WO2002100423A1

WO2002100423A1 - Method for extracting an active principle from salix, active principle obtained and cosmetic treatments

Info

Publication number: WO2002100423A1
Application number: PCT/FR2002/001985
Authority: WO
Inventors: Jean-Jacques Paufique
Original assignee: Societe Industrielle Limousine D'application Biologique (Silab)
Priority date: 2001-06-11
Filing date: 2002-06-11
Publication date: 2002-12-19
Also published as: FR2825632A1; FR2825632B1

Abstract

The invention concerns a method for extracting an active principle rich in polyphenols, in particular for increasing HSP 72 production against stresses generated by heat and/or UVB rays, from Salix. The invention also concerns the resulting active principle and adapted cosmetic treatments.

Description

METHOD FOR EXTRACTING AN ACTIVE INGREDIENT FROM S AUX, ACTIVE INGREDIENT OBTAINED AND COSMETIC TREATMENTS

The present invention relates to a method for extracting an active principle from Salix, salix alba or salix pυrpυrea or salix viminalis or salix daphnoides or salix fragilisow salix pentandra and also covers the active principle obtained which makes it possible to protect cells. skin and therefore an anti-aging effect. The invention also covers associated cosmetic treatments.

The skin is subjected to numerous stresses, in particular those caused by heat and UV radiation, during sun exposure that is too long and / or too intense.

All the cells of living organisms (humans, animals, plants, bacteria) respond in particular to this type of stress by the synthesis of specific proteins called heat shock proteins or Heat Shock

Proteins (HSP). These proteins constitute an important system of cellular protection in skin biology.

HSPs are classified into 5 classes according to their molecular weight and their sequence homology: HSP 110, HSP 90, HSP 70, HSP 60 and HSP 27.

The first function of these proteins is that of thermotolerance. Thus, when keratinocytes are subjected to a heat treatment of a hour at 40 ° C or 41 ° C, it causes an increase in the rate of mRNA and the synthesis of HSP.

Such a reaction makes it possible to limit denaturation during stress. These proteins play a major role in building and even repairing the damage caused.

These proteins not only provide a protective role but also the reproduction of functional proteins, the maintenance of cellular functions and the restoration of basic metabolic functions, while allowing the elimination of deleterious radicals. HSP 70s represent the most studied proteins because they are mainly synthesized by eukaryotic cells.

We distinguish in this category two forms one constitutive, HSP 73 or 70 and an inducible form, in particular under the effects of stress, HSP 72. We find this inducible form in fibroblasts and keratinocytes.

HSP 72 is induced by heat but also by UVB.

We also know (Muramatsu. T <& al, 1966, British Journal of Dermatology, 134: 1035-1038) that the inducibility of HSP 72 decreases with age. Immunohistochemical studies have shown a lower induction level on explants of skin from older donors than on explants from young donors.

In the more specific case of fibroblasts, a recent study

(Conrad-ôutsmann ά al, 1998, Experimental cell research 241: 404-413) has also shown that fibroblasts from young donors express higher HSP 72 levels than older donors, whether before or after heat stress.

The object of the present invention is to propose a process for extracting an active principle from salix which is rich in polyphenols (acid type phenolic), to stimulate the induction of HSP 72 with an anti-aging effect and to ensure the protection of skin cells.

To this end, the process for extracting an active principle rich in polyphenols, in particular for increasing the production of HSP 72 in the face of stresses generated by heat and / or UVB radiation, is characterized in that it comprises the succession of the following stages:

- solubilization of Salix, salix alba or salix pυrpυrea or salix viminalis or salix daphnoides or salix fragilis or salix pentandra in water,

- hydrolysis between 20 ° C and 50 ° C and at acidic pH in the presence of a protease,

- separation of the soluble and insoluble phases by any means or any combination of means, for example: decantation, filtration or centrifugation,

- sterilizing filtration to reach a total mesophilic flora of less than 100 germs / g, and an absence of yeasts and molds.

The active ingredient obtained is characterized by the following parameters:

- A dry matter content obtained by passing through an oven at 105 ° C for 16 hours, between 20 and 200 g / 1, more particularly between 40 and 100 g / 1. - a pH measured by the potentiometric method at room temperature, between 3.0 and 7.0, more particularly between 4.0 and 7.0.

- a rate of total polyphenols, expressed in gallic acid equivalent between 1.0 and 20.0 g / 1, more particularly between 1.0 and 10.0 g / 1 and specifically between 1.0 and 5.0 g / 1. The invention also covers cosmetic treatments.

The following description is made with reference to the appended drawings in which the various figures represent: FIG. 1, a table of the expression of HSP 72 obtained in the presence of the active principle according to the present invention,

- Figure 2, a comparative table of HSP 72 expression as a function of age, - Figure 3, a table of the results of anti-wrinkle effects

- Figure 4, a table of the results of fatigability of the skin,

- Figure 5, a table of anti-radical activity,

- Figure 6, a table of the rate of protection against UVA / UVB aggressions, and - Figure 7, a table of the results obtained by action of the active principle vis-à-vis the released interleukins.

1 / EXTRACTION PROCESS:

The method according to the present invention consists in carrying out the succession of the following steps:

- hydrolysis at around 20 ° C and at acidic pH, in the presence of a protease,

- decantation, - separation of the soluble and insoluble phases by any means or any combination of means, for example: decantation, filtration or centrifugation.

- possible concentration of the active fraction of at least 30%, and

- sterilizing filtration to reach a total mesophilic flora of less than 100 germs / g, and an absence of yeasts and molds, in particular by passage over a microporous membrane at 0.22 μm.

2 / CHARACTERISATTON OF THE ACTIVE PRINCIPLE OBTAINED Dry matter rate:

The dry matter is obtained by passing the active ingredient extracted into an oven at 105 ° C for 16 hours. The amount of dry matter obtained is between 20 and 200 g / l, more particularly between 40 and 100 g / l.

PH measurement:

The pH is measured by the potentiometric method at room temperature. The pH is between 3.0 and 7.0, more particularly between 4.0 and 7.0. Total polyphenols:

The determination is carried out by reaction of the polyphenols in the presence of potassium erricyanide and by subsequent detection of the colored complexes formed, at 715 nm.

It is then compared against a gallic acid standard covering the relevant range of measurements.

The following results are obtained, expressed in gallic acid equivalent: total polyphenol level between 1.0 and 20.0 g / 1, more particularly between 1.0 and 10.0 g / 1 and specifically a rate between 1 , 0 and 5.0 g / 1. Stability:

In addition, the active principle, at dosages of between 1 and 5% by volume, is completely stable in media having a pH of between 2.0 and 10.0 and stable in temperature up to 80 ° C for many hours.

3 / EFFECTS OF THE ACTIVE INGREDIENT ON THE SYNTHESIS OF HSP 72

The study is conducted on human keratinocytes with thermal induction and without thermal induction.

Study of the synthesis of HSP 72 with and without thermal induction. To quickly protect themselves from thermal and UV-induced damage, skin cells produce stress proteins. To verify and analyze it, keratinocytes are subjected to an evolving thermal stress. A positive control is produced by heating to 44 ° C., for 1 hour without active principle.

The thermal stress consists in bringing the human keratinocytes to 44 ° C for 15 minutes, then always to 44 ° C but for 30 minutes, in the absence and in the presence of the active principle as extracted previously, at a rate of 0.5 ; 1.0 and 2.0% by volume. In parallel, in order to allow a comparison and to verify that the polyphenols are indeed at the origin of the desired activity, we test a model mixture composed mainly of chlorogenic acid and p.coumaric acid, with a concentration of 2 % in volume.

In order to visualize the stress proteins thus produced, they are revealed by a brown coloring using a peroxidase system. The more intense the coloration, the higher the level of HSP 72 expression. The level is estimated under a microscope using a qualitative colorimetric scale, within the same study so as not to distort the interpretations.

The results are expressed by - or + knowing that the scale is as follows:

- no presence of HSP 72: -

- low presence of HSP 72: +

- average presence of HSP 72: ++

- strong presence of HSP 72: +++ - very strong presence of HSP 72: ++++ or +++++

The results are expressed in the table in FIG. 1. Without thermal induction, the active principle stimulates the synthesis of these HSP 72s, from 1%. The active principle according to the present invention gives substantially the same results as with the model mixture of polyphenols.

It is also noted that the greater the heat treatment, the higher the expression of HSP 72. It is also noted that, in the presence of active principle according to the present invention, the treated cells express these HSPs 72 more intensely.

In addition, there is an acceleration of the response since the rate of HSP 72 obtained for 30 minutes without active principle is similar to that obtained in 15 minutes from the presence of 1% of active principle.

Study of the expression of HSP 72 as a function of age:

We have just noted that the greater the thermal stress, the more significant the expression of HSP 72.

We can also compare the intensity of this response as a function of the donor's age.

The tests are carried out in the same way as previously but the skin explants have two origins, that of a young subject (19 years) and that of an elderly subject (77 years).

The tests are carried out with three increasing durations of thermal induction and the same temperature 44 ° C.

The results of FIG. 2 show that the expression is improved in both cases, which is particularly satisfactory for the elderly subject on whom it is shown that the active principle does indeed have an effect.

This expression is amplified on the young subject. The greater the heat stress, the more intense the HSP 72 response, however the intensity of this HSP 72 response is faster for the keratinocytes of the young donor. The active principle makes it possible to boost the synthesis activity and the speed of induction of HSP 72 for keratinocytes of the elderly donor.

The active ingredient compensates for the drop in activity due to cell aging.

4 / STUDY OF THE ANTI-WRINKLE EFFECT OF THE ACTIVE INGREDIENT

The in vivo quantification on 12 volunteers of the anti-wrinkle efficacy of the active principle of the present invention was evaluated in a cosmetic formula containing 5% of active principle. Footprints are taken before and after the twice-daily treatment for 28 days against placebo. The statistical test used is the Student test on paired data.

The results are analyzed with regard to the following parameters: total wrinkled surface, total length of wrinkles, number of wrinkles, average depth of wrinkles. These results are shown in the table in Figure 3.

There is a significant reduction in each of the parameters total wrinkled surface, length of wrinkles and number of wrinkles.

By way of nonlimiting examples, the following is a cosmetic product or composition comprising the active principle of the present invention, alone or combined with at least one other active principle.

EXAMPLE 1 Anti-Wrinkle Day Emulsion

- Montanov 68 3.0% (glucoside wax)

- Cetiol SB45 1.0% (shea butter)

- Tween 80 2.0% (polysorbate 80) - Stearic acid 1.0%

- Emulgin BI 1.0% (ceteareth-12)

- Lanol 1688 10% (cetearyl octanoate)

- Miglyol 812 2.0% (caprylic / capric triglyceride) - Isohexαdecαne 3.0%

- PPI 2.0% (isopropyl pαlmitαte)

- Sepigel 305 0.1% (polyαcrylαmide / isopαrαf f ineCi3-i4 / Iαureth7)

- Cαrbopol ETD2020 0.1% (cαrbomer) - Butylene glycol 10%

- Preservative 0.5%

- Active ingredient 5.0%

- Water qs 100%

5 / STUDIES OF BIOMECHANICAL PROPERTIES OF THE SKIN

The test is conducted on 20 volunteers, female and aged 31 to 62. This test is carried out with an emulsion containing 5% of the active principle against placebo.

The fatigability of the skin is measured before and after a twice-daily treatment. The statistical analysis used is the Student test on paired data.

The results are presented in the table in FIG. 4.

There is a significant decrease in the fatigability of the skin.

By way of nonlimiting example, a cosmetic product or composition is indicated below comprising, as the only active principle, that of the present invention.

Example 2 Firming Body Milk

- Montanov 202 3.0% (glucoside wax) - Ceteth 20 1.0%

- Sepigel 501 2.0% (isoparaffin C ₁₃ - 14 / isostearyl / isostearate / sodium polyacrylate / polyacrylamide / polysor ate 60) - Butylene glycol 5.0%

- DC 200 1.0% (dimethicone)

- Lαnol 1688 3.0% (ceteαryl octαnoαte)

- Lαnol 99 5.0% (isononyl isononαnoαte) - Preservative 1.0%

- Active ingredient 5.0%

- Water qs 100%

6 / PHOTOPROTECTOR ACTIVITY OF THE ACTIVE INGREDIENT Anti-free radical activity:

The tests are carried out on cell cultures of human keratinocytes in order to determine whether the active principle has a protective action against UVA and UVB radiation.

To this end, the cells are subjected to irradiation with an intensity of 5 to 20 joules / cm ² for UVA rays and from 0.05 to 0.40 joules / cm ² for UVB rays, preferably 9 joules / cm ² for UVA and 0.17 joules / cm ² for UVB.

The cell viability of these keratinocytes is then measured by an optical density measurement following staining with MTT, said dye being capable of being absorbed only by living cells. The results are grouped in the table in FIG. 5. There is a strong anti-radical activity, much higher than that obtained with the polyphenolic model, with respect to the free radicals UVA and UVB induced. This effect is dose dependent. Αnti-αpoptosis activity:

The study is carried out on skin explants and shows the photoprotective activity of the active principle according to the invention vis-à-vis the appearance of epidermal cells "sunburn" or called "sunburn cells". These cells have DNA damage and the characteristics of apoptotic cells, including an eosinophilic cytoplasm.

After irradiation with UVB, a "sunburn" cell count is ensured under a microscope, after staining of the histological sections of the explants.

The results mentioned in the table in FIG. 6 are obtained. The active principle has a strong protective activity and strongly limits the appearance of "sunburn" cells.

Anti-irritant activity:

Such activity can be advantageously measured by assaying the level of interleukin-la released in a culture medium for human keratinocytes subjected to oxidative stress caused by UVA + UVB rays.

The assay of the level of Interleukin-la released in the culture supernatant of keratinocytes subjected to UVA and UVB is carried out by the ELISA method. Values are given in pg / l. The comparison is again carried out with a polyphenolic model mixture.

The active principle according to the present invention causes a decrease in the level of interleukin-la.

The results are mentioned in the table in FIG. 7. This effect is also dose-dependent.

Example 3: Sun protection gel

- Lanol 99 3.0% (isononyl isononanoate) - Allαntoïne 0.1%

- Cαrbomer 0.5%

- Triethαnolαmine 3.0%

- TEA sαlicylαte 5.0% - Glycerol 5.0%

- DMDM hydαntoïne 0.3%

- Active ingredient 3.0%

- Preservative 1.0%

- Water Qsp 100%

The process has made it possible to extract a particular active principle and this active principle as it has just been characterized can be used for cosmetic treatments to ensure anti-aging cellular protection. In this case, 0.5% to 10.0% of active principle is mixed in a suitable dosage form: cream, gel, ointment, lotion, milk or emulsion or solution with suitable excipients, optionally and this preparation is applied to the skin.

More particularly, a cosmetic treatment can be carried out with 1.0 to 3.0% of active principle in the same dosage form as above.

It is thus possible to provide a cosmetic treatment to stimulate the synthesis of HSP 72 stress proteins and to provide protection against photo-induced damage by means of this same active principle by combining from 0.5% to 10.0% of active principle and more particularly from 1 to 3% in a suitable dosage form: cream, gel, ointment, lotion, milk or emulsion or solution with suitable excipients, optionally and this preparation is applied to the skin.

Claims

1. Method for extracting an active principle rich in polyphenols, of phenolic acid type, in particular for increasing the production of HSP 72 in the face of stresses generated by heat and / or UVB radiation, characterized in that it comprises the succession of the following stages: - solubilization of Salix, salix alba or salix pυrpυrea or salix viminalis or salix daphnoides or salix fragilis or salix pentandra, in water,

- hydrolysis between 20 ° C and 50 ° C and at an acid pH between 1.0 and 4.0 in the presence of a protease,

separation of the soluble and insoluble phases by any means or any combination of means, for example: decantation, filtration or centrifugation, and

2. Active ingredient obtained by aqueous solubilization of Salix, salix alba or salix purpurea or salix viminalis or salix daphnoides or salix fragilis or salix pentandra, and hydrolysis in the presence of a protease of this solution, characterized in that it has:

- A dry matter content obtained by passing through an oven at 105 ° C for 16 hours, between 20 and 200 g / 1, more particularly between 40 and 100 g / 1.

- a pH measured by the potentiometric method at room temperature, between 3.0 and 7.0, more particularly between 4.0 and 7.0.

- a rate of total polyphenols, expressed in gallic acid equivalents between 1.0 and 20.0 g / 1, more particularly between 1.0 and 10.0 g / 1 and specifically between 1.0 and 5.0 g / 1.

3. Cosmetic treatment to stimulate the synthesis of stress proteins HSP 72 and ensure an anti-aging action by means of the active principle of claim 3, characterized in that it consists in mixing from 0.5% to 10.0% active ingredient in a suitable dosage form: cream, gel, ointment, lotion, milk or emulsion or solution and with suitable excipients, possibly and to apply this preparation to the skin.

4. Cosmetic treatment according to claim 3, characterized in that it consists in mixing 2.0 to 5.0% of active principle in a suitable dosage form: cream, gel, ointment, lotion, milk or emulsion or solution and with suitable excipients, possibly and to apply it to the skin.

5. Cosmetic treatment to ensure cellular protection against photo-induced damage, by means of the active principle of claim 2, characterized in that it consists in mixing from 0.57o to 10% of active principle in a suitable dosage form : cream, gel, ointment, lotion, milk or emulsion or solution and with suitable excipients, possibly and to apply this preparation on the skin.

6. Cosmetic treatment according to claim 5, characterized in that it consists in mixing 2 to 5% of active principle in a suitable galenical form: cream, gel, ointment, lotion, milk or emulsion or solution and with suitable excipients, possibly and apply this preparation on the skin.

7. Cosmetic treatment to combat cellular aging of the skin by means of the active principle of claim 2, characterized in that it consists in mixing from 0.5% to 10.0% of active principle in a suitable galenical form : cream, gel, ointment, lotion, milk or emulsion or solution and with suitable excipients, possibly and to apply this preparation on the skin.

8. Cosmetic treatment according to claim 7, characterized in that it consists in mixing 2.0 to 5.0% of active principle in a suitable dosage form: cream, gel, ointment, lotion, milk or emulsion or solution and with suitable excipients, possibly and to apply it to the skin.

9. Cosmetic treatment to reduce wrinkles, by means of the active principle of claim 2, characterized in that it consists in mixing from 0.5% to 10.0% of active principle in a suitable galenical form: cream, gel , ointment, lotion, milk or emulsion or solution and with possibly suitable excipients and to apply this preparation to the skin.

10. Cosmetic treatment according to claim 9, characterized in that it consists in mixing 2 to 5% of active principle in a suitable dosage form: cream, gel, ointment, lotion, milk or emulsion or solution and with suitable excipients, possibly and apply it on the skin.