WO1999055301A1

WO1999055301A1 - Use of horseradish peroxidase in cosmetic or dermopharmaceutical compositions for preventing and/or repairing skin damage induced by oxygen radical forms

Info

Publication number: WO1999055301A1
Application number: PCT/FR1999/000757
Authority: WO
Inventors: Karl Lintner
Original assignee: Sederma
Priority date: 1998-04-27
Filing date: 1999-03-31
Publication date: 1999-11-04
Also published as: FR2777785B1; AU2941599A; FR2777785A1

Abstract

The invention concerns the use of horseradish peroxidase combined or not with caffeic acid or other substrates, in compositions for cosmetic or dermopharmaceutical use, for preventing and/or repairing harmful effects on the skin induced by oxygen radical forms produced by natural physiological reactions as well as by exposure to solar or artificial UV radiation, or any type of physical or chemical aggression. The product resulting from said combination is characterised in that it has a high detoxifying activity of the superoxide dismutase type on superoxide anion O2. and of catalatic type on hydrogen peroxide (H2O2).

Description

1

USE OF RAIFORT PEROXIDASE IN COSMETIC OR DERMOPHARMACEUTIC COMPOSITIONS FOR PREVENTING AND / OR CORRECTING SKIN DAMAGE INDUCED BY OXYGEN RADICAL FORMS

During life, both professional and private, exposure to the sun as well as various pollutions cause chemical and physical aggressions which induce the production, among other factors, of different radical forms of oxygen. The latter, especially in the skin, cause irreversible damage, the consequences of which are at least erythema, sunburn, premature aging of the skin, and, at most, skin cancer. The worsening of acne manifestations, the loss of skin suppleness, the drying of the skin and the appearance of wrinkles correspond to the first gross manifestations of aging. At the level of skin cells, the molecular modifications highlighted from the first sun exposures, which can only be detected by invasive and very sophisticated methods, are therefore even more pernicious since they are not detectable by the general population.

The sun is not solely responsible for the production of free oxygen radicals. A large number of vital biochemical reactions (such as the oxidation of amino acids or that of fatty acids, the synthesis of prostaglandins, etc.) quite naturally generate these hyper-reactive forms of oxygen, which are extremely toxic to fabrics in general and for the skin in particular.

Human beings are naturally protected against the deleterious effects of the radical forms of oxygen by physiological regulations, developed during evolution: progressive tanning from spring onwards against sub-erythematous skin consequences, presence of anti-radical enzymes such as than superoxide dismutases and catalases, reduction of the inflammatory and / or immune reaction, thickening of the epidermis ...

It is possible to act on some of these defense mechanisms, by amplifying or promoting them, for example, by using enzymes such as SOD (superoxide dismutase). Unfortunately, this is not enough to prevent actinic aging of the skin or physiological skin aging induced by free radicals. Indeed, as the following diagram shows, it is obvious that if the catalase, naturally present in the skin (Forestier JP (1992), Shindo al. (1994)), is either defective, or in insufficient quantity or having to do faced with an overproduction of superoxide anion, 2 hydrogen peroxide accumulates in the tissues, causing irreversible damage through the so-called termination reactions which correspond to the lipoperoxidation of the cell membranes of the skin.

UV SOD CATALASE 0 ₂ ⁺ 0 ₂ ^~ * - H ₂ O ₂ * H ₂ 0 + 0 ₂

Ion Superoxide Hydrogen Peroxide

As the catalase appears, under N ° 74, in the list of the substances which cannot enter in the composition of the cosmetic products of appendix 11 (1) of the European Directive 93/35 of June 14, 1993, it is not possible to use a SOD-catalase combination in cosmetic and dermopharmaceutical compositions.

The invention described here therefore proposes to strengthen the natural defenses of the skin or to alleviate their dysfunction (s) by promoting the elimination of the different free radicals by another biochemical system. Horseradish root or black radish, plant of the crucifer family, is known in Biochemistry to contain high concentrations of a very active peroxidase, horseradish peroxidase (EC 1.11.1.7, Horseradish peroxidase or HRP). This enzyme is extracted from Cochleasia Armoracia, a condiment used in Central Europe. Horseradish peroxidase is capable of detoxifying hydrogen peroxide (H 0). Its general properties have been the subject of numerous publications (for example: Halliwell B. & Alhuwallia S. (1976), Floris G. & al. (1984), Halliwell B. & Alhuwallia S.

(1976), Modi S. & Behere D.V. (1996), Buchanan I.D. & Nicell J.A. (1997), Goodwin D.C. & al. (1997).

The invention which is the subject of this patent consisted in using this peroxidase in place of the catalase prohibited in cosmetics. In addition, we have discovered that its action can be enhanced by combining it with caffeic acid, which acts as an electron donor substrate, thus making it possible to completely detoxify the radical forms of oxygen, both the superoxide anion (O ₂ ^! ) As hydrogen peroxide (H ₂ O ₂ ), produced by the various mechanisms mentioned above. Among the other substances which can be used as electron donor substrate, there may be mentioned ascorbic acid, phenols such as pyrogallol or guaiacol, polyphenols and among these hydroxy-cinnamic acids including chicoric acid, rosmanic acid, chlorogenic acid 3,5 dicaféylquinique acid, without this list being limiting. This latter class has many advantages such as, for example, better solubility and therefore better availability, satisfactory stability. 3 compared to those of tannins, a very weak coloring and harmlessness demonstrated daily by the large quantities of roasted coffee, fruits (apples, pears, cherries, blueberries) and fresh vegetables (chicory, artichoke) which are consumed in the world and which each contain at least one of these molecules. The effectiveness of the product resulting from the association of horseradish peroxidase with caffeic acid or other substances is demonstrated in the following three examples.

Example No. 1

This example demonstrates the inhibitory effect of horseradish peroxidase (Horse Radish

Peroxidase = HRP) on the production of the superoxide anion O ₂ * by the Xanthine Oxydase (XOD) / Hypoxanthine (HX) system according to the following reaction equation:

Xanthine Oxidase

Hypoxanthine ”. Uric acid + 0 ₂ ^s + H ₂ 0 ₂

This reaction is known to release the superoxide radical O ₂ * but, in the working concentrations used in Examples No. 1 and No. 3, there is production of the two free radicals, superoxide 0 ₂ ^! and hydrogen peroxide (H ₂ 0 ₂ ), the latter being the majority (Hausladen A. & Fridovich I. Arch. Biochem. Biophys. (93) 304: 479-482). The quantity of free radicals produced by this reaction is conventionally followed, at 560 nm for 40 minutes, by the evolution of the optical density of the reaction medium, in the presence of tetrazoiium nitroblue (NTB) which develops a coloration proportional to the quantity of formazan trained (Miura et al., 1987). The following results correspond to the mean ± SEM calculated on 5 different tests.

The control reaction comprising only the enzyme (XOD) and its substrate (HX) in the presence of NTB shows a variation of 0.888 ± 0.021 OD. In the presence of 0.33 * 10 ^"3 % of horseradish peroxidase (HRP), the variation observed is equal to 0.668 ± 0.025 OD, which represents an inhibition of the reaction of 25%. In the presence of 0.01% of 'caffeic acid, the variation is 0.629 ± 0.037 DO, i.e. an inhibition of 29%. In the presence of the association of 0.33 * 10 ^{' 3} % of RHP and 0.01% of caffeic acid, variation in OD is no longer equal to 0.092 ± 0.009, which corresponds to an inhibition of 90%, thus proving that under these conditions, the quantity of free radicals available in the medium was minimal with regard to the toxicity of the system .

However, we validated this test by making the hypothesis that this drop in production of free radicals could come from the inhibition of the enzyme-substrate reaction by the products tested. Tests have shown that this is not the case and that xanthine 4 oxidase was not inhibited in the systems described since the amount of uric acid formed was entirely comparable.

This example therefore illustrates the destructive effect of oxygen free radicals from horseradish peroxidase and caffeic acid in this system. In addition, this example demonstrates an important synergistic effect since the effect observed in the presence of the HPR and caffeic acid association (-90%) is much greater than that which could be expected and which should be close to l 'addition of the two effects observed in the presence of each of these products used alone (-26% + - 30% = -56%).

By varying the respective proportions of the two constituents of the association, or the amount of the association itself, it is even possible to highlight an effect whose intensity depends on the concentrations involved.

The same type of result can be observed if we replace caffeic acid with ascorbic acid, pyrogallol, guaiacol, chicoric acid, rosmanic acid, chlorogenic acid, 3,5 dicaféylquinic acid ; the latter substances being given only by way of non-limiting example.

Example 2

This example reports the destructive effect of the HRP + caffeic acid association carried out in the same proportions as in Example No. 1, with respect to the hydrogen peroxide H ₂ O _{2 alone} . The test consists in bringing together the products to be tested in the presence of H ₂ 0 ₂ and in quantifying their effect by the disappearance of H ₂ 0 ₂ by means of an electrode of

Clark who measures the amount of O ₂ released over time.

The specificity of the protocol used is verified because when we put the catalase in the presence of H ₂ O ₂ , a recorder connected to the Clark electrode then traces an ascending curve which it is possible to quantify with respect to calibrations carried out before. In the presence of horseradish peroxidase alone, or caffeic acid alone, at the same concentrations as in the previous example, no release of O ₂ is noted, which means that H ₂ O ₂ is not not degraded. If we then add catalase, we see a release of O ₂ , as in the previous control case. This proves that the products alone have not degraded the H ₂ O ₂ . Curiously, when the experiment is repeated in the presence of the product described in this patent, no release of O ₂ is still observed. On the other hand, if we then add the catalase, unlike the previous case, it is impossible to follow the appearance of H ₂ 0 ₂ , which clearly demonstrates that the product has completely degraded the H ₂ 0 ₂ put in its presence but did not release oxygen, as does catalase. 5 It is possible to deduce that the product detoxifies H ₂ O ₂ by a mechanism different from that of catalase and that there must be a transfer of electrons from H 0 ₂ from the acid caffeic. The same type of result can be observed if we replace caffeic acid with ascorbic acid, pyrogallol, guaiacol, chicoric acid, rosmanic acid, chlorogenic acid, 3,5 dicaféylquinic acid ; the latter substances being given by way of non-limiting example.

This example therefore demonstrates, indirectly, on the one hand that it is the association horseradish peroxidase with caffeic acid or other substances like those cited in this patent, without the list being exhaustive, which presents a detoxifying activity with respect to hydrogen peroxide and, on the other hand, this activity is of the catalase type since it is exerted against H 0 ₂ .

Example 3

We tested the above products, alone or according to the combination described above on cell mortality induced by an HX-XOD system. As in Example No. 1, the operating conditions used mean that the reaction releases the two free radicals, superoxide O ₂ * and hydrogen peroxide (H ₂ O ₂ ), the latter being in the majority.

Normal human fibroblates, in culture (20,000 cells / well) are placed in the presence of a buffer containing 80 μg / ml of hypoxanthine (HX). The test begins with the addition of 4mU / ml of xanthine oxidase (XOD). After 90 minutes, cell survival is conventionally estimated by revealing mitochondrial respiration using the MTT reagent ([3-

(4,5-dimethyl thiazol-2-yl) -2, '- diphenyltetrazolium bromide]) whose color changes from yellow to blue when the cell is alive.

In the presence of 0.01% caffeic acid, survival is improved by 6.0 ± 1.5% compared to its absence. Under the same conditions, in the presence of 0.33 * 10 ^"3 % of HRP, cell survival is 18.1 ± 3.2% higher than what is observed in its absence.

When the test is carried out in the presence of the combination of the two preceding products, at the same concentrations as above, the survival is improved by 60.5 ± 3.7% compared to the tests carried out without the combination. Additional tests have demonstrated, moreover, that on this system catalase was capable of improving cell survival

Identical experiments were carried out on normal human keratinocytes, with the same products and the same concentrations tested.

Survival is improved by 8.3 ± 1.8%, 12.5 ± 2.2% and 59.0 ± 2.4% in the presence, respectively, of caffeic acid, HRP and their combination. 6 This example demonstrates the relative activity of each of the products used on cell mortality induced by the two radical forms of oxygen, as well as the clear synergy that is obtained when these two products are used in combination.

The same type of result can be observed if we replace caffeic acid with ascorbic acid, pyrogallol, guaiacol, chicoric acid, rosmanic acid, chlorogenic acid 3,5 dicaféylquinic acid; the latter substances being given by way of non-limiting example.

In conclusion, the examples above demonstrate that the product resulting from the association of horseradish peroxidase with caffeic acid, or other substances such as those described in this patent without the list being exhaustive, has a powerful anti-radical effect in vitro, both of the superoxide dismutase type on the superoxide 0 ₂ * anion and of the catalase nature on hydrogen peroxide (H ₂ 0 ₂ ). This latter activity is however carried by a mechanism different from that conventionally described for catalase itself. In addition, it is obvious that, from this association results a significant synergy, since the effect measured is significantly greater than that expected when examining the effects of the only constituents when used alone.

It is also obvious that other peroxidases of similar structure can be isolated from other neighboring plants and can be used in place of the horseradish peroxidase described here.

When peroxidase is used alone, its concentration is between 0.01.10 ^"3 % and

20.10 ^"3 % (w / w), preferably between 0.1.10 ^{" 3} % and 3.0.10 ^"3 %, by weight in the cosmetic or dermopharmaceutical composition.

In the product resulting from the association of horseradish peroxidase and the electron donor substrate, such as caffeic acid, the concentration of horseradish peroxidase can vary between 0.01.10 ^"3 % and 50.10 ^{" 3} % (p / p), preferably between 0.1.10 ^"3 % and 10.10 ^{" 3} % (w / w); that of caffeic acid can vary between 0.003.10 ^"3 % and 10.10 ^{" 3} % (w / w), preferably between 0.01.10 °% and 1.0.10 ^"3 % (w / w).

The concentration of the product resulting from the association of horseradish peroxidase and the electron donor substrate, such as caffeic acid, can vary between 0.1% and 50% (w / w), preferably between 0.5% and 20%, by weight in the cosmetic or dermopharmaceutical composition.

The combination of horseradish peroxidase and caffeic acid, previously described and the subject of this patent, can be used in any dosage form used in cosmetics 7 or dermopharmacy: O / W and W / O emulsions, milks, lotions, gelling and viscous polymers, surfactants and emulsifiers, ointments, hair lotions, shampoos, soaps, sticks and pencils, sprays, body oils, without this list being exhaustive .

It is possible to incorporate the association, in cosmetic vectors such as liposomes, chylomicrons, macro-, micro- and nanoparticles as well as macro-, micro- and nanocapsules, to absorb them on powdery organic polymers, talcs, bentonites and other mineral supports.

The association can be combined in cosmetic or dermopharmaceutical compositions with any other ingredient usually used in cosmetics: extraction and / or synthetic lipids, gelling and viscosifying polymers, surfactants and emulsifiers, hydrosoluble or active ingredients, extracts of other plants, tissue extracts, marine extracts.

The product is used in cosmetic or dermopharmaceutical applications which correct and / or repair at the cutaneous level, the deleterious effects of the radical forms of oxygen produced during:

physiological reactions such as, for example, the oxidation of amino acids or fatty acids, the synthesis of prostaglandins,

- irradiation by solar UV radiation, normal and daily,

- irradiation by solar UV radiation, excessive during the holidays, or by artificial UV,

- all kinds of physical or chemical attacks such as atmospheric chemical pollution, air conditioning, combustion gases from automotive or industrial fuels, unbalanced food, stress, alcohol, tobacco, as well as for protection of hair, scalp, nails and mucous membranes without this list being exhaustive.

These cosmetics or dermopharmaceuticals are used for the preparation of drugs which correct and / or repair at the cutaneous level, the deleterious effects of the radical forms of oxygen produced during:

- irradiation by solar UV radiation, normal and daily,

- irradiation by solar UV radiation, excessive during the holidays, or by artificial UV, 8 - all kinds of physical or chemical attacks such as atmospheric chemical pollution, air conditioning, automobile or industrial fuel combustion gases, unbalanced food, stress, alcohol, tobacco, etc. protection of hair, scalp, nails and mucous membranes without this list being exhaustive.

Claims

9 CLAIMS

1. Product of vegetable origin containing horseradish peroxidase having a detoxifying action both of superoxide dismutase type on the superoxide anion O ₂ ^s and of catalase nature on hydrogen peroxide (H ₂ O ₂ ).

2. Cosmetic composition according to claim 1 characterized in that the peroxidase concentration is between 0.01.10 ^"3 % and 20.10 ^{" 3} % (w / w), preferably between

0.1.10 ^'3 % and 3.0.10 ^"3 % by weight.

3. Product resulting from the association of horseradish peroxidase according to 1 with electron donor substrates.

4. Product according to claim 3 characterized in that the substrate can come from different chemical classes such as phenols and polyphenols.

5. Product according to 3 to 4 characterized in that the substrate forming part of the hydroxy-cinnamic acids is particularly caffeic acid.

6. Product according to 3 to 5 characterized in that the final activity is characterized by the synergy provided by the association of the two components used.

7. Product according to 3 to 6 characterized in that, in the product, the concentration of horseradish peroxidase can vary between 0.01.10 ^'3 % and 50.10 ^"3 % (w / w), preferably between 0, 1.10 ^{" 3} % and 10.10 ^'3 % (w / w); that of caffeic acid can vary between 0.003.10 ^"3 % and 10.10 ^{" 3} % (w / w), preferably between 0.01.10 ^"3 % and 1.0.10 ^{" 3} % (w / w).

8. Cosmetic or dermopharmaceutical compositions characterized in that the concentration of the product according to claim 3 to 7, can vary between 0.1% and 50%

(w / w), preferably between 0.5% and 20%, by weight.

9. Cosmetic or dermopharmaceutical compositions characterized in that the product according to 1 to 2 or 3 to 8 is used in any dosage form used in cosmetics or dermopharmacy: O / W and W / O emulsions, milks, lotions, gelling and viscosifying polymers, surfactants and emulsifiers, ointments, hair lotions, shampoos, soaps, sticks and pencils, sprays, body oils.

10. Cosmetic or dermopharmaceutical compositions characterized in that the product according to 1 to 2 or 3 to 9 is incorporated in cosmetic vectors such as liposomes, chylomicrons, macro-, micro- and nanoparticles as well as macro-, micro- and nanocapsules, to absorb them on powdery organic polymers, talcs, bentonites and other mineral supports.

11. Cosmetic or dermopharmaceutical compositions characterized in that the product according to 1 to 2 or 3 to 10 is used with any other ingredient usually used in cosmetics: extraction and / or synthetic lipids, gelling and viscosifying polymers, 10 surfactants and emulsifiers, water or liposoluble active ingredients, extracts from other plants, tissue extracts, marine extracts.

12. Composition according to 2 or 8 to 11 for skin care, including against the deleterious effects of the radical forms of oxygen produced during: - physiological reactions such as, for example, the oxidation of amino acids or fatty acids, prostaglandin synthesis,

- irradiation by solar UV radiation, normal and daily,

- excessive irradiation by solar UV radiation during the holidays, or by artificial UV, - all kinds of physical or chemical attack such as atmospheric chemical pollution, air conditioning, fuel combustion gases automotive or industrial, unbalanced food, stress, alcohol, tobacco, as well as to promote the protection of hair, scalp, nails and mucous membranes. 13 Use of a composition according to 2 or 8 to 11 for the preparation of a medicament for skin care, including against the deleterious effects of the radical forms of oxygen produced during:

- physiological reactions such as, for example, oxidation of amino acids or fatty acids, synthesis of prostaglandins, - normal and daily irradiation by solar UV radiation,

- excessive irradiation by solar UV radiation during the holidays, or by artificial UV,

- all kinds of physical or chemical attacks such as atmospheric chemical pollution, air conditioning, combustion gases from automotive or industrial fuels, unbalanced food, stress, alcohol, tobacco, as well as for promote the protection of hair, scalp, nails and mucous membranes.