RU2794229C2 - Combination of retinoid and silybum marianum (l.) gaertn extract - Google Patents

Abstract

FIELD: dermatological industry.

SUBSTANCE: invention relates to the dermatological industry, namely to a dermatological or dermocosmetic composition for the treatment of diseases selected from acne, seborrhea, rosacea and/or seborrheic dermatitis. Dermatological or dermocosmetic composition for the treatment of diseases selected from acne, seborrhea, rosacea and/or seborrheic dermatitis, containing at least one dermatologically or dermocosmetically acceptable excipient and at least one combination containing retinoid and Silybum marianum (L.) Gaertn., moreover, the specified retinoid is retinaldehyde and is present in the composition in an amount of from 0.05% by weight to 5% by weight of the total mass of the composition and characterized in that the extract of Silybum marianum (L.) Gaertn. is an extract of achenes of Silybum marianum (L.) Gaertn., containing less than 0.2% by weight of silymarin, equivalent to the weight of the dry extract, is present in the composition in an amount of from 0.05% by weight to 10% by weight of the total weight of the composition and is obtained by the method, including the following successive stages:

(i) pressing achenes of Silybum marianum (L.) Gaertn. with obtaining oil;

(ii) extraction of oil obtained from seeds of Silybum marianum (L.) Gaertn. in stage (i) with an extraction solvent selected from subcritical water and a C₁-C ₃ alcohol, optionally mixed with water, to obtain an extraction phase and a lipid phase;

(iii) isolating the extraction phase obtained in sage (ii), and

(iv) partial or complete drying of the extraction phase to obtain a concentrated or dry extract.

EFFECT: exhibiting synergistic activity in the treatment of diseases selected from acne, seborrhea, rosacea and/or seborrheic dermatitis.

11 cl, 6 dwg

Description

FIELD OF TECHNOLOGY

The present invention relates to a combination of a retinoid and an extract of Silybum marianum (L.) Gaertn. (milk thistle), as well as a dermatological or dermocosmetic composition, preferably for external use, containing such a combination, in particular for the treatment of acne, seborrhea, rosacea and/or seborrheic dermatitis.

PRIOR ART

Latin name Silybum marianum (L.) Gaertn. refers to a plant (milk thistle) belonging to the family Asteraceae, annual or biennial, with a hard stem that can reach a height of more than one meter. Its large, glossy, alternate, stipuled leaves are covered with white spots and edged with hard, pointed spines. Flowers are united in apical inflorescences, often solitary. They are surrounded by large prickly bracts with very sharp tips. The five-lobed tubular flowers are purple-red. The fruits are glossy, black or yellow-marble achenes, surrounded by a ring of serrated bristles at the base. The popular name for this plant is milk thistle.

Achenes (often erroneously called seeds in the literature) Silybum marianum (L.) Gaertn. and preparations prepared from them are traditionally used orally for the symptomatic treatment of functional digestive disorders associated with impaired liver function.

According to the literature, the main active ingredient in the seeds of Silybum marianum (L.) Gaertn. is silymarin - a mixture of several flavonolignans (mainly silybin, isosilybin, silychristin and silydianin). Achenes contain up to 3 wt. % silymarin. They also contain oil (from 20 wt.% to 30 wt.%), mucus and proteins.

Vahlquist, А. (1985). Vitamin A in skin and serum - studies of acne vulgaris, atopic dermatitis, ichthyosis vulgaris and lichen planus. Br. J. Dermatol. 113(4), 405-413), его сниженный метаболизм описан в сально-волосяном эпителии (Everts, Н.В. (2012). Endogenous retinoids in the hair follicle and sebaceous gland. Biochim. Biophys. Acta 1821(1), 222-229). Ретинальдегид при наружном применении обеспечивает доставку различных компонентов витамина А в кожу человека (Sorg, О., Saurat, J.Н. (2014). Topical retinoids in skin ageing: A focused update with reference to sun induced epidermal vitamin A deficiency. Dermatology 228(4), 314-325) - свойство, которое уже использовали для профилактики и лечения кожи, предрасположенной к акне.In addition, vitamin A deficiency has been found in the skin of acne patients.

Vahlquist, A. (1985). Vitamin A in skin and serum - studies of acne vulgaris, atopic dermatitis, ichthyosis vulgaris and lichen planus. Br. J. Dermatol. 113(4), 405-413), its reduced metabolism has been described in the sebaceous epithelium (Everts, N.V. (2012). Endogenous retinoids in the hair follicle and sebaceous gland. Biochim. Biophys. Acta 1821(1), 222-229). Topical retinaldehyde delivers various components of vitamin A to human skin (Sorg, O., Saurat, J.H. (2014). Topical retinoids in skin aging: A focused update with reference to sun induced epidermal vitamin A deficiency. Dermatology 228 (4), 314-325) is a property that has already been used to prevent and treat acne-prone skin.

SUMMARY OF THE INVENTION

In this context, the present inventors have surprisingly demonstrated that a retinoid such as retinaldehyde (also referred to as retinal) in combination with Silybum marianum extract shows improved properties in the treatment of acne, seborrhea, rosacea or seborrheic dermatitis. In particular, the obtained research results demonstrated the synergistic role of retinaldehyde in combination with Silybum marianum extract in the production of retinoid acids by skin cells.

Therefore, the object of the present invention relates to a combination containing a retinoid and an extract of Silybum marianum (L.) Gaertn.

Another object of the present invention relates to a combination containing a retinoid and an extract of Silybum marianum (L.) Gaertn., for use as a drug.

Another object of the present invention relates to a combination containing a retinoid and an extract of Silybum marianum (L.) Gaertn., for use in the treatment of acne, seborrhea, rosacea and/or seborrheic dermatitis.

Another aspect of the present invention relates to the use of a combination containing a retinoid and an extract of Silybum marianum (L.) Gaertn. for the manufacture of a medicament for the treatment of acne, seborrhea, rosacea and/or seborrheic dermatitis.

Another object of the present invention relates to the use of a combination containing a retinoid and an extract of Silybum marianum (L.) Gaertn., in the treatment of acne, seborrhea, rosacea and/or seborrheic dermatitis.

Another object of the present invention relates to a method for the treatment of acne, seborrhea, rosacea and/or seborrheic dermatitis, comprising administering to a person in need of this an effective amount of a combination containing a retinoid and an extract of Silybum marianum (L.) Gaertn.

Another object of the present invention relates to a dermatological or dermocosmetic composition, preferably for external use, containing a retinoid and an extract of Silybum marianum (L.) Gaertn. in combination with at least one dermatologically or dermocosmetically acceptable excipient.

Another object of the present invention relates to a dermatological or dermocosmetic composition, preferably for external use, containing a retinoid and an extract of Silybum marianum (L.) Gaertn. in combination with at least one dermatologically or dermocosmetically acceptable excipient, for use in the treatment of acne, seborrhea, rosacea and/or seborrheic dermatitis.

Another object of the present invention relates to the use of a dermatological or dermocosmetic composition, preferably for external use, containing a retinoid and an extract of Silybum marianum (L.) Gaertn. in combination with at least one dermatologically or dermocosmetically acceptable excipient, for the treatment of acne, seborrhea, rosacea and/or seborrheic dermatitis.

Another object of the present invention relates to a method for the treatment of acne, seborrhea, rosacea and/or seborrheic dermatitis, comprising administering, preferably externally, to a person in need of an effective amount of a dermatological or dermocosmetic composition containing a retinoid and an extract of Silybum marianum (L.) Gaertn. in combination with at least one dermatologically or dermocosmetically acceptable excipient.

Another object of the present invention relates to a dermatological or dermocosmetic composition, preferably for external use, containing:

(i) a retinoid, and

(ii) an extract of Silybum marianum (L.) Gaertn.

in the form of a combination of products for simultaneous, separate or sequential use.

Another object of the present invention relates to a dermatological or dermocosmetic composition, preferably for external use, containing:

(i) a retinoid, and

(ii) an extract of Silybum marianum (L.) Gaertn.

in the form of a combination of products for simultaneous, separate or sequential use in the treatment of acne, seborrhea, rosacea and / or seborrheic dermatitis.

Another subject of the present invention relates to the simultaneous, separate or sequential use of a retinoid and an extract of Silybum marianum (L.) Gaertn. in the treatment of acne, seborrhea, rosacea and / or seborrheic dermatitis.

Another subject of the present invention relates to a method for the treatment of acne, seborrhea, rosacea and/or seborrheic dermatitis, comprising the simultaneous, separate or sequential application of an effective amount of a retinoid and an effective amount of Silybum marianum (L.) Gaertn extract. person in need.

INTELLIGENCE. CONFIRMING THE POSSIBILITY OF CARRYING OUT THE INVENTION

Definitions

In the present description, the plant Silybum marianum (L.) Gaertn. may be abbreviated as Silybum marianum and retinaldehyde may be abbreviated as RAL (from English: retinaldehyde).

When used in the context of the present invention, the term "fatty acid" means a carboxylic acid R1CO ₂ H, in which the chain R1 is a straight or branched hydrocarbon chain, saturated or containing C=C double bonds, and the carboxylic acid contains from 16 carbon atoms to 22 carbon atoms (including the carbon atom in the carboxylic acid functional group).

When used in the context of the present invention, the term "free" fatty acid (including linoleic acid) means a fatty acid that is not associated with other molecules (for example, glycerol or its derivatives to form glycerides or alcohol to form a fatty acid ester).

When used in the context of the present invention, the term "C ₁ -C ₃ alcohol" means an alcohol R2OH, in which the R2 chain is a saturated, linear or branched hydrocarbon chain containing from 1 carbon atom to 3 carbon atoms. The alcohol may be methanol, ethanol, n-propanol or isopropanol, in particular methanol, ethanol or isopropanol. Preferably it is isopropanol.

When used in the context of the present invention, the term "dermatologically or dermocosmetically acceptable" means that a substance can be used to prepare a dermatological or dermocosmetic composition that is generally safe, non-toxic and not biologically or otherwise undesirable, and which can be used for therapeutic or cosmetic purposes, in particular by external application.

When used in the context of the present invention, the term "about" means that the corresponding value may be 10%, in particular 5% or 1%, below or above the specified value.

When used in the context of the present invention, the term "dry extract" means an extract that does not contain the solvent used for extraction, or which contains only insignificant trace amounts of the solvent used for extraction. Accordingly, such a dry extract contains only material derived from Silybum marianum (L.) Gaertn. It may also contain minor traces of the solvent used for the extraction.

When used in the context of the present invention, the term "isosilybin" refers to the two diastereoisomers, isosilybin A and isosilybin B.

When used in the context of the present invention, the term "silybin", also referred to as "silibinin" in the art, means the four diastereomers - silybin A, silybin B, 2,3-cis-silybin A and 2,3-cis-silybin B.

When used in the context of the present invention, the term "silichristin" means two diastereomers - silychristin A and silychristin B.

When used in the context of the present invention, the term "silymarin" means a purified extract of Silybum marianum (L.) Gaertn. ., 2012). Therefore, the content of silymarin is less than 0.2 wt. % means that the total amount of silymarin components is less than 0.2 wt. %. This content can be determined, in particular, by high-performance liquid chromatography (HPLC; from English: high-performance liquid chromatography) or ultra-high-performance liquid chromatography (UPLC; from English: ultra-high-performance liquid chromatography) by calculating the total area of the peaks, corresponding to all components of silymarin, in particular using a reference sample of silymarin, which can be obtained, for example, from Sigma Aldrich, to determine the position of these peaks.

When used in the context of the present invention, the term "organic solvent immiscible with oil obtained from the achenes of Silybum marianum (L.) Gaertn." means an organic solvent which is not miscible or only partially miscible with the oil obtained from the Silybum marianum (L.) Gaertn. , in which at least two phases can be observed.

When used in the context of the present invention, the term "sterol (sterol)" means a molecule having a sterane core, the 3rd carbon atom of which carries the OH hydroxyl group; the sterane core has the following structure:

When used in the context of the present invention, the term "tocopherol" means α-tocopherol, β-tocopherol, γ-tocopherol and δ-tocopherol.

Retinoid

The retinoid used in the context of the present invention may be selected from retinol, retinaldehyde and retinoic acid in various isomeric forms (in particular trans, 13-cis and 9-cis). Preferably the retinoid is retinol or retinaldehyde. More preferably, the retinoid is retinaldehyde.

Extract of Silybum marianum (L.) Gaertn.

The extract used in the context of the present invention is an extract of Silybum marianum, and in particular an extract from the achenes of Silybum marianum.

According to a private embodiment of the present invention, the extract of the present invention contains less than 0.2 wt. % silymarin, preferably less than 0.1 wt. % silymarin, based on the weight of the dry extract.

According to a private embodiment of the present invention, the extract of the present invention contains at least 0.5 wt. % beta-sitosterol, preferably at least 1.0 wt. % beta-sitosterol, based on the weight of the dry extract. In particular, the extract of the present invention contains from 0.5 wt. % to 2.5 wt. % beta-sitosterol, in particular from 1.0 wt. % to 2.0 wt. % beta-sitosterol, for example - about 1.5 wt. % beta-sitosterol, based on the weight of the dry extract. Preferably, the extract of the present invention contains less than 0.2 wt. % silymarin, more preferably less than 0.1 wt. % silymarin, based on the weight of the dry extract. The weight ratio of silymarin/beta-sitosterol in the extract of the present invention may be less than 0.4, in particular less than 0.07. The extract of the present invention may also contain from 2 wt. % up to 7 wt. % sterols, in particular from 3 wt. % up to 6 wt. % sterols, for example - from 3 wt. % up to 5 wt. % sterols, based on the weight of the dry extract.

According to another particular embodiment of the present invention, the extract of the present invention contains at least 3 wt. % free linoleic acid, preferably at least 4 wt. % free linoleic acid, based on the weight of the dry extract. In particular, the extract of the present invention contains from 3 wt. % up to 15 wt. % free linoleic acid, in particular from 4 wt. % up to 10 wt. % free linoleic acid, in particular from 4 wt. % up to 6 wt. % free linoleic acid, for example - about 5 wt. % free linoleic acid, based on the weight of the dry extract. Preferably, the extract of the present invention contains less than 0.2 wt. % silymarin, more preferably less than 0.1 wt. % silymarin, based on the weight of the dry extract. The extract of the present invention may also contain from 10 wt. % up to 70 wt. % free fatty acids, in particular from 10 wt. % up to 30 wt. % free fatty acids, in particular from 15 wt. % up to 25 wt. % free fatty acids, based on the weight of the dry extract.

According to another particular embodiment of the present invention, the extract of the present invention contains from 0.5 wt. % to 2.5 wt. % beta-sitosterol, in particular from 1.0 wt. % to 2.0 wt. % beta-sitosterol, for example - about 1.5 wt. % beta-sitosterol, calculated on the weight of the dry extract, and from 3 wt. % up to 15 wt. % free linoleic acid, in particular from 4 wt. % up to 10 wt. % free linoleic acid, in particular from 4 wt. % up to 6 wt. % free linoleic acid, for example - about 5 wt. % free linoleic acid, based on the weight of the dry extract. Preferably, the extract of the present invention contains less than 0.2 wt. % silymarin, more preferably less than 0.1 wt. % silymarin, based on the weight of the dry extract. The weight ratio of silymarin-beta-sitosterol in the extract of the present invention may be less than 0.4, in particular less than 0.07. The extract of the present invention may also contain from 2 wt. % up to 7 wt. % sterols, in particular from 3 wt. % up to 6 wt. % sterols, for example - from 3 wt. % up to 5 wt. % sterols, calculated on the weight of dry extract, and from 10 wt. % up to 50 wt. % free fatty acids, in particular from 10 wt. % up to 30 wt. % free fatty acids, in particular from 15 wt. % up to 25 wt. % free fatty acids, based on the weight of the dry extract.

The extract of the present invention contains at least 0.01 wt. % tocopherols, in particular at least 0.05 wt. % tocopherols, based on the weight of the dry extract. In particular, the extract of the present invention contains from 0.01 wt. % to 0.5 wt. % tocopherols, in particular from 0.05 wt. % to 0.2 wt. % tocopherols, for example - about 0.1 wt. % tocopherols, based on the weight of the dry extract. Preferably, the extract of the present invention contains less than 0.2% silymarin, more preferably less than 0.1 wt. % silymarin, based on the weight of the dry extract. The weight ratio of silymarin/tocopherols in the extract of the present invention may be less than 1, in particular less than 0.1.

According to another particular embodiment of the present invention, the extract of the present invention contains less than 0.2 wt. % silymarin, preferably less than 0.1 wt. % silymarin, based on the weight of the dry extract; from 0.5 wt. % to 2.5 wt. % beta-sitosterol, in particular from 1.0 wt. % to 2.0 wt. % beta-sitosterol, for example - about 1.5 wt. % beta-sitosterol, based on the weight of the dry extract; from 3 wt. % up to 15 wt. % free linoleic acid, in particular from 4 wt. % up to 10 wt. % free linoleic acid, in particular from 4 wt. % up to 6 wt. % free linoleic acid, for example - about 5 wt. % free linoleic acid, based on the weight of the dry extract; and from 0.01 mass. % to 0.5 wt. % tocopherols, in particular from 0.05 wt. % to 0.2 wt. % tocopherols, for example - about 0.1 wt. % tocopherols, based on the weight of the dry extract. The extract of the present invention may also contain from 2 wt. % up to 7 wt. % sterols, in particular from 3 wt. % up to 6 wt. % sterols, for example - from 3 wt. % up to 5 wt. % sterols, calculated on the weight of dry extract, and from 10 wt. % up to 50 wt. % free fatty acids, in particular from 10 wt. % up to 30 wt. % free fatty acids, in particular from 15 wt. % up to 25 wt. % free fatty acids, based on the weight of the dry extract. The weight ratio of silymarin-beta-sitosterol in the extract of the present invention can be in particular less than 0.4, in particular less than 0.07. The weight ratio of silymarin/tocopherols in the extract of the present invention may in particular be less than 1, in particular less than 0.1.

Preferably the extract of the present invention is a dry extract.

The extract of the present invention can be obtained by extracting an oil derived from the achenes of Silybum marianum (L.) Gaertn. using an extraction solvent containing (in particular consisting of these components) an aqueous hydrotrope solution, subcritical water or an organic solvent immiscible with oil originating from achenes of Silybum marianum (L.) Gaertn., optionally mixed with water, in particular an organic solvent immiscible with an oil originating from achenes of Silybum marianum (L.) Gaertn., such as C ₁ -C ₃ alcohol, optionally mixed with water.

In particular, the extraction solvent contains (in particular consists of) methanol, ethanol or isopropanol, optionally mixed with water, in particular in an organic solvent/water volume ratio ranging from 80/20 to 100/0, in in particular from 85/15 to 95/5, in particular in a volume ratio of approximately 90/10.

The oil from the achenes of Silybum marianum can preferably be obtained by extraction from the achenes of Silybum marianum (L.) Gaertn. (the seeds may be whole or in the form of pieces), in particular by pressing, preferably by cold pressing (ie without heating, at room temperature).

The step of extracting the oil from the seeds of Silybum marianum can be carried out by mixing the oil with the extraction solvent for a period of time ranging from 1 hour to 12 hours, in particular at a temperature ranging from 15° C. to 25° C., in particular - at a temperature of approximately 20°C. The amount of extraction solvent used to carry out this extraction is preferably in the range of 0.5 g to 3 g per 1 g of oil, more specifically in the range of 1 g to 3 g per 1 g of oil.

The extraction phase thus obtained is then separated from the lipid phase, after which it is dried and evaporated, partially or completely, in particular under vacuum, in order to substantially remove the extraction solvent and obtain either a dry extract, if the solvent is completely removed, or a concentrated extract, which diluted with residual solvent.

Combination of the present invention

The combination of the present invention contains a retinoid as defined above and an extract of Silybum marianum (L.) Gaertn. as defined above.

The combination according to the present invention is preferably a combination of retinaldehyde as a retinoid with an extract of Silybum marianum, in particular with an extract from Silybum marianum achenes, essentially free of silymarin, that is, containing less than 0.2 wt. % silymarin, preferably less than 0.1 wt. % silymarin, based on the weight of the dry extract.

Dermatological and dermocosmetic compositions of the present invention

The subject of the present invention is also a dermatological or dermocosmetic composition containing at least one combination as defined above, in combination with at least one dermatologically or dermocosmetically acceptable adjuvant, more specifically intended for external use, in particular for application to the skin.

The dermatological and dermocosmetic compositions of the present invention may be presented in traditional topical forms, i.e. in particular in the form of lotions, foams, gels, dispersions, emulsions, sprays, serums, masks or creams, containing excipients providing, in in particular penetration through the skin to improve the properties and availability of the active principle. Preferably the composition is a cream.

These compositions typically contain, in addition to the ingredients of the combination of the present invention, a physiologically acceptable medium, usually based on water or a solvent such as alcohols, ethers or glycols. They may also contain surfactants, complexing agents, preservatives, stabilizers, emulsifiers, thickeners, gelling agents, humectants, emollients, trace elements, essential oils, fragrances, colorants, matting agents, chemical or mineral filters, moisturizers, thermal waters and etc.

These compositions may also contain other active ingredients leading to an additional or possible synergistic effect.

Preferably, the compositions of the present invention contain from 0.01 wt. % up to 10 wt. %, preferably from 0.05 wt. % up to 5 wt. %, extract of Silybum marianum according to the present invention (preferably in terms of the weight of the dry extract) of the total weight of the composition.

Preferably, the compositions of the present invention contain from 0.001 wt. % up to 5 wt. % retinoid, preferably from 0.01 wt. % to 1 wt. % retinoid based on the total weight of the composition.

Therapeutic Applications

The combinations of the present invention and the dermatological or dermocosmetic compositions of the present invention can be used to treat acne (for example, juvenile acne, also called teenage acne, or late acne, which may be cystic), seborrhea, seborrheic dermatitis and/or rosacea, preferably by means of external use, in particular - application to the skin.

The present invention is illustrated by the following non-limiting examples.

BRIEF DESCRIPTION OF GRAPHICS

Fig. 1 shows the UPLC chromatogram of Silybum marianum extract I prepared according to Protocol 1.

Fig. 2A, 2B and 2C are respectively UPLC chromatograms of Extract M, Extract E and Extract I of Silybum marianum prepared according to Protocol 2.

Fig. 3A, 3B, and 3C represent, respectively, the 10-22-minute zone (corresponding to the fatty acid zone) of the GC/MS chromatogram (GC/MS; from English: gas chromatography-mass spectrometry) of extract E, extract M, and extract I of Silybum marianum obtained under Protocol 3.

Fig. 4A, 4B and 4C represent, respectively, the 22-28 minute zone (corresponding to the sterol zone) of the GC/MS chromatogram (GC/MS; from English: gas chromatography-mass spectrometry) of extract E, extract M, and extract I of Silybum marianum, received under Protocol 3.

Fig. 5 represents the total amount of retinoids produced by skin cells as a function of the treatment received, i.e. vehicle alone (EtOH), 2 μM retinaldehyde (2 μM RAL), 50 μg/ml Silybum marianum extract (50 μg/ml SYL) or a combination of 2 μM retinaldehyde and 50 µg/ml extract of Silybum marianum (RAL + SYL).

Fig. 6 represents the total amount of retinoids produced by skin cells as a function of the treatment received, i.e. vehicle alone (EtOH), 2 μM retinol (2 μM ROL), 50 μg/ml Silybum marianum extract (50 μg/ml SYL) or a combination of 2 μM retinol and 50 µg/ml extract of Silybum marianum (ROL + SYL).

DESCRIPTION OF EXAMPLES OF CARRYING OUT THE INVENTION

I - Examples of preparations of the extracts of the present invention

Isopropanol extract 90 (Extract I) was obtained in the following way:

- cold pressing of achenes of Silybum marianum to obtain oil from achenes of Silybum marianum,

- extraction of the oil obtained from the seeds of Silybum marianum, with a mixture of isopropanol/water (volume ratio 90/10) using 1 gram of isopropanol/water per gram of oil for 2 hours at 20°C;

- isolation of the isopropanol phase, and

- evaporation of the solvent.

A methanol extract (Extract M) and an ethanol 90 extract (Extract E) were prepared in a similar manner by replacing isopropanol/water (90/10 v/v) with methanol and ethanol/water (90/10 v/v), respectively. Oil extraction from seeds of Silybum marianum was performed using 3 volumes of methanol and 3 volumes of ethanol/water (90/10 v/v) per volume of oil for 2 hours at 20°C, respectively.

These various extracts were characterized by ultra high performance liquid chromatography (UPLC) or gas chromatography combined with mass spectrometry (GC-MS) according to the protocols detailed below.

Protocols for evaluating the obtained extracts:

1. Protocol 1: Estimation of silymarin content by UPLC

• Preparation of test sample and control sample

- Control sample of silymarin: Prepare a solution of 5 mg of silymarin in 10 ml of a mixture of methanol/water (volume ratio 60:40).

- Test sample:

• Extract A: Prepare a solution of 100 mg of the extract to be analyzed in 10 ml of methanol/dichloromethane (70:30 v/v)

• Extracts M, E, I: Heat the dry extract to be analyzed to 35°C with stirring until a homogeneous and clear solution is obtained. Accurately weigh 200 mg (d) of the extract, solubilize it in 10 ml of a mixture of methanol/dichloromethane until the extract is completely solubilized and homogenize the solution. This mixture ranges from a methanol/dichloromethane volume ratio of 1:1 to pure methanol.

• Analysis conditions:

- Column: Acquity BEN Shield C18 150 mm × 2.1 mm - 1.7 µm (manufactured by Waters)

- Mobile phase:

• A: Water + 0.1% formic acid

• B: Acetonitrile + 0.1% formic acid

- Gradient:

- Column temperature: 40°C

- Volume flow rate: 0.4 ml/min

- Detection: 287 nm

- Injection volume: 1 µl

2. Protocol 2: Evaluation of linoleic acid content by UPLC

• Preparation of test sample and control sample

- Control sample of linoleic acid: Prepare a solution of 10 mg of linoleic acid in 10 ml of a mixture of methanol/dichloromethane (volume ratio 1:1).

- Test sample:

• Extracts M, E, I: Heat the dry extract to be analyzed to 35°C with stirring until a homogeneous and clear solution is obtained. Accurately weigh 50 mg (d) of the extract, solubilize it in 1 ml of a mixture of methanol/dichloromethane until the extract is completely solubilized, and homogenize the solution. This mixture ranges from a methanol/dichloromethane volume ratio of 1:1 to pure methanol.

• Analysis conditions:

- Column: Acquity BEN Shield C18 150 mm × 2.1 mm - 1.7 µm (manufactured by Waters)

- Mobile phase:

• A: Water + 0.1% formic acid

• B: Acetonitrile + 0.1% formic acid

- Gradient:

- Column temperature: 40°C

- Volume flow rate: 0.4 ml/min

- Detection: 215 nm

- Injection volume: 1 µl

3. Protocol 3: Evaluation of fatty acid and sterol content by GC-MS

• Sample preparation

- Heat the dry extract to be analyzed to 35°C with stirring until a clear homogeneous liquid is obtained.

- Solubilize 20 mg of the extract in 800 ml of methanol/dichloromethane (1:1 v/v).

- Add 200 µl Derivatization Reagent N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA; N,O-bis(trimethylsilyl)trifluoroacetamide) + trimethylchlorosilane (TMCS; trimethylchlorosilane) (99:1) (Supelco - manufactured by Sigma Aldrich).

- Stir with a vortex mixer for 1 minute.

• Gas Chromatography (GC) Conditions

- Column: DB-5ms (manufactured by Agilent technologies); 30 m × 0.25 mm; 0.25 µm.

- Injection: T=300°C; mode: thread division; division ratio = 100:1

- Thermostat: Temperature gradient (°C):

• Initial temperature = 150°C

• Gradient = 7°C/min to final temperature = 340°C

• Maintain a temperature of 340°C for 10 minutes

- Volume flow rate of carrier gas: 1 ml/min

- Detection: MS-EI (electron ionization mass spectrometry); T=300°C; scan time = 0.2 s; initial mass of full scan = 40; final mass of full scan=600

- Injection volume: 1 µl

Results:

Extract I contains predominantly substances with a retention time ranging from 13 minutes to 30 minutes in UPLC (see Fig. 1). It has the following characteristics:

UPLC and GC/MS chromatograms of 3 extracts (extracts I, M and E) of Silybum marianum were similar (see Fig. 2 and Fig. 3).

II - Examples of compositions containing a retinoid and/or extract of Silybum marianum according to the present invention

COMPOSITION 1 (cream) based on retinaldehyde = RAL1

COMPOSITION 2 (cream) based on retinaldehyde = RAL2

COMPOSITION 3 (cream) based on retinaldehyde = RAL3

COMPOSITION 4 (cream) based on retinaldehyde = RAL4

Retinaldehyde was added in an amount of 0.1 wt. % in a mixture containing:

5% glycerin

1.5% gelling agent

1% silica

Water - as needed.

COMPOSITION 5 (cream) based on Silybum marianum extract = SYL1

Extract I, obtained in Example 1, was added in the amount of 7 wt. % in a mixture of isopropanol/PEG 300 (mass ratio 1:1).

COMPOSITION 6 (cream) based on Silybum marianum extract = SYL2 (mass percent)

25% SYL1

5% glycerin

1.5% gelling agent

1% silica

Water - as needed.

COMPOSITION 7 (cream) containing a combination of Silybum marianum extract and retinaldehyde = ASSO1 (wt. percent)

25% SYL1

0.1% retinaldehyde

5% glycerin

1.5% gelling agent

1% silica

Water - as needed.

COMPOSITION 8 (cream) containing a combination of Silybum marianum extract and retinaldehyde = ASSO2 (mass percent)

12.5% SYL1

0.1% retinaldehyde

5% glycerin

1.5% gelling agent

1% silica

Water - as needed.

III - Clinical Studies

The various creams studied were applied to the face (cleansed skin) 2 times/day in the morning and in the evening by light massage, except for the compositions containing RAL: they were applied only in the evening.

Analysis of the obtained therapeutic effects was performed according to the Investigator Global Assessment (IGA) method approved by the US Food and Drug Administration (FDA; Food and Drug Administration) for acne and suitable for rosacea and seborrheic dermatitis, which is performed by dermatologists (Guidance for Industry Acne vulgaris: Developing Drugs for Treatment).

Using this method, the following IGA scores were established according to the severity of acne observed:

The results of various analyzes are presented below.

Analysis 1: Comparison of conditions before treatment, conditions after treatment with Silybum marianum extract without RAL and treatment with the combination of RAL + Silybum marianum extract

Analysis 2: Comparison of pre-treatment, post-treatment conditions with RAL without Silybum marianum extract and treatment with the combination of RAL + Silybum marianum extract

Conclusion

The results obtained with various treatments based respectively on the combination of retinaldehyde and Silybum marianum extract, retinaldehyde alone or Silybum marianum extract alone, in the clinical studies described above, show that retinaldehyde in combination with Silybum marianum extract provides a better clinical outcome. .

IV - In vitro studies: evaluation of the correction of vitamin A deficiency with a combination of retinaldehyde (RAL) or retinol (ROD + Silybum marianum extract (SYL)

Materials and methods:

Culture:

• 3 days before the experiment, A431 epidermoid carcinoma cells were cultured in 2 6-well plates at a density of 40,000 cells/well (2 ml/well).

Cell treatment (3 wells for each condition, 2 ml/well; final concentrations):

• Ethanol (solvent): 1% ethanol

• ROL: 10 µM ROL in 1% ethanol

• RAL: 2 µM RAL in 1% ethanol

• SYL: 50 µg/ml Silybum marianum Extract I in 1% ethanol

• ROL+SYL: 10 µM ROL + 50 µg/ml Silybum marianum extract in 1% ethanol

• RAL+SYL: 2 µM RAL + 50 µg/ml Silybum marianum extract in 1% ethanol

Incubation: 1 hour

Cell collection:

• Remove medium, wash twice with phosphate buffer solution (PBS; from English: phosphate saline buffer);

• Add 500 µl/well EDTA-NaOH (0.02% ethylenediaminetetraacetic acid (EDTA) + 200 µM NaOH), let stand for 10-15 minutes, then separate the cells with a rubber scraper and transfer to glass extraction tubes;

• Add 10 µl of 20 mM HCl solution, sonicate briefly, then transfer 10 µl of the suspension into 1.5 ml Eppendorf tubes for protein determination;

• In glass tubes, add 500 µl of 200 µM butylated hydroxytoluene (BHT) in ethanol, 20 µl of 20 µM retinyl acetate solution (internal standard), then 4 ml of hexane;

• Mix vigorously using a vortex mixer, then centrifuge the tubes (900g, 5 minutes);

• Transfer the supernatant to open glass tubes, evaporate to dryness under nitrogen atmosphere;

• Redissolve in 200 µl methanol, then transfer to high performance liquid chromatography (HPLC) vials.

Retinoid analysis by HPLC:

• Agilent 1100 HPLC chain with quaternary injection pump and diode array detector

• Macherey-Nagel Nucleodur C ₁₈ column, pyramidal, 3 µm

• Mobile phase:

- 0-6 minutes: 100% methanol

- 6-8 minutes: linear gradient to methanol/tetrahydrofuran (THF) (4:1)

- 8-18 minutes: methanol/THF (4:1)

-18-20 minutes: linear gradient to 100% methanol

- 20-30 minutes: 100% methanol

• Detection: 325 nm (retinol, retinol esters); 383 nm (retinal).

Results:

The results obtained are presented in Fig. 5 and FIG. 6.

Conclusion:

Silybum marianum extract alone did not produce significant retinoid production. On the other hand, skin cells produced significantly more retinoids in the presence of the combination of Silybum marianum extract with retinaldehyde or retinol than in the presence of retinaldehyde alone or retinol alone, demonstrating the synergistic effect of the combination of the present invention.

Claims (15)

1. Dermatological or dermocosmetic composition for the treatment of diseases selected from acne, seborrhea, rosacea and / or seborrheic dermatitis, containing at least one dermatologically or dermocosmetically acceptable excipient and at least one combination containing a retinoid and an extract of Silybum marianum (L. ) Gaertn., characterized in that the specified retinoid is retinaldehyde and is present in the composition in an amount of from 0.05 wt.% to 5 wt.% of the total mass of the composition and characterized in that the extract of Silybum marianum (L.) Gaertn. is an extract of seeds of Silybum marianum (L.) Gaertn., containing less than 0.2 wt.% silymarin in terms of the weight of dry extract, is present in the composition in an amount of from 0.05 wt.% to 10 wt.% of the total mass of the composition and is the resulting method comprising the following successive steps: (i) pressing the achenes of Silybum marianum (L.) Gaertn. with obtaining oil; (ii) extraction of the oil obtained from the seeds of Silybum marianum (L.) Gaertn. in step (i), an extraction solvent selected from subcritical water and a C ₁ -C ₃ alcohol, optionally mixed with water, to obtain an extraction phase and a lipid phase; (iii) isolating the extraction phase obtained in step (ii), and (iv) partial or complete drying of the extraction phase to obtain a concentrated or dry extract. 2. Dermatological or dermocosmetic composition according to claim 1, characterized in that the extract of Silybum marianum (L.) Gaertn. is an extract of seeds of Silybum marianum (L.) Gaertn., containing less than 0.1 wt.% silymarin, calculated on the weight of the dry extract. 3. Dermatological or dermocosmetic composition according to claim 1 or 2, characterized in that the extract of Silybum marianum (L.) Gaertn. contains from 0.5 wt.% to 2.5 wt.% beta-sitosterol, in particular from 1.0 wt.% to 2.0 wt.% beta-sitosterol, for example about 1.5 wt.% beta -sitosterol, based on the weight of the dry extract, preferably with a weight ratio of silymarin/beta-sitosterol of less than 0.4, in particular less than 0.07, and in particular from 2 wt.% to 7 wt.% sterols , in particular - from 3 wt.% to 6 wt.% sterols, for example - from 3 wt.% to 5 wt.% sterols, calculated on the weight of the dry extract. 4. Dermatological or dermocosmetic composition according to any one of paragraphs. 1-3, characterized in that the extract of Silybum marianum (L.) Gaertn. contains from 3 wt.% to 15 wt.% free linoleic acid, in particular from 4 wt.% to 10 wt.% free linoleic acid, in particular from 4 wt.% to 6 wt.% free linoleic acid, for example - about 5 wt.% free linoleic acid, calculated on the weight of the dry extract, and in particular from 10 wt.% to 50 wt.% free fatty acids, in particular from 10 wt.% to 30 wt.% free fatty acids, in particular from 15 wt.% to 25 wt.% free fatty acids, calculated on the weight of the dry extract. 5. Dermatological or dermocosmetic composition according to any one of paragraphs. 1-4, characterized in that the extract of Silybum marianum (L.) Gaertn. contains from 0.01 wt.% to 0.5 wt.% tocopherols, in particular from 0.05 wt.% to 0.2 wt.% tocopherols, for example, about 0.1 wt.% tocopherols, calculated on the mass dry extract, and preferably the mass ratio of silymarin/tocopherol is less than 1, in particular less than 0.1. 6. Dermatological or dermocosmetic composition according to any one of paragraphs. 1-5, characterized in that stage (i) is carried out by cold pressing the seeds of Silybum marianum (L.) Gaertn. 7. Dermatological or dermocosmetic composition according to any one of paragraphs. 1-6, characterized in that the C ₁ -C ₃ alcohol is selected from methanol, ethanol, isopropanol and mixtures thereof, preferably is isopropanol. 8. Dermatological or dermocosmetic composition according to any one of paragraphs. 1-7, characterized in that the extraction solvent is a mixture of isopropanol/water in a volume ratio of 90/10. 9. Dermatological or dermocosmetic composition according to any one of paragraphs. 1-8, characterized in that it contains from 0.05 wt.% to 5 wt.%, extract of Silybum marianum (L.) Gaertn., by weight of dry extract, in terms of the total weight of the composition. 10. Dermatological or dermocosmetic composition according to any one of paragraphs. 1-9, characterized in that it contains from 0.05 wt.% to 1 wt.% retinoid in terms of the total weight of the composition. 11. Dermatological or dermocosmetic composition according to any one of paragraphs. 1-10, characterized in that it is intended for external use, in particular for application to the skin.

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