ES2391211A1 - Synergic polyphenol combination - Google Patents

Abstract

The present invention relates to a combination of the polyphenols resveratrol, quercetin and catechin in a molar ratio of approximately 1:1:2 or, alternatively, approximately 1:1:5, which has synergic antioxidant power. This combination of polyphenols is suitable for incorporation into any type of pharmaceutical, cosmetic, foodstuff or veterinary formulation and may be used in the prevention and treatment of pathological or physiological conditions that can be treated with antioxidant and free-radical scavenger substances.

Description

"SYNERGIC POLYPHENOL COMBINATION"

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Technical field

The present invention relates to polyphenol compositions that have a synergistic antioxidant power.

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Prior art

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Polyphenolic compounds of natural origin are recognized antioxidant agents that have been shown to be effective as free radical scavengers. Free radicals are highly reactive molecules that are naturally generated in the tissues, and that can also be formed in an increased way due to environmental stress situations such as, for example, cigarette smoke, the presence of ionizing radiation, ultraviolet radiation, or environmental pollution, as well as other causes such as stress situations or the practice of a very intense exercise. Free radicals are capable of inducing the oxidation of nucleic acids, proteins and lipids of the organism, which can contribute to the acceleration of the aging process and trigger diseases associated with aging such as degenerative diseases, cancer, cardiovascular diseases and dermatological diseases. In the state of the art it is described that the antioxidant properties of naturally occurring polyphenols can be applied in different therapeutic areas, and can also contribute to delaying the signs of aging thanks to their ability to reduce cellular and tissue oxidation. Resveratrol is a polyphenolic compound present in the seed and in the skin of the grape (Vítís vinífera) and in other plant products. Its therapeutic interest has been reinforced since its possible involvement in the cardioprotective effects of red wine was postulated, as described in the article Siemann et al., Concentration of the phytoalexin resveratrol in wíne, Am. J. Eno. Vitic., Free. Rad. Res., 1992, 43, 49-52, and its possible participation in the so-called French paradox: The French population, which has a diet rich in saturated fats, has a lower mortality rate due to cardiovascular disease than other countries

developed. This reduction was associated with the higher consumption of red wine and the pre

Sentence of resveratrol in red wine. Numerous studies, conducted in experimental models and published over the last few years confirm that resveratrol:

-

It can be effective to prevent or decrease the progression of various patho

gias such as cancer, diabetes, and cardiovascular diseases,

-

It has antiviral and anti-inflammatory properties, showing itself as a

Good alternative for the treatment of chronic inflammation,

it has protective properties in different organs, especially in the

Ischemic heart disease associated with myocardial infarction, thanks to its capacity

to prevent the oxidation of low density lipoproteins (LDL, of

low density English and lipoproteins), and

-

slows the symptoms of aging in yeasts and some vertebrates

simple.

Also noteworthy is the study by Baur et al., Resveratrol ímproves health and survíval of mice on a hig-calorie díet, Nature, 2006, 444, 337-342, in which it was shown that rats with a high-fat diet had higher pathologies and less survival than others undergoing the same diet, but those that were also given resveratrol.

Although pharmacological effects have been observed for resveratrol even at the relatively low concentrations present in the usual sources of the diet, given its low bioavailability (Walle et al., Hígh absorptíon but very low bíoavaílabílíty of oral resveratrol in humans, Drug Metab. Disp., 2004, 32 (12), 13771382), it is desirable to administer higher doses to achieve higher plasma and tissue concentrations and to enhance their therapeutic effects. However, the possible adverse effects of the continued administration of high doses of resveratrol are unknown and, in any case, the economic cost would be very high.

Other polyphenols in the flavonoid family known for their antioxidant properties are quercetin and catechins as described, for example, in articles Zhang et al., Antioxídant propertíes of quercetin, Adv. Exp. Med. Biol., 2011, 915, 283-9, and Plumb et al., Antioxídant propertíes of catechíns and proanthocyanídíns: effect of polymerísatíon, galloylatíon and glycosylatíon, 1998, 29 (4), 351-8.

In the state of the art, combinations of resveratrol have been described with In the international patent application WO-A-02/081651 they are described as also part of the object of the invention is a composition which comprises

other polyphenols to increase their bioavailability.

For example, the combination with quercetin may contribute to increasing

bioavailability of resveratrol, as described in the article De Santi et al.,

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Sulphatíon of resveratrol, a natural compound present in wíne, and íts ínhíbítíon by

naturalflavonoíds, Xenobiotica, 2000, 30 (9), 857-866.

Some combinations of resve have been described in the state of the art

ratrol with other polyphenols, in particular with quercetin and catechins, in different

proportions

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International patent application WO-A-201 0/135589 is addressed specifically

citically to drinkable dietary supplements prepared on the basis of a suspe

sion of resveratrol in water to which, optionally, other ingredients are added,

for example, quercetin and / or catechins from an extract of green tea, and

which are mainly in epigallocatechin gallate form, as well as other anti

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oxidizers in the form of extracts or concentrates of plant origin, and also others

agents called "energy", also of plant origin. Not described

no preferred ratio between resveratrol and the rest of the components adi

Regional In particular, a specific example of a beverage is described, in which the

weight ratio between resveratrol, quercetin and catechins was found to be

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17.2: 3.4: 1, equivalent to a molar ratio of 21.8: 3.3: 1.

International patent application WO-A-03/015738 describes a

cosmetic composition that can be used for the fight against aging

of the skin and that includes a combination of grape seed extracts, leaves

of grape and grape skins. Such grape seed extracts contain polyphene

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les, especially catechins in amounts up to 15% by weight, extracts

of grape leaves contain quercetins in amounts of up to 5% by weight, and the

Grape skin extracts contain resveratrols in amounts up to 5% in

weight. An example is also described with a mixture of skin extracts, leaf

and from grape seeds, which represents a combination of resvera

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trol: quercetin: catechins in an approximate ratio of 1: 1: 3 by weight, equivalent

lens at an approximate molar ratio of 1.3: 1: 3, 1. It is described that said combi

nation has a synergistic effect in terms of metalloprotein inhibition

   Matrix sas (MMP).

positions with resveratrol, catechin, epicatechin and quercetin, alone or in combination

nation, for use as cardioprotectors, and its fibrinolytic activity is analyzed in

an in vitro model with human endothelial cells derived from the umbilical vein

5

(HUVEC) and in an in vivo rat model. The examples describe composition

nes in the form of capsules or orange juice in which resveratrol is combined,

quercetin and catechins, in a weight ratio of 1: 2.5: 7.5, equivalent to one

1: 1, 9: 5.9 molar ratio.

On the other hand, in US patent application US-A-201 0/0247670

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a dry extract obtained from black grape of the Vítís variety is described

brusque, containing resveratrol, quercetin, catechin and tannins. Also give

screen the extraction with hot water of the dry extract to obtain infusio

nes, which were effective for the treatment of various conditions, such as

common cold, hypercholesterolemia or hyperglycemia, or for dermato treatments

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Logical cosmetic type. The analysis of two of the infusions is also described

prepared, according to which the weight ratio between resveratrol, quercetin and ca

tequina is 1: 5: 15 and 1: 4: 63, equivalent respectively to mo ratios

lares of 1: 3.8: 11.8 and 1: 3: 49.5.

Similarly, in Spanish patent application ES-A-2217966 it was described

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Ben pharmaceutical, dietary and cosmetic compositions with antioxy properties

Dantes comprising an extract of skin, seeds and black grape rasp of the

Vítís vinífera species that contains polyphenols and anthocyanins. Among the polyphenols are

They find catechin, gallic acid, quercetin, and resveratrol. It is described that said

combination of polyphenols and anthocyanins has antioxidant properties, in the

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proportions in which it is obtained directly from the plant extract. Also give

Screen the result of the analysis of a black grape extract, in which resveratrol,

Quercetin and catechin are in an approximate ratio of 1: 3: 21, equivalent

at an approximate molar ratio of 1: 2.3: 16.5.

There remains, therefore, the need to have a beef composition

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Veratrol with improved antioxidant properties, which has an antioxy power

high even at relatively low doses.

Object of the invention

   The object of the invention is a synergistic combination of polyphenols.

turn on that combination.

The use of the combination for the preparation of a composition with antioxidant power is also part of the invention.

The combination for use as an antioxidant is also part of the invention.

Detailed description of the invention

The object of the present invention is a synergistic combination of polyethenols comprising resveratrol, quercetin and catechin, with a molar ratio between resveratrol, quercetin and catechin of approximately 1: 1: 2 or approximately

1: 1: 5.

The authors of this invention have developed a combination of the resveratrol, quercetin and catechin poly-phenols in such proportions that, surprisingly, it has a synergistic antioxidant power.

In a preferred embodiment, the combination of antioxidants comprises resveratrol, quercetin and catechin in a 1: 1: 2 or 1: 1: 5 molar ratio.

In a preferred embodiment, the molar ratio is 1: 1: 2.

In another preferred embodiment, the molar ratio is 1: 1: 5.

In the context of the invention, the term "approximately" means, for the value "1" of the molar ratio, a variation between 0.9 and 1.1, preferably between 0.95 and 1, 05; for the value "2" a variation between 1.8 and 2.2, preferably between 1, 9 and 2.1; and for the value "5" a variation between 4.5 and 5.5, preferably between 4.75 and 5.25, and more preferably between 4.9 and 5.5.

Throughout the description the percentages refer to percentages by weight and the relationships are molar ratios.

Within the framework of this invention, "dry extract" means the resulting product after subjecting the plant extract to a drying process whereby substantially all of the water it contains is removed.

In the context of this invention, "purified extract" means an extract having a polyphenol content equal to or greater than 90% by weight on the dried extract. In the case of resveratrol preferably equal to or greater than 95%, more preferably equal to or greater than 98%; in the case of quercetin preferably equal to or greater than 95% expressed as quercetin dihydrate, more preferably

remarkably equal to or greater than 98%; and in the case of the catechin a catechin content expressed as catechin preferably equal to or greater than 80%, more preferably equal to or greater than 85%.

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Antioxidants Antioxidants are substances that can protect cells against oxidation and the effects of free radicals, because they eliminate these radicals from the environment and because they inhibit oxidation by oxidizing themselves. Among the antioxidants are polyphenols, which are compounds widely distributed in the plant kingdom, and have an appropriate chemical structure to capture free radicals. In its molecule, polyphenols incorporate more than one phenol group. Its property as an antioxidant comes from its great reactivity as electron donors and the ability of the radical formed to stabilize and delocalize the missing electron. The main sources of polyphenols are red fruits, tea leaves, beer, grapes, wine, olive oil, chocolate, cocoa, nuts, and pomegranates, among others.

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Resveratrol In the context of the invention the term resveratrol refers to transresveratrol and cis-resveratrol interchangeably, and also includes the active substance and its possible hydrates and / or solvates. Trans-resveratrol is the common name that designates 5 - [(1 E) -2- (4hydroxyphenyl) ethenyl] -1,3-benzenediol, which can also be called 3,5,4'trihydroxystilbene, and has the following structure:

OH

HO

   OH

Cis-resveratrol is the common name that designates 5 - [(1 Z) -2- (4

hydroxyphenyl) ethenyl] -1,3-benzenediol, and has the double bond of the previous figure in

cis position

Trans-resveratrol is a phenolic compound found in nature

5

Leza, produced by various plants. For example, it is one of the constituents

of the skin and the grape seed and, consequently, one of the components of the wine,

Especially red wine.

The trans-resveratrol was isolated for the first time in the year 1940 from the

Hellebore root, as described in article M. J. Takaoka, Of the phenolíc

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substances of whíte hellebore (Veratrum grandíflorum Loes. fíl), J. Faculty Sci. Hok

Kaido Imperial University, 1949.3, 1-16.

Trans-resveratrol can be obtained as an extract from various species

Vegetable seeds such as skin and grape seed (Vítís), blueberries

(Vaccinum), Polygonum cuspídatum root, hops (Humulus lupulus), Cassia

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garrettíana, Cassía Quínquangulata, Gnetum klossíí, Pterolobíum hexapetallum, the

peanut (Arachís hypogaea), pistachio (Pístacía vera), leaves and rhubarb root

(Rheum rhabarbarum and Rheum Rhapontícum), the bark of Bauhínía racemosa, the

Veratrum grandiflorum leaves and Veratrum formosanum root, eucalyptus, el

Spruce and spruce, among others.

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Commercially you can find various plant extracts with

had trans-resveratrol, reaching values greater than 95% on the extract

dry.

For example, the Polygonum cuspídatum root extract from the company Se

Botanical lect, is a purified extract that has a water content as

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maximum of 8% by weight, and whose dry extract has a trans content

resveratrol greater than 98%, determined by HPLC.

Likewise, there are extracts from grape seed and root of

Polygonum cuspídatum generally, with very variable resveratrol contents

which can be comprised between 0.05% and 98% determined by HPLC.

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The determination of resveratrol content by HPLC can be done, for example

plo, as described in the article Kolouchová-Hanzlíková et al., Rapíd method

for resveratrol determination by HPLC wíth electrochemícal and UV detectíons in

   wínes, Food Chem., 2004.87 (1), 151-158.

Likewise, trans-resveratrol can be obtained in substantially form le), apples, onion, black grape, citrus, papaya, tomato, broccoli, cauliflower, chili

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pure through suppliers such as the Sigma-Aidrich company. Trans-resveratrol can also be synthesized, for example as described in the article Farina et al., An ímproved synthesís of resveratrol, in Nat. Prod. Res., 2006, 20 (3), 247-52. On the other hand, cis-resveratrol can be obtained, for example, from trans-resveratrol by exposure to ultraviolet radiation. In the combination of the invention, transresveratrol is preferably used. In the combination of the invention, resveratrol may be of synthetic or natural origin. When it is naturally occurring, preferably the source of resveratrol is a plant extract. More preferably as a source of resveratrol, an extract of plant origin with a high content of trans-resveratrol is used, preferably greater than 95% on the dry extract, determined by HPLC, more preferably a root extract of Polygonum cuspídatum, and even more preferably a purified extract of Polygonum cuspídatum root with a trans-resveratrol content greater than 98% on the dry extract, determined by HPLC.

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Quercetin Quercetin or 2- (3,4-dihydroxyphenyl) -3,5, 7-trihydroxy-4H-1-benzopyran-4-one, is a polyphenol that structurally belongs to the flavonoid group and responds to the following formula :

OH

HO

OH

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OH OR

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   Quercetin is widely distributed in nature, and is present in a variety of plants and fruits. Because of this, quercetin can be obtained as an extract, from various vegetables and fruits such as, for example, green tea and black tea (Meat / lees synensis), capers, mountain celery (Officin levisticum

(Capsícum annum), blueberries, or Sophora japoníca, among others.

Quercetin can be found commercially as an extract of origin

vegetable with various contents, which can be more than 98% over

5

the dry extract, determined by HPLC, expressed as quercetin dihydrate.

For example, the purified extract of acacia flowers from Japan or tree of pa

Godas (Sophora Japoníca), from the Select Botanical company, is presented in the form

of a yellow powder containing quercetin in the form of quercetin dihydrate,

with a water content that is a maximum of 12.5%, and that has a content

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minimum of 98% quercetin dihydrate on the dry extract, determined

by HPLC, for example as described in the article Beecher et al., Analysís of

tea polyphenols, Proc. Soc. Exp. Biol. Med., 1999, 220 (4), 267-70.

Quercetin is also found as onion extract (AIIíum strain)

with varying concentrations between 0.5% and 40%, and as ex

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tracts of apple, asparagus and grapes.

Quercetin can also be obtained commercially in sustan form

purely through suppliers such as Sigma-Aidrich.

On the other hand, quercetin can be synthesized, for example, as described.

Screen in the article Shakhova et al., Zh. Obshch Khím., 1962, 32, 390.

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In the context of the present invention, the term quercetin is understood as

broadly and includes the active substance and its possible hydrates and / or solvates.

In a preferred embodiment of this invention, quercetin is in the form of dihi.

drato.

In the combination of the invention, quercetin may be of synthetic origin.

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Co or natural When it is naturally occurring, preferably the source of quercetin

It is a plant extract. More preferably an extract of vegetative origin is used

such with a high quercetin content, such as for example greater than 95% or

The dry extract expressed as quercetin dihydrate, and more preferably

an extract of Sophora japoníca, and even more preferably a purified extract

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of Sophora japoníca with a content greater than 98% of quercetin dihydrate

   On the dry extract.

Catechin

Catechin is the common name by which it knows the product (2R, 3S) -2 (3,4-dihydroxyphenyl) -3,4-dihydro-2H-chromen-3,5,7-triol, a polyphenol also belonging to group of flavonoids, and that has the following structure:

~ OH

HO ,,,, ~

· ~ OH

OH

The catechin molecule has two asymmetric carbons or chiral centers in positions 2 and 3, as indicated in the previous structure and, therefore, there are actually four different stereoisomers: two of them in the cís configuration and two in the trans configuration The two trans forms correspond to the enantiomer (2R, 3S), represented in the above formula, and to the enantiomer (2S, 3R), in

The one that reverses the configuration of the two asymmetric carbons 2 and 3. The two enantiomers that are in cís configuration are usually called epicatechin. Thus, the name epicatechin corresponds to the product (2R, 3R) -2- (3,4-dihydroxyphenyl) -3,4-dihydro-2H-chromen-3,5,7 -triol, which has the following structure:

crOH

~

TODAY) '' '' ·· 'OH

1 2

or .. ,,,,

3 OH

OH

As for the trans isomer, epicatechin is understood as the assembly 25 formed by the two cis isomers, both the enantiomer (2R, 3R) represented arri

ba, as the corresponding enantiomer (2S, 3S), in which the configuration is invested

ration of the two asymmetric carbons 2 and 3.

Catechins are also found in nature in the form of dimers

and trimers, which are known by the name of proanthocyanidins or procyanidins or

5

oligomeric proanthocyanidins.

In the context of the present invention, any person is called catechin

of the 4 stereoisomers mentioned, that is, both the catechin and the epica

tequina and their respective enantiomers, to any of their possible dimers or

trimers in the form of proanthocyanidins type B (dimers formed from ca

1st

tequina and / or epicatechin) or proanthocyanidins type e (trimer formed from

epicatechin), to dimers in the form of gallate, and to any mixture of the ante

riores.

Commercially the source of catechin can be a plant extract with

different contents in catechins, which may be equal to or greater than

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90% on the dry extract expressed as catechin. For example, the extract

purified from Vítís vinífera of the company Select Botanical, which is presented in for

ma of an orange powder with a maximum water content of 8% and with

a catechin content of approximately 90% on the dry extract

expressed as catechin, determined by spectrophotometry, for example according to

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described in the article Peri et al., An Assay of Different Phenolíc Fractíons in Wí

nes, Am. J. Enol. Vitic., 1971, 22 (2), 55-58. Said extract contains a mixture of

trans-catechin, epicatechin, dimer in the form of gallate, and proanthocyanidins.

Other sources of catechins are extracts from green tea, cocoa, coffee

Green and blueberries.

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Commercially, the enantiomeric forms pu can also be obtained

flush of the catechin such as (2S, 3R) through suppliers such

as Sigma-Aidrich.

In a preferred embodiment of the present invention the catechist is used

na (2S, 3R) -2- (3,4-dihydroxyphenyl) -3,4-dihydro-2H-chromen-3,5,7-triol in sustan form

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purely pure.

In a more preferred embodiment of the present invention, the catechin is the

trans isomer, in any of its enantiomeric forms (2S, 3R) or (2R, 3S), or

either a mixture of the two in any proportion, and optionally containing

   also epicatechin in either of its two enantiomeric forms and / or proantho

cyanidines, or any mixture of the above in any proportion, prefer

It is obtained from vegetable extracts. In an even more preferable embodiment

rida, the source of catechin is a grape extract, that is, an extract of Vítís viní

preferably with a catechin content equal to or greater than 80% over

5

the dry extract expressed as catechin, and even more preferably used

a purified extract of vitis vinífera with a content of catechins equal or supe

less than 90% on the dry extract expressed as catechin.

In the present invention, plant extracts can be used as they were

tes of the antioxidants that contain different resveratrol contents, of

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quercetin, catechin, or extracts containing mixtures of two or more of es

cough polyphenols

Preferably a vegetable extract containing resveratrol is used.

so purely, that is, with a resveratrol content greater than 90% over

the dry extract, preferably greater than 92%, more preferably greater than

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95%, and even more preferably greater than 98%.

Preferably a vegetable extract containing quercetin is used.

so purely pure, that is, with a content greater than 90% on the extract

co, preferably greater than 92%, more preferably greater than 95%, and still

more preferably greater than 98% expressed as quercetin dihydrate.

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Preferably a vegetable extract containing catechin is used.

tancially pure, that is, with a catechin content greater than 80% over

the dry extract, preferably greater than 85%, and more preferably greater than

90% expressed as catechin.

In an especially preferred embodiment, the antioxidant combination

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comprises resveratrol, quercetin and catechin in a resvera molar relationship

trol: quercetin: catechin of approximately 1: 1: 2, the source of resveratrol is a

Polygonum cuspídatum root extract, preferably with a content of

trans-resveratrol greater than 95% on the dry extract, the source of quercetin is

an extract of Sophora japoníca, preferably with a dihydrate content of

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Quercetin greater than 98% on the dry extract, and the source of catechin is a

Vítís vinífera extract, preferably with an equal catechin content or

greater than 90% on the dry extract expressed as catechin.

In an especially preferred embodiment, the antioxidant combination

   comprises resveratrol, quercetin and catechin in a resvera molar relationship

trol: quercetin: catechin of approximately 1: 1: 5, the source of resveratrol is a

Polygonum cuspidatum root extract, preferably with a content of

trans-resveratrol greater than 95% on the dry extract, the source of quercetin is

an extract of Sophora japonica, preferably with a dihydrate content of

5

Quercetin greater than 98% on the dry extract, and the source of catechin is a

Vitis vinifera extract, preferably with an equal catechins content or

greater than 90% on the dry extract expressed as catechin.

In another especially preferred embodiment, the antioxidant combination

It consists essentially of resveratrol, quercetin and catechin in a molar relationship

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Resveratrol: Quercetin: catechin of approximately 1: 1: 2, the source of resveratrol

it is a root extract of Polygonum cuspidatum, preferably with a content

of trans-resveratrol greater than 95% on the dry extract, the source of quercetin

it is an extract of Sophora japonica, preferably with a dihydrate content

of quercetin greater than 98% on the dry extract, and the source of catechin is a

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Vitis vinifera extract, preferably with an equal catechins content or

greater than 90% on the dry extract, expressed as catechin.

In another especially preferred embodiment, the antioxidant combination

It consists essentially of resveratrol, quercetin and catechin in a molar relationship

Resveratrol: Quercetin: catechin of approximately 1: 1: 5, the source of resveratrol

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it is a root extract of Polygonum cuspidatum, preferably with a content

of trans-resveratrol greater than 95% on the dry extract, the source of quercetin

it is an extract of Sophora japonica, preferably with a dihydrate content

of quercetin greater than 98% on the dry extract, and the source of catechin is a

Vitis vinifera extract, preferably with an equal catechins content or

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greater than 90% on the dry extract expressed as catechin.

In a preferred embodiment, the antioxidant combination does not contain

routine.

The combination of polyphenols consisting of trans-resveratrol, catechin

((2R, 3S) -2- (3,4-dihydroxyphenyl) -3,4-dihydro-2H-chromen-3,5,7 -triol, its enantiomer or

30

mixtures of both), epicatechin ((2R, 3R) -2- (3,4-dihydroxyphenyl) -3,4-dihydro-2H

chromen-3,5,7-triol, its enantiomer, or mixtures of both), quercetin and rutin in

a 1: 1: 1: 1: 1 molar ratio, where each of the polyphenols has a

Chromatographic purity equal to or greater than 90%, such as those supplied by the com

   Pamaía Sigma-Aidrich, is not part of the invention.

Antioxidant power tests

The antioxidant power of the compositions of the present invention is

can be determined using an in vitro method designed to assess capacity

free radical scavenger of the tested substances. For example, the method

5

denominated TOSC (of the English Total Oxídant Scavengíng Capacíty), that allows the

Determination of the antioxidant power of the compounds at low concentrations.

With the TOSC method you can determine the antioxidant power of a com

set to a specific concentration, and can be applied to solutions with

pure antioxidants as in complex mixtures.

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In the TOSC method, the ability of the substance to be tested is determined

to inhibit oxidation of a-keto-and-methylthiobutyric acid (KMBA)

keto-gamma-methylthiobutyric acid) to ethylene by the action of free radicals such as,

for example, hydroxyl (HO ·), peroxyl (ROO ·) or peroxynitrite (ONOO} As per

writes in Winston et al., A rapid gas chromatograph assay for determining

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oxyradícal scavengíng capacíty of antioxídants and bíologícal fluíds, Free Rad. Biol

Med., 1998, 24 (3), 480-493, the reaction that takes place is as follows:

H3C-S-CH2-CH2-CO-COOH

KMBA Free radicals Ethylene

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Preferably, the TOSC test against peroxyl radicals was performed

(ROO ·) since they are more stable and have a longer half-life than radi

hydroxyl lime or peroxynitrite radicals. Peroxyl radicals can be obtained

25

by thermal decomposition and symmetric hydrolysis of the 2,2'-azobis compound (2

methylpropionamidine) (ABAP). In this way, the antioxidant power of sustan

The assays tested can be measured by the ability to inhibit the formation of ethylene

in the presence of peroxyl radicals compared to a control reaction, in which

an antioxidant substance is not used as an inhibitor.

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TOSC values are determined according to the methodology described in the articles

Asses Regoli et al., Quantifícatíon of total oxídant scavengíng capacíty of antioxídants

   for peroxynítríte, peroxyl radícals, and hydroxyl radícals, Toxicol. Appl. Pharm.,

1999, 156, 96-105 and Regoli et al., Total oxídant scavengíng capacíty (TOSC) of me

crosomal and cytosolíc fractíons from Antarctíc, Arctíc and Medíterranean scallops: differeníatíon between three potent oxídants, Aquat. Toxico l., 2000, 49 (1-2), 13-25.

Essentially, TOSC values are quantified by measuring ethylene formation, by comparison of the integrated areas for each tested substance and the control substance, according to the following equation:

, ._ '"' f '<; _"' :: -.;:, +. ~ "'" •> N ~ ·, ~ • "!: · X •; ::" "" ~~ "' :: - ·· · l '~ ...., ·, ..-._' !!, .- ,. · \

i} ·:> - ~ $ ~ · X.,? ¡... ,, · '' "'' ~ '".;', ,,,) li.l · XM. ~ ·

TOSC = 100-X 100}

'~~,. ~ ..: · ~ t, • -. · - ~~~ - ~ · ... {~~ f' ··· ::: · ~ ·.;, · F't "' '~' "}

\ ...., ..., .. ¡, ¡: _ .., _,.: E.:._~.• ..._ .., .._..... · or ;, - 4 ...... ~~ · 'l ._, __. \, <fl ...

l

..

Control substances, without the ability to eliminate free radicals, have a TOSC value of 0%, since they have the same area under the curve as the control. Compounds that completely prevent the formation of ethylene have an area under the curve equal to O and, therefore, a TOSC value of 1 00%.

Surprisingly, the authors of the present invention observed that the combination of the resveratrol, quercetin and catechin polyphenols in the molar proportions approximately 1: 1: 2 and approximately 1: 1: 5 show a synergistic anti-radical activity, since values of TOSC for combinations significantly greater than the sum of the TOSC values of its components, as described in Example 3.

As a result of this synergistic effect, it is possible to obtain a potent antioxidant effect without the need to ingest large amounts of resveratrol, or of the other individual antioxidants, which is advantageous in order to minimize possible risks of side effects.

Compositions A composition comprising the combination of polyphenols of the invention and an acceptable vehicle is also part of the object of the invention.

The composition is selected from a pharmaceutical composition, a cosmetic composition, a food composition, a dietary supplement, and a veterinary composition, and the vehicle is respectively pharmaceutical, cosmetic, food, dietary, or veterinarily acceptable.

Pharmaceutical forms suitable for incorporating the combination of polythene of the present invention can be formulated for oral, nasal, rectal, parenteral or topical administration. Both solid pharmaceutical forms and

semi-solids such as tablets, creams, hard capsules, soft capsules,

dragees, powder presentations; such as liquid or semi-liquid, such as

syrups or any type of aqueous or non-aqueous solution, suspension or emulsion.

The pharmaceutical compositions are formulated according to the procedures

5

while they are known to the expert in pharmaceutical technology, as

described, for example, in the book M. E. Aulton, Pharmacy. The science of design

pharmaceutical forms, second edition, Elsevier, Madrid, 2004. For example,

by mixing the antioxidant combination of the invention with at least one

pharmaceutically acceptable carrier or excipient and, optionally, with one or

1 o

more products with therapeutic or dietary functionalities, such as

vitamins, minerals, trace elements and other micronutrients.

Excipients suitable for use in pharmaceutical compositions

object of the present invention are well known to the technology expert

pharmaceutical and are described, for example, in the book R. C. Rowe, P. J. Sheskey and

fifteen

P.J. Weller, Handbook of Pharmaceutical Excipients, fourth edition, Pharmaceutical

Press, 2003.

For example, among the types of excipients and vehicles suitable for

be used in liquid formulations, in the form of solutions, suspensions or

emulsions are, for example, solvents, buffers, visco modifiers

twenty

sity, density modifiers, surfactants and emulsifiers, agen

flocculants, moisturizers, preservatives, sweeteners and flavorings, and mixtures

clas of the above. Similarly, between the types of excipients and / or vehicles

suitable for use in solid pharmaceutical compositions are, by

example, fillers, binders, non-stick agents, lubricate

25

tes, anti-caking agents, tablet coating materials,

gelatin for hard and soft capsules, plasticizers, stabilizers, agents with

servants, dyes, essences, and mixtures thereof.

The combination of polyphenols of the invention can also be incorporated.

in cosmetic compositions for external application, typically for the cui

30

hair or skin die. Cosmetic forms suitable for incorporating

Antioxidant combinations of the present invention are, for example, creams,

gels, milks, lotions, spray emulsions, and solid bars, among others.

Cosmetic compositions are formulated according to the procedures

   coughs that are known to the person skilled in the art, by mixing the com

polyphenol bination of the invention with at least one cosmetic vehicle

acceptable in which it dissolves, emulsifies, disperses, or suspends the same

ma. The vehicle is selected from water, a water miscible non-aqueous vehicle,

such as ethanol or isopropanol, and a non-aqueous non-miscible vehicle in

5

water, such as paraffin oil. Optionally, the compositions

Cosmetics may contain at least one additional cosmetic ingredient, which

it can be chosen, for example, from the group consisting of surfactants and

emulsifiers, lipid compounds and emollients, consistency factors and agen

thickeners, stabilizers, hydrotropes, preservatives, essences, colo

1O

rants, silicone compounds, fats, waxes, lecithins, phospholipids, factors

UV sun protection, film-forming, self-tanning, or tyrosine inhibitors (agen

depigmentation tes), or mixtures of the above.

Likewise, the combination of polyphenols of the present invention can

be incorporated into food products and dietary supplements, for example in

fifteen

form of nutraceutical products or functional foods or food supplements

Ticios for sports nutrition.

The elaboration of this type of products, incorporating the anti combination

oxidant of the invention, is performed by methods known to the former

expert in food technology.

twenty

Any type of food composition or dietary supplement is ade

to incorporate the combination of polyphenols object of the invention, by

for example, infusions, coffees, drinks in general such as juices or other drinks

Alcoholic or non-alcoholic, pastry or bakery products, jams, barri

Energy tastes, cereals, flours, dairy products such as cheese, milk, man

25

tequilla, yogurts or cream, chocolate-based products and candies such as candies,

chewing gums or ice cream, oils, margarine, preserves.

Optionally, the food product that incorporates the combination of po

lifenoles of the invention may additionally contain other products with fun

therapeutic or dietary requirements, preferably vitamins, minerals, oligoe

30

lements or other micronutrients, or other plant extracts source of polyphenols.

Also, the food product that contains the combination of polythene

The invention may additionally contain one or more ingredients, which are

   they choose among the usual authorized food additives, well known by the

expert in the field, for example of the type dyes, preservatives, condiments and

spices, flavor enhancers, thickeners and gelling agents. The combination of polyphenols of the invention can also be incorporated into veterinary compositions intended for animal feed, in particular to

5 feeding pets. Such combinations can be incorporated into pet food compositions, such as those described in patent applications EP-A-1514480 and WO-A-2005/110037, by methods well known to those skilled in the art.

The use of the combination for the preparation of a composition with antioxidant power is also part of the invention. The composition is selected from the group consisting of a pharmaceutical composition, a cosmetic composition, a food composition, a dietary supplement, and a veterinary composition. The combination of the invention for use as an antioxidant is also part of the invention. The combination of polyphenols object of the present invention can be used therapeutically to prevent or decrease the progression of all those

20 pathological conditions that can be treated with antioxidants and / or free radical scavengers, such as cancer, cardiovascular diseases, inflammatory diseases, coronary diseases, diabetes, Alzheimer's disease, multiple sclerosis or dermatological conditions such as psoriasis or the appearance of eczema , among other.

The combination of the invention is particularly useful for delaying the symptoms of aging, because of its ability to protect organs, tissues and cells against the harmful effects of oxidation. It is also appropriate, for example, to treat premature aging of the skin by preventing the breakdown of collagen fibers and, in general, all tissues.

The combination of the invention is also particularly suitable for those subjects most susceptible to generating free radicals in an increased manner in their organism, for example, smokers, people especially exposed to adverse conditions of environmental pollution, ionizing radiation, excess

of sun exposure, people subject to stress situations, or people who prac

5

Tican intense physical exercise. The following examples serve to illustrate the invention but should not be construed as limiting the same.

1 o

Example 1: Preparation of an antioxidant composition of resveratrol, guercetin and categorical molar ratio 1: 1: 2 based on resveratrol, guercetin and categorical origin from plant extracts

15 20

In a vessel equipped with a mixer for solid products, 253.16 g of purified extract of Polygonum cuspídatum root with a water content of 8% and a content of 98% of resveratrol were weighed on the dry extract, equivalent to 1 mol of resveratrol; 386.59 g of purified extract of Sophora japoníca with a water content of 12.5% and a content of 100% quercetin dihydrate on the dry extract, equivalent to 1 mol of quercetin; and 701.11 g of Vítís vinífera purified extract with a water content of 8% and a catechin content of 90% on the dry extract expressed as catechin, including 12.5% of monomeric catechins, the rest being dimeric procyanidins of type B and trimeric of type C, equivalent to 2 moles of catechin, and the whole was mixed until obtaining a complete homogenization.

25

Example 2: Preparation of an antioxidant composition of resveratrol, guercetin and categorical molar ratio 1: 1: 2 based on resveratrol and guercetin from vegetable extracts and categorical (2S, 3R)

30

   In a vessel equipped with a mixer for solid products, 253.16 g of purified extract of Polygonum cuspídatum root with a water content of 8% and a content of 98% of resveratrol were weighed on the dry extract, equivalent to 1 mol of resveratrol; 386.59 g of purified extract of Sophora japoníca with a water content of 12.5% and a content of 100% quercetin dihydrate on the dry extract, equivalent to 1 mol of quercetin; and 580.52 g of catechin (2S, 3R) substantially pure (Sigma), equivalent to 2 mo

of catechin, and the whole was mixed until homogenization was achieved

complete.

Example 3: Antioxidant Power Assay The antioxidant power of the compositions of the present invention was measured by the TOSC method, as described above. Solutions of 1 µm concentration of the substances or mixtures to be tested were used, and distilled water as a control, whose TOSC value is 0%. The following solutions were prepared, using distilled water as solvent:

1) Resveratrol (R): 253.16 mg of purified extract of Polygonum cuspídatum root was weighed, as used in Example 1, and dissolved in 1 1 of distilled water. 1 ml of the resulting solution was taken and diluted with distilled water to a volume of 1 l. The concentration of resveratrol was 1 µm.

2) Quercetin (Q): 386.57 mg of purified extract of Sophora japoníca were weighed, as used in Example 1, and dissolved in 1 1 of distilled water. 1 ml of the resulting solution was taken and diluted with distilled water to a volume of 1 l. The concentration of quercetin was 1¡tM.

3) Catechin (2S, 3R) (C): 290.26 mg of substantially pure catechin (2S, 3R) (Sigma) was weighed and dissolved in 1 1 of distilled water. 1 ml of the resulting solution was taken and diluted with distilled water to a volume of 1 l. The concentration of catechin was 1¡tM.

4) Catechin (Cextr): 350.56 mg of purified Vítís vinífera grape seed extract was weighed, as used in Example 1, and dissolved in 1 1 of distilled water. 1 ml of the resulting solution was taken and diluted with distilled water to a volume of 1 l. The concentration of catechin was 1¡tM.

5) Resveratrol-quercetin-catechin R + O + eextr (1: 1: 2): 0.5 ml of solution R, 0.5 ml of solution Q and 1 ml of solution eextr were taken, and mixed . This solution contained a concentration of 0.25 µm of resveratrol, 0.25 µm of quercetin and 0.5 µm of catechin. The total concentration of polyphenols was 1 µm.

6) Resveratrol-quercetin-catechin R + O + eextr (1: 1: 5): 0.5 ml of solution R, 0.5 ml of solution Q and 2.5 ml of solution eextr were taken, and They mixed. This solution contained a concentration of O, 143 µm of resveratrol, O, 143 µm of quercetin and O, 714 µm of catechin. The total concentration of polyphenols was 1 µm.

7) Resveratrol-quercetin-catechin (2S, 3R) R + O + e (1: 1: 2): 0.5 ml of solution R, 0.5 ml of solution Q and 1 ml of solution were taken e, and mixed. This solution contained a concentration of 0.25 µm of resveratrol, 0.25 µm of quercetin and 0.5 µm of catechin. The total concentration of polyphenols was 1 µm.

8) Resveratrol-quercetin-catechin (2S, 3R) R + O + e (1: 1: 5): 0.5 ml of solution R, 0.5 ml of solution Q and 2.5 ml of solution were taken solution e, and mixed. This solution contained a concentration of O, 143 µm of resveratrol, O, 143 µm of quercetin and O, 714 µm of catechin. The total concentration of polyphenols was 1 µm. Ethylene formation during the TOSe test was monitored by

gas chromatography, with an HP 5890-series 11 device, HP ehemsta Software

tion 5890, and was calculated by determining the area under the curve, by

the SRI PeakSimple® program. Linear regression was calculated using the program.

GraphPad Prism®. The slope of the regression was calculated in the linear margin of the

TOSe control curve against the concentration of the assets tested.

The results shown in Table 1 were obtained.

TABLE 1

Sample

Politeno les Experimental TOSC (%) TOSC calculated (%) Increase Power

one

R 22.00 - -

2

Q 11.00 - -

3

and 8.00 - -

4

Cextr 5.05 - -

5

R + O + C (1: 1: 2) 64.40 12.25 52.15 x5.3

6

R + O + C (1: 1: 5) 61.20 10.43 50.77 x5.9

7

R + O + Cextr (1: 1: 2) 60.00 10.78 49.23 x5.6

8

R + O + Cextr (1: 1: 5) 58.00 8.32 49.68 x7.0

5

The "Calculated TOSC" column represents the corresponding TOSC value

theoretically to samples of different antioxidant combinations you

taking into account only one additive effect of each of them. So the

calculated value is obtained by multiplying the concentration of each of the anti

oxidants in the sample by the experimental TOSC of each of the antioxidants

1O

individual and dividing by the concentration of the antioxidant in the individual sample

dual.

For example in the case of sample 5:

TOSC calculated =

(0.25 11M R x 22.00 / 1 11M) + (0.25 11M Q x 11.00 / 1 11M) +

+ (0.5 11M e x a, oo11 11M) = 12.25%

fifteen

The "Increment" column corresponds to the difference between the TOSC value

calculated and experimental, and allows quantifying the synergistic effect observed in the

antioxidant power of the combinations. It can be seen that the combination of

Resveratrol, quercetin and catechin of the invention, both in the 1: 1: 2 molar ratio,

   like in

The 1: 1: 5 molar ratio shows a remarkable synergistic effect. Likewise,

5

It can be seen that, although the synergistic effect with pure catechin (2S, 3R) is slightly higher than that of catechin from grape extract, a comparable synergistic effect can be seen in both cases, independent, therefore, of the catechins source used. The "Potency" column reflects the number of times the antioxidant power increases with the combinations object of the invention compared to the antioxidant power obtained from the separate addition of the antioxidant powers of resveratrol, quercetin and catechin.

1 o

Example 4: Preparation of a food supplement for the prevention of skin aging

fifteen

Soft gelatin (8/1 O oval) red capsules were prepared with the following filling per capsule:

Component

Amount (mg)

Purified Polygonum cuspídatum root extract of Example 1

33.53

Purified extract of Sophora japoníca from Example 1

52.18

Purified grape seed extract of Example 1

94.65

D acetate, L-a-tocopherol

10.00

Borage oil (> 20% y-linoleic acid)

150.00

Hydrogenated Soy Fat

10.00

   Bee wax

5.00

The molar ratio resveratrol: quercetin: catechin in this preparation is

1: 1: 2.04.

Example 5: Preparation of a food supplement for the improvement of the prevention of age-related macular degeneration

Soft gelatin capsules (14/16 oblong) of black color were prepared with the following filling per capsule:

Component

Amount (mg)

Vítís vinífera extract (8% water and 20% trans-resveratrol on the dry residue)

168.48

Purified extract of Sophora japoníca from Example 1

52.18

Purified grape seed extract of Example 1

94.65

Lutein 20% by weight

25.00

Blueberry dry extract (> 25% by weight of anthocyanidins)

325.00

Fish oil (50% by weight docosahexanoic acid)

325.00

Hydrogenated Soy Fat

20.00

Bee wax

10.00

The molar ratio resveratrol: quercetin: catechin in this preparation is 1: 0.97: 1, 98.

Example 6: Preparation of a food supplement for repair of expression wrinkles

Envelopes were prepared with the following filling per envelope:

Component

Quantity

Bovine Hydrolyzed Collagen

50.00 g

Maltodextrin

80.00 mg

Purified extract of Polygonum root cuspida tu m (8% water and 50% trans-resveratrol on the dry residue)

67.06 mg

Thereum strain extract (40% quercetin)

102.00 mg

Green tea extract (60% of catechins expressed as catechins)

130.62 mg

Pomegranate Seed Extract (40% ellagic acid)

87.08 mg

Aroma of red fruits

400.00 mg

Red beet coloring

150.00 mg

Sucralose

15.00 mg

Citric acid

200.00 mg

Sodium hyaluronate

10.00 mg

The molar ratio resveratrol: quercetin: catechin in this preparation is

1: 1: 2.

Example 7: Preparation of a functional coffee-based food for the prevention of cardiovascular disease

Single-dose sachets were prepared with the following filling per envelope:

Component

Quantity

Decaffeinated coffee

30.00 g

Purified Polygonum cuspídatum root extract of Example 1

37.61 mg

Purified extract of Sophora japoníca from Example 1

58.62 mg

Purified grape seed extract of Example 1

261.16 mg

Mocha aroma

50.00 mg

Citric acid

100.00 mg

The molar ratio resveratrol: quercetin: catechin in this preparation is

10 1: 1: 5.

Example 8: Preparation of a food supplement based on natural substances as an antioxidant in sports nutrition

15 Soft gelatin soft capsules (20 oblong) purple were prepared with the following filling per capsule:

Component

Amount (mg)

Coenzyme 01 O

100.00

Purified Polygonum cuspídatum root extract of Example 1

37.61

Purified extract of Sophora japoníca from Example 1

58.62

Purified grape seed extract of Example 1

261.16

RRR-alpha-tocopherol (67%)

15.00

Vitamin C

60.00

Beta-carotene dunaliella (95% retinol)

30.00

Wheat germ oil

500.00

Hydrogenated Soy Fat

20.00

Bee wax

10.00

5

1: 1: 5. The relationship cool resveratrol: quercetin: catechin in East prepared is

Example 9:

Preparation of a nutritional supplement for the improvement of the appearance of the skin and brightness of the fur in pets (mammals)

1 o

   Soft gelatin capsules (18/20 oblong) of red color were prepared with the following filling per capsule:

Component

Amount (mg)

Purified Polygonum cuspídatum root extract of Example 1

50.13

Purified extract of Sophora japoníca from Example 1

78.15

Purified grape seed extract of Example 1

353.61

Olive oil (30% oleic acid)

400.00

Borage oil (> 20% y-linoleic acid)

150.00

Hydrogenated Soy Fat

20.00

Bee wax

10.00

5

The molar ratio resveratrol: quercetin: catechin in this preparation is 1: 1: 5.09. Example 10: Preparation of a nutritional supplement to improve the fertility of stallions (mammals)

1 o

   Soft gelatin capsules (22 oblong) of red color were prepared with the following filling per capsule:

Component

Amount (mg)

L-carn itina tartrate

350.00

Purified Polygonum cuspídatum root extract of Example 1

18.80

Purified extract of Sophora japoníca from Example 1

29.28

Purified grape seed extract of Example 1

131.11

Fish oil (35% eicosapentaenoic acid and 25% docosahexaenoic acid)

550.00

Hydrogenated Soy Fat

20.00

Bee wax

10.00

The molar ratio resveratrol: quercetin: catechin in this preparation is

1: 1: 5.

Claims (16)

1.-Synergistic combination of polyphenols comprising resveratrol, quercetin and catechin, characterized in that the molar relationship between resveratrol, wants 5 tub and catechin is 0.9-1.1: 0.9-1.1: 1.8-2.2 or 0.9-1.1: 0.9-1.1: 4.5-5 , 5, with the proviso that the combination of polyphenols consisting of trans-resveratrol, catechin, epicatechin, quercetin and rutin in a 1: 1: 1: 1: 1 molar ratio, where each of the polyphenols have a purity Chromatographic equal to or greater than 90% is expressly excluded. 2. Combination according to claim 1, characterized in that the molar ratio is 1: 1: 2 or 1: 1: 5. 3. Combination according to claim 2, characterized in that the 15 molar ratio is 1: 1: 2. 4. Combination according to claim 2, characterized in that the molar ratio is 1: 1: 5. 5. Combination according to any one of claims 1 to 4, characterized in that the resveratrol is trans-resveratrol. 6. Combination according to any one of claims 1 to 5, characterized in that the resveratrol is of natural origin. 7. Combination according to claim 6, characterized in that the source of resveratrol is a plant extract. 8. Combination according to claim 7, characterized in that the vegetable extract is a root extract of Polygonum cuspidatum. 9. Combination according to any one of claims 1 to 8, characterized in that the quercetin is of natural origin. 10. Combination according to claim 9, characterized in that the source of quercetin is a plant extract. 11.-Combination according to claim 10, characterized in that the vegetable extract is an extract of Sophora japonica. 12.-Combination according to any one of claims 1 to 11, characterized in that the source of catechin is a plant extract. 13. A combination according to claim 12, characterized in that the plant extract is an extract of Vitis vinífera. 14.-Combination according to claim 1, characterized in that the resveratrol: quercetin: catechin molar ratio is 0.9-1.1: 0.9-1.1: 1.8-2.2, the source of beef 15 veratrol is a root extract of Polygonum cuspidatum, the source of quercetin is an extract of Sophora japonica, and the source of catechin is an extract of Vitis vinifera. 15.-Combination according to claim 1, characterized in that the relationship 20 molar resveratrol: quercetin: catechin is 0.9-1.1: 0.9-1.1: 4.5-5.5, the source of resveratrol is a root extract of Polygonum cuspidatum, the source of quercetin is an extract of Sophora japonica, and the source of catechin is an extract of Vitis vinífera. 16. Composition comprising the combination according to any one of claims 1 to 15 and an acceptable vehicle. 17. Composition according to claim 16, characterized in that the composition is pharmaceutical and the vehicle is pharmaceutically acceptable. 18. Composition according to claim 16, characterized in that the composition is cosmetic and the vehicle is cosmetically acceptable. 19. Composition according to claim 16, characterized in that the composition is a dietary supplement and the vehicle is dietically acceptable. 20. Composition according to claim 16, characterized in that the composition is food and the vehicle is food acceptable. 21. Composition according to claim 16, characterized in that the composition is veterinary and the vehicle is veterinarily acceptable. 22. Use of the combination according to any one of claims 1 to 15 for the preparation of a composition with antioxidant power. 23.-Combination according to any one of claims 1 to 15 for use as an antioxidant.