FR2785804A1 - COMPOSITIONS FOR COSMETIC OR DERMOPHARMACEUTICAL USE CONTAINING LARREA DIVARICATA OR LARREA TRIDENTATA EXTRACT FOR SLOWING SKIN AGING - Google Patents

Abstract

The invention concerns Larrea divaricata or Larrea tridentata extracts wherein have been identified quantities of nordihydroguaiaretic acid capable of being used industrially, and high skin anti-ageing activity have been isolated therein by means of an original operation on the Golgi apparatus. Said Larrea divaricata or Larrea tridentata extracts are used in cosmetic or dermopharmaceutical compositions, as such or for preparing medicines to delay the occurrence of, or for treating, skin-ageing symptoms of whatever origin (whether physiological, exposure to natural or artificial solar UV radiation), for slowing down hair loss and hair and body hair growth, as well as for treating the skin, mucous membranes, skin appendages of the scalp.

Description

As far as we can go back, from the origin of humanity to the present day, the study of successive civilizations shows that men have competed in imagination
and ingenuity to fight against aging itself and against its
physiological or purely aesthetic manifestations.

Nowadays, this permanent concern is exacerbated by fashion and by advertising that
always refer to youth, especially in their skin appearance
flexible, smooth and elastic.

Our current way of life (physical and chemical aggressions from pollution,
consumption of alcohol and tobacco, ...) promotes and worsens the aging process. It is the same for the perpetual quest for tanning with natural or artificial LTV.

The cosmetic industry is constantly looking for new ingredients capable of "repairing the irreparable outrage for years", and therefore, minimizing the main stigmas of wrinkles, dryness and loss of flexibility.

Until now, the discourse held, both scientific and marketing, was based on the fact that to remove the skin traces of aging, it was necessary to
suppress visible manifestations.

A large part of the solutions proposed consisted in promoting (growth factors, vitamins, trace elements, vitamins, etc.) the multiplication of skin cells. Due to their youth, these new cells, repelling and replacing older cells, had to give a youthful appearance to the skin concerned.

Other approaches, underpinned by the same type of reasoning, have been developed. This is how we have seen the peeling appear, which consists in removing, by chemical or mechanical abrasion, the surface layers of the skin which constitute dead or senescent cells.

Another proposed device consists of injecting biochemical components of the skin (such as collagen) to fill the empty spaces.

Various creams have also been proposed to hydrate the skin and thus make it look smooth and supple, in accordance with current criteria of beauty and youth.

The idea behind this patent is diametrically opposed.

It stems from reasoning based on a scientific theory, known as the Hayflick limit (Hayflick L. & Moorhead PS, (1961), Exp. Cell
Res. 25: 5585-621; Hayflick L. (1973) Amer. Daily Med. Sci. 265: 433-445) which is based on the following assumption: an animal cell is programmed for a certain number of multiplication. Without going into theoretical details, during its successive multiplications, there is a successive loss of genetic information on the DNA located at the chromosomal ends (Olovnikov (1996)
Exp. Geronto L31: 443-448). Thus, each cell would have a kind of internal molecular clock which would shell out the number of its possible replications.

It has been observed that the synthesis of type I collagen and enzymes, for example, gradually decreases in relation to the evolution of producer cells towards the stage of senescence (Kassem & al. (1997) Osteoporosis int. 7: 514- 524).

When we know that type I collagen fills tissue gaps, this theory, applied at the cutaneous level, takes on a crucial importance which, alone, can explain the cutaneous manifestations of aging which are wrinkles and loss of flexibility of the skin.

This therefore results in an approach completely opposite to the previous one.

To prevent the cutaneous manifestations of aging, cell proliferation should not be accelerated but, on the contrary, slow it down, just like in the dormancy of plant cells, a phenomenon which allows certain plants to resist the extreme climatic episodes encountered in certain deserts.

Thus, a cell made quescient by any system, will multiply less quickly than a cell whose activity is normal, even stimulated as in the previous approaches described at the beginning of this document; thus slowing the physiological aging proper to any living organism.

The object of this patent therefore lies in the industrial application of this hypothesis and in the practical solution that we bring to it because, after numerous studies, we have developed a plant extract which is capable of meeting this design, even when it is used topically; which therefore makes it possible to use it in Cosmetics and Dermopharmacy.

The plant used, Larrea divaricata or Larrea tridentata grows in America, the southern United States, Mexico and Peru. We have discovered that the main active ingredient present in the extracts of Larrea divaricata or Larrea tridentata which is the subject of this patent, is nordihydroguiairetic acid (NDGA).

NGDA is a product known for various inhibitory effects such as on prolactin secretion (Tagaya M. et al. (1993) FEBSLett. 324: 210-214) or on 5-lipoxygenase example (Morris HR & al. (1979) ) Br. J. Pharmacol. 66: 452-465) usable in certain pathologies involving allergic reactions such as asthma. Finally, patent WO 95/05156 describes the use of NDGA, in concentrations of 2 to 64%, in compositions for topical use, in the treatment of calluses, corns, warts and hyperkeratoses in general.

More recently, Fijiwara T et al. (R Biol. Chem. (1998) 273: 3068-3075) has demonstrated a reversible inhibitory effect of NDGA on the transport of proteins in the Golgi apparatus, the intracellular system involved in the secretion of proteins.

This could, at least partially, explain the effect of cell quiescence that we have discovered because in this way the cell will be able to save itself, without however stopping its normal physiological functioning. This hypothesis is supported by another discovery, which we made during the development of the extracts which are the subject of this patent is that effectively, in the presence of our extracts, the level of intracellular ATP is reduced, reflecting thus a slowing down of its energy metabolism.

To obtain the expected results, it is possible to use both the stem and the leaf or roots of Larrea divaricata or Larrea tridentata; with however a preference for the leaves.

Larrea divaricata or Larrea tridentata extracts can be obtained according to the following protocol. A quantity of 4.0 grams of dried and ground leaves of Larrea divaricata or Larrea tridentata is added to 100 ml of butylene glycol. The extraction is then carried out at 60 ° C. for one hour.

Naturally, provided that this proportion is respected, it is possible to use all the multiples of the values given here.

After filtration and / or sieving, the extract obtained is discolored by the activated carbon then, after elimination of the latter, the extract can be used as it is or can be concentrated under vacuum until a light brown powder is obtained. Depending on the origins of the plants and the extraction methods used, the analyzes performed by high performance liquid chromatography (HPLC) demonstrate the presence of
NDGA at concentrations varying between 0.5 and 5.0% (w / w).

The extraction solvents mentioned above are not limiting and can be chosen from water, propylene glycol, butylene glycol, glycerin, polyethylene glycol, methyl and / or ethyl ethers of diglycols, cyclic polyols , ethoxylated or propoxylated diglycols, alcohols (methanol, ethanol, propanol, butanol), or any mixture of these solvents.

Furthermore, it is possible to produce extracts of Larrea divaricata or Larrea tridentata by other methods such as, for example, simple decoction, leaching, extraction under reflux, extraction by means of ultrasound or microwaves or finally by means of counter-current techniques, without this list being exhaustive.

The incorporation of Larrea divaricata or Larrea tridentata extracts in cosmetic compositions is carried out by any type of process conventionally used in Cosmetology and Dermopharmacy.

Without being limiting, the following three examples give possible uses of the extracts obtained.

Example 1: CarbopolR 940 day cream 0.3
Triethanolamine 0.3
Sipol C16 / C18 S3 0 5
Amphisol 2.0
Tegin 5 1.5 Céraphil 494 6.5
Silicone DC 344 3.0
Glycerin 3.0
Larrea divaricata 5.0 extract
Perfume, preservatives qs. 100 ml
EXAMPLE 2 Anti-Wrinkle Cream
Polysorbate 60 3.8
Sorbitan stearate 2.0 Cetyl alcohol 1.5
Vaseline oil 13
Larrea tridentata 2.5 extract
Water & preservatives QSP 100g
Example 3: Deodorant body milk
Polysorbate 60 2.5
Oleic acid 0.9
Lanolin oil 2.5
Carbopol 940 0.3
Beeswax 2.0
Triethanolamine 0.1
Glycerin 5.0
Larrea tridentata 4.5 extract
Water & preservatives QSP I 00g
Only three beneficial biochemical and physiological effects highlighted during the development of extracts of Larrea divaricata or tridentata will be illustrated in the following examples, without this list being limiting.

Example Anti-IL-6 effect in vitro
The effect of Larrea tridentata extracts on the reduction of neo-synthesis of interleukin 6 (IL-6) was evaluated on fibroblasts (FH) and on normal human keratinocytes (KH) in culture, after irradiation with different doses of UV-B.

Schematically, the cells are cultured in a conventional DMEMc + 10% FCS culture medium for periods of 24 hours for FH and one week for KH. After removing the buffer and two successive rinses with a phosphate buffer solution, the cells are irradiated with UV-B at two standardized energy levels: 35 and 50 mJ. cm2. The buffer is then quickly eliminated and replaced by a culture medium identical to that used at the start of the experiment but containing the extracts to be tested at the pre-required concentrations or DMSO for the control series. After 24 hours, the culture medium is harvested and the determination of its IL-6 concentration is carried out, after freezing or not, using a standard Elisa method. An additional series is carried out according to the same protocol if not the absence of UV-B irradiation, in order to determine the base rate and to control the stability of the system studied. Finally, the tests are carried out in triplicate.

Under these conditions, in the absence of our Larrea tridentata extract, an increase in the release of IL-6 in the external medium is observed in keratinocytes equal to 212 7% and 439 + 11% respectively after irradiation with 35 and 50 mJ. cm2. Under the same conditions, the increases in IL-6 release observed are no longer only 155 3% and 313 7% respectively when our extract is diluted to 1/100 and 51 4% and 117 5% respectively in the presence of our extract diluted 1/10. Under the same conditions, similar results were observed on fibroblast cultures. Finally, the previous results are substantially reproduced identically if we use, under the same conditions, our extract of Larrea divaricata.

This example demonstrates on the one hand that the cellular system studied is correct since there is indeed the dose effect of irradiation / increase in the release of IL-6 expected and that, on the other hand, the extracts tested have , on the two cell types tested, a real efficacy which is dose-dependent.

EXAMPLE 5 Antiproliferative Effect, In Vitro
The method used here is based on the following principle. As the quantity of DNA is fixed from one cell to another, the measurement of the overall quantity of DNA corresponds to measuring the number of cells used for this measurement. This principle makes it possible not to routinely use finer but very cumbersome methodologies.

Different techniques have been developed on the basis of this protocol, including the one used here: by binding to DNA, according to a constant and known stoichiometry, the fluorophore Hoescht 33258 exhibits on the one hand increased fluorescence but also a shift in sound emission spectrum from 492 nm to 458 nm. By comparison with previously established calibration ranges, the combined monitoring of these two parameters makes it possible to quantify the quantity of DNA present in the cell samples studied.

The cell cultures (FH or KH) used are the same as described in the previous example. The Hoescht 33258 fluorophore is added at the end of manipulation, before the aliquots of cells are taken for the assay. A control series is carried out (absence of extract in the culture medium), in order to determine the basic cell proliferation in the system studied. Finally, the tests are carried out in triplicate.

Under these conditions, when fibroblasts are placed in the presence of our extract of Larrea tridentata diluted to 1/100 or 1/10, there is a decrease in the amount of DNA (and therefore the number of cells present) equal to respectively 5.9 1.2 and 16.3 1.5% compared to the control series, therefore without our extract from
Larrea tridentata. Under the same conditions, similar results were observed on fibroblast cultures. Finally, the previous results are substantially reproduced identically if we use, under the same conditions, our extract of Larrea divaricata.

This example demonstrates that in the presence of our extracts, cell multiplication is slowed down, on the two cell types studied. Our extracts therefore have real anti-proliferative efficacy which is dose-dependent.

EXAMPLE 6 Reduction of the Intracellular ATP Level in Vitro
The measurement of the concentration of intracellular ATP (or [ATP] s) is carried out on cells, fibroblasts or keratinocytes, in culture, by means of the conventional method of luminometry which uses the property of the luciferin / luciferase couple to emit a quantity of photons proportional to the quantity of ATP in the medium (for example: Doctor et al. Am. J Physiol. 266: C1803-1811). Each of the wells of the 96-hole plates used is seeded with 20,000 cells and then, after a period of 24 hours for FH or one week for KH, the cells are covered with the culture medium containing or not extracts of Larrea tridentata with concentrations used in Example N 4.

After 24 hours of incubation, elimination of the culture medium and rinsing of the cell layer, an aliquot of cells is taken to carry out the measurement of ATP with the luciferin / luciferase reaction, using a luminometer (Lumistar BMG in these experiences).

A control series is carried out by adding culture buffer without product to be tested, in order to determine the level of basic intracellular ATP. Finally, the tests are carried out in triplicate.

Under these conditions, when fibroblasts are placed in the presence of our extract of Larrea tridentata diluted 1/100 or 1/10, there is a decrease in 1 '[ATP] i of 5.3 0.7 and 18 respectively, 2 1.1% compared to the control series carried out without our Larrea tridentata extract. Under the same conditions, similar results were observed on fibroblast cultures. Finally, the previous results are substantially reproduced identically if we use, under the same conditions, our extract of Larrea divaricata.

This example demonstrates that the extracts tested exhibit, on the two cell types studied, a real efficiency in the setting of quiescence, which is dose-dependent.

In conclusion, the examples above demonstrate that the extracts of Larrea divaricata or of Larrea tridentata, have a real effect on the quiescence of skin cells, thus allowing them a longer life cycle, thus delaying skin aging. .

Due to their activities demonstrated above, such preparations have anti-aging and anti-wrinkle effects, slowing down hair loss as well as hair growth; protect the skin from solar or artificial UV aggressions, thus prolonging the suppleness and protective function of the skin.

The concentration of NDGA in the extracts of Larrea divaricata or of Larrea tridentata (called Extracts in the rest of this description) can vary between 0.05% and 10% (w / w), preferably between 0.5% and 5.0% ( p / p).

The concentration of the Extracts can vary between 0.01% and 50% (w / w), preferably between 0.1% and 10% (w / w) in the finished cosmetic or dermopharmaceutical composition.

The Extracts can be used in any dosage form used in cosmetics 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 extracts into 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 Extracts can be combined in cosmetic compositions with any other ingredient usually used in cosmetics: extraction and / or synthetic lipids, gelling and viscosifying polymers, surfactants and emulsifiers, water-soluble or liposoluble active principles, extracts from other plants, tissue extracts, marine extracts.

The Extracts are used as such, in cosmetic or dermopharmaceutical compositions as such or for the preparation of a medicament for skin care, including against the cutaneous manifestations of aging, whatever their origin (physiological , exposure to natural or artificial UV rays); as well as for all skin, mucous, dander or scalp care.

Claims (12)

 dander or scalp.  hair ; as well as for all skin care, mucous membranes,  to slow hair loss and hair growth and  the origin (physiological, exposure to natural or artificial UV rays),  or prevent the cutaneous manifestations of aging whatever  cosmetic or dermopharmaceutical compositions for eliminating, reducing  CLAIMS 1. Use of Larrea divaricata or Larrea tridentata extracts in 2. Extracts according to claim 1 characterized in that they contain between  0.05% and 10%, preferably between 0.5% and 5.0% (w / w) of acid  nordihydroguaiarétique (NDGA). 3. Process for obtaining cosmetic compositions or  dermopharmaceuticals according to one of claims 1 or 2, characterized in  that the extraction solvents used are chosen from water, propylene  glycol, butylene glycol, glycerin, polyethylene glycol, ethers  methyl or ethyl diglycols, cyclic polyols, diglycols  ethoxylated or propoxylated, alcohols (methanol, ethanol, propanol, butanol),  or any mixture of these solvents. 4. Method of obtaining according to one of claims 1 to 3 characterized in that  the extract is obtained by ethanolic extraction and then, after drying, the extract  dry is taken up in butylene glycol. 5. Method for obtaining cosmetic or dermopharmaceutical compositions  according to one of claims 1 to 4, characterized in that the extraction can be  replaced by maceration techniques or by other processes such as,  for example, simple decoction, leaching, refluxing,  Extraction using ultrasound or microwaves or finally  against the current. 6. Cosmetic or dermopharmaceutical compositions according to one of the  Claims 1 to 5, characterized in that the extract is obtained from the  whole plant or, preferably from the flowering and dry aerial part. 7. Cosmetic or dermopharmaceutical compositions according to claim 6  characterized in that the extract is used either in liquid form or in  dry form obtained by conventional precipitation, drying techniques,  evaporation, atomization or lyophilization. 8. Cosmetic or dermopharmaceutical compositions according to one of the  Claims 6 to 7, characterized in that the concentration of the extract can  vary between 0.1% and 50% (w / w), preferably between 0.1% and 10% (w / w)  in the finished product. 9. Cosmetic or dermopharmaceutical compositions according to one of the  Claims 6 to 8, characterized in that they are in any form  dosage used in cosmetics or dermopharmacy: O / W emulsions and  W / O, 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 according to one of the  Claims 6 to 9, characterized in that the extract is incorporated into  cosmetic vectors such as liposomes, chylomicrons, macro-,  micro- and nanoparticles as well as macro-, micro- and nanocapsules,  absorb on powdery organic polymers, talcs, bentonites and others  mineral supports.  tissues, marine extracts.  hydro-or liposoluble active ingredients, extracts from other plants, extracts  synthetic, gelling and viscosifying polymers, surfactants and emulsifiers,  other ingredient usually used in cosmetics: extraction lipids and / or  Claims 6 to 10, characterized in that the extract is used with all  11. Cosmetic or dermopharmaceutical compositions according to one of the 12. Use of the extract according to one of claims 1 to 5 or of a composition  according to one of claims 6 to 11 for the preparation of a medicament  for skin care, including against skin manifestations of  aging whatever its origin (physiological, exposure to natural or artificial UV rays), to slow down hair loss as well as hair growth; as well as for all skin care, mucous membranes, dander or scalp.