A PUNICA GRANATUM EXTRACT AND ITS COSMETIC USES

Abstract

The present invention concerns an extract of Punica granatum comprising: i. at least 10% by weight of the total weight of the dry extract, of punicalagins; and ii. at least 10% by weight of the total weight of the dry extract, of ellagic acid; and wherein the weight ratio [ellagic acid/punicalagins] is ranging from 0.5 to 2.

The present invention also relates to a method of preparation of the said extract

Another object of the present invention is a preparation comprising the said extract and at least one water-miscible organic solvent preferably chosen from lower alcohols containing from 2 to 8 carbon atoms and in particular from 2 to 6 atoms.

The present invention also concerns a composition comprising the said extract or preparation; a process for the cosmetic treatment of keratin materials, comprising a step of applying the said composition and cosmetic uses of the said extract.

Claims

1. An extract of Punica granatum comprising: i. at least 10% by weight of the total weight of the dry extract, of punicalagins; and ii. at least 10% by weight of the total weight of the dry extract, of ellagic acid-; and wherein the weight ratio [ellagic acid/punicalagins] is ranging from 0.5 to 2.

2. The extract, according to claim 1, which comprises: i. from 10% to 15% by weight of the total weight of the dry extract, of punicalagins; and ii. from 10% to 15% by weight of the total weight of the dry extract, of ellagic acid.

3. The extract, according to claim 1, which further comprises from 1% to 5% by weight of the total weight of the dry extract, of punicalins.

4. The extract according to claim 1, wherein the weight ratio [ellagic acid/punicalagins] ranges from 0.6 to 1.6.

5. The extract according to claim 1, wherein the said extract is an ethanolic extract or a dry extract.

6. A preparation comprising an extract as defined in claim 1 and at least one water-miscible organic solvent.

7. The preparation according to claim 6, wherein the said at least one water-miscible organic solvent is chosen from glycerin, ethanol, butylene glycol, dipropylene glycol, ethoxydiglycol, and their mixtures.

8. The preparation according to claim 6, wherein the weight ratio [solvent(s)/dry extract] ranges from 95:5 to 99.9999:0.0001.

9. The preparation according to claim 6, wherein the said extract represents from 0.0001% to 5% by weight in dry extract of the total weight of the preparation and the said at least one solvent represents from 95% to 99.9999% by weight of the total weight of the preparation.

10. A composition comprising in a physiologically acceptable medium an extract according to claim 1 or a preparation comprising the extract and at least one water-miscible organic solvent.

11. The composition according to claim 10; wherein the composition further comprises at least one ingredient selected from silicone fats; non-silicone fatty substances; fatty acids having 8 to 32 carbon atoms; esters and synthetic ethers; linear or branched hydrocarbons of mineral or synthetic origin; fatty alcohols having from 8 to 26 carbon atoms; water; C2-C6 alcohols; glycols; thickeners, emulsifiers, surfactants, gelling agents, cosmetic active ingredients, perfumes, fillers, dyestuffs, moisturizers, and vitamins, polymers.

12. The composition according to claim 10, wherein the extract represents from 0.0001% to 5% by weight in dry extract of the total weight of the composition.

13. The composition according to claim 10, wherein the preparation represents from 0.1% to 60% by weight of the total weight of the composition.

14. A method for decreases or preventing oxidation of chemical compounds in a cosmetic composition which comprising including in the cosmetic composition the extract according to claim 1, as an antioxidant or antiradical agent.

15. A cosmetic process for preventing and/or treating the signs of skin aging which comprises contacting the skin with the extract according to claim 1, as an anti-aging agent.

16. A cosmetic process for preventing and/or reducing the dull and/or heterogeneous appearance of complexion of skin which comprises contacting the skin with the extract according to claim 1.

17. A process for the cosmetic treatment of keratin materials comprising a step of applying a composition according to claim 10 onto the keratin materials.

18. A method of preparation of a Punica granatum extract according to claim 1 comprising at least the following steps: i. providing a Punica granatum pericarp; ii. extracting from the said pericarp, at least punicalagins and ellagic acid, with ethanol of at least 99.5% degree at a temperature ranging from 37° C. to 45° C. during 2 to 5 hours; iii. filtering and repeating the step ii. at least two more times; and iv. optionally drying the filtrate obtained at the end of the step iii. at a temperature ranging from 35° C. to 40° C. under vacuum.

19. The extract according to claim 2, which further comprises from 1% to 5% by weight of the total weight of the dry extract, of punicalins.

20. The extract according to claim 2, wherein the weight ratio [ellagic acid/punicalagins] ranges from 0.6 to 1.6.

Description

FIGURES

[0128] FIG. 1: Schema of the method of preparation of a Punica granatum extract according to the invention.

[0129] FIG. 2: Decrease of the lipid peroxidation with a Punica granatum extract according to the invention compared to Ellagic acid alone. Note: The value in % illustrated over each bar (in the graph) is the % protection offered by the raw materials tested vs long UV A exposure.

EXAMPLES

Example 1: Preparation of the Punica granatum Extract According to the Invention

[0130] Dried pericarp of Punica granatum (1 kg) was extracted at 42° C. with 5 Litres of Ethanol (Proof 99.5%) with stirring at 800 rpm for a brief period of 4 h. The ethanolic extract obtained was filtered through a Buchner funnel containing cotton cloth (mesh size: 20 micron). The filtered extract was evaporated under vacuum at 38° C., to afford a brown amorphous powder. The extraction is repeated successively for two consecutive times under the conditions explained above. The combined dried extract consists of the major phenolic compounds ie: Ellagic acid and Punicalagins. The schema of the preparation method of a Punica granatum extract according to the invention is presented in FIG. 1.

[0131] The specifications of the extract according to the invention are as follows:

TABLE-US-00001 TABLE 1 Specifications of the extract Parameters Values in % w/w Punicalagins 14.44%  Punicalins 3.28% Total polyphenols (punicalins, .sup. 42% punicalagins and ellagic acid, gallic acid, epi-catechin gallate, gallagic acid, ellagic acid hexoside/glycoside) Total fats  2.6% Total protein 0.73% Total sugars 19.1% Moisture  6.5%

[0132] The ratio of these two compounds namely Ellagic acid and Punicalagins vary with different organic extraction solvent used ie: Absolute alcohol: 99.5% and rectified spirit: 95.0-96.0%, Methanol, ethyl acetate and aqueous acetone. Therefore, the extraction was performed with different organic solvents. The results indicate that the absolute alcohol (proof: 99.5%) provides an extract with the appropriate quantities and ratio of Ellagic acid/Punicalagins.

TABLE-US-00002 TABLE 2 *Comparison of different organic solvents Impact of Ellagic weight ratio extraction acid Punicalagins [Ellagic acid/ solvents w/w % w/w % Punicalagins] Ethanol; Rectified spirit   .sup. 6% .sup. 22% 0.27 (polarity index 5.1; Proof: 95.0-96.0%) OUTSIDE OF THE INVENTION Ethanol; Absolute Alcohol  .sup. 11.35% 14.44%  0.79 (polarity index 4.4; Proof: 99.5%) ACCORDING TO THE INVENTION Methanol (Polarity 7.5-8.0% 20.2% 0.40 Index 5.1) OUTSIDE OF THE INVENTION Ethyl acetate  .sup. 4.81% — 0 (Polarity Index 4.7) OUTSIDE OF THE INVENTION 70% Aqueous Acetone — 22.0% — OUTSIDE OF THE INVENTION

[0133] Furthermore, the ratio of these two compounds namely Ellagic acid and Punicalagins vary in function of the temperature of the extracting step and of the temperature of the drying step. Therefore, the extracting step and drying step was performed at different temperatures (° C.).

TABLE-US-00003 TABLE 3 Comparison of different extraction temperatures Impact of Ellagic Weight ratio extraction acid Punicalagins [Ellagic acid/ temperature w/w % w/w % Punicalagins] 35° C. 3.51% 10.42% 0.34 OUTSIDE OF THE INVENTION 42° C. 11.35% 14.44% 0.79 ACCORDING TO THE INVENTION 50° C. 16.45% 2.65%% 6.2 OUTSIDE OF THE INVENTION 70° C. 35.5% 0.71% 50 OUTSIDE OF THE INVENTION

TABLE-US-00004 TABLE 4 Comparison of different drying temperatures Impact of Drying Ellagic weight ratio Temperature acid Punicalagins [Ellagic acid/ (Under Vaccum) w/w % w/w % Punicalagins] 38° C. 11.35% 14.44% 0.79 ACCORDING TO THE INVENTION 45° C. 8.52% 3.85% 2.21 OUTSIDE OF THE INVENTION

Example 2: Antioxidant Activity of the Extract According to the Invention Obtained in Example 1

[0134] a. Peroxidation of Lipids Test

[0135] An evaluation of the protection potential against UVA-induced lipid peroxidation of Punica granatum extract according to the invention obtained according to Example 1 was made and compared to the protection against UVA-induced lipid peroxidation of pure ellagic acid.

[0136] Normal human fibroblasts are treated either with the Punica granatum extract according to the invention at 0.001% or with pure ellagic acid at 3 μM for 24 h and then exposed to a UVA dose inducing no more than 20% cell death.

[0137] A control was performed consisting in normal human fibroblasts exposed to a UVA dose inducing no more than 20% cell death.

[0138] 8-iso-Prostanes release is assayed 2 h30 after the exposure. The results are presented in FIG. 2.

[0139] The control (UV exposed) shows a release of 100% of 8-iso-Prostanes, with 0% of protection against lipid peroxidation while the treatment with the Punica granatum extract according to the invention shows a lower release of 58% of 8-iso-Prostanes, with a higher protection against lipid peroxidation of 42%

[0140] Ellagic acid shows a release of 65% of 8-iso-Prostanes, with 35% of protection against lipid peroxidation.

[0141] Therefore, the Punica granatum extract according to the invention shows a better protection against lipid peroxidation than ellagic acid alone.

Example 3: Evaluation of the Antioxidant Activity of the Extract According to the Invention (Extraction Made with Ethanol 99.5%) Compared to the Extract Outside of the Invention (Extraction Made with Ethanol 95%)

[0142] The technique for evaluating the antioxidant activity of the compounds used according to the invention is carried out in accordance with a well-known method (J. of Photochemistry and Photobiology B: Biology 57 (2000) 102-112 TOBI et al.: Glutathione modulates the level of free radicals produced in UVA-irradiated cells). This technique uses a fluorescent probe, a marker of the intracellular overall oxidative stress, 2′-7′-dichlorofluorescin diacetate (DCFH-DA).

[0143] Principle

[0144] The use of DCFH-DA as a marker of the oxidative stress is based on its physicochemical properties. It is an apolar and nonionic molecule capable of diffusing through cell membranes. Once inside the cell, DCFH-DA will be hydrolysed by intracellular esterases to give a non-fluorescent compound: DCFH or 2,7-dichlorofluorescin. In the presence of activated oxygen species, DCFH is rapidly oxidized to give a highly fluorescent compound: DCF or 2,7-dichlorofluorescein.

[0145] Procedure:

Treatment of the Keratinocytes with an Extract According to the Invention Obtained in Example 1 (Extraction Made with Ethanol 99.5%) and an Extract Outside of the Invention Obtained in Example 1 (Extraction Made with Ethanol 95%)

[0146] At confluence, the keratinocytes are incubated in the presence of the extracts to be tested for 24 hours at 37° C., 5% CO.sub.2, in the culture medium.

Incorporation of DCFH-DA

[0147] The keratinocytes, pretreated with the each of the extracts, are rinsed then incubated in the presence of DCFH-DA in the dark.

Exposure to UVA

[0148] After this incubation, the DCFH-DA solution is removed, the cells are then exposed to 2 J/cm.sup.2 of UVA. Observation: an unexposed control plate is stored in the dark at room temperature.

Measurement of the Fluorescence

[0149] The fluorescence of DCF is evaluated immediately after the exposure to UVA, by spectrofluorimetry (excitation: 480 nm; emission: 530 nm).

Results

[0150] The photoprotective efficacy results are expressed in Table 4 below, as % decrease in fluorescence relative to the control cells exposed to UV A. The measurement is carried out on two samples and the average value is determined.

TABLE-US-00005 TABLE 5 results Mean (% of Extracts tested fluorescence inhibition) Extract according to the invention % Inhibition: 52% obtained in Example 1 (extraction (n = 2) made with ethanol 99.5%) Active Concentration : 0.0500 g/L Extract outside of the invention % Inhibition: 13.1% obtained in Example 1 (extraction (n = 2) made with ethanol 95%) Not Active Concentration : 0.100 g/L
The above results reveal a better antioxidant activity of the extract from Example 1 obtained with ethanol 99.5% with respect to the UVA-induced oxidative stress, with a higher fluorescence inhibition of DCF than those obtained with the extract from Example 1 extracted with ethanol 95%.
Thus, the Punica granatum extract according to the invention induce a better active photoprotection of the skin with respect to the untreated control and the comparative extract outside of the invention.

Example 4: Cosmetic Composition Comprising the Extract According to the Invention Obtained in Example 1

[0151] An anti-ageing aqueous gel for the skin is prepared, comprising (% by weight):

TABLE-US-00006 Carbopol    .sup. 4% Extract of Punica granatum obtained according to Example 1   .sup. 0.5% Water qs. 100%
After application to the face, the composition makes it possible to protect the skin from UVA-induced stress.