, afzelechin,
epiafzelechin, catechin, epicatechin, gallocatechin, epigallocatechin,
cinchonain 1 a, mahuanin D,
gambiriin C, proanthocyanidins, fisetinidol, teasinensin A, kandelin A-1,
silandrin, silimarin,
eriocitrin, neoeriocitrin, 2,3-dihydrogossypetin, 6-methoxitaxifolin, 3-0-
taxifolin acetate, astilbin
(neoastilbin), silichristin, hesperidin, kolaflavanone, maniflavanone,
neohesperidin, 6-
hydroxicyanidin, rosinidin, capensinidin, hirsutidin, awobanin,
malonylawobanin, gentiodelphin, 8-
hydroxikaempferol, 6-hydroxikaempferol, 3-methoxiapigenin, kaempferide, morin,
3,3',4',5,7,8-
hexahydroflavone, 3-0-methylquercetin, azaleatin, isorhamnetin, pinoquercetin,
quercetagetin,
rhamnetin, sexangularetin, tamarixetin, 3,7-di-0-methylquercetin, 3,3',4',5,7-
pentahydroxi-6-
methoxiflavone, patuletin, laricitrin, 3,7,4'-tri-O-methylquercetin, 3',4',5,6-
tetrahydroxi-3,7-
dimethoxiflavone, axilarin, pachyipodol, santin, tambulin, siringetin, 3-
quercetin sulfate, 3',4',5-
trihydroxi-3,6,7-trimethoxiflavone, 3',4',5-trihydroxi-3,7-dimethoxiflavone,
chrysosplenol C,
oxyayanin A, oxyayanin B, chrysosplenetin, veloquercetin, 8-(1,1-
dimethylallyl)-kaempferide,
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34
3,3'-quercetin bis-sulfate, lilalin, quercitrin, gossypetin, trifolin,
isotrifolin, hyperin, myricitrin,
quercimeritrin, 3,3',7-quercetin tris-sulfate, 3,4',7-quercetin sulfate,
gossypin,
sophoraflavonoloside, baimaside, robinin, 6-methoxiaromadendrin 3-0-acetate,
phellamurin,
sanggenon C, sanggenon D, vitexin, genkwanin, isoscutellarein, scutellarein,
chrysoeriol,
apigenin 7,4'-dimethylether, diosmetin, hispidulin, 6-hydroxiluteolin,
hipolaetin, tricetin, pedalitin,
cirsiliol, tricin, cirsilineol, eupatilin, nevadensin, acerosin, timonin.
sinensetin, tangeretin, nobiletin,
filospadin, morusin, isoorientin, orientin, isoscoparin, swertiajaponin,
agatisflavone,
amentoflavone, cupressuflavone, hinokiflavone, calycopterones, neoschaftoside,
robustaflavone,
schaftoside, carlinoside, neocarlinoside, violantin, saponarin, ginkgetin,
vicenin-2, lucenin-2,
sciadopitisin, kuwanone G, kuwanone H, the glycosylated, dimeric, trimeric and
oligomeric
derivatives thereof, and the like; quinones, phenols, phenolcarboxylic acids,
hydroxicinnamic
acids, tannins, stilbenes, phenylanthrenes, pterocarpans, phenylpropanoids,
furocoumarins and
the like.
In the other hand, the present invention also may improve the plant resistance
against pests and
pathogens. Specifically, the improved resistance is due to the increase in the
concentration of
polyphenolic compounds or their metabolic derivatives in plant tissues when
subjected to a
treatment as described, specifically, when this increase in resistance is
accompanied by the
changes (effects) previously mentioned. Especially, this increase in
resistance is focused on a
higher resistance against fungi, bacteria and insects attack.
Likewise, a higher resistance of the plant product treated as previously
described against fungi,
bacteria and insects after harvest and during storage, conservation and/or
processing may be
achieved. Specifically, this resistance is due to the accumulation
(concentration) of polyphenolic
compounds and/or metabolic derivatives thereof in the plant products treated
as previously
described, especially flavonoids, such as those mentioned before.
The present invention also provides functional foods that include a higher
content of antioxidant
species, especially polyphenols, flavonoids and anthocyanins, more
specifically those
polyphenolic and flavonoid derivatives mentioned above. Specifically, these
functional foods are
fruits and infrutescences, flowers and inflorescences and/or other plant parts
obtained by means
of the abovementioned procedures, i.e. flowers and inflorescences, fruits and
infrutescences
and/or other plant parts treated with the compositions of the invention that
result in an increased
content of antioxidant species, especially the polyphenolic compounds,
flavonoids and
anthocyanins mentioned previously.
The present invention is also related to plants, plant parts, fruits, flowers
and/or propagating
material treated with the compositions of the invention.
The present invention is illustrated by means of formulation examples and test
examples, without
limiting the scope and spirit of the present invention as it has been
described.
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Examples
Different formulations were prepared to test the effects that said
formulations have on Royal Gala
apples during ripening. The compounds used for this were: caffeic acid,
coumaric acid and ferulic
acid. In a first example, a formulation was prepared (formulations A) that
contained only ferulic
acid, which was directly sprayed over the fruit in a single application, 5
weeks before harvest. In a
second example, a second type of formulations (formulations B) were prepared,
which contained
one or more active compounds. In this second opportunity, these formulations
were directly
sprayed over the fruit twice, 7 and 3 weeks before harvest. In both cases,
formulations were
applied during morning hours, so the formulation could dry over the fruits
before the hours of
highest solar irradiation.
Example 1: Preparation of diluted formulations of ferulic acid
As previously mentioned, formulations containing only ferulic acid in a
suitable range of working
concentrations were prepared. For this, 3 diluted formulations were prepared
(designated
"formulations A"), each in a final volume of 0.5 liters. The first
formulation, formulation A-I,
contained a final concentration of 10 mM ferulic acid, the second formulation,
formulation A-II,
contained a final concentration of 20 mM ferulic acid, and the last
formulation, formulation A-III,
contained a final concentration of 40 mM ferulic acid. The composition of the
final diluted
formulations is shown in the following table.
Table 4: Composition of diluted formulations A
A-I A-II A-III
Ferulic acid 10 mM 20 mM 40 mM
Ethanol 100 ml 100 ml 100 ml
Zoom 50 0.25 ml 0.25 ml 0.25 ml
KH2PO4 50 mM 50 mM 50 mM
Water 400 ml 400 ml 400 ml
Formulations A-I, A-II and A-III were prepared as follows. First, equal
suitable amounts of water
and pure ethanol (Merck S.A., Santiago, Chile) were mixed to form a 20%
ethanol-water solution.
In this mixture, a suitable amount of KH2PO4 (Merck S.A., Santiago, Chile) was
dissolved to
obtain a 50 mM KH2PO4 solution in 20% ethanol. To 500 ml of this solution 0.25
ml of Zoom 50
(commercially available from ANASAC, S.A.C.I., Santiago, Chile) was added.
Zoom 50 is a non-
ionic surfactant co-adjuvant that acts as humectant-adherent and emulsifier
agent. Zoom 50 is
composed by alkylphenol ether and polyethyleneglycol, with a concentration of
440 g/l. For the
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36
purposes of this invention, it has been found that the use of said surfactant
agent allows a better
application and a better penetration of the active compounds in the tissues of
treated plants, as
explained above. Finally, to 500 ml of the resulting solution a suitable
amount of 99.5% ferulic
acid (Sigma-Aldrich Co., St. Louis, MO) was added to achieve the desired
concentration for each
formulation. The resulting suspension was stirred until completely dissolving
the solid ferulic acid
at room temperature (10 minutes), with no additional heating to avoid inducing
degradative
processes. These formulations were applied over treated plant parts by
spraying, achieving a
total coverage of the treated parts.
Example 2: Preparation of concentrated formulations of ferulic
acid, p-coumaric acid and caffeic acid and mixtures of ferulic and
p-coumaric acids and caffeic and p-coumaric acids
Subsequently, a new set of formulations was prepared (designated "formulations
B"), which
include mixtures of some active compounds of the invention. The formulations
also comprise
different concentration levels for the active compounds used alone or as part
of a mixture. As a
comparative reference with respect to the previous set of formulations
(formulations A), a
formulation including only ferulic acid in two different concentrations was
used. Formulations B
were initially prepared as concentrated solutions of the compound(s) of the
invention in a suitable
solvent. In Formulations B, dimethylformamide (DMF) was used as the solvent in
an amount
slightly in excess over the amount required to solubilize the required amount
of active compound,
without heating. In this way, the desired final concentration could be
obtained by suitably diluting
the concentrated solution before application.
To prepare the required concentrated solution to obtain 500 ml of diluted
solution in each case,
an appropriate amount of the active compound(s) was mixed with 7 ml of
dimethylformamide. The
resulting suspension was stirred for a time enough to achieve complete
dissolution of the active
compound(s) at room temperature (10 minutes), without additional heating to
avoid inducing
degradative processes.
Following this procedure, the concentrated formulation mixtures of Table 5
were prepared.
Although an equal amount of solvent (DMF) was always used in the example shown
with the
purpose of comparing the different effects of each formulation on the fruit,
the amount of solvent
might be suitably varied for each of the formulations, according to the
requirements of each case.
Table 5: Concentrated formulation
Formulation
B-I B-II B-III B-IV B-V B-VI B-VII B-VIII
Compound
Ferulic ac. (g) - 1.9420 - - 3.8840 - 1.9420 -
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- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - -
p-Coumaric ac.
1.6416 - - 3.2832 - - 1.6416 1.6416
(g)
Caffeic ac. (g) - - 1.8016 - - 3.6032 - 1.8016
DMF (ml) 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0
This new set of formulations was mmediately diluted in a carrier liquid that
contained the
remaining components of the formulation, as exposed in the following Example,
just before
spraying them over apples.
Example 3: Preparation of dilution liguid and diluted formulations of
ferulic acid, p-coumaric acid and caffeic acid and mixtures of ferulic
and p-coumaric acids and caffeic and p-coumaric acids from
concentrated solutions
The concentrated Formulations B of Table 5 were diluted immediately before
application in a
dilution liquid prepared from the components and with the proportions
indicated in Table 6. The
dilution liquid comprises a humectant-adherent and emulsifying agent (Zoom 50
, from ANASAC
S.A.C.I., Santiago, Chile), that allows also a better penetration of the
active compounds in the
plant tissues, and a volatile pH buffer (ammonium bicarbonate), both
solubilized in water.
Table 6: Formulation of the dilution liquid
Compound Amount
Zoom 50 0.25 ml
(NH4)HCO3 3.45 g
Water Up to 500 ml
The dilution liquid was prepared by dissolving 3.45 g of ammonium bicarbonate
in 450 ml of
water, with stirring. Once the solid completely dissolved, 0.25 ml of Zoom 50
were added and
the solution was homogenized during 1 additional minute. Finally, water was
added in a sufficient
amount to reach 500 ml.
Using this dilution liquid that contributes the remaining components of
Formulations B, a diluted
solution was prepared, with the suitable volume proportion between the
concentrated formulation
and the dilution liquid indicated in Table 7. For this, the entire
concentrated solution previously
prepared was used and dilution liquid was added up to 500 ml, just before
application by spraying
over the plants, looking for a total coverage of the treated parts.
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Table 7: Preparation of the diluted formulation for application
Solution Volume (ml)
Concentrated formulation 7-12
Dilution liquid up to 500 ml
In this way, the final composition of each of the diluted formulations used in
the present
Example to be applied directly over the plants is presented in Table 8.
Table 8: Composition of diluted formulations B
Formulation
Component B-I B-II B-III B-IV B-V B-VI B-VII B-VIII
Ferulic ac. - 20 - 40 20 -
(mM)
p-Coumaric ac. 20 - - 40 - - 20 20
(mM)
Caffeic ac. - - 20 - - 40 - 20
(mM)
DMF (ml) 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0
Zoom 50 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
(% v/v)
Ammonium
bicarbonate 100 100 100 100 100 100 100 100
(mM)
Water up to up to up to up to up to up to up to up to
500 ml 500 ml 500 ml 500 ml 500 ml 500 ml 500 ml 500 ml
Example 4: Preparation of diluted formulations of ferulic acid
containinp another aprochemical product
A new set of formulations was also prepared (designated "formulations C"),
said formulations
including mixtures of an active compound of the invention and other
agrochemical compounds.
The active compound selected for these formulations was ferulic acid, which
was combined in a
first formulation (designated "Formulation C-I") with a fungicide
(tebuconazole), and in a second
formulation (designated "Formulation C-II") with an insecticide (diazinon). As
a comparative
reference for the effect of ferulic acid with this two agrochemical compounds,
previously
described Formulation B-II, which contained 20 mM ferulic acid, was used.
Diluted formulations C were prepared from the concentrated solutions
previously prepared
according to the method of Example 2, using a suitable amount of ferulic acid
and
dimethylformamide. Concentrated formulations were diluted in the dilution
liquid described in
Example 3. An appropriate amount of the corresponding additional agrochemical
compound
contained in a suitable commercial formulation was added to the obtained
solution. The obtained
solution was immediately applied by spraying over the plants looking for a
total coverage of the
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treated parts, including leaves and branches to keep the effect of the other
agrochemical
components.
Suitable commercial formulations were used to prepare Formulations C. TACORA
25 WP, a
wettable powder formulation including 25% by weight of tebuconazole (ANASAC
S.A.C.I.,
Santiago, Chile), was used to include tebuconazole in the formulation, while
DIAZINON 40 WP, a
commercial formulation including 40% by weight of diazinon (ANASAC S.A.C.I.,
Santiago, Chile),
was used to include diazinon in the mixture. The commercial formulations of
said products were
used according to the manufacturer's indications for each corresponding
product.
In this way, the final composition of each of the diluted formulations used in
the present Example
that were applied directly over the plants is presented in Table 9.
Table 9: Composition of diluted formulations C
Formulation
Component C-I C-II
Ferulic ac. 20- 20
(mM)
0.2
Tacora 25 WP (g) (equivalent to 0.05 g -
of tebuconazole)
0.6
Diazinon 40 WP (g) - (equivalent to 0.24 g
of diazinon)
DMF (ml) 7.0 7.0
Zoom 50 (% v/v) 0.5 0.5
Ammonium bicarbonate 100 100
(mM)
Water up to 500 ml up to 500 ml
Example 5: Results of the application of the formulations over
Royal Gala apples
Results for Formulations A.
Many concentrations of ferulic acid were initially tested to determine a
concentration
range that was interesting for the preparations of the present invention.
Formulation A-I was
prepared including 10 mM ferulic acid, Formulation A-II including 20 mM
ferulic acid, and
Formulation A-III including 40 mM ferulic acid. All Formulations A were
compared with a "control"
formulation that did not contain ferulic acid or any other active compound
described in the present
invention, but including the remaining components of Formulations A in the
abovementioned
concentrations. In Figure 1, results are shown for fruit color when using each
of the three
formulations. For the purpose of analyzing the obtained results, an acceptable
fruit color was
defined as red color covering more than 35% of the total surface of the apple.
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Obtained results, expressed as percentage based on treated apples, are shown
in Figure 1 and
in Table 10.
Table 10: Summary for color results
Summary table for color results
Level > 35% < 35%
Formulation A-I 61% 39%
Formulation A-II 93% 7%
Formulation A-III 69% 31%
Control 58% 42%
Regarding the obtained results, it can be observed that:
0 Formulation A-I had little effect to improve the color
OFormulation A-I1 produced an increase of more than 30% of well-colored
apples, i.e. apples
having more than 35% of their surface covered with red color, and a related
reduction of poorly-
colored apples.
OFormulation A-I II produced a moderate increase in apple color.
When all three Formulations A were assessed as protectants against sunburn
damage intensity,
the obtained results are shown in Figure 2 and in Table 11, expressed as
percentage based on
treated apples.
Table 11: Summary of results for sunburn damage intensity
Summary table of results for sunburn damage intensity
Level No damage Damaged
Formulation A-I 49% 51%
Formulation A-II 48% 52%
Formulation A-III 68% 32%
Control 54% 46%
Regarding the obtained results, it can be observed that:
0 Formulations A-I and A-II were not significanty effective to reduce sunburn
intensity.
OFormulation A-III produced a moderate increase in the percentage of apples
with no sunburn
damage.
Likewise, Figure 3 and Table 12 show the results of the tests performed with
Formulations A to
assess their effectiveness to decrease the total apple surface affected by
sunburn damage. The
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results are shown as percentage based on treated apples, by categorizing
results in three classes
according to the total percentage of surface affected: 0% damaged surface
(Nothing), less than
15% damaged surface (Slight) and more than 15% of damaged surface (Remainder).
Furthermore, the sum of apple percentages without sunburn damage and with
little surface
damage caused by sunburn (Nothing + Slight) is shown.
Table 12: Summary of results for total surface affected by sunburn damage
Summary table of results for total surface affected by sunburn damage
0%
(Nothin
Level g) (Slight) (Nothing + Slight) Remainder
Formulation A-I 49% 34% 83% 17%
Formulation A-II 48% 39% 87% 13%
Formulation A-III 68% 17% 85% 15%
Control 54% 20% 74% 26%
Regarding the obtained results, it can be observed that:
OFormulations A-I, A-II and A-III produce a moderate decrease of the surface
damaged by
sunburn. In the cases when said formulations does not avoid sunburn
occurrence, they protect
against sunburn damage extended over more than 15% of the fruit surface.
To determine whether the desired effects are produced by the applied
formulations and not due
to random effects, the obtained results were subjected to 3 different
statistical tests: X2 analysis
for homogeneity or independence, Mann-Whitney-Wilcoxon test for independence
(MWW) and
Kolmogorov-Smirnov test for independence (KS). In the following Table 13, the
summary of
results for Formulations A for the three studied characteristics (increased
color, decreased
sunburn damage intensity and decreased surface affected by sunburn damage) and
the statistical
validity found for each treatment with respect to the control are shown.
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Table 13: Summary of results of statistical tests for the significance of the
treatments
Levels of significance (p) of the population equality hypothesis
Formulation Statistical test
Color X2 MWW KS
Formulation A-I < 0.01 0.09 n.s.
Formulation A-1I < 0.01 < 0.01 < 0.01
Formulation A-III < 0.01 0.02 < 0.01
Intensity X2 MWW KS
Formulation A-I < 0.01 0.06 n.s.
Formulation A-1I < 0.01 0.03 < 0.1
Formulation A-III < 0.01 < 0.01 < 0.01
Surface X2 M W W KS
Formulation A-I < 0.01 n.s. n.s.
Formulation A-1I < 0.01 n.s. < 0.05
Formulation A-III < 0.01 < 0.01 < 0.01
n.s.: non significant
The statistical validity of the differences detected when applying the
different treatments over the
fruit and the improvement obtained from the use of said treatments is thus
demonstrated.
Results for Formulations B.
Given that the concentrations required to achieve a good color are different
than those required to
obtain a good protection against sunburn damage when using compositions that
use only ferulic
acid, a new set of formulations (Formulations B) were designed to improve the
observed results.
The new set of formulations also compared the effects and properties of p-
coumaric, caffeic and
ferulic acids either individually or in combination at 20 mM and 40 mM
concentrations, in order to
determine their ability to improve color and to quantify their efficiency to
decrease the intensity
and extension of sunburn damage. Therefore, Formulation B-I including 20 mM p-
coumaric acid,
Formulation B-II including 20 mM ferulic acid, Formulation B-III containing 20
mM caffeic acid,
Formulation B-IV containing 40 mM p-coumaric acid, Formulation B-V including
40 mM ferulic
acid, Formulation B-VI containing 40 mM caffeic acid, Formulation B-VII
including a mixture of p-
coumaric and ferulic acids 20 mM each, and Formulation B-VIII containing a
mixture of p-
coumaric and caffeic acids 20 mM each were prepared.
The effects of Formulations B-I to B-VIII over apple categorization and
commercial valuation
when compared to a control formulation that do not contain any of the active
compounds
described by the present invention, but including the remaining components at
the same
concentration, were assessed. The commercial categories of apples according to
their color,
form, lack of russet and lack of visible damage are: Juice, Commercial,
Choice, Fancy, Extra
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Fancy and Premium, being Juice the category corresponding to worst color and
lower price and
Premium the apple with best color and higher price. The results obtained for
the categorization
change of apples are shown in Figure 4 and Table 14.
Table 14: Results for the commercial categorization of apples obtained when
using Formulations
B
Category
Commercia
Product Premium Extra Fancy Fancy Choice I Juice
Control 0.6% 23.9% 35.0% 16.7% 8.7% 15.1%
B-I 0.7% 28.4% 23.9% 22.4% 13.4% 11.2%
B-II 3.3% 60.0% 15.0% 16.7% 3.3% 1.7%
B-III 12.7% 46.0% 14.3% 15.9% 3.2% 7.9%
B-IV 0.0% 32.3% 31.3% 13.5% 7.3% 15.6%
B-V 4.2% 45.3% 25.3% 17.9% 4.2% 3.2%
B-VI 0.0% 26.7% 40.0% 20.0% 3.3% 10.0%
B-VII 0.0% 29.4% 27.9% 18.4% 14.4% 10.0%
B-VIII 0.0% 53.3% 28.0% 12.0% 2.7% 4.0%
From the former data, it is possible to conclude that:
OThe application of these Formulations produces a systematic increase of the
proportion of Extra
Fancy and Premium apples, i.e. the proportion of apples with a desirable color
and without
sunburn damage increases.
OThe best results are obtained with the formulations that include ferulic acid
and caffeic acid
alone, and with the mixture of p-coumaric and caffeic acids, Formulations B-
II, B-III and B-VIII,
respectively.
0 Medium concentrations (20 mM) resulted more effective than high
concentrations (40 mM).
Results for Formulations C.
To exemplify the use of formulations of the invention that contain other
agrochemical products,
Formulations C were prepared and applied over apples as in the Examples above.
The result of
the effect over commercial categorization of apples of this assay was compared
with the result
obtained for Formulation B-II. Additionally, the fungicide effect or
insecticide effect of the
additional compound in the presence of ferulic acid was compared, using a
control formulation
without ferulic acid as a reference, but containing all the remaining
components of the
corresponding Formulation C, specifically the corresponding additional
agrochemical compound.
The effect of Formulations C-I and C-II on the commercial categorization of
apples was studied
as for Formulations B. The results of this assay were the following:
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ONo significant differences between the results of commercial categorization
of apples given by
Formulations C-I and C-II and those produced by Formulation B-II, according to
a X2 analysis for
homogeneity or independence, a Mann-Whitney-Wilcoxon test for independence
(MWW) and a
Kolmogorov-Smirnov test for independence (KS). Therefore, the effect of the
compound of the
invention is not altered by the presence of other agrochemical compounds.
mAdditionally, no significant differences were assessed for the effectiveness
of the other
agrochemical compounds when composing the formulation of the invention, in
comparison with a
control formulation that did not contain the compound of the invention.
