(en)The present invention concerns various cosmetic uses of compositions comprising an essential oil or one of its constituents, preferably a major constituent, in the targeted treatment and prevention of benign keratoses, and more particularly keratoses induced by solar radiation on the skin of a subject. More particularly, the present invention concerns compositions based on the essential oil of oregano or of rosewood, or on linalool, thymol or carvacrol. The present invention also concerns therapeutic applications of a composition as described in the prevention or targeted treatment of actinic keratoses, essentially for prophylactic purposes.

1.ApplicationNumber: US-201113580979-A
1.PublishNumber: US-2015150929-A1
2.Date Publish: 20150604
3.Inventor: MARROT LAURENT
SOEUR JÉRÉMIE

4.Inventor Harmonized: MARROT LAURENT(FR)
SOEUR JÉRÉMIE(FR)

5.Country: US
6.Claims:
(en)The present invention concerns various cosmetic uses of compositions comprising an essential oil or one of its constituents, preferably a major constituent, in the targeted treatment and prevention of benign keratoses, and more particularly keratoses induced by solar radiation on the skin of a subject. More particularly, the present invention concerns compositions based on the essential oil of oregano or of rosewood, or on linalool, thymol or carvacrol. The present invention also concerns therapeutic applications of a composition as described in the prevention or targeted treatment of actinic keratoses, essentially for prophylactic purposes.

7.Description:
(en)The present invention relates to the field of cosmetics, and more particularly relates to the skin. More generally, it relates to the context of the prevention and targeted treatment of actinic keratosis.
It is known that chronic exposure to sun induces dermatological lesions of the actinic keratosis type and photo-aging of the skin (Ortonne, J. P. 2002).
Dermatologically, keratoses appear as zones of epithelial dysplasia which are observed in regions of the body that are frequently exposed to the sun, and are often associated with photo-aging. The majority of such keratoses regress spontaneously (Ortonne, J. P. 2002). In addition, keratoses are unsightly in appearance, which may bring affected subjects to request that they be treated in order to eliminate the keratoses on esthetic grounds.
In the skin, it has been shown that actinic keratoses are frequently constituted by keratinocytes carrying a mutation in the p53 gene (Taguchi, M. et al. 1994). The gene coding for the p53 protein is frequently damaged by UV and in some cases is subjected to mutations, in particular CC to TT mutations. The mutated cells may lose the specific response to UV controlled by the p53 protein: thus, repair is less effective and the presence of damage to the DNA no longer arrests proliferation: replication can occur in the presence of lesions, thereby increasing the risk of errors (mutations), leading in time to high genetic instability. Since mutated cells enter the apoptosis process less easily, they finish up by manifesting a growth advantage compared with normal cells. This process of clonal expansion of abnormal cells creates pre-cancerous patches in the affected organs.
In order to study these mechanisms, it is possible to use keratinocytes carrying mutations in the p53 gene. This is the case with the HaCat line, which was originally described by Fusenig et al. (Fusenig, N. E. et al. 1998) and which is now very frequently employed in various dermatological research laboratories. Thus, for example, HaCat cells have been integrated into an organotypic model of the skin in the presence of normal keratinocytes: in such a system, it was possible to mimic the promoter effects of exposure to the sun by demonstrating the capacity of UVB to stimulate clonal expansion of HaCat cells (Mudgil, A. V. et al. 2003) mutated at p53. More recently, it has been reported that HaCat cells exposed to UVA and reimplanted into mice could be the source of skin tumors (Wischermann, K. et al. 2008). Thus, there is a need for products inducing significantly more intense cytotoxicity in HaCat cells compared with normal human keratinocytes (or keratinocyte lines not mutated at p53) which would be potentially capable of treating actinic keratosis. For this reason, the work described below consisted of comparing and understanding the cytotoxic effect of essential oils on normal human keratinocytes, on HaCat keratinocytes (carrying a mutation at p53) and on immortalized keratinocytes derived from carcinoma.
The induction of cell death by apoptosis, preferably in cells with problems as regards proliferation and differentiation, while conserving the integrity of healthy cells, constitutes an effective treatment against actinic keratosis type lesions.
Apoptosis is a complex process which may have various origins, but it is currently accepted that mitochondria play an important role therein. Various molecular signals, such as the Bcl2/Bax equilibrium, for example, possibly competing with p53, are capable of modifying the permeability of the mitochondrial membrane and of promoting release of cytochrome C into the cytosol. That process may initiate activation of the caspase pathway, caspases being the enzymes in charge of the degradation of certain intracellular compounds, which irreversibly results in apoptosis (Gogvadze et al., 2008). Further, it has also been demonstrated that permeabilization of the mitochondrial membrane can release various pro-oxidizing mitochondrial molecules into the cytosol, allowing the induction of apoptosis.
Essential oils (EO) and their derivative products are currently very popular products with consumers. This popularity, specifically for products of natural origin (agroalimentary, cosmetology, pharmacology, etc.) or arising from the “natural” concept, is the consequence of a general demand for products of a durable development type (green chemistry, bio-agriculture). This interest in “nature” and its “soft medicine” products (aromatherapy, phytotherapy) also has its origin in the limitations of conventional medicine (viral resistance, bacterial resistance, cancer treatment, etc.). The absence of substantial secondary effects in the context of well-documented, reasoned aromatherapy treatments is a major advantage in the use of essential oils.
Essential oils are complex, volatile products with a powerful odor, characteristic of the part of the plant used for its manufacture. Of approximately 800000 recorded plant species, only aromatic plants are used to obtain essential oils. These are plants with sufficient cells synthesizing and secreting those aromatic molecules, i.e. approximately 3000 plants of biological interest. The major portion of the essential oils on the market is represented by approximately 300 essential oils.
Essential oils correspond to a complex mixture of secondary metabolites (molecules not essential to survival of the plant) synthesized and secreted by specialized organs: epidermal glandular hairs, glandular pouches and channels (schizogens or schizolysigens). Such secondary metabolites are represented by a very wide diversity of chemical molecules. The most common are terpenes (mono-, sesqui- and diterpene: C10, C15 and C20 respectively) and other aromatic molecules (cyclic molecules). All chemical functions are present in the essential oils: aldehydes, ketones, alcohols, peroxides, lactones, ethers, esters, etc.
Essential oils are obtained after hydro-distillation of plant material of the aromatic plant. Currently, various extraction methods are used; examples that may be cited are supercritical CO 2 extraction and solvent extraction.
Essential oils have a recognized cosmetic and therapeutic potential and are principally used for their bactericidal, virucidal, antioxidant and anti-inflammatory activities, but also for their fragrant character, which is capable of causing a feeling of well-being. Several modes of using essential oils are possible: inhalation, ingestion or application to the skin. Pure essential oils are almost never applied directly to the skin as they are often irritants, but are diluted in other vegetable oils (olive oil, sunflower seed oil, etc.). Application to the skin is carried out in the context of massage, local treatments (infections) or when using perfumes (major constituents).
In addition to their cosmetic interest, essential oils are also used in the context of certain antibiotic treatments for their ability to enhance their efficiency, or to combat infections of the respiratory tracts. In the same manner, a non-negligible proportion (approximately 35%) of European patients with cancer for whom conventional treatment has not provided a cure have turned to complementary methods such as aromatherapy as an alternative to conventional medicine (Molassiotis, A. et al. 2005). Some studies have demonstrated the anticancer and/or antiproliferative properties of essential oils as regards certain cancer lines. Thus, recent work has demonstrated potentially anti-carcinogenic or antiproliferative activities of certain essential oils on leukemia cell lines (Kumar, A. et al. 2008), (Verma, M. et al. 2008), colon cancer lines (Sharma, P. R. et al. 2008), breast cancer cells (Diaz, C. et al. 2008), or melanoma cells (Loizzo, M. R. et al. 2008).
Other studies have also demonstrated an antiproliferative activity of certain compounds isolated from essential oils, such as alpha santalol on an epidermoid carcinoma line (Kaur, M et al. 2005), or that of terpinee-4-ol, from the essential oil of tea tree, on melanoma cells (Calcabrini, A. et al. 2004).
Regarding keratinocytes, the cytotoxicity of certain essential oils has been studied as regards the keratinocyte line HaCat mutated at p53 (Koba K. et al. 2009) or of the keratinocyte line SVK14 (Itharat, A. et al. 2004).
In general, the invention concerns any essential oil or one of its constituents for a cosmetic use in the prevention or targeted treatment of keratoses, and more particularly actinic keratoses.
More particularly, the invention concerns any essential oil extracted from different parts (roots, stems, bark, leaves) of plants of the oregano family (Lamiaceae) or rosewood (Lauraceae) as well as any essential oil containing at least one of the compounds identified in the essential oils from compact oregano and rosewood. Still more preferably, the invention concerns the essential oil of Origanum compactum and that of Aniba rosaeodora , as well as linalool, thymol and carvacrol.
The present inventors have surprisingly demonstrated that essential oils with very different molecular compositions, namely the essential oil from compact oregano ( Origanum compactum , Lamiaceae family) and the essential oil from rosewood ( Aniba rosaeodora, Lauraceae family) can induce cell death by apoptosis in a targeted manner in cancerous and pre-cancerous (mutated at p53) human keratinocytes compared with normal keratinocytes.
The compositions of these two essential oils are very different from a chemical viewpoint, as are the major constituents, and so the effects obtained for these two essential oils may thus be considered to be representative of the effects obtained with any essential oil or its constituents.
In accordance with the present invention, any composition containing an essential oil may thus be used to prevent, treat and reduce actinic keratoses known to be formed very often from keratinocytes carrying mutations in the p53 gene induced by UV. This treatment also has the advantage of not generating inflammation, since apoptosis is not a pro-inflammatory process.
More specifically, the invention concerns various cosmetic applications of compositions comprising an essential oil in the prevention or treatment of actinic keratoses. The cosmetic applications of the invention are preferably for human beings.
In a first aspect, said cosmetic methods or applications are preferably without any therapeutic purposes.
The cosmetic applications relate to treatments intended to modify the appearance of the skin, its esthetic properties, with no therapeutic or prophylactic aim, i.e. a physician would not recommend such a treatment as it intended only to modify the skin esthetically.
In the context of this aspect of the invention, the keratoses to be treated are benign, free of tumor cells; in particular, they are not in the transformation phase. In this situation, a physician would not recommend excision of the keratosis for therapeutic reasons, given that the large proportion of keratoses disappear naturally with time without entering the transformation phase. The keratoses under consideration are preferably small, and do not present any danger of becoming cancerous in the short or medium term.
The term “keratosis” (or keratoderma or hyperkeratosis) means a hyperplasia of the epidermal stratum corneum. The term “actinic keratosis” or “solar keratosis” means a keratosis induced by exposure to solar radiation. In the context of the present invention, keratoses that are particularly preferred for cosmetic applications are actinic keratoses. The term “actinic keratoses” is to be understood as lesions free of tumor cells.
The keratoses and actinic keratoses under consideration may be constituted or primarily comprise cells mutated at p53.
Because of their unattractive appearance, there is a strong desire for prevention and/or treatment of keratoses for purely cosmetic rather than therapeutic reasons.
In accordance with this aspect, the invention concerns the use of a composition comprising an essential oil or at least one of its constituents, for a cosmetic application in the prevention or treatment of keratoses. The keratoses under consideration are in particular free of tumor cells. Preferably, it is a major constituent of an essential oil.
The term “major constituent” or “preponderant constituent” of an essential oil means either a constituent the proportion of which in that essential oil is more than 10% by weight, or one of the five constituents the proportion by weight of which is the highest in that essential oil. Preferably, in the context of the present invention, a “major or preponderant constituent” means one of the five most abundant constituents, by weight, in the essential oil.
The term “prevention of keratoses” in particular means obtaining a preventative effect such that the keratoses do not develop, or that those that already exist do not develop further.
The term “treatment of keratoses” in particular means an effect such that the keratoses that already exist stop growing and regress, or even disappear entirely.
The prevention or treatment of keratoses is particularly appropriate in the case of skin that has been exposed to a strong UV radiation or to repeated exposure to UV. In such an application, the present invention in particular envisages being used as an after-sun preparation. “Prevention” may also involve application to the skin before exposure to the sun, in particular before sustained exposure, in order to prevent the development of a keratosis, after prior verification, however, that the compositions or essential oils used are not phototoxic.
The prevention or treatment of keratoses in a cosmetic application in accordance with the invention is also particularly appropriate in the case of keratoses in the development phase, i.e. the keratoses are already present on the skin and they are becoming larger. In such an application, the use of the invention can stop the growth of keratoses and reduce their size; they might disappear completely.
One of the major advantages of the cosmetic applications described resides in the targeted action of the essential oils. In fact, in one range of concentration, the essential oil has a targeted action against pre-cancerous cells (mutated at p53, mimicking cells from actinic keratosis), which may be hyperproliferative; normal cells are hardly affected by the cytotoxicity. It is thus possible to treat all of the skin of an individual without any deleterious effect for the normal keratinocytes. This property is particularly appreciated in the context of a cosmetic application. It should be noted that the inventors are in fact the first to have carried out a comparative study of the impacts of essential oils on keratinocytes mutated at p53 and on normal keratinocytes. No data relating to such a comparison of cytotoxicity had been available before the present invention. The targeted action of the essential oils and their constituents thus had never been brought to light before the invention. In view of the foregoing, then, the present invention can be used for the prevention or targeted treatment of keratoses, i.e. a lower toxicity of this treatment as regards normal cells, in particular normal keratinocytes, surrounding the keratosis.
The term “targeted cytotoxicity” as used in the invention means that the property of cytotoxicity is preferentially induced in hyperproliferative cells and/or p53-mutated cells (for example HaCat and A431) compared with non-hyperproliferative cells, not mutated at p53 (for example HEK001 or NHEK894), i.e. the toxicity levels are at least 1.5 times higher in mutated cells than in non-mutated cells, preferably at least 2 times higher. Preferably, the viability of the non-hyperproliferative, non-mutated cells remains of the order of at least 40%, or even 50%, preferably 60%. The inventors have demonstrated the existence of ranges of concentrations for which such a targeted cytotoxicity, saving healthy cells, could be obtained.
Because of the targeted action of the essential oils and their constituents, the preventative cosmetic treatment of the invention is not dangerous to healthy cells not mutated at p53. As a consequence, the treatment may, for example, be carried out again before or after each exposure to the sun.
In the compositions of the present invention, the essential oil under consideration or the constituent of the essential oil under consideration constitutes the active principle of the composition or one of the active principles because the inventors have demonstrated the targeted cytotoxicity of the essential oils and their constituents against mutated keratinocytes representative of keratinocytes derived from keratoses.
In accordance with a preferred embodiment of the present invention, the essential oil contains at least one of the constituents of the essential oils of Origanum compactum or of Aniba rosaeodora ; preferably, it comprises at least 10%, or at least 15% or 20% by weight of linalool, carvacrol or thymol. Preferably, it has a composition that comprises an essential oil or a mixture of oils such that the essential oil or the mixture comprises said proportions of linalool, carvacrol or thymol. The invention also concerns compositions in which said percentages correspond to chromatographic percentages.
In the experimental section, the cytotoxicity of two essential oils has been studied in particular: the essential oil of compact oregano ( Origanum compactum , Lamiaceae family) and the essential oil of rosewood ( Aniba rosaeodora , Lauraceae family), on human epidermal cells: the keratinocyte line HaCat (mutated at p53 and spontaneously immortalized), the A431 line (from a spino-cellular carcinoma, mutated at p53) as well as normal human primary epidermal keratinocytes.
The essential oil of compact oregano is primarily composed of two monophenols (terpenoids): thymol and carvacrol. This essential oil is recommended in the context of the treatment of colitis and pulmonary disorders. It can combat fatigue, but it is primarily a broad spectrum bactericide.
The essential oil of rosewood is composed of 80% linalool. Its properties are used to treat irritated, fatigued and wrinkled skin. It is also anti-infectious (less powerful than the essential oil of oregano), immunostimulating and tends to improve depressive behavior. It is very slightly irritant and slightly toxic.
The present invention preferentially concerns the use of compositions comprising the essential oil of Origanum compactum and/or of Aniba rosaeodora.
Although the cytotoxic activity of an isolated constituent is less than that of an essentially complete oil, in accordance with another embodiment of the invention, the use more specifically concerns at least one constituent of an essential oil, preferably at least one of the constituents of the essential oils of Origanum compactum or of Aniba rosaeodora . These two essential oils and their constituents are in fact preferred in the context of the present invention, for the reasons given above.
The various constituents of the essential oils are well known to the skilled person and may be obtained from firms marketing said essential oils.
Regarding the essential oils of Origanum compactum or of Aniba rosaeodora, Example 1 of the present application provides an analytical characterization of the various constituents that have been detected.
Particularly preferably in the context of the present invention, the essential oil constituent that is used is not linalool oxide, and preferably not an oxide at all.
Preferably, the component used is an alcohol or a phenol, more particularly preferably a terpenol or terpenic alcohol such as linalool, terpineol, nerol, geraniol, citronellol, or menthanol, or a terpenoid such as citral, menthol, carvacrol or thymol; other terpenes that may also be envisaged are p-cymene, terpinene, myrcene and beta caryophyllene. Particularly preferred constituents are linalool, carvacrol and thymol.
In other preferred embodiments of cosmetic uses as described above, the constituent is selected from 1,8-cineole, terpinolene, linalool, alpha-terpineol, geraniol, alpha-copaene, alpha and beta-selinene, benzyl benzoate, alpha-thujene, alpha pinene, myrcene, alpha-phellandrene, alpha terpinene, para-cymene, beta-phellandrene, gamma-terpinene, thymol, carvacrol and beta caryophyllene. They are in fact constituents of one or the other of the essential oils of oregano and of rosewood, wherein the proportion in the volatile part of the oil is more than 0.5% (as a chromatographic percentage).
In accordance with another embodiment of a cosmetic application, an essential oil is used comprising at least 0.5% by weight or as a chromatographic percentage, preferably at least 1%, or even 5% of at least one of the following compounds: 1,8-cineole, terpinolene, linalool, alpha-terpineol, geraniol, alpha-copaene, alpha and beta-selinene, benzyl benzoate, alpha-thujene, alpha pinene, myrcene, alpha-phellandrene, alpha terpinene, para-cymene, beta-phellandrene, gamma-terpinene, thymol, carvacrol and beta caryophyllene. Preferably, it is a chromatographic percentage applied to the volatile portion of the essential oil.
Preferably, in the cosmetic applications of the invention, at least one essential oil constituent is used selected from the following components: linalool, thymol and carvacrol. Linalool is the major compound of the essential oil of rosewood and thymol and carvacrol are the two major compounds in the essential oil of oregano.
In a preferred embodiment, the invention also concerns the use of an essential oil comprising at least one of the following constituents: linalool, thymol and carvacrol. Preferably, at least one of these components is present in a proportion of more than 0.5%, preferably more than 1%, 5% or even 10%, 15% or 20% by weight. Alternatively, it may concern chromatographic percentages. Such an essential oil is, for example, the essential oil of thyme, wild thyme or savory (large proportion of thymol or carvacrol) or the essential oil of basil, of thyme (with linalool, Thymus vulgaris linaloliferum), of lavender or of Shiu wood (also known as Ho wood).
More particularly, the present invention concerns the cosmetic use of compositions comprising at least 10%, preferably at least 15% or at least 20% of one of the following constituents: linalool, carvacrol and thymol. Other compositions that may also be envisaged are such that the cumulative quantity of linalool, carvacrol and thymol is more than 10%, preferably more than 15%, or even more than 20% by weight. Alternative, they may be chromatographic percentages.
In accordance with another embodiment of the invention, the component used in the cosmetic applications is selected from alpha pinene, camphene, myrcene, alpha terpinene, para-cymene, trans-ocimene, gamma-terpinene, linalool, terpinen-4-ol, beta caryophyllene, alpha-humulene and caryophyllene oxide. They are in fact components which are common to the two essential oils that are particularly preferred in the context of the present invention, namely the essential oil of oregano and the essential oil of rosewood.
In another embodiment, the invention also concerns the use of an essential oil comprising at least one of the following components: alpha pinene, camphene, myrcene, alpha terpinene, para-cymene, trans-ocimene, gamma-terpinene, linalool, terpinen-4-ol, beta caryophyllene, alpha-humulene and caryophyllene oxide. Preferably, at least one of these components is present in a proportion of more than 0.5%, preferably more than 1%, 5% or even 10%, 15% or 20% by weight. Alternatively, they may be chromatographic percentages.
In accordance with another embodiment envisaged in the context of the present invention, the essential oil or the essential oil constituent is extracted from plants of the Lamiaceae or Lauraceae family: Origanum, Acinos, Agastache, Ajuga, Calamintha, Glechoma, Hyssopus, Lamium, Lavandula, Leonurus, Lycopus, Melissa, Melittis, Mentha, Micromeria, Monarda, Ocimum, Orthosiphon, Perilla, Perovskia, Phlomis, Physostegia, Pogostemon, Prostanthera, Prunella, Rosmarinus, Salvia, Satureja, Scutellaria, Sideritis, Solenostemon, Stachys, Teucrium and Thymus.
Preferably, it is from the genus Origanum.
In accordance with preferred uses in the context of the present invention, the essential oil or the constituent is an extract of oregano, lavender, mint, rosemary, savory, sage, thyme, spotted deadnettle, black horehound, bugle weed, basil, hyssop, marjoram, deadnettle, ground ivy, wood betony, prunella, wild thyme or lemon balm.
More particularly preferably, in the cosmetic applications of the present invention, an essential oil or a constituent of an essential oil extracted from the species Origanum compactum is used, in particular thymol or carvacrol.
In accordance with another embodiment of the invention, the essential oil or the essential oil constituent is extracted from plants deriving from one of the following botanical genuses of the Lauraceae family: Aniba, Cinnamomum, Laurus, Persea, Ravensara or Umbellularia.
Preferably, it is from the genus Aniba.
In accordance with preferred uses in the context of the present invention, the essential oil or the essential oil constituent is extracted from rosewood, cinnamon, laurel, horseradish ( Ravensara ), camphor or avocado.
More particularly preferably, in the cosmetic applications of the present invention, the essential oil or essential oil constituent extracted from the species Aniba rosaeodora is used.
In accordance with another embodiment of the invention, an essential oil or a constituent of an essential oil is used that is capable of generating a pro-oxidizing endogenous situation that is specifically produced in keratinocytes that are mutated at p53. Particular essential oils with this property are the essential oil of compact oregano and the essential oil of rosewood. Results in this regard are shown in Example 2 of the experimental section. The detailed molecular analysis in the experimental section shows that these essential oils cause destabilization of the mitochondrial membrane and release of reactive oxygen species, at the origin of the cytotoxicity targeted against cells mutated at p53.
The examples also define simple tests for demonstrating the generation of a pro-oxidizing situation, in particular by detecting the production of the mitochondrial superoxide anion.
The essential oil or the essential oil constituent used in accordance with the invention may be extracted from roots, stems, bark or leaves of said plant. Reliable techniques are known for extracting essential oils, in particular cold extraction, which consists of placing the plant matter under high pressure using a hydraulic press, steam entrainment, which consists of forming steam that passes through the plants and picks up aromatic molecules, and dry distillation. The essential oil obtained by distillation is a plant essence modified by oxidation and hydrolysis. A managed temperature and a low pressure are essential in conserving aromatic quality and a chemical composition as close as possible to the plant essence to be extracted. The majority of essential oils are obtained by distillation and steam entrainment (hydro-distillation).
Other extraction techniques are well known and the skilled person will know, as a function of the plant under consideration, which technique is the most appropriate. The skilled person will also know which technique is the most appropriate as a function of the part of the plant that will be used for extraction of the essential oil.
Clearly, it is preferable to use techniques that produce the highest purity and guarantee the absence of solvents in the extract obtained (essential oil or essential oil constituent).
In the context of the various cosmetic applications of the present invention, it is preferable to use an essential oil or an essential oil constituent in combination with other compounds. The other compounds may in particular be vegetable oils. Particularly preferred vegetable oils in combination with the essential oils of the invention or their constituents that may be cited include grapeseed oil, sweet almond oil, and also hazelnut oil, macadamia nut oil, sunflower seed oil and olive oil. In particular embodiments, the essential oil or the essential oil constituent may be used in combination with a sunscreen, or in combination with a moisturizing solution or a vegetable oil, or with both.
It may be a combination of at least two distinct essential oils, for example an essential oil extracted from a plant of the Lamiaceae family and an essential oil extracted from a plant of the Lauraceae family.
It may also be a combination comprising an essential oil of a plant with a constituent of that same essential oil, which results in modifying the natural proportions of the various constituents of said essential oil; as an example, Origanum compactum essential oil may be used in association with thymol or carvacrol; or the Aniba rosaeodora essential oil in association with linalool.
Alternatively, it may be a combination comprising an essential oil of a plant with a constituent originating from another essential oil, extracted from a different plant, in particular the essential oil of Origanum compactum in association with linalool; or the essential oil of Aniba rosaeodora in association with thymol or carvacrol.
There may be two distinct constituents originating from different essential oils which are not naturally together in any essential oil, or which are not found naturally in these proportions in any essential oil.
Other additional compounds may be selected as a function of the desired texture and the desired mode of application for the essential oils or their constituents in the cosmetic applications of the invention.
Furthermore in the present invention, the inventors have demonstrated the existence of a specific range of concentration of essential oil or essential oil constituent for which the cytotoxicity is specifically targeted towards cells mutated at p53, as opposed to normal cells, and more particularly to keratinocytes mutated at p53 compared with normal keratinocytes. The cosmetic applications envisaged in the context of the present invention preferably employ a concentration range allowing targeted cytotoxicity towards hyperproliferative cells and/or cells mutated at p53 and providing a viability of at least 40%, or even at least 50% or 60% for the normal non-mutated cells. Carrying out the protocols detailed in Examples 2 and 3 of the experimental section allows the skilled person to determine the concentration ranges that are suitable for any essential oil or one of its components.
In the case of in vitro culture, the inventors have demonstrated (see Example 4) that said concentration ranges are: from 0.0125% to 0.0175% for the essential oil of oregano and from 0.035% to 0.045% for the essential oil of rosewood, in order to obtain a targeted cytotoxicity in cells mutated at p53.
As a function of this data, the skilled person will know how to adapt the composition of the invention in order to obtain a comparable concentration range. In particular, the skilled person will be able to adapt the composition of the invention into the form of a patch to guarantee a long-term contact. It is also possible to envisage repeated applications, for example every 24 h for a week or a month or more.
The expression “various cosmetic uses of the invention” means uses under conditions such that they induce a preferential cytotoxicity in keratinocytes derived from or mimicking an actinic keratosis type lesion, in particular mutated at p53, compared with normal or non-hyperproliferative keratinocytes.
In the context of this invention, the uses mentioned are principally envisaged for topical skin applications. This is the mode of administration that not only provides the best efficacy, but also better targeting of the cells to be treated. A formulation in the form of a patch is particularly preferred. Other modes of administration may be envisaged, however, such as ingestion or inhalation.
A composition used in the context of the present invention comprises a concentration of essential oil or essential oil constituent of approximately 0.03% to 0.15%, preferably approximately 0.03% to 0.1% of the essential oil of oregano, or approximately 0.06% to 0.15% of the essential oil of rosewood, for application to the skin.
In the case of preventative applications, the compositions of the invention may be formulated into the form of a milk, for example as a sun milk, in the form of a pomade or in the form of an oil.
Preferably, the essential oil selected or one of its constituents is formulated in combination with a vegetable oil, in particular to allow it to be diluted and thus to reduce any irritant effect.
Further, since the inventors have demonstrated specific targeting of the cytotoxic effect of the essential oils or their constituents, in particular the major constituents, against hyperproliferative cells such as cells mutated at p53, the present invention also concerns any cosmetic use of an essential oil or one of its constituents, preferably a major constituent, in the treatment or targeted prevention of unsightly zones of the skin due to hyperproliferation of cells of the dermis or epidermis, for example due to hyperproliferation of melanocytes, in particular in the context of nevi, or of unsightly zones in the context of scars.
A subject who might be able to be treated using the cosmetic methods of the invention is a human being, man or woman, of any age. Preferably, it is an adult, but the invention is not limited to adults and also encompasses the treatment of adolescents, preferably over 15 years of age, for the various envisaged cosmetic applications.
In a second aspect, the present invention also concerns various cosmetic methods, in particular cosmetic methods for the prevention or treatment of actinic keratoses, comprising applying at least one essential oil or one of its constituents to the skin. Said methods are purely cosmetic and are only intended to modify the esthetic appearance of the skin. Said keratoses may be free of tumor cells.
The various preferred embodiments in the context of these cosmetic methods are identical to the embodiments detailed above concerning the various uses for cosmetic applications.
In particular, as has already been explained, more particularly preferred essential oils or essential oil constituents are extracts of plants from the Lamiaceae or Lauraceae family.
Still more preferably, it is an essential oil extracted from Origanum compactum or from Aniba rosaeodora , or one of the constituents of these essential oils. Said constituents are listed in Example 1 of the experimental section.
Ideally suited constituents in the context of the cosmetic methods of the invention are linalool, carvacrol and thymol.
In the context of the present invention, the various applications described above, which are preventative or for the treatment of keratoses, are also envisaged not only for a purely cosmetic esthetic purpose, but also for a prophylactic purpose, if a change of the keratosis to a pre-cancerous lesion is suspected or envisaged, for example from family history or the history of that same subject. In this case, treatment of a keratosis may be envisaged not only for cosmetic reasons but also for medical reasons, as long as that keratosis is not a tumor and does not have any tumor cells.
In this case, the present invention is also directed to therapeutic applications of the compositions described above, comprising an essential oil or at least one of its constituents, preferably a major constituent.
More particularly, in this aspect, the invention concerns a composition comprising an essential oil or at least one of its constituents for therapeutic use in the prevention or targeted treatment of keratoses, preferably in human beings. Said keratoses essentially means actinic keratoses, free of tumor cells.
Preferably, as for the applications described already in the invention, the essential oil contains at least one of the components of the essential oils of Origanum compactum and of Aniba rosaeodora , preferably thymol, carvacrol and/or linalool.
Although the cytotoxic activity of an isolated constituent is less than that of a complete essential oil, the invention in particular concerns a composition comprising a constituent of the essential oil Origanum compactum or of Aniba rosaeodora , for a therapeutic use in the prevention or targeted treatment of keratoses. This constituent is preferably not an oxide, in particular not an oxide of linalool.
Preferably, the constituent is selected from 1,8-cineole, terpinolene, linalool, alpha-terpineol, geraniol, alpha-copaene, alpha and beta-selinene, benzyl benzoate, alpha-thujene, alpha pinene, myrcene, alpha-phellandrene, alpha terpinene, para-cymene, beta-phellandrene, gamma-terpinene, thymol, carvacrol and beta caryophyllene. More particularly preferably, the constituent is selected from linalool, thymol and carvacrol.
More particularly, the present invention concerns compositions comprising at least 10%, preferably at least 15% or at least 20% by weight of one of the following constituents: linalool, carvacrol and thymol. Other compositions that may be envisaged are those such that the cumulative linalool, carvacrol and thymol content is more than 10%, preferably more than 15%, or even more than 20% by weight. Alternatively, they may be chromatographic percentages.
Other constituents envisaged in the context of the present invention have already been described in the context of the other aspects of the invention. These particular embodiments are all also applicable to therapeutic applications.
In particular, as mentioned already, the present invention more specifically concerns compositions comprising an essential oil or one of its constituents, or said essential oil is extracted from plants of the Lamiaceae or Lauraceae family. The list of the various preferred genuses in the context of the invention in these families has already been given and is also applicable to this aspect of the invention.
More particular genuses for consideration in the context of the present invention are Origanum, Acinos, Agastache, Ajuga, Calamintha, Glechoma, Hyssopus, Lamium, Lavandula, Leonurus, Lycopus, Melissa, Melittis, Mentha, Micromeria, Monarda, Ocimum, Orthosiphon, Perilla, Perovskia, Phlomis, Physostegia, Pogostemon, Prostanthera, Prunella, Rosmarinus, Salvia, Satureja, Scutellaria, Sideritis, Solenostemon, Stachys, Teucrium and Thymus . The genus Origanum is more particularly preferred.
In particular, in accordance with a particular embodiment, the present invention concerns a composition comprising an essential oil or one of its constituents, preferably one of its major constituents, where said oil or said constituent is extracted from oregano, lavender, mint, rosemary, savory, sage, thyme, spotted deadnettle, black horehound, bugle weed, basil, hyssop, marjoram, deadnettle, ground ivy, wood betony, prunella, wild thyme or lemon balm.
In a preferred embodiment of the invention, the composition comprises an essential oil or one of its constituents, preferably a major constituent, which is extracted from the species Origanum compactum.
Alternatively, other genuses may also be considered in the context of the invention, namely the genuses Aniba, Cinnamomum, Laurus, Persea, Ravensara and Umbellularia. The genus Aniba is particularly preferred.
In particular, in a particular embodiment, the present invention concerns a composition comprising an essential oil or one of its constituents, preferably one of its major constituents, where said oil or said constituent is extracted from rosewood, cinnamon, laurel, horseradish ( Ravensara ), camphor or avocado.
In a preferred embodiment of the invention, the composition comprises an essential oil or one of its constituents, preferably major, which is extracted from the species Aniba rosaeodora.
As for the various applications already described here, the composition comprising the essential oil or one of its constituents is preferably used in combination with other compounds, in particular therapeutically acceptable excipients or vegetable oils. Particularly preferred additional compounds have already been described in respect of other aspects of the invention and are applicable to the therapeutic uses.
For the various envisaged therapeutic purposes of the present invention, topical application of the composition is also envisaged, as was the case for the purely cosmetic applications.
In a particularly preferred embodiment of the present invention, the keratoses are actinic keratoses.
The preferred range of concentrations of essential oil or of essential oil constituent are also those that provide targeted cytotoxicity while preserving the viability of healthy non-mutated cells, as was described for the other aspects of the invention.
The compositions of the present invention are preferably such that they may be used to induce preferential cytotoxicity in keratinocytes derived from or mimicking an actinic keratosis type lesion mutated at p53 compared with normal and/or non-hyperproliferative keratinocytes.



KEY TO FIGURES
FIG. 1 : FIG. 1 represents the measurement of cell viability (MTT test) on A431, HaCat and NHEK cells treated for 4 h with the EO of oregano. The double line ( ) corresponds to the 50% lethal dose (LD50);
FIG. 2 : FIG. 2 represents the measurement of cell viability (MTT test) on A431, HaCat and NHEK cells treated for 4 h with the EO of rosewood. The double line ( ) corresponds to the 50% lethal dose (LD50);
FIG. 3 : FIG. 3 represents the measurement of cell viability (MTT test) on A431, HaCat and NHEK cells treated for 20 h with the EO of oregano. The double line ( ) corresponds to the 50% lethal dose (LD50);
FIG. 4 : FIG. 4 represents the measurement of cell viability (MTT test) on A431, HaCat and NHEK cells treated for 20 h with the EO of rosewood. The double line ( ) corresponds to the 50% lethal dose (LD50);
FIG. 5 : FIG. 5 represents the measurement of apoptosis (AV/PI test) in A431, HaCat and NHEK cells after 12 h of treatment with the EOs of oregano and rosewood;
FIG. 6 : FIG. 6 represents the measurement of fluorescence (caspase activity) by flow cytometry. The non-tagged cells, the untreated tagged cells and the treated (by EO) and tagged cells are labeled;
FIG. 7 : FIG. 7 represents the fluorescence (caspase activity) by fluorescence microscopy;
FIG. 8 : FIG. 8 illustrates the quantification of the number of A431 and HEK001 cells (non-mutated keratinocytes) that have undergone a drop in the mitochondrial membrane potential after 6 h of treatment with the EOs of oregano and rosewood;
FIG. 9 : FIG. 9 illustrates the detection of oxidizing stress induced after incubation for 4 h with the OEs of oregano and rosewood in A431, HaCat and NHEK894 cells by measuring (flow cytometry) the fluorescence of the DHR 123 probe;
FIG. 10 : FIG. 10 represents the measurement of apoptosis (AV/PI test), in A431 and HEK001 cells (immortalized non-mutated keratinocytes) after 12 h of treatment with the following essential oil constituents: linalool, carvacrol and thymol.



EXPERIMENTAL SECTION
Example 1
Analytical Study of Essential Oils (EO) of Oregano and Rosewood
The choice of the EO of oregano for study was determined from the work of Dr Averbeck's laboratory, which showed an anti-genotoxic activity of this EO in yeast (Bakkali, F. et al. 2006). The EO of rosewood was selected in the light of its beneficial properties on the skin.
The present example concerns the characterization of the volatile portion of 2 commercially available bio-essential oils: an essential oil of rosewood and an essential oil of oregano.
The applied methodology involved GC-MS analysis on 2 columns with different polarities (apolar OV1 column and VF WAX polar column).
Apparatus and Methods
1. Apparatus
The analyses were carried out on Agilent instruments with 2 types of columns:
apolar capillary column: HP-1 capillary column, length: 50 m, internal diameter: 0.2 mm, film: 0.33 μm; polar capillary column: VF-WAX capillary column, length: 60 m, internal diameter: 0.25 mm, film: 0.25 μm.

It is important to emphasize that all of the percentages obtained are chromatographic percentages and not real percentages of the compounds present in the volatile phase. In order to quantify the constituents exactly, an internal standard had to be used in order to discount variations due to the instrumentation.
2. Identification of ingredients from a gas chromatograph on two columns with different polarities, coupled to a mass spectrometer detector (GC/MS).
Identification of the molecules was carried out with the aid of libraries of mass spectra specific to perfumes.
The sensitivity of the instrumentation meant that compounds with a relative percentage of 0.001% or more could be quantified.
The presentation of the results and the quantification was carried out in GC/FID on an apolar column; quantification of the co-eluted constituents was carried out on a polar column.
3. Samples Analyzed




EO of rosewood;
EO of oregano.




Results:
Essential Oil of Rosewood:
In light of the results, it appears that:
99.12% of the total area of the chromatogram could be identified with certainty; 62 molecules were identified and quantified; Table 1 of the composition (as chromatographic percentages) established for the essential oil of rosewood is recorded below:









TABLE 1






Composition


ROSEWOOD
%, GC











6 METHYL 5 HEPTENE 2
0.12


ONE


LINALOYL OXIDE =
0.33


LIMETOL


BETA PINENE
0.14


MYRCENE
0.05


HERBOXIDE (isomer) =
0.02


DESOXYDE


BENZYL ALCOHOL
0.01


ALPHA TERPINENE
0.02


PARA-CYMENE
0.08


LIMONENE
0.46


1,8 CINEOLE
0.57


CIS BETA OCYMENE
0.03


5 DIMETHYL 2,2
0.05


TETRAHYDROFURAN =


CITROXIDE


TRANS BETA OCIMENE
0.09


GAMMA TERPINENE
0.02


CIS LINALOOL OXIDE
1.9


TRANS LINALOOL OXIDE
1.28


TERPINOLENE
0.52


LINALOOL
78.93


HOTRIENOL
0.2


ALPHA FENCHOL
0.02


MYRCENOL
0.06


CIS OCIMENOL
0.14


NEROL OXIDE
0.03


TRANS OCIMENOL
0.16


DELTA CADINENE
0.28


TRANS NEROLIDOL
0.27


SPATHULENOL
0.11


CARYOPHYLLENE OXIDE
0.07


alcohol sesquit C15H24O
0.23


HP1: 1637


alcohol sesquit C15H24O
0.15


HP1: 1646


7 EPI ALPHA EUDESMOL
0.12


alcohol sesquit C15H24O
0.27


EPOXY LINALOOL 1 = CIS
0.15


LINALOOL OXIDE


(PYRANOID)


EPOXY LINALOOL 2 =
0.25


TRANS LINALOOL OXIDE


(PYRANOID)


BORNEOL
0.06


TERPINEN 4 OL
0.2


ALPHA TERPINEOL
4.52


GAMMA TERPINEOL
0.04


NEROL
0.38


NERAL
0.04


GERANIOL
1.47


GERANIAL
0.05


METHYL ANISOATE
0.02


GERANYL ACETATE
0.02


ALPHA COPAENE
1.15


BETA ELEMENE
0.13


ALPHA GURJUNENE
0.15


TRANS BETA
0.04


CARYOPHYLLENE


ALPHA GUAIENE
0.05


ALPHA HUMULENE
0.02


ALLO AROMADENDRENE
0.06


GAMMA MUUROLENE
0.04


SELINA 4,11 DIENE
0.14


BETA SELINENE
0.82


ALPHA SELINENE
0.77


GAMMA CADINENE
0.12


HP1: 1689


alcohol sesquit C15H24O
0.29


HP1: 1697


alcohol sesquit C15H24O
0.4


HP1: 1701


BENZYL BENZOATE
0.6


TOTAL (%)
99.12



































































































the major compound in the essential oil is linalool (78.93%);
the compounds present in the majority in the EO, after the linalool, are alpha terpineol (4.52%), linalool cis oxide (1.90%), geraniol (1.47%), trans linalool oxide (1.28%) and alpha copaene (1.15%);
the minor compound is benzyl alcohol (0.01%).




The percentages by weight of the major compounds of the essential oil of rosewood are as follows:
linalool: 78.93% alpha terpineol: 4.91%.

Essential Oil of Oregano:
In the light of the results, it appears that:
97.69% of the total area of the chromatogram could be identified with certainty; 20 molecules were identified and quantified; Table 2 of the composition (as chromatographic percentages) established for the essential oil of oregano is below:









TABLE 2






COMPOSITION


OREGANO
% GC











Alpha thujene
0.87


Alpha pinene
0.64


Camphene
0.12


1 octen-3-ol
0.23


Octanone 3
0.17


Myrcene
1.86


Alpha phellandrene
0.23


Alpha terpinene
1.47


Para-cymene
12.65


Beta phellandrene
0.56


Trans ocimene
0.07


Gamma terpinene
15.45


Sabinene trans hydrate
0.19


Linalool
1.66


Teipinen-4-ol
0.61


Thymol
20.23


Carvacrol
38.61


Beta caryophyllene
1.88


Alpha humulene
0.1


Caryophyllene oxide
0.09


TOTAL (%)
97.69













































the major compounds of the EO are carvacrol (38.61%), thymol (20.23%), gamma terpinene (15.45%) and para-cymene (12.65%);
the minor compound is trans ocimene (0.07%);
there are 12 molecules common to the EO of oregano and the EO of rosewood: alpha pinene, camphene, myrcene, alpha terpinene, para-cymene, trans ocimene, gamma terpinene, linalool, terpineno-4-ol, beta caryophyllene, alpha humulene and caryophyllene oxide. These compounds are principally terpenes.




The percentages by weight of the major compounds of the essential oil of oregano were as follows:










para-cymene: 12.27%


gamma terpinene: 16.10%,


thymol: 18.65%


carvacrol: 31.83%.


















Conclusions:
The precise chemical composition of each EO was determined by gas chromatographic analysis (see Tables 1 and 2). Observation of the chemical composition of each EO revealed a very substantial qualitative difference if the number of major compounds was considered. Although the essential oil of rosewood has a larger number of different chemical molecules (approximately 62), it has one compound that is very much in the majority: linalool, more than 78% in the analyzed fraction. In contrast, the EO of oregano, with its 20 detected molecules, possessed four major molecules: carvacrol (38%), thymol (20%), gamma terpinene (15%) and para-cymene (12%). To this end, the EO of rosewood is termed “mono-molecular” and the essential oil of oregano is termed “poly-molecular”. It should also be noted that the EO of oregano is principally composed of mono-phenol (carvacrol and thymol), while the EO of rosewood is primarily constituted by terpenic alcohol (linalool).
Example 2
In the present work, the cytotoxicity of two essential oils was studied in particular: the essential oil of compact oregano ( Origanum compactum , Lamiaceae family) and the essential oil of rosewood ( Aniba rosaeodora , Lauraceae family) on human epidermal cells: the HaCat keratinocyte line (mutated at p53 and spontaneously immortalized), the A431 line (from spino-cellular carcinoma) as well as on normal human primary epidermal keratinocytes.
These three models were used to evaluate the potential biological effects of these two essential oils on normal epidermal cells mutated at p53 (that could be likened to pre-cancerous) and cancerous, the whole representing, to a certain extent, the various situations between healthy skin and skin with a risk of non-melanoma cancer and/or skin from keratosis.
Measurement of Cell Viability:
The inventors compared the cytotoxic effect of the essential oils on normal, pre-cancerous mutated (HaCat) and mutated cancerous (A431) keratinocytes.
Cell viability was determined by the MTT test after a 4 h and 20 h treatment with different concentrations of EO. This test (MTT) was based on the mitochondrial enzyme activity of dehydrogenase succinate. Each viability point is represented by three independent experiments (NHEK: normal human epidermal keratinocyte).
FIGS. 1 and 2 indicate that:
the EO of oregano is more toxic than the EO of rosewood, independent of time (comparison between 4 h and 20 h of treatment) and of the cell line used; normal cells (NHEK) are more resistant than cancer cells (A431) and pre-cancerous mutated cells (HaCat) after 4 h of treatment; the cytotoxicity profile is identical for the A431 and HaCat cells after 4 h of treatment (with the essential oils of oregano and rosewood).

FIGS. 3 and 4 confirm that this difference in toxicity between the A431/HaCat cells and the NHEK cells was conserved after 20 h of treatment, although at this time a difference in sensitivity was observed between the HaCat and A431 cells.
These results demonstrate a targeted toxicity of the OEs for cancer cells and mutated pre-cancerous cells compared with normal cells at concentrations of 150 nL/mL and 400 nL/mL of the OE of oregano and of rosewood respectively.
Detection of Apoptosis:
Apoptosis is defined as the induction of characteristic biochemical events leading to programmed cell death. These characteristic events are, for example, translocation of a particular membrane phospholipid (phosphatidylserine) of the internal plasma membrane, or activation of certain proteases (caspases) involved in the protein degradation process, leading to cell death.
The cell viability results brought to light a concentration for each EO where a difference is revealed in the sensitivity of cancer and mutated pre-cancerous cells compared with normal cells: 150 nL/mL for the EO of oregano and 400 nL/mL for the EO of rosewood. These concentrations will be used in the context of a 16 h treatment (4 h and 12 h treatments were also earned out).
FIG. 5 indicates that after 12 h of treatment:
there was a significantly higher viability for normal cells (NHEK: viability>70%) compared with the A431 cancer line and HaCat pre-cancerous mutated line (viability<15% in A431 and HaCat lines); there was a substantial induction of apoptosis in the A431 cancer line (more than 60% of positive annexin V cells); there was late apoptosis or necrosis in the mutated pre-cancerous HaCat cells.

These results confirm the higher sensitivity to the toxicity of the essential oils for the mutated pre-cancerous HaCat and cancerous A431 cells compared with the normal NHEK cells. They also bring to light an induction of cell death by apoptosis.
In order to confirm the induction of cell death by apoptosis by the essential oils, a second test based on the detection of the activity of caspases, a family of proteins specific to the apoptosis mechanism, was carried out on cells treated with the essential oils. Caspase activation is an earlier phenomenon than translocation of phosphatidylserine detected previously by annexin V. The principle of the test is based on the use of a substrate for these enzymes coupled to a fluorochrome. Cells in apoptosis, where the caspases are activated, will degrade the substrate and emit fluorescence. Non-apoptotic cells do not emit fluorescence. The fluorescence was measured by flow cytometry and fluorescence microscopy after 4 h of treatment with the essential oils.
FIG. 6 shows up a displacement towards the right (and thus an increase in the fluorescence intensity) of the curve representing the cells treated with the essential oils compared with the curve representing the tagged but untreated cells in the A431 and HaCat cells only. This increase in fluorescence, which was confirmed by fluorescence microscopy ( FIG. 7 ), indicates caspase activation and confirms the following:
induction of apoptosis in the cancer cells and HaCat cells by both essential oils, at the concentrations used; that this induction is observed only in A431 and HaCat cells; the normal cells are thus more resistant than the cancer cells and pre-cancerous mutated cells for the same treatment with the essential oils of oregano and of rosewoods.

Effect of Essential Oils on Mitochondria
After treatment with the essential oils of oregano and of rosewood, an analysis of the integrity of the mitochondrial membrane of the cancerous mutated A431 keratinocytes and non-mutated keratinocytes was carried out by flow cytometry (monitoring fluorescence linked to the accumulation of TMRM in the integrated mitochondrial membrane). It can be seen in FIG. 8 that in the selected concentration range, there was indeed a destabilizing effect on the mitochondria linked to treatment by the essential oils, but this effect was particularly marked in the A431 cells compared with the non-mutated keratinocytes. This impact on the mitochondria is very probably associated with the development of the apoptotic process (cause and/or consequence).
Intracellular Generation of Reactive Oxygen Species Following Treatment with Essential Oils
One of the consequences of mitochondrial destabilization is the release of pro-oxidizing molecules into the cytosol; this phenomenon can be detected in flow cytometry by the DHR123 probe, which fluoresces in the case of an oxidizing stress. FIG. 9 shows the change in the number of fluorescent cells (which are thus subject to the generation of reactive oxygen species that can be detected by the DHR123) after various treatments with the essential oils. It will be seen that at the concentrations used, the normal keratinocytes were not affected, while a dose-dependent increase in the intracellular pro-oxidizing state could be detected in the mutated HaCat or A431 cells. Thus, the essential oils are capable of producing a significant and specific oxidizing stress in pre-cancerous or cancerous mutated keratinocytes, that stress probably being strongly implicated in the targeted cytotoxicity described in FIGS. 1 to 4 .
Conclusions
The data presented here demonstrates a sensitivity to the cytotoxicity of the essential oils of oregano and of rosewood that is significantly higher in cancerous and mutated pre-cancerous human keratinocytes compared with normal keratinocytes.
The analysis of the mechanisms occurring has shown that this difference in sensitivity can be explained by the induction of death by apoptosis in the cancerous and mutated pre-cancerous cells, while the normal cells had cell viabilities of more than 70% for the same concentration of essential oil.
The molecular analysis shows that this specific toxicity by the essential oils involves modifications to mitochondrial integrity and the generation of an endogenic oxidizing stress. Thus, it can be envisaged that mutated pre-cancerous keratinocytes (in particular in keratoses or in zones of the skin damaged by UV) can be targeted by the essential oils and thus can specifically eliminate them.
Example 3
Measurement of the Pro-Apoptotic Activity of the Major Isolated Compounds of the Essential Oils of Oregano and Rosewood
The essential oil of rosewood is composed of a primary molecule (more than 80%): linalool (terpene alcohol). The EO of compact oregano contains two primary compounds (monoterpenol): carvacrol and thymol, present in amounts of 40% and 20% respectively.









These three molecules were incubated for 12 h with non-mutated immortalized keratinocytes (HEK001) and mutated cancerous keratinocytes (A431). The percentage of cells in apoptosis was measured by the annexin V/propidium iodide test. The concentrations of the molecules used were equal to those present during the treatments with the essential oils. Example: the concentration of linalool used here represents the equivalent of 80% of linalool present in the essential oil of rosewood. In fact, the linalool was used in a concentration of 320 nL/mL (see FIG. 10 ), i.e. 80% of 400 nL/mL of essential oil of rosewood, which is the concentration shown to be the most toxic for HaCat and A431 cells, while leaving a maximum of viable NHEK and HEK001 cells (see Example 2, conclusions on the section “Measurement of cell viability” and FIGS. 3 and 4 as well as the section “Detection of apoptosis” and FIG. 5 ). This was the same for the carvacrol and thymol of the essential oil of oregano.
The apoptosis results are presented in FIG. 10 .
The results of this experiment, expressed as the % of cells—living, in apoptosis or in late apoptosis and necrosis, show the following:
lower cytotoxicity and pro-apoptotic cytotoxicity of the major molecules isolated compared with that induced by the essential oils in their complexity (70% apoptosis induced in cancer cells at the equivalent dose of the essential oil of oregano, i.e. 150 nL/mL). In fact, FIG. 5 highlights, for example, that more than 60% of the A431 cells are apoptotic after a treatment with essential oil of oregano in a concentration of 150 nL/mL. FIG. 10 , in contrast, shows that approximately only 25% of the A431 cells were apoptotic after 12 h of treatment with carvacrol in a concentration of 60 nL/mL (corresponding to 40% of 150 nL/mL) and approximately 15% of apoptotic cells with a treatment with 30 nL/mL thymol (corresponding to 20% of 150 nL/mL); the viability of the non-mutated cells (HEK001) was higher than that observed in the mutated cells (A431). Preferential targeting of mutated cells was thus conserved.

This experiment shows that death by targeted apoptosis of mutated cancer cells is qualitatively conserved if the principal compounds of rosewood (linalool) or oregano (carvacrol and thymol) are used. In contrast, the cytotoxic efficacy is lower than with complex essential oils. Thus, synergistic effects with the other constituents probably exist.
This point is confirmed by the observation by the inventors that the mixture of several molecules isolated from the same essential oil reproduces the activity observed with the essential oil, the activity being higher than with simple addition of the activities of the isolated molecules taken separately.
Apparatus and Methods for Examples 2 and 3
Thirty six hours before the treatment, the cells were distributed into a 96-well plate (5×10 5 cells/mL).
EO treatment solution: the essential oils were diluted to 1/10 th a first time in 100% ethanol. Next, a second dilution was carried out in culture medium in order to obtain the desired concentrations.
The viability measurement was carried out using the MTT test (1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan, Sigma-Aldrich). Briefly, after the desired treatment time, the treatment medium was replaced by a medium containing the MTT solution (0.25 μg/mL final concentration). The 96-well plate containing the cells was then incubated for 4 h at 37° C. before eliminating the MTT solution and adding 100 μl of DMSO. The absorbance measurement was then carried out at 540 nm after homogenizing the violet precipitate.
The measurement of the oxidizing stress was carried out using a DHR 123 fluorescent probe (Molecular Probe). After 4 h of treatment with the essential oils, the medium was eliminated and the cells were harvested (by trypsinization), centrifuged then rinsed in 4 mL of PBS 1X. The cells were then incubated for 30 min (27° C.) in culture medium containing the probe (5 μM). Before measuring the fluorescence by cytometry (excitation: 488 nm/emission: 530 nm), the cells were rinsed in 4 mL of PBS 1X, centrifuged and taken up in 0.5 mL of PBS.
Detection of Apoptosis:
Annexin V/Propidium Iodide Test:
One of the markers for apoptosis is the translocation of phosphatidyl-serine from the internal face towards the external face of the plasma membrane (translocation, allowing apoptotic bodies to be recognized and phagocyted by the macrophages).
The AV/PI test can be used to detect this event because of the affinity of the protein annexin V for phosphatidyl-serine. Propidium iodide, which targets DNA, can be used to distinguish early apoptotic cells from cells in late apoptosis and/or in necrosis.
After the desired treatment time, the medium was eliminated, the cells were harvested (by trypsinization), centrifuged and rinsed in 4 mL of PBS 1X. Tagging was carried out following the instructions of the manufacturer (Kit Vybrant, Molecular Probe). The cells were rapidly taken up in 100 μl of tagging buffer, to which 6 μl of annexin V and 3 μg/mL of propidium iodide per sample was added. The cells were then incubated for 30 min in the dark (at ambient temperature). Four hundred μL of tagging buffer was added before measuring the fluorescence of the annexin V (at 530 nm) and of the propidium iodide (at 610 nm).
Caspase Activity:
Caspase activity is a second marker of apoptosis. The caspase family encompasses the proteins principally involved in regulating cell death by apoptosis. The caspase activity was detected using a VAD (valine-alanine-aspartic acid) substrate coupled to a fluorochrome (FITC. Emission: 530 nm). Detection of this activity was carried out by following the instructions with the CasPACE™ kit (Promega). After treatment, the cells were harvested (by trypsinization), centrifuged and rinsed in 4 mL of PBS 1X. Next, the cells were incubated for 20 min in a culture medium containing the reagent (10 μM), 20 min in the dark and at ambient temperature. The cells were then rinsed twice in 4 mL of PBS 1X before being fixed for 30 min in a formalin solution. Before measuring the fluorescence by flow cytometry, the cells were rinsed (3 times 5 min) in PBS 1X then taken up in 0.5 mL of PBS.
Mitochondrial Permeabilization:
Permeabilization of mitochondrial pores is also an early marker of apoptosis. A fluorescent probe (TMRM: tetramethyl rhodamine methyl ester; Molecular Probe) becomes localized in the mitochondria, and will reduce in intensity when it migrates in the cytoplasm following opening of the mitochondrial pores. A fall in fluorescence is thus measured by flow cytometry.
After 4 h of treatment, the cells were trypsinated and rinsed in PBS 1X before they were incubated for 30 min at 37° C. in a culture medium containing the fluorescent probe (50 nM final). After this incubation, the cells were rinsed once for 5 min in PBS 1X then taken up in 0.5 mL of PBS before the cytometric measurement.
Example 4
Range of Essential Oil Concentration: Targeted Cytotoxicity
The ranges of concentrations of essential oil (as % of essential oil per volume of culture medium) for which the cytotoxicity property was observed to be preferentially induced in the mutated cells (HaCat and A431) compared with non-mutated cells (HEK001 and NHEK894) are:
from 0.0125% to 0.0175% for the essential oil of oregano;
from 0.035% to 0.045% for the essential oil of rosewood.
In these concentration ranges, the degrees of toxicity are twice as high in the mutated cells than in the non-mutated cells where the viability remains of the order of 60%.
Further, it should be pointed out that the studies have shown good skin penetration of the principal ingredients of the essential oils of rosewood and of oregano.
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