HK40004167B - Novel silybum marianum achene extract and uses thereof in dermatology and dermo-cosmetics - Google Patents

Description

New Silybum marianum achene extract and its use in dermatology and dermocosmetics

Technical Field

The present invention relates to a novel Silybum marianum achene extract, more particularly for topical application, as well as to pharmaceutical or cosmetic compositions comprising it, a process for its preparation and its use in dermatology or cosmetics, in particular for the treatment of acne, seborrhea, seborrheic dermatitis and/or rosacea.

Background Art

The scientific name Silybum marianum (L.) Gaertn. refers to a plant belonging to the Asteraceae family. It is an annual or biennial with a strong stem that can reach a meter in height. Its large, glossy, alternating leaves lack stipules, are spotted with white, and are surrounded by hard, sharp spines. The flowers are clustered in heads and are usually solitary. They are surrounded by large, spiny bracts with very sharp ends. The flowers, with their small tubes and five lobes, are violet-purple. The fruits are shiny, black or yellow-spotted achenes, topped with a pappus with toothed bristles arranged in a ring at the base. The common name for this plant is milk thistle.

The achenes of Silybum marianum (often incorrectly referred to as seeds in the literature) and preparations thereof are commonly used orally for the symptomatic treatment of digestive disorders attributed to a hepatic origin.

The main active ingredient in milk thistle achenes is silymarin, a mixture of flavonolignans (primarily silybin, isosilybin, silybin, and silymarin). Achenes contain up to 3% silymarin by weight. They also contain oil (20-30% by weight), mucilage, and protein.

Silymarin has been the subject of numerous studies (in vitro, in vivo, and clinical) that have demonstrated its antioxidant, hepatoprotective, digestive, or anti-inflammatory properties.

Currently, silymarin-rich Silybum marianum achene extracts are present in a variety of pharmaceutical preparations for the treatment of various liver and bile diseases, e.g.

Furthermore, silymarin has been the subject of investigation for the treatment of acne (Sahib et al., 2012) and erythematoteleangiectatic rosacea associated with methylsulfonylmethane (MSM) (Berardesca et al., 2008).

Milk thistle oil is rich in omega-6 and vitamin E and is primarily used in cooking. It is typically obtained from the achenes by cold pressing.

However, studies on the antioxidant and hepatoprotective properties of this orally administered milk thistle oil have been conducted in rats or mice (Hermenean et al. 2015; Zhu et al. 2014).

Summary of the Invention

Summary of the Invention

The applicant has surprisingly demonstrated that a Silybum marianum achene extract low in silymarin exhibits very interesting properties for the treatment of acne, seborrhea, rosacea or seborrheic dermatitis.

One subject of the present invention is therefore an extract of Silybum marianum achenes comprising less than 0.2% by weight, preferably less than 0.1% by weight, of silymarin relative to the weight of the dry extract, for use in the treatment of acne, seborrhea, rosacea and/or seborrheic dermatitis.

Another subject of the present invention is the use of an extract of Silybum marianum achenes, containing less than 0.2% by weight, preferably less than 0.1% by weight, of silymarin relative to the weight of the dry extract, for the preparation of a pharmaceutical product for the treatment of acne, seborrhea, rosacea and/or seborrheic dermatitis.

Another subject of the present invention is the use of milk thistle for treating acne, seborrhea, rosacea and/or seborrheic dermatitis, said milk thistle comprising less than 0.2% by weight, preferably less than 0.1% by weight, of silymarin relative to the weight of the dry extract.

Another subject of the present invention relates to a method for treating acne, seborrhea, rosacea and/or seborrheic dermatitis, comprising administering to a subject in need thereof an effective amount of an extract of Silybum marianum achenes, said extract comprising less than 0.2% by weight, preferably less than 0.1% by weight, of silymarin relative to the weight of the dry extract.

Another subject of the present invention is an extract of Silybum marianum achenes comprising less than 0.2% by weight, preferably less than 0.1% by weight, of silymarin relative to the weight of the dry extract.

Another subject of the present invention is an extract of Silybum marianum achenes comprising less than 0.2% by weight, preferably less than 0.1% by weight, of silymarin relative to the weight of the dry extract, for use as a pharmaceutical product.

Another subject of the present invention relates to a pharmaceutical (especially dermatological) or cosmetic (especially dermocosmetic) composition comprising an extract of Silybum marianum achenes containing less than 0.2% by weight, preferably less than 0.1% by weight, of silymarin relative to the weight of the dry extract, in admixture with at least one pharmaceutically or cosmetically acceptable excipient.

Another subject of the present invention relates to a pharmaceutical (especially dermatological) or cosmetic (especially dermocosmetic) composition comprising an extract of Silybum marianum achenes, in admixture with at least one pharmaceutically or cosmetically acceptable excipient, said extract comprising less than 0.2% by weight, preferably less than 0.1% by weight, of silymarin relative to the weight of the dry extract, for the treatment of acne, seborrhea, rosacea and/or seborrheic dermatitis.

Another subject of the present invention relates to the use of a pharmaceutical (especially dermatological) or cosmetic (especially dermocosmetic) composition for the treatment of acne, seborrhea, rosacea and/or seborrheic dermatitis, said composition comprising an extract of Silybum marianum achenes, said extract comprising less than 0.2% by weight, preferably less than 0.1% by weight, relative to the weight of the dry extract, of silymarin, in admixture with at least one pharmaceutically or cosmetically acceptable excipient.

Another subject of the present invention relates to a method for treating acne, seborrhea, rosacea and/or seborrheic dermatitis, comprising administering to a subject in need thereof, preferably by topical route, an effective amount of a pharmaceutical (particularly dermatological) or cosmetic (particularly dermocosmetic) composition comprising an extract of Silybum marianum achenes, said extract comprising less than 0.2% by weight, preferably less than 0.1% by weight, of silymarin relative to the weight of the dry extract, in admixture with at least one pharmaceutically or cosmetically acceptable excipient.

Another subject of the present invention relates to a process for preparing an extract of milk thistle achenes comprising a step of extracting the oil obtained from the milk thistle achenes, said extract comprising less than 0.2% by weight, preferably less than 0.1% by weight, of silymarin relative to the weight of the dry extract, with an extraction solvent comprising, in particular consisting of, a hydrophilic aqueous solution, subcritical water or an organic solvent immiscible with the oil obtained from milk thistle, said organic solvent optionally being mixed with water.

Detailed Description of the Invention

In this specification, the milk thistle (Silybum marianum (L.) Gaertn.) plant may be referred to using the abbreviation Silybum marianum.

According to the present invention, the term "silymarin" refers to a purified Silybum marianum achene extract comprising primarily (at least 95% by weight) a mixture of the following four flavonolignans: silybin, isosilybin, silychristin, and silydianin (Kuki et al., 2012). A silymarin content of less than 0.2% by weight means that the total amount of silymarin components is less than 0.2% by weight. Such a content can be determined by HPLC (high performance liquid chromatography) or UPLC (ultra-performance liquid chromatography) by calculating the total area of the peaks corresponding to all components of silymarin, in particular using a reference silymarin sample (e.g., available from Sigma Aldrich) to determine the positions of these peaks.

According to the present invention, the term "silybin", also known in the art as silibinin, refers to the four diastereoisomers silybin A, silybin B, 2,3-cis-silybin A and 2,3-cis-silybin B.

According to the present invention, the term "isosilybin" refers to the two diastereomers isosilybin A and isosilybin B.

According to the present invention, the term "silymarin" refers to the two diastereoisomers silymarin A and silymarin B.

In this specification, the term "about" means that the value under consideration may be 10%, particularly 5%, especially 1% less or greater than the indicated value.

According to the present invention, the term "dry extract" refers to an extract that contains no extraction solvent or only insignificant trace levels of extraction solvent. Thus, such a dry extract contains only substances obtained from milk thistle. It may also contain insignificant traces of extraction solvent.

According to the present invention, the term "organic solvent immiscible with the oil obtained from milk thistle achenes" means an organic solvent olefin that is not miscible or only partially miscible with the oil obtained from milk thistle achenes, so that the mixture of the organic solvent and the oil obtained from milk thistle achenes results in a heterogeneous mixture in which at least two separate phases can be observed.

Extraction according to the present invention

The extract according to the invention is a Silybum marianum achene extract comprising less than 0.2% by weight, preferably less than 0.1% by weight, of silymarin relative to the weight of the dry extract.

According to a particular embodiment, the extract according to the invention comprises at least 0.5% by weight, preferably at least 1.0% by weight, of β-sitosterol, relative to the weight of the dry extract. In particular, the extract according to the invention comprises 0.5% to 2.5% by weight, particularly 1.0% to 2.0% by weight, for example about 1.5% by weight, of β-sitosterol, relative to the weight of the dry extract. In particular, the extract according to the invention has a silymarin/β-sitosterol mass ratio of less than 0.4, in particular less than 0.07. The extract according to the invention may further comprise 2 to 7% by weight, particularly 3 to 6% by weight, for example 3 to 5% by weight, of sterols, relative to the weight of the dry extract.

According to the present invention, the term "sterol" means a molecule having a sterane nucleus in which carbon 3 carries a hydroxyl group OH, the sterane having the following structure:

According to a particular embodiment, the extract according to the invention comprises at least 3% by weight, preferably at least 4% by weight, of free linoleic acid relative to the weight of the dry extract. In particular, the extract according to the invention comprises 3% to 15% by weight, particularly 4% to 10% by weight, especially 4% to 6% by weight, for example about 5% by weight, of free linoleic acid relative to the weight of the dry extract. The extract according to the invention may further comprise 10% to 70% by weight, particularly 10% to 30% by weight, and especially 15% to 25% by weight of free fatty acids.

According to the invention, the term "fatty acid" means a carboxylic acid _R1CO2H , wherein the chain R1 is a linear or branched hydrocarbon chain, saturated or containing a double C=C bond, said carboxylic acid containing from 16 to 22 carbon atoms (including the carbon atoms of the carboxylic acid function).

According to the present invention, the term "free" fatty acids (including linoleic acid) refers to fatty acids that are not bound to other molecules, such as glycerol or its derivatives to produce glycerides or alcohols to produce fatty esters.

According to a further specific embodiment, the extract according to the invention comprises 0.5% to 2.5% by weight, in particular 1.0% to 2.0% by weight, for example about 1.5% by weight, of β-sitosterol, relative to the weight of the dry extract, and 3% to 15% by weight, in particular 4% to 10% by weight, especially 4% to 6% by weight, for example about 5% by weight, of free linoleic acid, relative to the weight of the dry extract. In particular, the extract according to the invention may have a silymarin/β-sitosterol mass ratio of less than 0.4, in particular less than 0.07. The extract according to the invention may further comprise 2% to 7% by weight, in particular 3% to 6% by weight, for example 3% to 5% by weight, relative to the weight of the dry extract, of sterols, and 10% to 50% by weight, in particular 10% to 30% by weight, and in particular 15% to 25% by weight, relative to the weight of the dry extract, of free fatty acids.

According to a particular embodiment, the extract according to the invention comprises at least 0.01% by weight, in particular at least 0.05% by weight, of tocopherol relative to the weight of the dry extract. In particular, the extract according to the invention comprises from 0.01% to 0.5% by weight, in particular from 0.05% to 0.2% by weight, for example about 0.1% by weight, of tocopherol relative to the weight of the dry extract. In particular, the extract according to the invention may have a silymarin/tocopherol mass ratio of less than 1, in particular less than 0.1.

According to the present invention, the term "tocopherol" denotes alpha-tocopherol, beta-tocopherol, gamma-tocopherol and delta-tocopherol.

According to a further specific embodiment, the extract according to the invention comprises 0.5% to 2.5% by weight, in particular 1.0% to 2.0% by weight, for example, about 1.5% by weight of β-sitosterol, relative to the weight of the dry extract; 3% to 15% by weight, in particular 4% to 10% by weight, especially 4% to 6% by weight, for example, about 5% by weight of free linoleic acid, relative to the weight of the dry extract; and 0.01% to 0.5% by weight, in particular 0.05% to 0.2% by weight, for example, about 0.1% by weight of tocopherol, relative to the weight of the dry extract. The extract according to the invention may further comprise 2% to 7% by weight, in particular 3% to 6% by weight, for example, 3% to 5% by weight of sterols, relative to the weight of the dry extract, and 10% to 50% by weight, in particular 10% to 30% by weight, and in particular 15% to 25% by weight of free fatty acids, relative to the weight of the dry extract. In particular, the extract according to the invention may have a silymarin/β-sitosterol mass ratio of less than 0.4, in particular less than 0.07. In particular, the extract according to the invention may have a silymarin/tocopherol mass ratio of less than 1, in particular less than 0.1.

Preferably, the extract according to the invention is a dry extract.

According to a preferred embodiment, the extract according to the invention is suitable for being obtained by the process according to the invention described hereinafter.

Method for preparing the extract according to the invention

A method for preparing an extract according to the invention, comprising a step of extracting the oil obtained from the achenes of milk thistle with an extraction solvent comprising, in particular consisting of, a hydrophilic aqueous solution, subcritical water or an organic solvent immiscible with the oil obtained from the achenes of milk thistle, optionally mixed with water.

According to a particular embodiment, the organic solvent comprises, in particular consists of, an organic solvent that is immiscible with the oil obtained from Silybum marianum achenes, optionally mixed with water.

The organic solvent immiscible with the oil obtained from the achenes of milk thistle may in particular be a C ₁ to C ₃ alcohol.

The extraction solvent may especially be a C ₁ to C ₃ alcohol, optionally mixed with water.

According to the invention, the term " _C1 to _C3 alcohol" refers to an alcohol R2OH in which the chain R2 is a saturated, linear or branched hydrocarbon chain containing 1 to 3 carbon atoms. It may be composed of methanol, ethanol, n-propanol or isopropanol, in particular methanol, ethanol or isopropanol. Preferably, it is isopropanol.

Organic solvents immiscible with the oil obtained from the achenes of Silybum marianum, in particular _C1 to _C3 alcohols such as methanol, ethanol or isopropanol, may be used in admixture with water, in particular in an organic solvent/water volume ratio of 80/20 to 100/0, in particular 85/15 to 95/5, in particular about 90/10.

The extraction solvent may in particular be chosen from methanol, methanol/water mixtures, ethanol, ethanol/water mixtures, isopropanol and isopropanol/water mixtures.

According to a preferred embodiment, the extraction solvent is methanol, an ethanol/water mixture with a volume ratio of about 90/10 or an isopropanol/water mixture with a volume ratio of about 90/10, preferably an isopropanol/water mixture with a volume ratio of about 90/10.

In particular, the step of extracting the oil obtained from the milk thistle achenes is carried out by mixing the oil obtained from the milk thistle achenes with an extraction solvent for 1 to 12 hours, in particular at a temperature of 15 to 25° C., in particular about 20° C. The amount of extraction solvent used to carry out this extraction is in particular 0.5 to 3 g, in particular 1 to 3 g, per 1 g of the oil obtained from the milk thistle achenes.

At the end of this extraction, an extract phase and a lipid phase are then obtained. The extract phase will advantageously be separated from the lipid phase and recovered, then dried partially or completely, in particular in vacuo, to remove more or less of the extraction solvent and obtain a dry extract (if the solvent has been completely removed) or a concentrated extract diluted in a residual solvent.

The oil obtained from milk thistle achenes can advantageously be obtained by extraction from the achenes (the achenes may be whole or in pieces), in particular by pressing, advantageously by cold pressing (ie at ambient temperature without heating).

According to one embodiment of the present invention, the method according to the present invention will comprise the following two consecutive steps:

(i) extracting oil from milk thistle achenes, and

(ii) extracting the oil obtained from the milk thistle achenes with an extraction solvent comprising, in particular consisting of, a hydrophilic aqueous solution, subcritical water or an organic solvent immiscible with the oil obtained from the milk thistle achene extract, optionally mixed with water.

According to a preferred embodiment of the present invention, the method according to the present invention will comprise the following consecutive steps:

(i) optionally extracting oil from milk thistle achenes, and

(ii) extracting the oil obtained from the milk thistle achenes with an extraction solvent comprising, in particular consisting of, a hydrophilic aqueous solution, subcritical water or an organic solvent immiscible with the oil obtained from the milk thistle achene extract, optionally mixed with water.

(iii) recovering the extract phase obtained in step (ii), and

(iv) Partial or complete drying of the extracted phase to produce a concentrated extract or a dry extract according to the invention.

Step (i) will advantageously be carried out by cold pressing the milk thistle achenes (whole or in pieces).

Step (ii) will advantageously be carried out with an extraction solvent as defined above, in particular chosen from methanol, methanol/water mixtures, ethanol, ethanol/water mixtures, isopropanol and isopropanol/water mixtures.

Organic solvents immiscible with the oil obtained from the achenes of milk thistle, in particular _C1 to _C3 alcohols such as methanol, ethanol or isopropanol, can be used mixed with water, in particular in an organic solvent/water ratio by volume of 80/20 to 100/0, in particular 85/15 to 95/5, in particular about 90/10. An advantageous extraction solvent is an isopropanol/water mixture in a volume ratio of about 90/10.

The extraction step (ii) can be carried out, in particular, at a temperature of 15 to 25° C., in particular at about 20° C., by mixing the oil obtained from the milk thistle achenes with an extraction solvent for 1 to 12 hours. The amount of extraction solvent used to carry out this extraction is advantageously between 0.5 and 3 g, in particular between 1 and 3 g, per 1 g of the oil obtained from the milk thistle achenes. This extraction step (ii) ultimately makes it possible to obtain the desired extract phase and lipid phase.

Step (iii) is advantageously carried out by separating the extract phase from the lipid phase.

Step (iv) will advantageously be carried out in vacuo.

The pharmaceutical or cosmetic composition according to the invention

The pharmaceutical or cosmetic composition according to the invention comprises an extract according to the invention as defined above, mixed with at least one pharmaceutically or cosmetically acceptable excipient. It is preferably a topical composition.

In the present invention, the term "pharmaceutically or cosmetically acceptable" means that it can be used to prepare a pharmaceutical or cosmetic composition, that is generally safe, non-toxic, neither biologically nor otherwise undesirable, and that it is acceptable for pharmaceutical or cosmetic use, especially dermatological or skin cosmetic use, especially by topical application.

The compositions according to the invention are advantageously used for topical application, in particular on the skin.

Thus, the composition according to the invention may be in the form commonly known for topical application, i.e. in particular lotions, foams, gels, dispersions, emulsions, sprays, serums, masks or creams, wherein the excipients are particularly suitable for skin penetration, thereby enhancing the properties and accessibility of the active ingredient. Advantageously, it is a cream.

In addition to the extracts according to the invention, these compositions generally contain a physiologically acceptable medium, generally based on water or solvents 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, mattifying agents, chemical or mineral filters, moisturizers, mineral water, etc.

According to a particular embodiment, the composition according to the invention comprises at least one pharmaceutically or cosmetically acceptable excipient chosen from oil-control agents, moisturizers and mixtures thereof. According to another embodiment, the composition according to the invention comprises at least one oil-control agent and/or at least one moisturizer and optionally at least one chemical or mineral filter (UV filter).

Moisturizers increase the moisture content of the skin, keeping it soft and smooth. They can be, for example, urea, amino acids, glycerol (also known as glycerin), propylene glycol, butylene glycol, sorbitol, xylitol, maltitol, mannitol, polydextrose, collagen, elastin, hyaluronic acid and its salts (e.g., sodium or potassium salts), pectin, gelatin, chitosan, aloe vera juice, honey, or a mixture thereof.

An oil-control agent is an ingredient that makes the skin matt, which prevents the skin from being shiny. It can be, for example, talc, silica, rice flour or a mixture thereof, especially in micronized form.

UV filters are compounds that block or absorb ultraviolet (UV) light, in order to protect the skin in particular from the sun's UV. They can be, for example, UVA filters, UVB filters, broad-spectrum filters or mixtures thereof.

According to another particular embodiment, the composition according to the invention comprises isopropyl alcohol, polyethylene glycol (PEG) or a mixture thereof as a pharmaceutically or cosmetically acceptable excipient. Thus, advantageously, the composition according to the invention will comprise, in particular consist of, an extract according to the invention, isopropyl alcohol and polyethylene glycol (PEG). Said composition may also comprise an extract according to the invention, isopropyl alcohol, polyethylene glycol (PEG) and at least one ingredient chosen from oil-control agents, moisturizers, UV filters and mixtures thereof. Said composition may also comprise an extract according to the invention, isopropyl alcohol, polyethylene glycol (PEG), at least one oil-control agent and/or at least one other moisturizer, and optionally at least one chemical or mineral filter (UV filter). Oil-control agents, moisturizers and UV filters are as defined above.

The mass ratio of isopropanol/PEG is advantageously 1/2 to 2/1, in particular 1/1.5 to 1.5/1, in particular about 1/1.

The average molecular weight of the polyethylene glycol is in particular 200 to 600 g/mol, in particular 200 to 500 g/mol, in particular 200 to 400 g/mol, for example 250 to 350 g/mol, in particular about 300 g/mol. Thus, it may in particular be PEG 300.

These compositions may further contain other active ingredients which produce complementary or, optionally, synergistic effects.

Advantageously, the composition according to the invention will comprise at least 0.001% by weight, especially 0.001 to 15% by weight, 0.001 to 10% by weight or 0.001 to 5% by weight, particularly 0.01 to 15% by weight, 0.01 to 10% by weight or 0.01 to 5% by weight, preferably 0.1 to 10% by weight and especially 0.1 to 5% by weight of an extract according to the invention relative to the total volume of the composition.

Preferably, the composition according to the invention as defined above does not contain any vegetable oil, in particular any oil.

Therapeutic applications

The extracts according to the invention and pharmaceutical compositions comprising the same are suitable for the treatment of acne (e.g. juvenile acne, also known as teenage acne, or adult acne, which may be cystic), seborrhea, seborrheic dermatitis and/or rosacea, preferably for topical application, in particular on the skin.

BRIEF DESCRIPTION OF THE DRAWINGS

1A , 1B and 1C respectively show UPLC chromatograms of silymarin (Sigma Aldrich) obtained according to Scheme 1 , a silymarin achene extract I according to the present invention, and a silymarin achene extract A according to the prior art.

2A , 2B and 2C respectively show UPLC chromatograms of the methanol extract M according to the present invention, the ethanol 90° extract E according to the present invention and the isopropanol 90° extract I according to the present invention obtained according to Scheme 2 .

3A , 3B and 3C respectively show UPLC chromatograms of linoleic acid obtained according to Scheme 2, the milk thistle achene extract I according to the present invention, and the milk thistle achene extract A according to the prior art.

4A , 4B and 4C respectively show the 10-28 minute region (corresponding to the fatty acid and sterol region) of the GC-MS chromatograms of the ethanol 90° extract E according to the present invention, the methanol extract M according to the present invention, and the isopropanol 90° extract I according to the present invention obtained according to Scheme 3.

Figures 5A, 5B, and 5C represent the 22-28 minute region (corresponding to the sterol region) of the chromatograms in Figures 4A, 4B, and 4C, respectively.

6A, 6B, and 6C represent the 10-22 minute region (corresponding to the fatty acid region) of the chromatograms in FIGs. 4A, 4B, and 4C, respectively.

7 shows the mean therapeutic effect determined by the IGA method in 7 patients suffering from acne after sequential treatment with a Silybum marianum achene extract according to the invention low in silymarin (extracts I, M and E) and a Silybum marianum achene extract according to the prior art rich in silymarin (extract A).

FIG8 shows the therapeutic effect of 11 patients with adult acne after treatment with the Silybum marianum achene extract according to the present invention, as determined by the IGA method.

FIG9 shows the therapeutic effect of 10 patients suffering from adolescent acne after treatment with the Silybum marianum achene extract according to the present invention, as determined by the IGA method.

FIG10 shows the therapeutic effect measured by the IGA method in 9 patients with rosacea associated with seborrheic dermatitis after treatment with the Silybum marianum achene extract according to the invention.

FIG. 11 shows the average inhibition (fold) of two esterase-producing genes, FADS2 and SCD1, by the milk thistle achene extract according to the present invention and the milk thistle extract according to the prior art.

DETAILED DESCRIPTION

Example

1. Extraction Method

Method A: Method according to the prior art, which produces an extract A containing a high silymarin content:

Extract milk thistle achenes with 96% ethanol at reflux for 1 hour, using 10 ml of ethanol per gram of achene.

● Filter the mixture,

● recovery of the ethanol phase, and

●Evaporate the solvent.

Method B: A method according to the invention which produces an isopropyl alcohol 90 extract (extract I) containing a low silymarin content:

● Cold pressing of milk thistle achenes to obtain oil from milk thistle achenes,

The oil obtained from the achenes of Silybum marianum was extracted with an isopropanol/water mixture (90/10 v/v) at 1 g of isopropanol/water mixture per gram of oil for 2 hours at 20°C.

● recovery of the isopropanol phase, and

●Evaporate the solvent.

Methanol (extract M) and ethanol (extract E) extracts were obtained in a similar manner by replacing the isopropanol/water mixture (90/10 v/v) with methanol and ethanol/water mixture (90/10 v/v), respectively. The oil obtained from Silybum marianum achenes was extracted with 3 volumes of methanol and 3 volumes of ethanol/water mixture (90/10 v/v) per 1 volume of oil at 20° C. for 2 hours.

These different extracts were characterized by UPLC (Ultra Performance Liquid Chromatography) or by GC-MS (Gas Chromatography-Mass Spectrometry) according to the protocols detailed below.

Evaluation scheme of the obtained extracts :

Protocol 1: Silymarin content assessment by UPLC

○Sample and standard preparation:

- Silymarin standard: a solution containing 5 mg of silymarin was prepared in 10 ml of a methanol/water mixture (60:40) (v/v).

-sample:

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

Extracts M, E, and I: Heat the dried extract to be analyzed to 35°C with stirring until a clear, homogeneous solution is obtained. Accurately weigh 200 mg (pe) of the extract and dissolve it in 10 mL of a methanol/dichloromethane mixture, ensuring complete dissolution and a homogeneous solution. This mixture can be varied from a 1:1 (v/v) methanol/dichloromethane ratio to pure methanol.

○Analysis conditions:

-Column: Acquity BEH Shield C18 150mm x 2.1mm-1.7μm (Waters)

-Mobile phase:

○A: Water + 0.1% formic acid

○B: Acetonitrile + 0.1% formic acid

-gradient:

T(min) A(%) B (%) 0 90 10 15 60 40 20 0 100 39.5 0 100 40 90 10 45 90 10

- Column temperature: 40℃

-Flow rate: 0.4ml/min

-Detection: 287nm

-Injection volume: 1 μl

Protocol 2: Assessment of Linoleic Acid Content by UPLC

○Sample and standard preparation:

- Linoleic acid standard: a solution containing 10 mg of linoleic acid in 10 ml of a methanol/dichloromethane mixture (1:1) (v/v) was prepared.

-sample:

Extracts M, E, and I: Heat the dry extract to be analyzed to 35°C with stirring until a clear, homogeneous solution is obtained. Accurately weigh 50 mg (pe) of the extract and dissolve it in 1 ml of a methanol/dichloromethane mixture, ensuring complete dissolution and a homogeneous solution. This mixture can be varied from a 1:1 (v/v) methanol/dichloromethane ratio to pure methanol.

○Analysis conditions:

-Column: Acquity BEH Shield C18 150mm x 2.1mm-1.7μm (Waters)

-Mobile phase:

○A: Water + 0.1% formic acid

○B: Acetonitrile + 0.1% formic acid

-gradient:

T(min) A(%) B (%) 0 50 50 1 50 50 10 0 100 15 0 100 15.5 50 50 20 50 50

- Column temperature: 40℃

-Flow rate: 0.4ml/min

-Detection: 215nm

-Injection volume: 1 μl

Protocol 3: Assessment 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.

- 20 mg of the extract were dissolved in 800 μL of a methanol/dichloromethane mixture (1:1) (v/v).

- Add 200 μL of the derivatizing agent N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) + trimethylchlorosilane (TMCS) (99:1) (Supelco–Sigma Aldrich)

- Vortex for 1 minute

○ Gas chromatography (GC) conditions

-Column: DB-5ms (Agilent technologies); 30m x 0.25mm; 0.25μm

- Injection: T = 300 ° C; Mode = Split; Split ratio = 100:1

-Furnace: Temperature gradient (℃):

○ Initial temperature = 150℃

○ Gradient = 7°C/min until final temperature = 340°C

○ Maintain at 340℃ for 10 minutes

-Carrier gas flow rate: 1ml/min

-Detection: MS–EI; T = 300°C; Scan time = 0.2 sec.; Full scan start mass = 40; Full scan end mass = 600

-Injection volume: 1 μl

result:

The silymarin-rich Silybum marianum achene extract A according to the prior art substantially contains silymarin as a component, which has a retention time of 6 to 14 minutes by UPLC (see FIG. 1C and FIG. 1A ).

The Silybum marianum achene extract I according to the present invention, which is low in silymarin, mainly contains substances having a retention time of 13 to 30 minutes by UPLC (see FIG. 1B ).

Therefore, method A favors the extraction of polar compounds, especially silymarin flavonolignans, while method B favors the extraction of lipophilic compounds, such as free fatty acids, sterols, tocopherols and other non-polar compounds.

The silymarin content of extracts A and I was determined by UPLC after calibration with a standard commercial silymarin solution (Sigma Aldrich) (see Figure 1).

Extract A contained 28% by mass of silymarin.

Extract I contained 0.06% by mass of silymarin.

The content of free fatty acids (more particularly linoleic acid) and sterols (more particularly β-sitosterol) in Extract I was determined by UPLC (see FIG3B ) and GC (see FIG4C , 5C and 6C ):

We were able to confirm that the UPLC (see Figure 2) and GC (see Figures 4, 5 and 6) of the three achene extracts according to the present invention obtained by methanol extraction (Extract M), ethanol 90 extraction (Extract E) and isopropanol 90 extraction (Extract I) were similar.

in conclusion:

Various analyses by UPLC and GC-MS made it possible to demonstrate the following characteristics.

● "Chromatogram of Silymarin" (see Figure 1): Comparison of these chromatograms shows:

→ The isopropyl alcohol 90% achene extract (extract I) according to the invention is practically free of silymarin, in particular polar flavonolignans;

→ The ethanol extract according to the prior art (extract A) is itself rich in silymarin. The titration is 28% by mass of silymarin.

• "Linoleic acid chromatogram" (see Figure 3): Linoleic acid is the peak at 7.742 min. It should be noted that, unlike the isopropanol 90 extract according to the invention (Extract I), the ethanolic achene extract according to the prior art (Extract A) contains virtually no linoleic acid.

• Furthermore, we were able to confirm that the UPLC (see Figure 2) and GC (see Figures 4, 5 and 6) of the three achene extracts according to the invention obtained by methanol, ethanol 90 and isopropanol 90 extraction (extracts M, E and I) were similar.

2. Compositions according to the present invention

The extract prepared in Example 1 was formulated in an isopropyl alcohol/PEG 300 mixture (1:1) (w/w) at 7% (w/v). The resulting composition was used in various clinical studies (see Examples 3 to 6 below) by topical application on the skin.

The extract according to the invention can also be formulated, for example, in the form of a serum according to the following formula:

3. Comparative clinical studies on acne treatment

Seven patients suffering from acne were treated sequentially with a composition comprising a Silybum marianum achene extract according to the invention low in silymarin (Extract I, M or E) or a Silybum marianum achene extract according to the prior art enriched in silymarin (Extract A) from Example 2 according to the following schedule:

- Phase 1: 18 weeks of treatment with a composition comprising a Silybum marianum achene extract M according to the invention low in silymarin (5.64±0.01 μg/g), then

- Phase 2: 12 weeks of treatment with a composition comprising Silybum marianum achene extract A according to the prior art, rich in silymarin (23.21 ± 2.03 mg/g), then

- Phase 3: 18 weeks of treatment with a composition comprising a Silybum marianum achene extract E according to the invention low in silymarin, then

- Phase 4: 6 weeks of treatment with a composition comprising a Silybum marianum achene extract A according to the prior art, rich in silymarin, then

- Phase 5: 6 weeks of treatment with a composition comprising a Silybum marianum achene extract I according to the invention low in silymarin.

The analysis of the therapeutic effects obtained was performed according to the global clinical status analysis method IGA (Investigator Global Assessment) accepted by the FDA (Food and Drug Administration) and implemented by clinical dermatology experts (Guidance for Industry Acne Vulgaris: Developing Drugs for Treatment).

Using this approach, the following IGA grades are assigned based on the severity of the observed acne:

The average results obtained for the seven patients are shown in Figure 7 .

The graph shows that during the first treatment phase with the Silybum marianum achene extract according to the invention, low in silymarin, a significant improvement in the clinical state of acne was observed, corresponding to a "success" according to FDA criteria, this effect being remarkably stable given the particular duration of treatment (18 weeks).

When switching to the second treatment phase with the silymarin-rich Silybum marianum achene extract according to the prior art, a loss of efficacy with the return of lesions was observed, corresponding to a "failure" according to FDA criteria.

The same observations were made in subsequent phases 3, 4, and 5.

These clinical observations clearly indicate that the observed clinical therapeutic effects are independent of silymarin, as compositions containing infinitesimal silymarin concentrations exert significant clinical therapeutic effects, whereas compositions containing high silymarin concentrations tend to worsen the clinical state, leading to relapse.

Thus, treatment with the silymarin-rich Silybum marianum achene extract according to the prior art resulted in regression of the clinical status, whereas treatment with the silymarin-low Silybum marianum achene extract according to the invention provided a significant improvement.

Thus, the observed clinical results demonstrate a better efficacy of the extract according to the invention containing negligible amounts of silymarin compared to the prior art extract rich in silymarin, which resulted in the reversal of acne.

4. Clinical research on the treatment of adult acne

Eleven patients with adult acne were treated for 18 weeks with the composition from Example 2 comprising a Silybum marianum achene extract according to the invention that is low in silymarin.

The analysis of the therapeutic effect obtained was performed as described in Example 3 according to the global clinical status analysis method IGA (Investigator Global Assessment) accepted by the FDA (Food and Drug Administration) and implemented by clinical dermatology experts (Industry Guidelines for Acne Vulgaris: Developing Drugs for Treatment).

The results obtained are shown in Figure 8 and in the following table:

These results indicate a significant improvement in the clinical status of adult acne, with a decrease of two grades in 7 of 11 cases, corresponding to "success" according to FDA criteria, and a remarkable stability of this effect given the particular duration of treatment (18 weeks).

These clinical observations demonstrate the indisputable clinical therapeutic effect of the extract according to the invention on adult acne.

5. Clinical research on the treatment of adolescent acne

Ten adolescent patients (15-18 years) suffering from adolescent acne were treated for 18 weeks with the composition from Example 2 comprising a Silybum marianum achene extract according to the invention that is low in silymarin.

The analysis of the therapeutic effect obtained was performed as described in Example 3 according to the global clinical status analysis method IGA (Investigator Global Assessment) accepted by the FDA (Food and Drug Administration) and implemented by clinical dermatology experts (Industry Guidelines for Acne Vulgaris: Developing Drugs for Treatment).

The results obtained are shown in Figure 9 and in the following table:

These results demonstrate a significant improvement in the clinical status of adolescent acne, with a decrease of two grades in 8 of 10 cases, corresponding to "success" according to FDA criteria, an effect with remarkable stability given the particular duration of treatment (18 weeks).

These clinical observations demonstrate the indisputable clinical therapeutic effect of the extract according to the invention on juvenile acne.

6. Clinical study on the treatment of rosacea (optionally with seborrheic dermatitis)

Nine patients suffering from rosacea (optionally with seborrheic conditions) were treated for 18 weeks with the composition from Example 2 comprising a Silybum marianum achene extract according to the invention that is low in silymarin.

The analysis of the therapeutic effect obtained was performed as described in Example 3 according to the FDA (Food and Drug Administration) accepted global clinical status analysis method IGA (Investigator Global Assessment) for acne and applied to rosacea by clinical dermatology experts (Industry Guide for Acne Vulgaris: Developing Drugs for Treatment).

The results obtained are shown in Figure 10 and in the following table:

These results demonstrate a significant improvement in the clinical status of rosacea in all patients, and given the exceptional duration of treatment (18 weeks), this effect is remarkably stable. This overall improvement corresponds to a significant effect on various clinical parameters of this condition (Wilkin et al., 2004): vascular, inflammatory, papulopustular, and seborrheic components, reflecting in these cases the "mixed facial dermatitis" type of seborrheic dermatitis (SD). This seborrheic dermatitis is associated with rosacea lesions in the typical areas of this condition, i.e., the facial ridges, as well as seborrheic dermatitis lesions in the folds, as described by B. Cribier in Traité Francophone de Dermatologie, 5th edition (Cribier B. Dermatoses faciales page 861 in Dermatologie et IST, 5th edition Elsevier Masson 2009).

These clinical observations demonstrate the indisputable clinical therapeutic effect of the extract according to the invention on rosacea, optionally accompanied by seborrheic conditions.

7. In vitro comparative studies

In this study, an isopropanol Silybum marianum achene extract according to the present invention and Silybum marianum achene extract A according to the prior art were tested in vitro at a concentration of 0.03% (w/v) in culture medium on primary human sebocyte cultures (Zenbio, Inc. Research Triangle Park, NC) for 3 consecutive days (with daily medium changes). The cells were harvested and RNA was extracted 24 hours after the final treatment.

PCR (polymerase chain reaction) analysis allowed the measurement of the expression levels of two sebogenic enzyme genes, FADS2 and SCD1. These two enzymes are involved in the synthesis of sebum-specific lipids, and their inhibition is known to induce a decrease in sebaceous gland activity. The average inhibition results (fold change) of these two genes relative to the solvent (isopropanol, at a concentration of 1% in the culture medium), obtained from at least five experiments, are shown in FIG11 , with a 2-fold inhibition being considered significant.

Therefore, these results show that, unlike the silymarin-rich ...

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Claims (47)

1. Use of an extract of Silybum marianum (L.) Gaertn. achene in the preparation of a medicament for treating acne, seborrhea, rosacea and/or seborrheic dermatitis, the extract comprising less than 0.2% by weight of silymarin relative to the weight of the dry extract; the extract being obtained by a process comprising the step of extracting oil obtained from Silybum marianum achenes with an extraction solvent selected from subcritical water and a _C1 to _C3 alcohol optionally mixed with water. 2. Use according to claim 1, characterized in that the extract contains less than 0.1% by weight of silymarin relative to the weight of the dry extract. 3. Use according to claim 1, characterized in that the extract contains 0.5% to 2.5% by weight of β-sitosterol relative to the weight of the dry extract. 4. Use according to claim 3, characterized in that the extract contains 1.0% to 2.0% by weight of β-sitosterol relative to the weight of the dry extract. 5. Use according to claim 4, characterized in that the extract contains 1.5% by weight of β-sitosterol relative to the weight of the dry extract. 6. The use according to claim 3, characterized in that the mass ratio of silymarin to β-sitosterol in the extract is less than 0.4. 7. The use according to claim 6, characterized in that the mass ratio of silymarin to β-sitosterol in the extract is less than 0.07. 8. Use according to claim 3, characterized in that the extract contains 2% to 7% by weight of sterols relative to the weight of the dry extract. 9. Use according to claim 8, characterized in that the extract contains 3% to 6% by weight of sterols relative to the weight of the dry extract. 10. Use according to claim 9, characterized in that the extract comprises 3% to 5% by weight of sterols relative to the weight of the dry extract. 11. Use according to claim 1, characterized in that the extract contains 3% to 15% by weight of free linoleic acid relative to the weight of the dry extract. 12. Use according to claim 11, characterized in that the extract contains 4 to 10% by weight of free linoleic acid relative to the weight of the dry extract. 13. Use according to claim 12, characterized in that the extract contains 4 to 6% by weight of free linoleic acid relative to the weight of the dry extract. 14. Use according to claim 13, characterized in that the extract contains 5% by weight of free linoleic acid relative to the weight of the dry extract. 15. Use according to claim 11, characterized in that the extract comprises 10% to 50% by weight of free fatty acids relative to the weight of the dry extract. 16. Use according to claim 15, characterized in that the extract comprises 10% to 30% by weight of free fatty acids relative to the weight of the dry extract. 17. Use according to claim 16, characterized in that the extract comprises 15 to 25% by weight of free fatty acids relative to the weight of the dry extract. 18. Use according to claim 1, characterized in that the extract contains 0.01% to 0.5% by weight of tocopherol relative to the weight of the dry extract. 19. Use according to claim 18, characterized in that the extract comprises 0.05% to 0.2% by weight of tocopherol relative to the weight of the dry extract. 20. Use according to claim 19, characterized in that the extract contains 0.1% by weight of tocopherol relative to the weight of the dry extract. 21. The use according to claim 18, characterized in that the mass ratio of silymarin to tocopherol in the extract is less than 1. 22. The use according to claim 21, characterized in that the mass ratio of silymarin to tocopherol in the extract is less than 0.1. 23. The use according to claim 1, characterized in that the extract is a dry extract. 24. The method according to claim 1, wherein the method comprises the following consecutive steps: (i) optionally extracting oil from milk thistle achenes, (ii) extracting the oil obtained from the milk thistle achenes with an extraction solvent, (iii) recovering the extract phase obtained in step (ii), and (iv) Partially or completely drying the extract phase to produce a concentrated extract or a dry extract. 25. The use according to claim 24, characterized in that step (i) is carried out by pressing milk thistle achenes. 26. The use according to claim 24, characterized in that step (i) is carried out by cold pressing the milk thistle achenes. 27. The use according to claim 1, wherein the _C1 to _C3 alcohol is selected from methanol, ethanol, isopropanol and mixtures thereof. 28. The use according to claim 1, wherein the _C1 to _C3 alcohol is isopropyl alcohol. 29. The method according to claim 1, wherein the extraction solvent is an isopropyl alcohol/water mixture in a volume ratio of 90/10. 30. Use of a composition comprising an extract as defined in any one of claims 1 to 29 in admixture with at least one pharmaceutically or cosmetically acceptable excipient for the preparation of a medicament or cosmetic for the treatment of acne, seborrhea, rosacea and/or seborrheic dermatitis. 31. The use according to claim 30, characterized in that the composition is for topical application. 32. The use according to claim 30, wherein the composition is for topical application on the skin. 33. The use according to claim 30, characterized in that the composition comprises the extract as defined in any one of claims 1 to 29, isopropyl alcohol and polyethylene glycol (PEG). 34. Use according to claim 33, characterized in that the average molecular weight of the polyethylene glycol is 200 to 600 g/mol. 35. Use according to claim 34, characterized in that the average molecular weight of the polyethylene glycol is 200 to 500 g/mol. 36. Use according to claim 35, characterized in that the average molecular weight of the polyethylene glycol is 200 to 400 g/mol. 37. The use according to claim 36, characterized in that the average molecular weight of the polyethylene glycol is 250 to 350 g/mol. 38. The use according to claim 37, characterized in that the average molecular weight of the polyethylene glycol is 300 g/mol. 39. The use according to claim 30, characterized in that the composition consists of the extract as defined in any one of claims 1 to 29, isopropyl alcohol and polyethylene glycol (PEG). 40. The use according to claim 39, characterized in that the average molecular weight of the polyethylene glycol is 200 to 600 g/mol. 41. The use according to claim 40, characterized in that the average molecular weight of the polyethylene glycol is 200 to 500 g/mol. 42. Use according to claim 41, characterized in that the average molecular weight of the polyethylene glycol is 200 to 400 g/mol. 43. Use according to claim 42, characterized in that the average molecular weight of the polyethylene glycol is 250 to 350 g/mol. 44. Use according to claim 43, characterized in that the average molecular weight of the polyethylene glycol is 300 g/mol. 45. Use according to claim 30, characterized in that the composition comprises 0.001% to 15% by weight of the extract as defined in any one of claims 1 to 29, relative to the total volume of the composition. 46. Use according to claim 45, characterized in that the composition comprises from 0.01% to 15% by weight of an extract as defined in any one of claims 1 to 29, relative to the total volume of the composition. 47. Use according to claim 46, characterized in that the composition comprises from 0.1% to 10% by weight of the extract as defined in any one of claims 1 to 29, relative to the total volume of the composition.