Combination of myrtle extract and tripterygium extract for controlling inflammation caused by propionibacterium acnes

Abstract

The present invention relates to a combination comprising an extract of Myrtus communis and an extract of Tripterygium wilfordii, in particular for the treatment of inflammation caused by Propionibacterium acnes and for the treatment of acneic skin.

Description

Technical Field

[0001] This invention relates to a novel combination comprising myrtle extract and Tripterygium wilfordii extract, and compositions comprising the combination, particularly for use in the field of acne, especially in the treatment of inflammation caused by Propionibacterium acnes. Background Technology

[0002] Acne is a common, multifactorial skin disease of the hair follicles and sebaceous glands that causes the formation of comedones and pimples, affecting the face, shoulder area, arms, and chafed areas. It is the most common and leading cause of skin diseases. It is important not to underestimate this disease and to treat it properly, as it can have disabling psychosocial outcomes, particularly due to scarring.

[0003] There are many forms of acne, and a common factor in all of them is the attack on the sebaceous gland follicles. These include, for example, acne vulgaris, acne conglobata, keloid acne of the neck, drug-induced acne, recurrent folliculitis, necrotizing acne, neonatal acne, premenstrual acne, occupational acne, senile acne, and solar acne.

[0004] Acne vulgaris, also known as common blackheads or whiteheads, is the most common type of acne and consists of four stages:

[0005] - Stage 1 corresponds to comedo-type acne, characterized by a large number of open and / or closed comedones and microcysts;

[0006] Stage 2, or papulopustular acne, is a mild to moderate severity characterized by the presence of open and / or closed comedones, microcysts, and red papules and pustules. It primarily affects the face and leaves some scarring;

[0007] Stage 3, or papular comedonal acne, is more severe and extends to the back, chest, and shoulders. It is accompanied by extensive scarring.

[0008] Stage 4 or nodular cystic acne is characterized by extensive scarring. It manifests as nodules and large, painful, purple pustules.

[0009] Acne affects almost everyone in its mildest form. Its frequency is highest during adolescence, but it can first appear as early as 7-9 years old and even after age 40. Acne can still occur frequently after age 25. Furthermore, acne affects both men and women equally. During adolescence, influenced by hormone secretion, particularly androgens, and various external factors, excessive sebum production, known as hyperspermia, is observed. In acne-prone subjects, this environment favors the growth of the dominant acne bacterium, *Propionibacterium acnes* (C. acnes) (formerly known as *Propionibacterium acnes*). This bacterium metabolizes skin triglycerides into irritating fatty acids by attacking lipases in the hair follicle wall and surrounding dermis. It also produces various enzymes and chemical inducers for immune phagocytes, and stimulates various cell types (especially sebaceous cells, keratinocytes, and monocytes) to produce pro-inflammatory cytokines, which exacerbate inflammation. Combating this bacterial species has long been considered a key focus in acne treatment. Various topical antibacterial agents are still widely used today (benzoyl peroxide, erythromycin, triclosan). However, researchers have recently learned that the goal should not be to eradicate *Propionibacterium acnes* as a symbiotic bacterium (which is essential for tissue homeostasis), but rather to re-establish the balance, because acne is associated with the loss of phylogenetic diversity of *Propionibacterium acnes*, with the pathogenic phylogenetic type IA1 becoming dominant. Contrary to long-held beliefs, acne is not associated with the proliferation of *Propionibacterium acnes*, but rather with changes in the proportion of phylogenetic types and the loss of richness of these phylogenetic types.

[0010] Similarly, depending on the severity of the condition, long-term systemic antibiotic therapy (tetracycline, doxycycline) is sometimes combined with treatment. Dermatologists are now increasingly turning to topical anti-inflammatory drugs and sebum regulators.

[0011] All these treatments have been observed to fail frequently, usually due to a high proportion of drug-resistant strains of *Propionibacterium acnes*. This resistance may be a result of the bacterial population organizing into biofilms. Biofilms are bacterial cell communities contained in the extracellular matrix secreted by microorganisms, composed of sugar polymers and called glycocalyx. Attached bacteria (associated with biofilms) differ phenotypedly and physiologically from planktonic (free) bacteria. Studies have confirmed *Propionibacterium acnes*' ability to form biofilms as well as in vitro (Holmberg et al., Clin. Microbiol. Infect. 2009, 15, 787-795) and on medical devices (Craig et al., J. Am. Acad. Dermatol. 2007, 722-724).

[0012] This bacterium plays a crucial role in acne, particularly by stimulating local inflammatory responses (Dagnelie et al., Journal of the European Academy of Dermatology 2019, 33(12), 2340-2348). Contrary to long-held belief, *Propionibacterium acnes* does not proliferate in the sebaceous follicles of acne-prone skin, but a loss of richness / diversity of its different phylogenetic types was observed, with phylogenetic type IA1 being highly dominant. This phylogenetic type is propathogène and has a strong ability to organize into biofilms, which enhance the bacterium's virulence. This virulence can be measured by quantifying the virulence factors produced by *Propionibacterium acnes*.

[0013] Propionibacterium acnes IA1 stimulates a local inflammatory response by acting directly on sebaceous follicle cells, particularly keratinocytes, sebaceous cells, and immune cells (monocytes). It exerts its effect by stimulating these cells to produce pro-inflammatory cytokines, especially interleukins 6 and 8. This is followed by an immune-inflammatory cascade, specifically the recruitment and differentiation of primitive CD4+ T lymphocytes into LTH17. Recent pathological studies in acne have demonstrated that these LTH17 cells specifically produce interleukin-17 from the subclinical stage of acne to the most inflammatory lesions.

[0014] Therefore, there remains a need for more effective acne treatments that do not have adverse side effects on patients. In particular, there are currently no agents that directly target the biofilm of Propionibacterium acnes and the inflammatory response it generates. Summary of the Invention

[0015] The object of this invention is to respond to these needs. Indeed, the inventors have unexpectedly demonstrated that the combination of myrtle extract and Tripterygium wilfordii extract has the ability to reduce the production of inflammatory cytokines, inflammation stimulated by the presence of Propionibacterium acnes. More specifically, the combination according to the invention has the advantage of synergistically acting on the immune inflammatory cascade mediated by Propionibacterium acnes, and therefore can be used to treat acne.

[0016] Therefore, the object of the present invention is a combination comprising myrtle extract and Tripterygium wilfordii extract.

[0017] Another object of the present invention is a cosmetic or dermatological composition comprising: a combination of myrtle extract and tripterygium extract; and at least one excipient acceptable in cosmetic or dermatological terms.

[0018] Another object of the present invention is the use of the combination according to the invention, in other words, a combination comprising myrtle extract and tripterygium extract, for the treatment of inflammation caused by Propionibacterium acnes.

[0019] The present invention also relates to the use of the combination according to the invention in the preparation of cosmetic or dermatological compositions for treating inflammation caused by Propionibacterium acnes.

[0020] The present invention also relates to the use of the combination according to the invention in the treatment of inflammation caused by Propionibacterium acnes.

[0021] The present invention also relates to a method for treating inflammation caused by Propionibacterium acnes, comprising administering an effective amount of the combination according to the invention to a person in need.

[0022] Another object of the present invention is the use of the combination according to the invention for treating acne or acne-prone skin.

[0023] The present invention also relates to the use of the combinations according to the invention in the preparation of cosmetic or dermatological compositions for treating acne or acne-prone skin.

[0024] The present invention also relates to the use of the combination according to the invention in the treatment of acne or acne-prone skin.

[0025] The present invention also relates to a method for treating acne or acne-prone skin, comprising administering an effective amount of the combination according to the invention to a person in need.

[0026] Another object of the present invention is the use of a cosmetic composition or dermatological composition according to the present invention, in other words, a cosmetic composition or dermatological composition comprising a combination of myrtle extract and tripterygium extract and at least one cosmetically or dermatologically acceptable excipient for the treatment of inflammation caused by Propionibacterium acnes.

[0027] The present invention also relates to the use of cosmetic or dermatological compositions according to the invention in the preparation of medicaments intended for the treatment of inflammation caused by Propionibacterium acnes.

[0028] The present invention also relates to the use of cosmetic or dermatological compositions according to the invention in the treatment of inflammation caused by Propionibacterium acnes.

[0029] The present invention also relates to a method for treating inflammation caused by Propionibacterium acnes, comprising administering an effective amount of the cosmetic composition or dermatological composition according to the invention to a person in need.

[0030] Another object of the present invention is the use of the cosmetic or dermatological compositions according to the invention for treating acne or acne-prone skin.

[0031] The present invention also relates to the use of the cosmetic or dermatological compositions according to the invention in the preparation of medicaments for treating acne or acne-prone skin.

[0032] The present invention also relates to the use of cosmetic or dermatological compositions according to the invention in the treatment of acne or acne-prone skin.

[0033] The present invention also relates to a method for treating acne or acne-prone skin, comprising administering an effective amount of the cosmetic composition or dermatological composition according to the invention to a person in need.

[0034] Detailed description of the invention

[0035] According to a first aspect, the present invention relates to a combination comprising myrtle extract and Tripterygium wilfordii extract.

[0036] Myrtle extract

[0037] Myrtus communis L., a common plant in the Myrtaceae family, is a shrub. The myrtus extract according to the invention is more particularly derived from the leaves of Myrtus communis. It preferably involves the nonpolar fractions of the aerial parts of Myrtus communis, and more particularly the nonpolar fractions of the leaves.

[0038] The term "nonpolar fraction" should refer to fractions of nonpolar extracts, typically fractions of nonpolar extracts that have undergone activated carbon treatment to remove chlorophyll.

[0039] The term "nonpolar extract" should be understood to mean an extract that can be obtained using a nonpolar extraction solvent such as ethyl acetate, isopropyl acetate, or a mixture thereof.

[0040] According to the present invention, the term "above-ground part" should mean the part of a plant that is above ground, such as leaves, stems, petioles and / or flowers, especially leaves.

[0041] The extract according to the invention can be obtained by extraction using a solvent or solvent mixture selected from the following (referred to as the extraction solvent) or by extraction using supercritical CO2:

[0042] - Alcohols, such as ethanol, methanol, and isopropanol.

[0043] - Ketones, including acetone and methyl ethyl ketone.

[0044] -hexane,

[0045] -Dichloromethane

[0046] - Isopropyl ether,

[0047] - Ethyl acetate or isopropyl acetate

[0048] - and their mixtures.

[0049] According to one embodiment, myrtle extract can be obtained by extraction using isopropyl acetate, preferably from the aerial parts of myrtle, especially the leaves.

[0050] Therefore, the plant (or any part thereof) is brought into contact with the extraction solvent. After extraction, the plant / extraction solvent mixture is filtered to separate the solvent phase from the plant residue (called the pomace). The resulting pomace is typically washed with the same solvent as the extraction solvent, and the resulting wash solvent is mixed with the solvent phase to obtain the extract.

[0051] Advantageously, the extract obtained after filtration and rinsing of the residue is bleached by adding activated carbon, which removes chlorophyll.

[0052] The extract can also be stabilized by adding antioxidants such as butylated hydroxytoluene or α-tocopherol, in particular in amounts of 0.05% to 1% by weight of the dry extract.

[0053] The extract according to the invention is particularly characterized by containing, and especially being rich in, myrtucommulone and ursolic acid. Myrtucommulone A, B', D, B, isos (isosemimyrtucommulone) and S (semimyrtucommulone) are advantageously present in the extract, and particularly are myrtucommulone present as the predominant component.

[0054] Advantageously, the total content of myristic phloroglucinol in the extract is 3% to 10% by weight of the dry extract.

[0055] The content of ursolic acid, based on the weight of the dry extract, is 10% to 30%, preferably ≥15%.

[0056] These molecules possess, at least in part, the activity required in the context of this invention.

[0057] In a particular embodiment of the invention, the myrtle extract is preferably as described in patent application EP 1 112 079 (the teachings of which are incorporated herein by reference) or as described in Example 1 of this application. EP 1 112 079 describes the antimicrobial properties of myrtle extract and its use in cosmetic and dermatological compositions.

[0058] The myrtle extract used in this invention is also the subject of patent FR 2 992 862, which describes the anti-biofilm activity of the myrtle extract against Propionibacterium acnes.

[0059] Tripterygium wilfordii extract

[0060] Tripterygium wilfordii is a medicinal plant belonging to the Celastraceae family. Terpenes are among the most active components of this plant, primarily located in its roots. These terpenes specifically include pentacyclic triterpenes, such as Tingénine A (also known as Tingénone or Mayténine), Tingénine B (also known as 22-β-hydroxy-Tingénone), Célastrol, Pristimérine, and Tripterygone. These molecules are described in patent application EP 3 454 875 (the teachings of which are incorporated herein by reference).

[0061] In one embodiment, the Tripterygium wilfordii extract may be an extract containing, in particular, pentacyclic triterpenes such as euonymus triterpenoid A, euonymus triterpenoid B, and / or triptolide. The extract may optionally contain plant-derived components (including active components) other than pentacyclic triterpenes.

[0062] According to the present invention, the term "pentacyclic triterpenoid" should mean pentacyclic triterpenoids naturally produced by the cells of the plant Tripterygium wilfordii, particularly triptolide of formula I, euonymus triterpenoid A (also known as euonymus ketone or maytansine) of formula II, euonymus triterpenoid B (also known as 22-β-hydroxyeuonymus ketone) of formula III, euonymus ketone of formula IV and / or triptolide of formula V, preferably triptolide, euonymus triterpenoid A and / or euonymus triterpenoid B, especially triptolide.

[0063] [Chemical Formula I]

[0064]

[0065] [Chemical Formula II]

[0066]

[0067] [Chemical Formula III]

[0068]

[0069] [Chemical Formula IV]

[0070]

[0071] [Chemical Formula V]

[0072]

[0073] The term "crude extract" should refer to extracts obtained directly from the plant (in this case, Tripterygium wilfordii).

[0074] The term “enriched extract” of Tripterygium wilfordii should mean an extract in which the amount of pentacyclic triterpenes, particularly euonymus triterpenes A, euonymus triterpenes B and / or triptolide, is greater than 30% by weight, and particularly greater than 50% by weight, relative to the amount of pentacyclic triterpenes in the dry crude extract.

[0075] In one embodiment, the Tripterygium wilfordii extract, particularly the enriched extract, used according to the invention comprises 90% to 100% by weight of pentacyclic triterpenes relative to the total weight of the dry extract, particularly the enriched dry extract.

[0076] Extracts, especially crude or enriched extracts, can be obtained from any part of the Tripterygium wilfordii plant, particularly the roots, seeds, or aerial parts.

[0077] Alternatively, Tripterygium wilfordii extract can be obtained from plant cell cultures of these plants. In this case, the extract can be obtained specifically from the supernatant, suspension, or biomass of the cell culture, as particularly described in Plant Cell Tiss Organ Cult by Coppede et al. (2017, 118, 33-43).

[0078] In a preferred embodiment, the Tripterygium wilfordii extract is an extract containing, and particularly rich in, pentacyclic triterpenes, which can be obtained by the following methods:

[0079] (i) The proliferation stage of Tripterygium wilfordii cells in proliferation medium.

[0080] (ii) An induction phase is achieved by adding an induction mixture to the cell culture obtained in step (i), the induction mixture comprising at least one monocarboxylic acid compound inducer and at least one biological inducer, and

[0081] (iii) Prepare an extract from the cell culture obtained in step (ii), the extract containing, in particular, pentacyclic triterpenes.

[0082] According to the present invention, the term "Tripterygium wilfordii cell" should refer to the cell of any part of the plant: seed, root, above-ground part, especially above-ground part, and even more so to leaf.

[0083] According to the present invention, the term "proliferation phase of Tripterygium wilfordii cells" refers to the stage in which Tripterygium wilfordii cells are suspended in a proliferation medium under conditions suitable for their proliferation. These cells are specifically obtained from callus tissue (cals) before suspension. If necessary, the cell suspension can be periodically re-inoculated to maintain proliferation conditions.

[0084] According to the present invention, the term "callus" should refer to a cluster of undifferentiated cells (also known as stem cells or meristematic cells).

[0085] In this specification, the term "about" shall mean that the relevant value may be 10% smaller or larger than the value shown, particularly 5%, and especially 1%.

[0086] Callus can be induced by any method known to those skilled in the art. Callus according to the invention can be obtained, in particular, in the manner described below. Induction of callus from explants derived from plant parts, especially aerial parts, such as leaves of *Tripterygium wilfordii*, is well known to those skilled in the art. Callus induction can be particularly carried out by the following methods:

[0087] - Obtain explants of plant tissue, for example, approximately 1 cm in size. 2 A leaf,

[0088] - Culture explants in agar proliferation medium (e.g., by adding 4 to 12 g / L of agar, such as about 8 g / L of agar, to the proliferation medium according to the invention).

[0089] - Especially in the dark, at a temperature of about 25 to 30°C, for example, about 27 to 28°C.

[0090] Step (i): Cell proliferation stage

[0091] Those skilled in the art familiar with cell cultures of plants such as Tripterygium wilfordii can readily determine the composition of the proliferation medium required for its proliferation. Preferably, this proliferation medium enables cells to proliferate in an undifferentiated form, in other words, in a totipotent form. Maintaining the undifferentiated form can be achieved, in particular, by using a specific cytokinin / auxin ratio in the proliferation medium. The proliferation medium may specifically comprise:

[0092] - At least one macroelement, particularly selected from NH4NO3, KNO3, CaCl2·2H2O, MgSO4·7H2O, KH2PO4 and mixtures thereof, having a total macroelement concentration of, for example, 1000 to 9000 mg / L of growth medium, for example, 3000 to 8000 mg / L of growth medium; during the growth phase, the total macroelement concentration of the growth medium is particularly 3000 to 5500 mg / L of growth medium;

[0093] - At least one trace element, particularly selected from KI, H3BO3, MnSO4·4H2O, ZnSO4·H2O, Na2MoO4·2H2O, CuSO4·5H2O, CoCl2·6H2O, FeSO4·7H2O, Na2EDTA·2H2O, and mixtures thereof, wherein the total trace element concentration is, for example, 10 to 200 mg / L of growth medium, particularly 50 to 150 mg / L of growth medium;

[0094] - At least one vitamin, particularly selected from inositol, niacin, pyridoxine hydrochloride, thiamine hydrochloride and mixtures thereof, wherein the total vitamin concentration may be in the range of 0.01 to 3 g / L of growth medium, particularly 0.05 to 1 g / L of growth medium;

[0095] - At least one amino acid, particularly glycine, whose total amino acid concentration may, for example, be in the range of 0.15 to 5 mg / L of proliferation medium, particularly 1 to 4 mg / L of proliferation medium; during the proliferation phase, the amino acid concentration of the proliferation medium is particularly 1 to 2.5 mg / L of proliferation medium;

[0096] - At least one carbon source, particularly sucrose, with a total carbon source concentration of, for example, 10 to 70 g / L of growth medium, for example, about 30 g / L;

[0097] - At least one plant hormone (also known as plant growth hormone, plant growth factor, or plant growth regulator), particularly selected from one or more cytokinins (especially kinetin and / or 6-furfurylaminopurine), one or more auxins (especially 2,4-dichlorophenoxyacetic acid (2,4-D) and / or naphthaleneacetic acid (NAA)), and mixtures thereof. During the proliferation phase, the proliferation medium particularly contains at least one cytokinin and at least one auxin. The plant growth hormone is added to the proliferation medium, particularly at a concentration and proportion that enables cells to proliferate in an undifferentiated form. The plant growth hormone is particularly selected from kinetin, 6-furfurylaminopurine, 2,4-D acid, NAA acid, and mixtures thereof; especially from kinetin, 2,4-D acid, NAA acid, and mixtures thereof. It may particularly contain a mixture of kinetin, 2,4-D acid, and NAA acid.

[0098] The concentration of auxin is specifically 0.001 to 10 mg / L of proliferation medium, for example 0.1 to 3 mg / L of proliferation medium.

[0099] The concentration of cytokinin is particularly 0.001 to 0.5 mg / L of proliferation medium, for example 0.05 to 0.15 mg / L of proliferation medium.

[0100] In one embodiment, the auxin / cytokinin hormone ratio is 0.2 to 2.5 / 0.01 to 0.5, particularly 1 to 2 / 0.05 to 0.2, especially about 1.5 / 0.1.

[0101] The proliferation medium according to the invention specifically contains 1.5 mg / L of auxin (especially 2,4-D acid and NAA acid) and 0.1 mg / L of cytokinin (especially kinetin).

[0102] The proliferation medium is advantageously sterile, and its pH is preferably close to neutral.

[0103] Examples of proliferation media suitable for cell proliferation from Tripterygium wilfordii plants according to the present invention are particularly described by Murashige & Skoog (Physiologia Plantarum, 1962, 15: 473-497) or according to Example 2 of this application.

[0104] For example, such a proliferation medium may have the following composition (concentrations expressed relative to the volume of cell-free proliferation medium): macroelements: 1650 mg / L NH4NO3, 1900 mg / L KNO3, 440 mg / L CaCl2·2H2O, 370 mg / L MgSO4·7H2O, 170 mg / L KH2PO4; microelements: 0.83 mg / L KI, 6.2 mg / L H3BO3, 22.3 mg / L MnSO4·4H2O, 6.6 mg / L ZnSO4·H2O, 0.25 mg / L Na2MoO4·2H2O. O, 0.025 mg / L CuSO4·5H2O, 0.025 mg / L CoCl2·6H2O, 27.8 mg / L FeSO4·7H2O, 37.3 mg / L Na2EDTA·2H2O; Vitamins: 100 mg / L inositol, 0.5 mg / L niacin, 0.5 mg / L pyridoxine hydrochloride, 0.5 mg / L thiamine hydrochloride; Amino acids: 2 mg / L glycine; Carbon source: 30 g / L sucrose; and Plant hormones: 1 mg / L NAA acid, 0.5 mg / L 2,4-D acid, 0.1 mg / L kinetin. Adjust the overall pH to 6 and then sterilize, for example, by autoclaving at 121°C for 20 minutes or filtering through a 0.2 μm filter.

[0105] Optionally, the proliferation medium may have the following composition (concentrations expressed relative to the volume of cell-free proliferation medium): macroelements: 1650 mg / L NH4NO3, 2500 mg / L KNO3, 440 mg / L CaCl2·2H2O, 370 mg / L MgSO4·7H2O, 130 mg / L KH2PO4; microelements: 0.41 mg / L KI, 6.2 mg / L H3BO3, 22.3 mg / L MnSO4·4H2O, 7.5 mg / L ZnSO4·H2O, 0.25 mg / L Na2MoO 4.2H2O, 0.025 mg / L CuSO4·5H2O, 0.025 mg / L CoCl2·6H2O, 19.85 mg / L FeSO4·7H2O, 26.64 mg / L Na2EDTA·2H2O; Vitamins: 50 mg / L inositol, 0.25 mg / L niacin, 0.25 mg / L pyridoxine hydrochloride, 0.25 mg / L thiamine hydrochloride; Carbon source: 30 g / L sucrose; and Plant hormones: 0.35 mg / L NAA acid, 0.575 mg / L 2,4-D acid, 0.083 mg / L kinetin.

[0106] Optionally, the proliferation medium may have the following composition (concentration expressed relative to the volume of cell-free proliferation medium): macroelements: 20 mM NH4NO3, 19 mM KNO3, 3 mM CaCl2·2H2O, 1.5 mM MgSO4·7H2O, 1.2 mM KH2PO4, 0.005 mM KI, 0.1 mM H3BO3, 0.1 mM MnSO4·4H2O, 0.04 mM ZnSO4·H2O, 0.001 mM Na2MoO4·2H2O. O, 0.0001mM CuSO4·5H2O, 0.0001mM CoCl2·6H2O, 0.1mM FeSO4·7H2O, 0.1mM Na2EDTA·2H2O, 0.5mM inositol, 0.004mM nicotinic acid, 0.002mM pyridoxine hydrochloride, 0.0015mM thiamine hydrochloride, 0.03mM glycine, 87.6mM sucrose, 0.005mM NAA acid, 0.002mM 2,4-D acid, 0.0005mM kinetin.

[0107] Inoculate the proliferation medium with a suspension of callus cells at a concentration of 20 to 300 g in 1 L of proliferation medium, preferably 100 to 200 g in 1 L of proliferation medium, for example, about 150 g in 1 L of proliferation medium.

[0108] The proliferation stage takes place under biomass proliferation conditions.

[0109] According to the present invention, the term "biomass proliferation conditions" specifically refers to the temperature, duration, agitation, and light conditions required for cell proliferation in a suspension. Those skilled in the art familiar with Tripterygium wilfordii cell cultures can readily determine the biomass proliferation conditions. In one embodiment of the invention, the biomass proliferation step is carried out in the dark at 20°C to 35°C, particularly 27°C to 28°C, especially at about 27°C or 28°C, particularly with simultaneous agitation at 100 to 200 rpm, particularly at about 125 rpm (22.5 mm track), and the culture duration is 10 to 30 days, particularly 15 days.

[0110] In this step, the cells can be "passaged" or propagated, for example, every 7 to 15 days. Cell passaging is well known to those skilled in the art and can specifically involve diluting a portion of the cell culture in fresh, concentrated medium. For example, one-fifth of the culture is suspended in a volume of fresh medium corresponding to the volume of the initial culture. This allows the cell line to remain in a proliferative state in liquid medium.

[0111] Step (ii): Induction Phase

[0112] Following the proliferation phase, the cells contained in phase (i) are induced by adding an induction mixture. The proliferation medium to which the induction mixture is added is called the "induction medium". The induction phase is the stage that brings the cells into a physiological state conducive to the biosynthesis of secondary metabolites such as pentacyclic triterpenes.

[0113] During the induction phase, the production of pentacyclic triterpenes occurs in the cytosol of the cells and can partially diffuse into the induction medium. Therefore, within the meaning of this invention, the induction phase corresponds to the production (biosynthesis) of pentacyclic triterpenes, and more particularly triptolide, euonymus triterpenoid A, and euonymus triterpenoid B.

[0114] Induction is preferably carried out after a proliferation phase of 7 to 21 days, particularly 12 to 20 days, especially 15, 16, or 17 days (without passage culture). Optionally, the induction mixture may be added when the cell concentration obtained during the proliferation phase is twice, particularly more than twice, the initial cell concentration in the proliferation medium. In particular, the induction mixture may be added when the cell concentration is greater than 200 g / L, for example 200 to 400 g / L, particularly about 300 g / L (based on the number of cells per liter of proliferation medium). In one embodiment, the induction mixture is added to a culture in which the cell concentration changes from 150 to 200 g / L at the beginning of the proliferation phase to 300 to 400 g / L at the end of the proliferation phase.

[0115] After the proliferation phase, plant cells have consumed most or even all of the elements contained in the proliferation medium, especially carbon sources such as sucrose. Therefore, it may be necessary to reconstitute the composition of the medium before or simultaneously with the addition of the induction mixture.

[0116] To reconstitute the composition of the culture medium, the cell culture obtained after the proliferation step can be concentrated (e.g., by decantation or filtration), and then new proliferation medium can be added to the resulting cells. In this case, the cells are resuspended in new proliferation medium to obtain a concentration of 200 to 400 g / L, for example 250 to 350 g / L, particularly about 300 g / L (based on the number of cells per liter of proliferation medium).

[0117] Optionally, a concentrated mixture can be added to the cell culture to restore the elemental concentrations of the proliferation medium. Specifically, the concentrated mixture can be added before, after, or simultaneously with the induction mixture. For example, an equal volume of proliferation medium concentrated five times can be used to replace one-fifth of the cell culture.

[0118] It is believed that the concentrations of elements in the proliferation medium approach or equal to zero after 14, 15, or 16 days of the proliferation phase. In particular, it is believed that the concentrations of mineral elements (especially macro and micro elements) and carbonaceous elements (especially carbon sources) approach or equal to zero after 14, 15, or 16 days of the proliferation phase.

[0119] In one embodiment, the proliferation medium according to the invention comprises, in particular, the following at the beginning of the induction phase:

[0120] - At least one macroelement, particularly selected from NH4NO3, KNO3, CaCl2·2H2O, MgSO4·7H2O, KH2PO4 and mixtures thereof, wherein the total macroelement concentration is, for example, 5000 to 8000 mg / L of growth medium, preferably greater than 6000 mg / L of growth medium, advantageously 6000 to 8000 mg / L;

[0121] - At least one trace element, particularly selected from KI, H3BO3, MnSO4·4H2O, ZnSO4·H2O, Na2MoO4·2H2O, CuSO4·5H2O, CoCl2·6H2O, FeSO4·7H2O, Na2EDTA·2H2O, and mixtures thereof, wherein the total trace element concentration is, for example, 10 to 200 mg / L of growth medium, particularly 50 to 150 mg / L of growth medium;

[0122] - At least one vitamin, particularly selected from inositol, niacin, pyridoxine hydrochloride, thiamine hydrochloride and mixtures thereof, wherein the total vitamin concentration may be in the range of 0.01 to 3 g / L of growth medium, particularly 0.05 to 1 g / L of growth medium;

[0123] - At least one amino acid, particularly glycine, at a concentration of, for example, 3 to 4 mg / L of proliferation medium;

[0124] - At least one carbon source, particularly sucrose, with a total carbon source concentration of, for example, 10 to 70 g / L of proliferation medium, such as about 30 g / L.

[0125] In one embodiment, the proliferation medium at the beginning of the induction phase does not contain cytokinins and auxins, or contains negligible amounts, particularly less than 0.001 g / mL of cytokinins and auxins.

[0126] The proliferation medium during the induction phase is advantageously sterile, and its pH is preferably close to neutral.

[0127] During the induction phase, the proliferation medium may specifically have the following composition: macroelements: 2.8 g / L NH4NO3, 3 g / L KNO3, 0.45 g / L CaCl2·2H2O, 74 mg / L MgSO4·7H2O, 34 mg / L KH2PO4; microelements: 0.16 mg / L KI, 6.2 mg / L H3BO3, 18.5 mg / L MnSO4·4H2O, 6.6 mg / L ZnSO4·H2O, 0. 25 mg / L Na₂MoO₄·2H₂O, 0.025 mg / L CuSO₄·5H₂O, 0.025 mg / L CoCl₂·6H₂O, 28 mg / L FeSO₄·7H₂O, 37 mg / L Na₂EDTA·2H₂O; Vitamins: 250 mg / L inositol, 1.7 mg / L niacin, 1 mg / L pyridoxine hydrochloride, 1 mg / L thiamine hydrochloride; Amino acids: 4 mg / L glycine; Carbon source: 30 g / L sucrose.

[0128] According to the present invention, the term "induction mixture" should mean a mixture capable of stopping cell division. Such an induction mixture comprises at least one monocarboxylic acid compound inducer and at least one biological inducer. For example, a concentrated stock solution is used to introduce the induction mixture into the culture medium.

[0129] According to the invention, the term "monocarboxylic acid compound inducer" should more specifically refer to an inducer selected from 5-chlorosalicylic acid, salicylic acid, acetylsalicylic acid, methyl esters (especially methyl jasmonic acid), and mixtures thereof. In one embodiment of the invention, the monocarboxylic acid compound inducer is methyl jasmonic acid, salicylic acid, and / or 5-chlorosalicylic acid, especially methyl jasmonic acid. The monocarboxylic acid compound inducer is added specifically to obtain a final concentration of 0.005 to 0.1 g / L of induction medium, particularly 0.01 to 0.05 g / L, especially 0.002 to 0.004 g / L.

[0130] According to the present invention, the term "biological inducer" should more specifically refer to a biological inducer selected from substances comprising, preferably composed of, N-acetylglucosamine (especially chitin, chitosan), extracts of microorganisms or fungi, and oligosaccharides (polysaccharides, pectin, cellulose). In one embodiment, the biological inducer is chitin. Chitin is a linear polymer having repeating units of β-1,4N-acetyl-D-glucosamine. The biological inducer is added specifically to obtain a final concentration of 0.05 to 50 g / L of induction medium, for example 0.1 to 10 g / L, for example 0.5 to 7 g / L, and particularly 1 to 5 g / L.

[0131] In one embodiment, the induction mixture comprises methyl jasmonate and chitosan, wherein the final concentration of methyl jasmonate in the induction medium is particularly 0.002 to 0.005 g / L, and the final concentration of chitosan is particularly 1 to 4 g / L of induction medium.

[0132] In one embodiment, the induction mixture according to the invention further comprises at least one cell differentiation factor for plant cells and / or at least one precursor of the terpene synthesis pathway.

[0133] According to the present invention, "cell differentiation factors for plant cells" may be particularly selected from cell differentiation factors comprising, preferably, the following substances: cytokinins (especially benzylaminopurine), abscisic acid, kinetin, thidiazuron, 6-γ,γ-dimethylallylaminopurine (or isopentenyladenine) or zeatin, gibberellins, and mixtures thereof, especially benzylaminopurine and / or 6-γ,γ-dimethylallylaminopurine, especially 6-γ,γ-dimethylallylaminopurine.

[0134] According to the present invention, the "terpene synthesis precursor" is particularly selected from terpene synthesis precursors comprising, preferably, sodium pyruvate, potassium pyrophosphate, mevalonic acid, geraniol, farnesol, isopentenyl, dimethylallyl (including their pyrophosphated forms), sodium acetate, pyruvate, and mixtures thereof, especially geraniol, farnesol, sodium pyruvate, potassium pyrophosphate, and mixtures thereof, such as sodium pyruvate and / or potassium pyrophosphate.

[0135] Benzylaminopurine (BAP) can be used, specifically, in induction medium at a final concentration of 0.01 to 5 mg / L, for example, 0.5 to 5 mg / L.

[0136] 5-Chlorosalicylic acid (5-chloroSA) can be used specifically in induction medium at a final concentration of 0.1 to 15 mg / L.

[0137] Salicylic acid can be used, specifically, in induction medium at a final concentration of 0.1 to 100 mg / L, for example 20 to 60 mg / L, such as about 45 mg / L.

[0138] Farnesol can be used, particularly in induction medium, at a final concentration of 1 to 100 mg / L, for example 15 to 30 mg / L, such as about 30 mg / L.

[0139] Geraniol can be used, particularly in induction medium, at a final concentration of 1 to 100 mg / L, for example, 20 to 30 mg / L.

[0140] Sodium pyruvate can be used, specifically, in induction medium at a final concentration of 100 to 5000 mg / L, for example, 500 to 2000 mg / L.

[0141] Potassium pyrophosphate can be used, particularly in induction media, at a final concentration of 1 to 2000 mg / L, for example, 100 to 1000 mg / L.

[0142] 6-γ,γ-dimethylallylaminopurine (also known as 2iP or isopentenyladenine) can be used, particularly in induction media, at a final concentration of 0.005 to 10 mg / L, for example 0.01 to 3 mg / L, or for example 0.1 to 2 mg / L.

[0143] In one embodiment of the invention, the inducing mixture comprises methyl jasmonate, chitosan, sodium pyrophosphate, or a mixture thereof.

[0144] In one embodiment of the invention, the inducing mixture comprises methyl jasmonate, chitosan, sodium pyruvate, potassium pyrophosphate, and optionally benzylaminopurine and / or 6-γ,γ-dimethylallylaminopurine.

[0145] In one embodiment of the invention, the induction mixture comprises or consists of the following substances (concentrations given in parentheses correspond to concentrations in the induction medium, and the initial mixture can be more or less concentrated depending on the intended dilution): sodium pyruvate (500 to 2000 mg / L), potassium pyrophosphate (100 to 1000 mg / L), 6-γ,γ-dimethylallylaminopurine (0.1 to 2 mg / L), methyl jasmonate (0.002 to 0.005 g / L), and chitosan (1 to 4 g / L).

[0146] In one embodiment of the invention, the induction mixture comprises or consists of the following substances (concentrations given in parentheses correspond to concentrations in the induction medium; the initial mixture can be more or less concentrated, depending on the intended dilution): benzylaminopurine (0.5 to 5 mg / L, particularly 0.5 to 3 mg / L), 5-chlorosalicylic acid (2 to 6 mg / L, particularly 3 to 5 mg / L, for example about 3 mg / L or about 5 mg / L), acetylsalicylic acid and / or salicylic acid (20 to 60 mg / L, particularly 30 to 50 mg / L, especially 33 to 45 mg / L), methyl jasmonic acid (0.002 to 0.05 g / L, particularly 10 to 40 mg / L), chitosan (1 to 4 g / L), and farnesol (19 to 40 mg / L) and / or geraniol (20 to 30 mg / L).

[0147] In one embodiment of the invention, the induction mixture comprises or consists of the following substances (concentrations given in parentheses correspond to concentrations in the induction medium, and the initial mixture can be more or less concentrated depending on the intended dilution): sodium pyruvate (500 to 2000 mg / L), potassium pyrophosphate (100 to 1000 mg / L), 6-γ,γ-dimethylallylaminopurine (0.1 to 1 mg / L), methyl jasmonate (0.002 to 0.05 g / L, particularly 10 to 40 mg / L), and chitosan (1 to 4 g / L).

[0148] In one embodiment of the invention, the induction mixture comprises or consists of the following substances (concentrations given in parentheses correspond to concentrations in the induction medium, and the initial mixture can be more or less concentrated depending on the intended dilution): sodium pyruvate (about 1.5 g / L), potassium pyrophosphate (about 0.4 g / L), 6-γ,γ-dimethylallylaminopurine (about 0.4 mg / L), methyl jasmonate (about 0.03 g / L), and chitosan (about 2 g / L).

[0149] During the induction phase, after the addition of the induction mixture, the culture is stirred at a temperature of 20°C to 35°C, particularly about 27°C, and advantageously at a dissolved oxygen content of 2% to 40%, preferably about 16%, in the culture medium, at a speed of 50 to 200 rpm, particularly about 125 rpm, for a duration of 3 to 30 days, particularly 10 to 25 days, particularly 12 to 15 days. If necessary, especially in the dead volume of the bioreactor or by diffusion in the culture medium, a supply of adequately oxygen-rich sterile air can be used. The induction phase (ii) is preferably carried out in the dark. During the induction phase, the cell culture is preferably not passaged.

[0150] Step (iii): The stage of preparing extracts containing, in particular, pentacyclic triterpenes.

[0151] Following the induction phase, the method includes the step of preparing an extract containing, in particular, pentacyclic triterpenes.

[0152] Extracts can be obtained specifically by separating biomass and culture supernatant. Separation can be performed specifically by direct cell filtration (0-50 μm), centrifugation, or decantation.

[0153] In one embodiment, the extract used according to the invention may consist of culture supernatant recovered in this manner.

[0154] Extracts can also be obtained after lysing biomass. For example, the cells contained in the recovered biomass can be lysed by physical methods (ultrasonic treatment or grinding) or chemical methods (acid lysis), and then the lysate can be extracted with a solvent (called an extraction solvent). The organic phase, particularly containing triterpenes from the cytosol, is then recovered, especially by decantation or centrifugation. The solvent is particularly an ester solvent, more particularly an alkyl acetate (alkyl groups are more particularly straight-chain or branched chains having 1 to 6 carbon atoms), especially ethyl acetate or isopropyl acetate. The volume of solvent used per weight of biomass is particularly 2 volumes.

[0155] Preferably, the extract used according to the invention corresponds to the organic phase thus recovered, wherein the organic phase is optionally partially or completely concentrated, i.e., the extraction solvent is partially or completely evaporated.

[0156] Alternatively, the extract can be obtained by evaporating the solvent and optionally replacing it (particularly by a carrier suitable for the application of the extract in cosmetic or pharmaceutical fields), especially by vegetable oils or, in particular, pentylene glycol (or pentiol) or Myritol. (obtained after solvent).

[0157] The resulting extract can be optionally purified to obtain a purified extract of one or more pentacyclic triterpenes. The extracts according to the invention, and more particularly the purified extracts, advantageously contain 90% or more, especially 95% or more, and especially 98% or more of one or more pentacyclic triterpenes according to the invention, by weight relative to the total weight of the dry extract.

[0158] In one embodiment, the extract according to the invention, particularly the enriched or purified extract, contains 60% or more, particularly 80% or more, particularly 85% or more, particularly 90% or more, particularly 95% or more, particularly 98% or more, particularly 100% of triptolide relative to the total weight of the extract. It may particularly contain 50% to 98%, particularly 70% to 90%, particularly 76% to 84% of triptolide relative to the total weight of the dry extract.

[0159] In one embodiment, the extract according to the invention, particularly the enriched or purified extract, contains 1% to 20%, particularly 5% to 15%, especially 8% to 12% of Euonymus triterpenoid A by weight relative to the total weight of the dry extract.

[0160] In one embodiment, the extract according to the invention, particularly the enriched or purified extract, contains 1% to 20%, particularly 5% to 15%, especially 8% to 12% of Euonymus triterpenoid B by weight relative to the total weight of the dry extract.

[0161] In one embodiment, the extract according to the invention, particularly an enriched or purified extract, comprises:

[0162] - 50% to 98%, particularly 70% to 90%, and especially 76% to 84% of the total weight of the dry extract by weight;

[0163] - 1% to 20%, particularly 5% to 15%, and especially 8% to 12% of the total weight of the dry extract by weight;

[0164] - 1% to 20%, particularly 5% to 15%, and especially 8% to 12% of the total weight of the dry extract by weight of Euonymus triterpenoid B.

[0165] Purification of the extract according to the invention can be carried out in particular by the following steps: separation of one or more pentacyclic triterpenes, particularly triptolide, euonymus triterpenoid A and / or euonymus triterpenoid B, particularly by HPLC (high performance liquid chromatography), wherein the peaks of triptolide, euonymus triterpenoid A and euonymus triterpenoid B at 426 nm appear at 19.75 min, 18.03 min and 16.1 min, respectively.

[0166] According to a second aspect, the present invention also relates to a cosmetic or dermatological composition comprising a combination according to the invention and at least one cosmetically or dermatologically acceptable excipient, said combination comprising myrtle extract and tripterygium extract.

[0167] In one particular embodiment, the combination according to the invention is the sole active ingredient of the cosmetic composition or dermatological composition according to the invention.

[0168] The present invention is preferably intended for use in cosmetic or dermatological compositions according to the invention in a form suitable for topical application.

[0169] Therefore, the cosmetic or dermatological compositions according to the invention can be in forms commonly known for topical application, in other words, particularly lotions, shampoos, creams, foams, gels, dispersants, emulsions, sprays, serums, masks, creams, or sticks, having excipients, some of which are particularly capable of penetrating to improve the properties and ease of use of the active ingredients.

[0170] The cosmetic or dermatological compositions according to the invention advantageously comprise a myrtle extract, said myrtle extract being a nonpolar fraction comprising phloroglucinol and ursolic acid. Specifically, phloroglucinol includes phloroglucinol derivatives A, B', D, B, iso-semi-phloroglucinol, and semi-phloroglucinol.

[0171] The cosmetic or dermatological compositions according to the invention advantageously comprise Tripterygium wilfordii extract, said extract comprising at least one pentacyclic triterpenoid as defined above, said pentacyclic triterpenoid being particularly selected from euonymus triterpenoid A, euonymus triterpenoid B, triptolide, sclerotin, triptolide, and mixtures thereof, particularly selected from euonymus triterpenoid A, euonymus triterpenoid B, triptolide, and mixtures thereof, especially mixtures of euonymus triterpenoid A, euonymus triterpenoid B, and triptolide. The Tripterygium wilfordii extract is advantageously composed of, particularly rich in, pentacyclic triterpenoids such as euonymus triterpenoid A, euonymus triterpenoid B, and / or triptolide, and more particularly rich in, particularly rich in pentacyclic triterpenoids obtainable by the method described above, said method comprising the following steps:

[0172] (i) The stage of proliferating Tripterygium wilfordii cells in proliferation medium.

[0173] (ii) An induction phase is achieved by adding an induction mixture to the cell culture obtained in step (i), the induction mixture comprising at least one monocarboxylic acid compound inducer and at least one biological inducer, and

[0174] (iii) Prepare an extract from the cell culture obtained in step (ii), the extract containing, in particular, pentacyclic triterpenes.

[0175] The cosmetic or dermatological compositions according to the invention particularly comprise 0.01% to 1% (by weight of dry extract) relative to the total weight of the composition, particularly 0.05% to 0.8% (by weight of dry extract), particularly 0.1% to 0.5% (by weight of dry extract), and especially 0.2% to 0.4% (by weight of dry extract) relative to the total weight of the composition. According to a preferred embodiment, the cosmetic or dermatological compositions according to the invention comprise about 0.3% (by weight of dry extract) of Tripterygium wilfordii extract relative to the total weight of the composition.

[0176] Tripterygium wilfordii extract advantageously comprises 90% or more of pentacyclic triterpenes relative to the total weight of the dry extract. In one advantageous embodiment, Tripterygium wilfordii extract comprises 1% to 20% of Euonymus triterpenoid A, 1% to 20% of Euonymus triterpenoid B, and at least 60% of triptolide relative to the total weight of the dry extract.

[0177] The cosmetic or dermatological compositions according to the invention advantageously comprise 0.01% to 1% (by weight of the dry extract) relative to the total weight of the composition, particularly 0.05% to 0.8% (by weight of the dry extract), particularly 0.08% to 0.4% (by weight of the dry extract), and especially 0.08% to 0.2% (by weight of the dry extract). According to a preferred embodiment, the cosmetic or dermatological compositions according to the invention comprise about 0.1% (by weight of the dry extract) relative to the total weight of the composition.

[0178] Myrtle extract advantageously contains 3% to 10% by weight of phloroglucinol relative to the total weight of the dry extract and / or 10% to 30% by weight of ursolic acid relative to the total weight of the dry extract.

[0179] According to a third aspect, the present invention relates to a cosmetic composition or dermatological composition comprising a combination of myrtle extract and tripterygium extract, or comprising such a combination and at least one cosmetically or dermatologically acceptable excipient, for the use in treating inflammation caused by Propionibacterium acnes.

[0180] The present invention also relates to the use of cosmetic or dermatological compositions comprising a combination of myrtle extract and tripterygium extract, or comprising such a combination and at least one cosmetic or dermatologically acceptable excipient, for the treatment of acne or acne-prone skin.

[0181] The following examples illustrate the present invention, but do not limit the scope of the invention. Attached Figure Description

[0182] Figure 1 The HPLC chromatogram of the Tripterygium wilfordii “PCC” extract obtained according to Example 2 is shown, the extract containing the following pentacyclic triterpenoids: triptolide, euonymus triterpenoid A and euonymus triterpenoid B. Detailed Implementation

[0183] Example

[0184] Example 1: Preparation of Myrtle Extract

[0185] 1 kg of ground myrtle leaves were extracted with 5 volumes of isopropyl acetate and stirred under reflux for 1 hour. After filtration and rinsing of the residue, the extract was bleached by adding activated charcoal. After filtration, the bleached filtrate was concentrated to 2 liters and then dried with ethanol until isopropyl acetate was removed. The resulting aqueous phase was then deodorized by heat treatment and subsequently dried by lyophilization.

[0186] One kilogram of myrtle leaves yields approximately 25g of dried myrtle extract. It contains 7% phloroglucinol and 25% ursolic acid.

[0187] Example 2: Obtaining Tripterygium wilfordii plant cell culture (PCC) extract rich in pentacyclic triterpenes

[0188] Cultures were generated in a 5L Wave reactor from Sartorius Stedim Biotech (Germany). The reactor was inoculated with a suspension of Tripterygium wilfordii cells from an Erlenmeyer flask. The proliferation medium had, for example, the composition shown below:

[0189] Major elements: 1650 mg / L NH4NO3, 2500 mg / L KNO3, 440 mg / L CaCl2·2H2O, 370 mg / L MgSO4·7H2O, 130 mg / L KH2PO4;

[0190] Trace elements: 0.41 mg / L KI, 6.2 mg / L H3BO3, 22.3 mg / L MnSO4·4H2O, 7.5 mg / L ZnSO4·H2O, 0.25 mg / L Na2MoO4·2H2O, 0.025 mg / L CuSO4·5H2O, 0.025 mg / L CoCl2·6H2O, 19.85 mg / L FeSO4·7H2O, 26.64 mg / L Na2EDTA·2H2O;

[0191] Vitamins: 50 mg / L inositol, 0.25 mg / L niacin, 0.25 mg / L pyridoxine hydrochloride, 0.25 mg / L thiamine hydrochloride;

[0192] Carbon source: 30 g / L of sucrose;

[0193] Plant hormones: 0.35 mg / L NAA acid, 0.575 mg / L 2,4-D acid, and 0.083 mg / L kinetin.

[0194] Adjust the pH of the culture medium to pH 6 ± 0.5 (by adding KOH, 1M), and then perform appropriate sterilization treatment, such as autoclaving at 121°C for at least 20 minutes or sterilizing by filtration at 0.2 μm.

[0195] After obtaining the maximum amount of biomass in approximately 17 days with continuous stirring, induction was performed. The induction mixture was then added to the proliferation medium in an Erlenmeyer flask using a mother liquor produced in dimethyl sulfoxide. The composition of the inducing mixture was such that the following concentrations could be obtained in the induction medium (+cells): sodium pyruvate 1.5 g / L, potassium pyrophosphate 0.44 g / L, 2iP 0.0004 g / L, methyl jasmonate 0.036 g / L, and chitosan 2 g / L. Culture was stopped after 15 days of induction. Most of the biomass was recovered by filtering the cell suspension through a nylon filter (20–50 μm). Approximately 1925 g of biomass was recovered from 5 L of suspension. This biomass was extracted with ethyl acetate (or isopropyl acetate) at a weight-to-volume ratio of 2:1 (volume:weight) (in this case, 3850 mL of solvent for 1925 g of biomass). The biomass / solvent mixture was then physically extracted by sonication or grinding. The organic phase was then recovered after stirring and impregnation. The addition of solvent (after stirring and impregnation and recovery of the organic phase) was repeated twice. The solvent was concentrated under vacuum, and the concentrate was then dissolved in pentanediol, and the mixture was placed under vacuum to remove residual organic solvent. A solution of an extract called "PCC" was then obtained, containing the following pentacyclic triterpenes: triptolide, euonymus triterpenoid A, and euonymus triterpenoid B, as described by… Figure 1 The results were determined by the HPLC chromatogram shown.

[0196] HPLC chromatographic conditions: Alliance liquid chromatography system (Waters 2695 model 2.03); Sunfire column C18. 5 μm (4.6 mm × 150 mm); water / acetonitrile solvent gradient, flow rate 3 ml / min; triterpenes were detected at λ460 nm.

[0197] Example 3: Biological properties of the combination according to the present invention

[0198] The aim of this study was to evaluate the anti-inflammatory properties of myrtle extract and tripterygium extract against inflammation induced by tissue-formed Propionibacterium acnes or by membrane extracts of Propionibacterium acnes. To demonstrate this hypothesis, suboptimal concentrations of myrtle extract, tripterygium extract, and combinations thereof were evaluated in response to the immune inflammatory cascade induced upstream of the Th17 pathway in response to the propathogenic germline of Propionibacterium acnes (germline IA1), and the production of pro-inflammatory cytokines by monocytes.

[0199] method:

[0200] The experiment was conducted on immature dendritic cells derived from monocytes (final concentration 1.10). 5The procedure was performed on a biofilm or co-culture of germline IA1 Propionibacterium acnes (cells / ml / well) and membranes. Mononuclear cells were purified from human blood via negative selection (EasySep). TM Human mononuclear cell enrichment kit (stem cells).

[0201] On day 0, monocytes were incubated in differentiation medium (Gibco Roswell Park Memorial Institute (RPMI) containing 10% v / v fetal bovine serum (FBS) with complement depleted (heated to 56°C for 30 min), 50 ng / ml IL-4, and 100 ng / ml recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF)). On day 3, half of the medium was replaced with fresh medium. On day 6, immature dendritic cells were characterized by flow cytometry and stimulated with biofilms or membranes of Propionibacterium acnes.

[0202] Preparation of biofilms:

[0203] Culture medium used:

[0204] -Colombian agar + 5% sheep blood (COS)

[0205] -A biofilm culture medium composed of 0.005 g / L FeSO4·7H2O + 12.5 g / L Na2HPO4 + 5 g / L KH2PO4 + 1 g / L vitamin tyrosine + 0.25 g / L lactose + 1 g / L yeast extract.

[0206] By adjusting the spectrometer to approximately 54% transmittance at 640 nm, at approximately 10 8 A suspension of Propionibacterium acnes P52 was prepared directly in a biofilm culture medium at a concentration of CFU / ml.

[0207] The initial suspension was counted by continuously diluting it to 1 / 10, up to a count of 10. -6 Diluent, spread 100 μL of 10-diluted solution onto COS agar. -5 Up to 10 -6 Diluent. Incubate under anaerobic conditions at 36°C for 72 hours.

[0208] To obtain biofilms, 2 mL of biofilm culture medium (containing 10% biofilm) was inoculated into all wells of a 24-well microplate. 8CFU / mL of Propionibacterium acnes was incubated anaerobically at 36°C. After 24 hours of incubation, the medium was replaced; the wells were emptied and then gently rinsed once with 2 mL of SDW (sterile distilled water). 2 mL of biofilm medium was introduced into each well, and the plate was returned to the microplate and incubated anaerobically at 36°C. After 48 hours of incubation, the medium was replaced again under the same conditions.

[0209] The biofilms prepared therefrom were washed with culture medium (RPMI + 10% v / v FBS) before being incubated with dendritic cells.

[0210] membrane:

[0211] The Propionibacterium acnes membrane was purchased from Icare Laboratories (Saint-Beauzire, France).

[0212] The stimulation of immature dendritic cells derived from monocytes by the membrane of Propionibacterium acnes was carried out using a 1 / 20 dilution of the membrane solution at a concentration of 0.15 mg / ml in the culture medium (RPMI + 10% v / v FBS).

[0213] The stimulation of dendritic cells derived from monocytes by the biofilm of Propionibacterium acnes is achieved with an infection multiplicity (MOI) of nearly 100.

[0214] MOI = [Propionibacterium acnes] / [Dendrical cells derived from monocytes].

[0215] The myrtle extract used in this study was prepared according to Example 1. This extract was dissolved and stored in ethanol at a concentration of 20 mg / ml (10 mg of the dry extract prepared according to Example 1 is in 1 ml of ethanol). The tested concentration was 10 μg / ml.

[0216] The Tripterygium wilfordii extract used in this study (hereinafter referred to as Trip extract) was prepared according to Example 2. This extract was dissolved and stored at 10 mg / ml in culture medium (RPMI + 10% v / v FBS). The tested concentration was 25 μg / ml.

[0217] The study also tested a positive control for inhibiting the production of inflammatory cytokines; it contained dexamethasone at a concentration of 300 ng / ml dissolved in water.

[0218] The supernatant was recovered 24 hours after stimulation.

[0219] The results of the three independent experiments were averaged.

[0220] This study evaluated a variety of cytokines, including IL-6, IL-8, IL-10, and IL-12p40; these cytokines were quantified using Luminex technology (Bioplex 200, Biorad) in multiple channels.

[0221] result:

[0222] Stimulation by the membrane of Propionibacterium acnes

[0223] The results of inhibition of interleukin-6 (IL-6) production by dendritic cells derived from monocytes due to membrane stimulation by Propionibacterium acnes are presented in Table 1 below.

[0224] [Table 1]

[0225]

[0226] Mb: membrane; Trip: Tripterygium wilfordii; NS: not significant.

[0227] In the absence of stimulation, dendritic cells derived from monocytes do not produce IL-6. However, the membrane of *Propionibacterium acnes* significantly induces IL-6 production in dendritic cells derived from monocytes. The positive control of 300 ng / ml dexamethasone significantly inhibited this production; these expected results validate the assay.

[0228] A concentration of 10 μg / ml of *Myrtle* extract significantly inhibited IL-6 production by dendritic cells derived from monocytes. A concentration of 25 μg / ml of *Tripterygium wilfordii* extract did not alter IL-6 release relative to the irritant condition of *Propionibacterium acnes* membranes. Conversely, the combination of *Myrtle* and *Tripterygium wilfordii* extracts at the same concentrations previously showed a strong and significant (p<0.01) inhibition of IL-6 production, exhibiting a synergistic effect (Table 1). In fact, the inhibition of IL-6 production reached 60% in the presence of both extracts, compared to 42% when the effects of the extracts used individually were combined.

[0229] The results of inhibition of interleukin-8 (IL-8) production by dendritic cells derived from monocytes due to membrane stimulation by Propionibacterium acnes are presented in Table 2 below.

[0230] [Table 2]

[0231]

[0232] Mb: membrane; Trip: Tripterygium wilfordii; NS: not significant.

[0233] In the absence of stimulation, dendritic cells derived from monocytes do not produce IL-8. However, the membrane of *Propionibacterium acnes* significantly induces IL-8 production in dendritic cells derived from monocytes. The positive control of 300 ng / ml dexamethasone significantly inhibited this production; these expected results validate the assay.

[0234] A single 10 μg / ml dose of myrtle extract was insufficient to inhibit IL-8 production by dendritic cells derived from monocytes. Similarly, a single 25 μg / ml dose of triptolide extract did not alter IL-8 release relative to the irritant status of Propionibacterium acnes membranes. Conversely, the combination of the same concentrations of myrtle extract and triptolide extract significantly (p<0.05) inhibited IL-8 production, revealing a synergistic effect (Table 2).

[0235] The results of inhibition of interleukin-10 (IL-10) production by dendritic cells derived from monocytes due to membrane stimulation by Propionibacterium acnes are presented in Table 3 below.

[0236] [Table 3]

[0237]

[0238] Mb: membrane; Trip: Tripterygium wilfordii; NS: not significant.

[0239] In the absence of stimulation, dendritic cells derived from monocytes do not produce IL-10. However, the membrane of *Propionibacterium acnes* significantly induces IL-10 production in dendritic cells derived from monocytes. The positive control of 300 ng / ml dexamethasone significantly inhibited this production; these expected results validate the assay.

[0240] 10 μg / ml of myrtle extract significantly inhibited IL-10 production by dendritic cells derived from monocytes. 25 μg / ml of triptolide extract did not alter IL-10 release relative to the irritant condition of Propionibacterium acnes membranes. Conversely, the combination of myrtle and triptolide extracts at the same concentrations previously used strongly and very significantly (p<0.001) inhibited IL-10 production, exhibiting a synergistic effect (Table 3). More specifically, IL-10 production was reduced by more than two-thirds in the presence of both extracts, compared to less than half the effect if the effects of the extracts used individually were combined.

[0241] The results of inhibition of interleukin-12p40 (IL-12p40) produced by dendritic cells derived from monocytes due to membrane stimulation by Propionibacterium acnes are presented in Table 4 below.

[0242] [Table 4]

[0243]

[0244] Mb: membrane; Trip: Tripterygium wilfordii; NS: not significant.

[0245] In the absence of stimulation, dendritic cells derived from monocytes do not produce IL-12p40. However, the membrane of *Propionibacterium acnes* significantly induces IL-12p40 production in dendritic cells derived from monocytes. The positive control of 300 ng / ml dexamethasone significantly inhibited this production; these expected results validate the assay.

[0246] A concentration of 10 μg / ml of myrtle extract significantly inhibited IL-12p40 production by dendritic cells derived from monocytes, with an inhibition rate of almost 50%. A concentration of 25 μg / ml of triptolide extract alone tended to reduce IL-12p40 release, but this was not statistically significant relative to the irritation of the Propionibacterium acnes membrane. Conversely, the combination of the same concentrations of myrtle extract and triptolide extract synergistically and very significantly (p<0.001) inhibited IL-12p40 production (Table 4).

[0247] Stimulation by the biofilm of Propionibacterium acnes

[0248] The results regarding the production of IL-10 by dendritic cells derived from monocytes due to biofilm stimulation by Propionibacterium acnes are presented in Table 5 below.

[0249] [Table 5]

[0250]

[0251] Bf: biofilm; Trip: Tripterygium wilfordii; NS: not significant.

[0252] In the absence of stimulation, dendritic cells derived from monocytes do not produce IL-10. However, the biofilm of Propionibacterium acnes significantly induces IL-10 production in dendritic cells derived from monocytes. The positive control of 300 ng / ml dexamethasone significantly inhibited this production; these expected results make the test valid.

[0253] The combination of myrtle extract (10 μg / ml) and Tripterygium wilfordii extract (25 μg / ml) inhibited IL-10 production even more strongly than dexamethasone (p<0.01) (Table 5).

[0254] In summary, the inventors have demonstrated that myrtle extract and Tripterygium wilfordii extract, used alone, exhibit relatively general anti-inflammatory activity in these models by inhibiting the production of various interleukins. Conversely, the inventors have demonstrated that combining these two extracts produces a synergistic effect of anti-inflammatory activity.

Claims

1. A cosmetic or dermatological combination comprising 0.08% to 0.2% by weight of dried myrtle extract and 0.2% to 0.4% by weight of dried triptolide extract relative to the total weight of the composition, wherein the myrtle extract is a nonpolar fraction comprising phloroglucinol and ursolic acid, the triptolide extract comprises at least one pentacyclic triterpenoid selected from euonymus triterpenoid A, euonymus triterpenoid B, triptolide, and mixtures thereof, wherein the myrtle extract is obtained by extracting myrtle leaves using isopropyl acetate, and wherein the triptolide extract is obtained by the following method: (i) The proliferation stage of Tripterygium wilfordii cells in proliferation medium, (ii) An induction phase by adding an induction mixture to the cell culture obtained in step (i), said induction mixture comprising at least one monocarboxylic acid compound inducer and at least one biological inducer, and (iii) Prepare an extract containing pentacyclic triterpenes using the cell culture obtained in step (ii).

2. The cosmetic or dermatological combination according to claim 1, characterized in that, The myristic resorcinol includes myristic resorcinol A, B', D, B, iso-semi-myristic resorcinol, and semi-myristic resorcinol.

3. The cosmetic or dermatological combination according to claim 1, characterized in that, The total content of phloroglucinol from myrtle wood is 3% to 10% by weight relative to the total weight of dried myrtle extract.

4. The cosmetic or dermatological combination according to claim 1, characterized in that, The content of ursolic acid is 10% to 30% by weight relative to the total weight of dried myrtle extract.

5. The cosmetic or dermatological combination according to claim 1, characterized in that, The total content of myristic resorcinol is 3% to 10% by weight relative to the total weight of dried myristic extract, and the content of ursolic acid is 10% to 30% by weight relative to the total weight of dried myristic extract, and the myristic resorcinol includes myristic resorcinol A, B', D, B, iso-semi-myristic resorcinol and semi-myristic resorcinol.

6. The cosmetic or dermatological combination according to claim 1, characterized in that, Tripterygium wilfordii extract contains Euonymus triterpenoid A, Euonymus triterpenoid B and triptolide.

7. The cosmetic or dermatological combination according to claim 1, characterized in that, The Tripterygium wilfordii extract contains at least one pentacyclic triterpenoid at 90% by weight relative to the total weight of the dry extract.

8. The cosmetic or dermatological combination according to claim 1, characterized in that, The Tripterygium wilfordii extract comprises, by weight, 1% to 20% of Euonymus triterpenoid A, 1% to 20% of Euonymus triterpenoid B, and at least 60% of Tripterygium oleoresin, relative to the total weight of the dry extract.

9. The cosmetic or dermatological combination according to claim 1, characterized in that, The myrtle extract is a nonpolar fraction containing phloroglucinol and ursolic acid, wherein the total content of phloroglucinol is 3% to 10% by weight relative to the total weight of the dried myrtle extract, and the content of ursolic acid is 10% to 30% by weight relative to the total weight of the dried myrtle extract. The phloroglucinol includes phloroglucinol A, B', D, B, iso-semi-phloroglucinol, and semi-phloroglucinol. The Tripterygium wilfordii extract contains 1% to 20% by weight of Euonymus triterpenoid A, 1% to 20% by weight of Euonymus triterpenoid B, and at least 60% by weight of Tripterygium oleoresin, relative to the total weight of the dry extract.

10. Use of the cosmetic or dermatological combination according to any one of claims 1 to 9 in the preparation of a cosmetic or dermatological composition for treating inflammation caused by Propionibacterium acnes.

11. Use of the cosmetic or dermatological combination according to any one of claims 1 to 9 in the preparation of a cosmetic or dermatological composition for treating acne-prone skin.

12. A cosmetic or dermatological composition comprising a cosmetic or dermatological composition according to any one of claims 1 to 9 and at least one cosmetically or dermatologically acceptable excipient.

13. The composition according to claim 12, characterized in that, The composition is in a form suitable for topical application.

14. Use of the composition according to claim 12 in the preparation of a medicament for treating inflammation caused by Propionibacterium acnes.

15. Use of the composition according to claim 12 in the preparation of a medicament for treating acne-prone skin.

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