Composition for improving aesthetic appearance of hair and skin appendages

Abstract

The present invention relates to a composition comprising a combination of bioactive ingredients that inhibits glycogen phosphorylase and improves glycogen metabolism in hair follicles, thereby stimulating hair growth and improving the aesthetic appearance of hair.

Description

Technical Field

[0001] The present invention relates to a composition for improving the aesthetic appearance of hair and skin appendages.

[0002] The present invention originates from the field of products for the care and treatment of keratin structures, preferably hair and skin appendages.

[0003] Specifically, provided herein is a composition containing a synergistic combination of bioactive ingredients that inhibits glycogen phosphorylase and enhances glycogen metabolism in hair follicles, stimulating hair growth and improving their aesthetic appearance. Background Art

[0004] The life cycle of the hair bulb includes a growth phase called anagen, a degeneration phase called catagen, and a resting phase called telogen.

[0005] During the anagen phase, the dermal papilla generates chemical signals that activate and direct stem cells in the hair bulb to form the hair matrix by migrating to the base of the follicle. As they "migrate," the bulb stem cells form a cellular "channel" that will develop into the outer root sheath, or ORS.

[0006] In response to further signals from the dermal papilla, stem cell-derived stromal cells proliferate and initiate a differentiation process, moving upward to form the shaft and inner sheath of the hair follicle.

[0007] The onset of catagen is characterized by the cessation of cell proliferation and apoptosis of stromal cells. During catagen, the dermal papilla migrates downward toward the hair bulb. This close proximity of the papilla to the hair bulb is believed to be crucial for initiating a new hair growth cycle. This allows quiescent hair bulb cells to interact and activate, initiating a new cycle of hair and skin appendage growth.

[0008] After the catagen / telogen transition, some hair bulb cells migrate to the papilla, come into contact with it, and form hair germs.

[0009] Telogen hairs contain a population of cells at their base, actually called a hair germ, located near the dermal papilla. Near the end of the telogen phase, the hair germ becomes activated and begins to proliferate, even before the hair bulb, forming a new bulb matrix surrounding the papilla.

[0010] Hair follicles (HFs) consume energy and metabolites to maintain high proliferation rates, hair fiber formation, and hair cycle-related remodeling.

[0011] Human hair follicles have a particularly pronounced demand for energy and precursors for biosynthesis during the anagen phase, a demand that is primarily met by aerobic glycolysis, as highlighted in the publication “Characterisation of cell cycle arrest and terminal differentiation in a maximally proliferative human epithelial tissue: Lessons from the human hair follicle matrix.” Eur. J. Cell Biol. 2017;96:632–641. doi:10.1016 / j.ejcb.2017.03.011 by Purba TS et al.

[0012] According to the publications Vidali S, Knuever J, Lerchner J, Giesen M, Bíró T. Hypothalamic–pituitary–thyroid axis hormones stimulate mitochondrial function and biogenesis in human hair follicles. J. Invest. Dermatol. 2014;134:33–42. doi:10.1038 / jid.2013.286.; Lemasters JJ, et al. Compartmentation of mitochondria and oxidative metabolism in growing hair follicles: Aring of fire. J. Invest. Dermatol. 2017;137:1434–1444. doi:10.1016 / j.jid.2017.02.983, it is clear that, despite the presence of a large number of mitochondria, hair follicles preferentially obtain the energy they need for proliferation from aerobic glycolysis. This need is confirmed by the publication Williams R, Philpott MP, Kealey T. Metabolism of freshly isolated human hair follicles capable of hair elongation: Aglutaminolytic, aerobic glycolytic tissue. J Investig. Dermatol. 1993; 100: 834–840. doi: 10.1111 / 1523-1747.ep12476744).

[0013] The importance of aerobic glycolysis to hair follicle function has also been confirmed in animal models studying the key role of lactate dehydrogenase and the process of glucose metabolism to lactate in activating hair follicle (HF) epithelial stem cells (HFSCs), as described in Flores A, et al. Lactate dehydrogenase activity drives hair follicle stem cell activation. Nat. Cell Biol. 2017; 19: 1017–1026. doi: 10.1038 / ncb3575.

[0014] Hair follicles contain high levels of glycogen and glucose polymers and are the main energy storage sites in the human body. Glycogen, a glucose polysaccharide, is the main form of energy storage in mammals and is also present at high levels in hair follicles during the anagen growth phase (Figlak K, Williams G, Bertolini M, Paus R, Philpott MP. Human hair follicles operate an internal Cori cycle and modulate their growth via glycogen phosphorylase. Sci Rep. 2021 Oct 21; 11(1): 20761. doi: 10.1038 / s41598-021-99652-8. PMID: 34675331; PMCID: PMC8531296). These glucose levels decrease during the catagen growth phase, while glycogen is completely absent during the telogen phase.

[0015] Human hair follicles have the ability to synthesize glycogen from lactate through their own internal Cori cycle.

[0016] The growth phase of hair follicles consumes a lot of energy; for this reason, it is crucial to stimulate hair follicles to maximize energy acquisition / storage. In vitro studies of hair follicles have demonstrated that inhibiting a key enzyme involved in energy storage, glycogen phosphorylase (PYGL), via glycogen increases their elongation and prolongs their growth phase, thereby delaying the transition to the catagen phase (Figlak K, Williams G, Bertolini M, Paus R, Philpott MP. Human hair follicles operate an internal Cori cycle and modulate their growth via glycogen phosphorylase. Sci Rep. 2021 Oct 21; 11(1): 20761. doi: 10.1038 / s41598-021-99652-8. PMID: 34675331; PMCID: PMC8531296).

[0017] An object of the present invention is to provide a composition comprising bioactive ingredients, which improves the appearance of hair follicles by regulating and promoting the physiological growth of hair follicles and intervening in the energy metabolism of hair follicles.

[0018] Another object of the present invention is to provide a composition that promotes glycogen metabolism in hair follicles and can be formulated into a dietary supplement and a topical preparation. The dietary supplement is for oral administration, and the topical preparation is for application to parts of the body that need to stimulate the growth of keratin structures, particularly hair and skin appendages.

[0019] Yet another object of the present invention is to provide a composition that is suitable both for cosmetic use to improve the aesthetic appearance of human keratin structures and for therapeutic use to prevent or treat hair growth disorders. Summary of the Invention

[0020] In summary, the present invention provides novel uses of combinations of bioactive ingredients in the fields of cosmetology and trichology.

[0021] According to some aspects of the present invention, the inventors have discovered that the combination of selected bioactive ingredients inhibits the activity of glycogen phosphorylase, increases the energy content of hair follicles and promotes their glycogen metabolism, thereby stimulating the growth of keratin structures (particularly mammalian hair and skin appendages, preferably human hair).

[0022] In the present invention, when the term "skin appendages" is used, it is intended to include beards, eyelashes, eyebrows and nails.

[0023] According to a first aspect, the present invention relates to the non-therapeutic, cosmetic use of a composition for improving the aesthetic appearance of mammalian keratinous structures selected from hair and skin appendages, in particular human hair, comprising a combination of maslinic acid and a Swertia plant, in particular Swertia chirata, and a physiologically acceptable carrier.

[0024] Advantageously and preferably, a particular object of the present invention is the non-therapeutic, cosmetic use of a composition comprising masleric acid and an extract of a plant of the genus Swertia, in particular Swertia indica, for promoting hair growth and / or preventing and / or reducing hair loss in mammals, in particular humans.

[0025] According to some specific aspects of the present invention, the hair loss reducing activity of the compositions described herein is effective in individuals who are significantly affected by physiological hair loss and the resulting thinning of the scalp.

[0026] According to another aspect, the combination composition described herein can be used in the cosmetic field to improve aspects of the appearance of hair and skin appendages, such as their volume, their shine and / or their tightness, their light-reflecting ability and the vividness of their color.

[0027] According to some embodiments, the composition of the present invention is a cosmetic composition suitable for topical application.

[0028] According to some embodiments, the composition for cosmetic use is selected from a topical composition and a dietary supplement that can be incorporated into the dietary regimen of an individual wishing to improve the appearance of hair and skin appendages.

[0029] According to another aspect, the present invention relates to a composition comprising a combination of masleric acid and an extract of a plant of the genus Swertia, preferably Swertia indica, for use in treating diseases or disorders in the growth of keratinous structures selected from hair and skin appendages, in particular hair.

[0030] For example, the compositions described herein for therapeutic-trichological uses are suitable for treating conditions that cause or exacerbate the loss of keratinous structures such as hair and skin appendages.

[0031] Advantageously, it has been discovered that the compositions described herein, when administered topically or systemically to a subject suffering from thinning or loss of hair or skin appendages, can induce a gradual thickening and improvement in the keratin structure comprising the hair or skin appendages.

[0032] According to some aspects, there is provided a composition comprising masleric acid and an extract of a plant of the genus Swertia (preferably Swertia indica) and a physiologically acceptable carrier for use in treating hair and skin appendage growth disorders.

[0033] For example, the composition comprising masleric acid and an extract of a plant of the genus Swertia, preferably Swertia indica, for therapeutic use described herein is shown to be useful in treating alopecia, one of the most prevalent disorders in the growth of keratinous structures, in particular hair.

[0034] In particular, the compositions can be used to treat androgenic alopecia (AGA). In individuals affected by androgenic alopecia, hair follicles formed during the early neogen phase decrease in size (miniaturization) over time, gradually causing new hair to grow thinner than the previous hair. The result is the formation of extremely fine hair.

[0035] The compositions described herein are also suitable for treating other forms of hair loss besides androgenic alopecia, such as telogen effluvium, alopecia areata, tinea, cicatricial alopecia, and hair loss due to excessive use of cosmetics.

[0036] Some aspects of the invention are described with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying figure shows an exemplary bar graph comparing the results of the following extracts: 2 μg / mL Swertia indica extract alone, 6 μg / mL Maslinic acid alone, 6 μg / mL Maslinic acid + 2 μg / mL Swertia indica extract, and an olive leaf and fruit extract containing 75 wt% Maslinic acid (6 μg / mL).

[0038] Example 5 relates to the detection of glycogen phosphorylase (PYGL) gene expression by qRT-PCR. The bar graph highlights that a solid mixture of olive leaf and fruit extracts (containing 75 wt% Maslinic acid) and a combination of Maslinic acid and Swertia indica exerted a synergistic and significantly (p < 0.005) higher inhibition of PYGL gene expression compared to the positive control and the individual components. DETAILED DESCRIPTION

[0039] According to some aspects of the present invention, the inventors have discovered that the combination of masliic acid and an extract of a Swertia plant (preferably Swertia indica) promotes cellular glycogen metabolism in hair follicles by stimulating the growth of keratin structures (particularly mammalian hair and skin appendages, preferably human hair).

[0040] Therefore, the subject of the present invention is the use of a composition as defined in the appended claim 1 , comprising masleric acid and an extract of a plant of the genus Swertia, preferably Swertia indica, and a physiologically acceptable excipient.

[0041] According to this aspect, the compositions described herein are thus useful for improving the aesthetic appearance or external characteristics of keratinous structures selected from the group consisting of hair and skin appendages, in particular hair.

[0042] In the present invention, when the term "skin appendages" is used, it is intended to include hair, beard, eyelashes, eyebrows and nails.

[0043] In the context of the present invention, the term "keratinous structures" refers to hair and skin appendages, such as beards, eyelashes, eyebrows and nails, preferably of mammals, such as humans; or hair, preferably of mammals, such as animals, e.g. dogs or cats or other pets or companion animals.

[0044] According to the present invention, the composition according to any one of the embodiments described herein can be used in the cosmetic field to improve the aesthetic appearance of hair and skin appendages, such as hair pigmentation.

[0045] Generally, the composition comprising masleric acid and a plant of the genus Swertia, preferably Swertia indica, can stimulate the physiological growth of hair and thicken the hair, thereby improving its appearance.

[0046] This effect is attributed to the combined composition's inhibitory effect on glycogen phosphorylase expression, thereby increasing the duration of the hair follicle's anagen phase.

[0047] According to a second aspect of the present invention, there is provided a composition comprising a combination of masriic acid and an extract from a plant of the genus Swertia (preferably Swertia indica), for use in treating a growth disorder of keratinous structures (selected from hair and skin appendages of a mammal). Preferably, the composition comprises a combination of masriic acid and an extract from a plant of the genus Swertia (preferably Swertia indica), for use in treating a hair growth disorder in a human.

[0048] According to this use, hair growth disorders or conditions that may be treated include alopecia and telogen effluvium, which disorders affect both male and female individuals. The composition is also suitable for treating or preventing female pattern baldness or androgenic alopecia.

[0049] Regardless of the cosmetic and / or therapeutic use, the composition of the present invention comprises masleric acid and an extract of a plant of the genus Swertia (preferably Swertia indica) as bioactive ingredients or components.

[0050] This combination produces a synergistic inhibitory effect on glycogen phosphorylase.

[0051] One of the components of the composition of the present invention is masleric acid, also known as crategolic acid or (2α,3β)-2,3-dihydroxyolean-12-ene-28-oic acid (IUPAC), CAS No. 4373-41-5, which has the following chemical structure:

[0052]

[0053] Maslilic acid belongs to the class of pentacyclic triterpenes known as oleanane.

[0054] This compound is widely distributed in the plant kingdom and can be derived from wax on the olive skin and / or from dried olive pomace oil (a by-product of olive oil extraction). Typically, it is a by-product of extracting olive oil from the fruit of a tree, particularly Olea europaea.

[0055] According to some embodiments, Maslinic acid is of natural origin and is contained in a solid mixture extracted from leaves and / or fruits of the olive tree (Olea europaea).

[0056] Masliic acid may be present in the composition in the form of a plant extract obtained from olive leaves and / or olives (particularly their peels).

[0057] According to some embodiments, Maslinic acid is contained in the composition in solid form (such as tablet form), and the content of Maslinic acid is 0.18 mg to 180 mg, preferably 3 mg to 100 mg, for example 18 mg; Maslinic acid is contained in the composition in liquid form, and the content of Maslinic acid is 6 μg / mL to 6 mg / mL, preferably 100 μg / mL to 3 mg / mL, for example 0.06 g / 100 mL.

[0058] Within the scope of the present invention, suitable plant extracts comprising Masliic acid can be obtained from the fruit of the olive tree, in particular the olive tree. For the use according to the present invention, the extract is preferably obtained from the fruit of the olive tree, ie olives.

[0059] In one embodiment, Masliic acid can be obtained from a solid mixture extracted from olive leaves and fruits.

[0060] Preferably, the oil extracted from olives may contain Maslinic acid in an amount greater than or equal to 60% (w / w), more preferably in an amount ranging from 60% to 75% (w / w), even more preferably in an amount ranging from 65% to 70% (w / w), for example 67.5% (w / w).

[0061] Plant extracts comprising Masliic acid can be obtained by extraction from olives using physiologically acceptable solvents as extraction medium.

[0062] The term "physiologically acceptable solvent" refers to a solvent that does not produce significant adverse reactions when introduced into or administered to the human body. Suitable solvents for obtaining plant extracts are physiologically acceptable liquids in which the biologically active components of the selected plant are soluble and do not undergo changes that render them inactive.

[0063] In some embodiments, the physiologically acceptable solvent is a protic polar solvent, preferably selected from water, acetic acid, ethanol, n-butanol ethyl acetate, n-butanol, isopropanol, n-propanol, and mixtures thereof.

[0064] Preferably, the extraction solvent is a polar protic solvent selected from water, ethanol, ethyl acetate and mixtures thereof; more preferably, the extraction solvent is a mixture of water and ethyl acetate.

[0065] To obtain plant extracts from olive trees, one or more bioactive components can be isolated / extracted from the plant tissues of the tree using well-known extraction techniques (eg, solid-liquid extraction).

[0066] In some embodiments, extraction of one or more bioactive components is performed by extracting a portion of olive or a plant matrix, such as extracts of olive fruit and leaves, in a suitable solvent as described herein.

[0067] According to a preferred and advantageous embodiment, the method for extracting suitable Masliic acid from olive fruit comprises the following steps:

[0068] a) extraction from olives using a protic polar solvent, preferably a mixture of water and ethyl acetate;

[0069] b) removing the solvent and drying the extract obtained in step a).

[0070] Advantageously, there is a purification step of the olive extract product between steps a) and b).

[0071] For example, a suitable extract can be obtained by immersing or soaking a portion of olives in a mixture of water and ethyl acetate for a suitable period of time to allow the solvent to enrich one or more bioactive components. Under these conditions, extraction of the bioactive components from the plant tissue of the selected plant is primarily achieved by diffusion and / or osmosis. The immersion time of the plant part in the solvent can vary, for example, from 1 hour to 48 hours. The extracted material is purified and subsequently dried.

[0072] According to some embodiments, the preparation of a suitable extract from olives (particularly the peel) comprises the following steps:

[0073] i) crushing olives or olive skins;

[0074] ii) adding an extraction solvent, such as a water-ethyl acetate mixture, to the pulverized product of step i) to obtain a mixture having a solid / water-alcohol solvent ratio of about 1:10 w / w to about 1:50 w / w;

[0075] iii) impregnating the mixture of step ii);

[0076] iv) extracting bioactive components;

[0077] v) filtering the bioactive component;

[0078] vi) concentrating the filtrate, for example by evaporating the hydroalcoholic solvent under reduced pressure, to obtain an extract;

[0079] vii) drying the extract thus obtained.

[0080] According to another embodiment, the method of extraction from olives comprises the following steps:

[0081] A) crushing the olives to obtain a powder;

[0082] B) transferring the obtained powder into a suitable percolator;

[0083] C) performing diafiltration, for example, using an amount of extraction solvent to provide a solids / solvent weight ratio of about 1:20 to about 1:100;

[0084] D) recycling a portion of the leachate until the extract is exhausted to obtain a vegetable bed;

[0085] E) squeezing the extracted plant material bed to recover all of the extraction solvent;

[0086] F) filtering the leachate;

[0087] G) Concentrating the filtrate, for example by evaporating the solvent under reduced pressure.

[0088] According to some embodiments, in a final step of removing the solvent by evaporation, a solid carrier (such as starch or maltodextrin) is added to obtain the extract in the form of a dry powder.

[0089] The extracts obtained from olives can be fluid, soft or dry.

[0090] Olive extracts with varying polarity can be prepared.

[0091] In some embodiments, the extraction is accomplished using a solvent and a plant matrix in a weight ratio of 1:10 to 10:1.

[0092] Preferably, the extract obtained from olive fruit and / or leaves comprises masleric acid and / or oleanolic acid; more preferably, the total amount of masleric acid and oleanolic acid in the extract is 10 wt%-80 wt%, 30 wt%-80 wt%; even more preferably 75 wt%-80 wt%.

[0093] According to some embodiments, the olive fruit extract comprises triterpenes, wherein the content of masleric acid and oleanolic acid is greater than 75 wt %. For example, by HPLC titration, masleric acid and oleanolic acid account for 75%-80% of the total pentacyclic triterpenes, of which masleric acid accounts for 60%-68% and oleanolic acid accounts for 12%-15%.

[0094] In the composition described herein, Masriic acid is combined with an extract of a plant of the genus Swertia, preferably Swertia indica.

[0095] Swertia japonica belongs to the Gentianaceae family and is considered a Himalayan medicinal herb used in traditional medicine to treat liver disease, malaria and diabetes.

[0096] Suitable Swertia plant extracts can be obtained from the whole plant.

[0097] Within the scope of the present invention, suitable plant extracts can be obtained from any part of the Swertia plant, such as roots, leaves or flowers. For the use according to the present invention, the extract is preferably obtained from the aerial parts of the Swertia plant, usually the leaves.

[0098] According to some embodiments, the plant extract of the present invention is obtained by extraction from a plant or a part of its tissue using a physiologically acceptable solvent as an extraction medium.

[0099] The term "physiologically acceptable solvent" refers to a solvent that does not produce significant adverse reactions when introduced into or administered to the human body.

[0100] Suitable solvents for obtaining plant extracts are physiologically acceptable liquids in which the biologically active ingredients of the selected plant are soluble and in which they do not undergo changes that render them inactive.

[0101] In some embodiments, the physiologically acceptable solvent is selected from water, ethanol, ethyl acetate and mixtures thereof. For example, the solvent is a hydroalcoholic solution of water / ethanol.

[0102] In order to obtain the plant extract of Swertia genus, solid-liquid extraction technology can be used to separate / extract one or more bioactive components from the plant tissue of the plant.

[0103] In some embodiments, extraction of one or more bioactive components is performed by macerating a Swertia plant portion or a plant matrix thereof in a suitable solvent (eg, a water-alcohol mixture).

[0104] For example, a suitable extract can be obtained by immersing or soaking a portion of the aerial parts of a Swertia plant in a water-ethanol mixture for an appropriate period of time to allow the solvent to be enriched with one or more bioactive components. Under these conditions, extraction of the bioactive components from the plant tissue of the selected plant is primarily achieved by diffusion and / or osmosis.

[0105] The time for immersion of the plant material in the solvent can vary, for example from 1 hour to 48 hours.

[0106] According to some embodiments, the preparation of a suitable Swertia plant extract comprises the following steps:

[0107] - Crushing plant leaves;

[0108] - adding an extraction solvent, such as a water-ethanol or ethyl acetate mixture, to obtain a plant macerate / hydroalcoholic solvent ratio of about 1:10 to about 1:50 w / w;

[0109] - impregnation of aerial parts;

[0110] - Extraction of bioactive ingredients;

[0111] -filter;

[0112] - concentrating the filtrate, for example by evaporating the hydroalcoholic solvent under reduced pressure;

[0113] - optionally, continuing the evaporation until the solvent is removed;

[0114] - Optionally, drying the extract thus obtained.

[0115] In some embodiments, the extraction step can be repeated two or three times.

[0116] In a final step of removal of the solvent by evaporation, a solid support (such as, as non-limiting examples, starch or maltodextrin) may optionally be added to obtain the extract in the form of a dry powder.

[0117] According to another embodiment, the method of extracting from Swertia plants comprises the following steps:

[0118] - crushing, for example, of aerial parts of plants;

[0119] - Transfer the obtained powder into a suitable percolator;

[0120] - performing diafiltration, for example, using an amount of extraction solvent to provide a drug-solid / solvent weight ratio of about 1:20 to about 1:100;

[0121] - Recirculate part of the leachate until the material to be extracted is exhausted;

[0122] - Pressing the extracted plant bed to recover all the extraction solvent;

[0123] - Filter the leachate;

[0124] - concentrating the filtrate, for example by evaporating the solvent under reduced pressure;

[0125] - optionally, continuing the evaporation until the solvent is removed;

[0126] - Optionally, drying the extract thus obtained.

[0127] According to some embodiments, in a final step of removing the solvent by evaporation, a solid carrier (such as starch or maltodextrin) is added to obtain the extract in the form of a dry powder.

[0128] Typically, the extract obtained from the Swertia plant can be fluid, soft or dry.

[0129] For example:

[0130] - In a fluid extract, 1 mL of extract contains the bioactive ingredient dissolved in 1 g of the plant's active ingredient;

[0131] - in the case of soft extracts, the solvent is partially evaporated, in particular until the extract wets the filter paper;

[0132] - In dry extract, the solvent evaporates almost completely and a powder is obtained.

[0133] Swertia plant extracts with varying polarity can be prepared.

[0134] For example, a highly polar extract can be obtained using a polar solvent (such as a hydroalcoholic solution), a moderately polar extract can be obtained using a less polar solvent (such as ethyl acetate), or a non-polar extract can be obtained using supercritical CO2, thereby extracting some components in the plant complex that have inhibitory activity against the 5-α-reductase type 2 enzyme.

[0135] In some embodiments, the extraction is accomplished using a solvent and a plant matrix in a weight ratio of 1:10 to 10:1.

[0136] Alternative extraction techniques may be used to extract bioactive components from Swertia plants, such as digestion, maceration, pressing, decoction, percolation, countercurrent extraction, Soxhlet extraction, extraction with supercritical gas or ultrasound.

[0137] The bioactive components extracted from Swertia plants have not yet been identified, but may include xanthones, lignans, alkaloids, flavonoids, terpenes, iridoids, palmitic acid, oleic acid, and stearic acid.

[0138] Chiratin is one of the most representative xanthones in the extract. Other active components that may be present in Swertia plant extracts include amarogentin, swertiamarin, mangiferin, swerchirin, and mixtures thereof.

[0139] According to some embodiments, the weight ratio of the Swertia plant extract in the composition is 0.5-6, for example, 1-3, relative to the weight of Maslinic acid.

[0140] The compositions described herein for cosmetic or therapeutic-trichological use are suitable for topical or systemic administration.

[0141] According to some embodiments, the composition for oral administration is or is contained in a functional food, dietary supplement or nutritional product.

[0142] Functional food refers to any food or modified food ingredient that contains, in addition to the traditional nutrients it contains, a beneficial effect or protective effect against a disease or physiological state.

[0143] Regardless of the administration route, the cosmetic composition enhances the activity of hair follicle cells during the growth phase and activates dormant cells of keratin structures (preferably the scalp), stimulates the metabolism of hair follicles, and promotes the growth of new hair or skin appendages.

[0144] In some embodiments, the composition of the present invention may further comprise one or more other ingredients having trichological activity.

[0145] In some embodiments, the compositions for cosmetic and therapeutic uses further comprise other ingredients, such as vitamins, eg, vitamin A, vitamin C, additional B vitamins, niacin, and mixtures thereof.

[0146] According to some embodiments, the composition of the present invention further comprises amino acids, particularly sulfur-containing amino acids (such as L-cysteine ​​or L-methionine), preferably for preparing a formulation for oral administration.

[0147] According to some embodiments, the composition of the present invention further comprises one or more micronutrients and / or minerals or micronutrients (such as Mg, Zn, Ca, Fe, Cr, Se, etc.), preferably for preparing a formulation for oral administration.

[0148] In some embodiments, the composition may further comprise other substances, such as folic acid, calcium pantothenate, mucopolysaccharides (such as hyaluronic acid), or soybean derivatives (such as soybean isoflavones).

[0149] In some embodiments, the compositions of the present invention for cosmetic and medical use comprise a physiologically and / or pharmaceutically acceptable carrier, diluent, or excipient.

[0150] Typically, the physiologically acceptable carrier in the composition of the present invention is an excipient, carrier or diluent suitable for local administration and / or systemic administration. The physiologically acceptable carrier and the pharmacologically acceptable carrier can be the same carrier.

[0151] In the context of this invention, the term "carrier" refers to an excipient, carrier, diluent or adjuvant that may be present in the composition of the present invention. In the application of the plant extracts or active ingredients described herein, any carrier and / or excipient suitable for the desired dosage form can be used.

[0152] In the context of the present specification, the term "combination" is intended to mean that the substances Masleric acid and the extract of a plant of the genus Swertia (preferably Swertia indica) are both present in the composition (e.g. in the form of a mixture), however, the two substances do not interact at the chemical level and / or no chemical bond is formed between them.

[0153] Within the scope of the present invention, and as stated above, preferably the term "keratin structure" is intended to mean human hair, beard, eyelashes, eyebrows or the hair of a mammal, such as a dog or cat or other pet or companion animal.

[0154] Typically, suitable carriers are physiologically, edible or pharmaceutically acceptable carriers.

[0155] Typically, compositions for oral administration may include one or more edible carriers.

[0156] The composition of the present invention includes any composition prepared by mixing masleric acid and an extract of a Swertia plant (preferably Swertia indica) and a physiologically acceptable carrier. Such a composition is suitable for use in nutrition, medicine or diet of mammals (especially humans).

[0157] The carrier can take a wide variety of suitable forms depending on the desired form of preparation for oral and topical administration.

[0158] Thus, the physiologically and / or pharmaceutically acceptable carrier, diluent or excipient may be selected based on the intended route of administration of the composition.

[0159] Compositions for oral administration may be in solid or liquid form.

[0160] Typically, solid form compositions include tablets, capsules, powders, granules, and pills. If desired, tablets can be coated with suitable polymers or agents by conventional techniques to achieve sustained release / delayed release.

[0161] Exemplary excipients that can be used in solid forms include cellulose derivatives such as hydroxymethylcellulose, hydroxypropylmethylcellulose, methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, carboxyethylcellulose, ethylhydroxyethylcellulose, cellulose acetate butyrate, cellulose acetate phthalate, and mixtures thereof.

[0162] Examples of suitable excipients also include lactam polymers (usually pyrrolidone and its derivatives, such as polyvinylpyrrolidone, polyvinylpolypyrrolidone and mixtures thereof), inorganic salts (such as calcium phosphate or dicalcium phosphate), lubricants (such as magnesium stearate, triglycerides and mixtures thereof).

[0163] Tablets, pills, capsules, etc. may also contain a binder such as gum tragacanth, gum arabic, corn starch or gelatin; an excipient such as dicalcium phosphate; a disintegrant such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetener such as sucrose, lactose, saccharin or other sweeteners. When the unit dosage form is a capsule, in addition to the above-mentioned materials, a liquid carrier (such as a fatty oil) may also be included.

[0164] In some embodiments, the composition of the present invention further comprises one or more other components, such as additives, fillers, stabilizers, emulsifiers, structurants, film formers, plasticizers, wetting agents, and thickeners.

[0165] Various other materials can be used as coatings, or to adjust the physical shape of the dosage unit. For example, tablets can be coated with shellac, sugar, or both shellac and sugar. In addition to the active ingredient, the syrup or elixir can contain sucrose as a sweetener, methylparaben and propylparaben as preservatives, coloring agents, and flavorings (such as cherry or orange flavoring). To prevent rupture during passage through the upper gastrointestinal tract, the composition can be an enteric-coated preparation.

[0166] Typically, liquid forms of compositions for oral administration include solutions, suspensions, syrups, emulsions, and gels.

[0167] In these administration forms, suitable carriers include water, glycols, or oil-in-water or water-in-oil emulsions containing the biologically active ingredient dispersed therein.

[0168] In some embodiments, the composition of the present invention is administered topically. Compositions for topical administration can be in solid, semi-liquid, or liquid form. Typically, topical compositions are applied to the skin, particularly the scalp, in an effective amount.

[0169] For example, for cosmetic use, a cosmetically effective amount of the composition of the present invention may be applied directly to the scalp once or more daily, conveniently for a period of 2-3 months, alternating with rest periods.

[0170] Compositions for topical administration may be in solid, semisolid or fluid form.

[0171] Suitable solid forms of formulations include creams, gels, pastes, ointments.

[0172] In other embodiments, the formulation for topical administration is in the form of a fluid, such as a lotion, gel, shampoo, suspension, or emulsion.

[0173] For fluid or semi-fluid formulations, the biologically active ingredient can be diluted in a physiologically acceptable liquid carrier such as water, alcohol, hydroalcoholic or glycerol solution, or mixed with other liquids suitable for topical administration.

[0174] In the form of a solution, suspension, or dispersion, the compositions of the present invention may comprise from about 1% to 99.9% liquid (e.g., water mixed with an alcohol). In some embodiments, the water is present in an amount of 5% to 95% by weight. In other embodiments, the water is present in an amount of 10% to 90% by weight.

[0175] Typically, the topical composition for topical use is a hydroalcoholic solution (water and ethanol) comprising dissolved therein a combination of Maslinic acid and an extract of a plant of the genus Swertia, preferably Swertia indica.

[0176] For example, a liquid form of the composition of the present invention can be prepared by dissolving the soluble components in water and the remaining components in ethanol, and then combining the components under stirring. The resulting mixture can then be buffered to a suitable pH range, preferably between 5 and 7 to be compatible with the pH of the scalp, and then filtered and packaged in a suitable container (such as a bottle or vial).

[0177] In some embodiments, the compositions of the present invention are in the form of a topical lotion.

[0178] Typically, the bioactive ingredients masleric acid and Swertia plant (preferably Swertia indica) extract in the composition are formulated into dosage units. Calculated per dosage unit of the finished product for daily administration, each dosage unit may contain 0.1 mg to 1000 mg of the active ingredient.

[0179] In some embodiments, the effective amount of the formulation depends on the severity of the disorder or condition to be treated. In some embodiments, the dosage is from 0.001% to about 60% by weight of the formulation.

[0180] According to some preferred aspects, the present invention relates to topical compositions that can be used to stimulate hair follicles, thereby beneficially treating hair growth disorders (such as hair loss), particularly in women. Treatment methods for related populations are also a feature of the present invention.

[0181] According to another aspect of the present invention, a beauty care method is provided, comprising applying the composition of the present invention to the area to be cared for (preferably the scalp), wherein the composition is an effective amount of the above-mentioned type of composition.

[0182] The dosage and frequency of administration of the composition will depend on the type and severity of the trichological condition to be treated.

[0183] For example, for topical use, a cosmetically or pharmaceutically active amount of the composition of the invention is applied directly to the area to be treated (preferably the scalp) once or more daily, conveniently for a period of 2-3 months alternating with rest periods.

[0184] The following examples are provided only to illustrate the present invention and should not be construed as limiting the scope of protection as indicated in the appended claims.

[0185] Example 1

[0186] A composition for oral administration, comprising an olive extract containing Masriic acid, having the following formula:

[0187]

[0188] Example 2

[0189] Supplements for oral administration, having the following formulations:

[0190]

[0191]

[0192] Example 3

[0193] The composition in liquid form has the following formula:

[0194]

[0195] Example 4

[0196] The composition has the following formula:

[0197]

[0198]

[0199] Example 5

[0200] Experimental testing

[0201] In the context of this test, the Maslinic acid used is contained in a mixture called Naturolive, based on an extract of olive fruit (olive oil) with a terpene content of at least 75% by weight, wherein the Maslinic acid content is greater than 60% (w / w) and the mixture of Maslinic acid and oleanolic acid is greater than 75% (w / w). Maslinic acid is combined with a dry extract of Swertia indica on maltodextrin in a ratio of 3:1.

[0202] Materials and methods

[0203] Cell culture

[0204] use Human hair follicle dermal papilla cell (HFDPC) cell line was used to test the inhibitory activity of masleric acid and Swertia indica extract (dry extract on maltodextrin) alone or in combination against PYGL.

[0205]

[0206] HFDPC was isolated from the scalp dermis of healthy subjects.

[0207] HFDPCs (DP; PromoCell GmbH, Heidelberg, Germany, batches 325Z017.1 and 322Z030.1) were cultured in basal medium (hair follicle dermal papilla cell growth medium) (PromoCell GmbH) supplemented with 4 μL / mL bovine pituitary extract (bovine pituitary extract), 0.04 mL / mL fetal serum, 1 ng / mL recombinant human fibroblast growth factor, and 5 μg / mL recombinant human insulin (PromoCell). Cells were treated with 50 mg / mL Primocin (Invivogen, Toulouse, France, #ant-pm-2) and cultured in a humidified incubator at 37°C with 5% CO2.

[0208] Hair Follicle Dermal Papilla Cell Growth Medium contains all the growth factors and components required for optimal growth of dermal papilla cells, as shown in the table below:

[0209]

[0210] comparison

[0211] Positive control: cells treated in hair follicle dermal papilla cell growth medium without serum (starvation) under the same culture conditions.

[0212] Negative control: untreated cells in hair follicle dermal papilla cell growth medium under the same culture conditions.

[0213] Reagents and instruments used

[0214]

[0215]

[0216]

[0217] RNA extraction and qPCR analysis

[0218] PYGL gene expression was investigated by RT-PCR. Analysis was performed using Taqman probes according to the methods reported below (Gibson et al., 1996; Heid et al., 1996).

[0219] a) Day 1: Cell seeding

[0220] When the cells (HFDPC) reached about 80% confluence, trypsin / EDTA was used to detach the cells and 1×10 6 The cells were seeded at a density of 100 cells / mL in a 12-well plate and cultured at 37°C under 5% CO2 for 24 h.

[0221] b) Day 2-3: Processing

[0222] Once 80% confluence was reached, the spent medium was removed and the cells were treated with serum-free hair follicle dermal papilla cell growth medium (starvation) for 24 h under the same culture conditions. Cells treated with complete medium served as negative controls.

[0223] After 24 h, cells were treated with the substances to be evaluated:

[0224] #1: Maslinic acid (solid mixture of olive tree leaf and fruit extracts containing 75 wt% of Maslinic acid): 6 μg / mL

[0225] #2: Indian Swertia (SWT-7 TM )2μg / mL

[0226] #3: pure Maslic acid: 6 μg / mL;

[0227] #4: Maslinic acid (solid mixture of olive tree leaf and fruit extracts containing 75 wt% of Maslinic acid) 6 μg / mL + Swertia indica (SWT-7 TM )2μg / mL

[0228] #9: Pure Masriic acid 6μg / mL + Swertia indica (SWT-7 TM )2μg / mL

[0229] Pure substances were dissolved in DMSO (1000X stock solution) and diluted to the final concentration in human hair follicle dermal papilla cell growth medium.

[0230] Positive and negative controls were included in the experiments.

[0231] The cells were cultured at 37°C and 5% CO2 for 24 h.

[0232] c) RT-PCR

[0233] The gene expression of different markers was assessed by relative quantitative RT-PCR (quantitative reverse transcription-polymerase chain reaction-qRT-PCR).

[0234] This analysis involves 3 consecutive steps:

[0235] -Total RNA extraction;

[0236] -Reverse transcription to synthesize cDNA;

[0237] -qRT-PCR.

[0238] Total RNA was extracted using the RNeasy mini kit according to the method described by Chomczynski and Mackey (6). At the end of the extraction, the extracted RNA was quantified using a QiaExpert instrument (Qiagen) and the concentration of the extracted total RNA was calculated (μg / mL) at a wavelength of 260 nm.

[0239] Finally, the integrity of RNA (2 μg / mL) was assessed by 1% agarose gel electrophoresis.

[0240] Total RNA is converted into cDNA (complementary DNA) using an enzyme capable of synthesizing DNA molecules using RNA strands as templates; this RNA-dependent DNA polymerase is called reverse transcriptase.

[0241] It binds to the 3' end of single-stranded RNA and synthesizes cDNA strands using random primers and deoxynucleoside triphosphates (DNTPs).

[0242] For this purpose, the commercial kit "PrimeScript TM RT Reagent Kit (perfect RealTime)" (Takara Bio Inc., Japan), which includes: 5X PrimeScript buffer (for real-time), PrimeScript reverse transcriptase mix 1, Oligo dT primer, random 6mers, and RNase-free dH2O.

[0243] Extracted and quantified RNA was diluted to a concentration of 2 μg / mL and reverse transcribed into cDNA. Prepare 10 μL of a master mix (containing 5X PrimeScript Buffer (for real-time), PrimeScript Reverse Transcriptase Mix 1, Oligo dT Primer 50 μM, and Random Hexamer 100 μM) and add 10 μL of RNA (2 μg / mL).

[0244] The samples were placed in a thermal cycler (Stratagene Mx3000P Real-Time PCR System, Agilent Technologies Italia, Milan, Italy) and reverse transcription was performed under the following conditions:

[0245] 37°C, 15 minutes;

[0246] 85°C, 5 seconds;

[0247] 4℃, maintain.

[0248] At the end of reverse transcription, 30 μL DEPC water was added to the sample to obtain a final cDNA concentration of 40 ng / μL.

[0249] qRT-PCR is a method that amplifies and quantifies the generated amplicons in real time by monitoring the fluorescence emitted during the reaction.

[0250] The probe system (Applied Biosystems) was used for RT-PCR amplification. The following TaqMan probes were used: Hs00958087_m1 (PYGL) and Hs99999905_m1 (GAPDH). GAPDH was used as a control gene (housekeeping gene).

[0251] Taqman probe is a probe that can produce fluorescence during the amplification process. TM A fluorescent dye (a reporter group) is attached to the 5' end of the probe, while a quencher group is attached to the 3' end of the probe. The proximity of the reporter group and the quencher group eliminates the emission of the fluorescent signal. The fluorescent signal is only detectable in the presence of a thermostable DNA polymerase (Taq polymerase) with 5' exonuclease activity. The cycle-by-cycle increase in reporter fluorescence allows for the assessment of amplification product accumulation.

[0252] For qRT-PCR, prepare the master mix as follows:

[0253] 2× Premix Ex Taq, 10 μL;

[0254] ·20×TaqMan Gene Expression Assays (including 2 primers and fluorescent probe FAM labeled with fluorescent group) TM ), 1 μL;

[0255] ROX II Passive Reference Dye, 0.4 μL;

[0256] DEPC water, 5 μL.

[0257] Add 4 μL of target gene cDNA and 1 μL of housekeeping gene cDNA to the premix.

[0258] Amplification was performed for 40 cycles under the following conditions:

[0259] 95°C, 30 seconds (Amplitaq activation);

[0260] 95°C, 5 seconds (denaturation);

[0261] 60°C, 20 seconds (annealing-extension);

[0262] Each analysis was performed in duplicate.

[0263] Use 2 -ΔΔCT The data obtained can be analyzed by the method, so the relative expression of the target gene can be calculated, normalized with the housekeeping gene and calibrated on the control sample (untreated cells):

[0264] ΔΔCt=ΔCt 目标基因-管家基因 (Control)-ΔCt 目标基因-管家基因 (treated cells)

[0265] Assuming the amplification efficiency is 100%, calculate 2 -ΔΔCT value.

[0266] d) Statistical analysis

[0267] Statistical analysis was performed using GraphPad Prism 6.00 for Windows (GraphPad Software, La Jolla, CA, USA, www.graphpad.com).

[0268] The Student's t test was used to compare the mean values ​​and relative standard errors of all test preparations with those of the positive control group, with a confidence interval set at 95%.

[0269] result

[0270] As shown in the figure, Maslinic acid (6 g / mL) was able to significantly inhibit (p < 0.005) the expression of the PYGL gene in dermal papilla cells. This inhibitory effect was significant in both the test using Maslinic acid alone and the solid mixture of olive leaf and fruit extract containing 75 wt% of Maslinic acid ( Figure 1 Swertia indica (2 μg / mL) also exerted a significant (p<0.005) inhibitory effect on PYGL gene expression ( Figure 1 Surprisingly, the combination of a solid mixture of olive leaf and fruit extracts containing 75 wt% of Maslinic acid and Swertia indica extract, as well as the combination of Maslinic acid and Swertia indica extract, showed a synergistic effect and significantly (p<0.005) higher inhibition of PYGL gene expression compared to the positive control and the single components (Figure).

Claims

1. A composition for non-therapeutic, cosmetic use for improving the appearance or aesthetic features of keratinous structures of a mammal and / or for promoting the physiological growth of keratinous structures, wherein the keratinous structures are selected from the group consisting of hair and skin appendages, wherein the composition comprises a combination of masleric acid and an extract of a plant of the genus Swertia, preferably Swertia indica, and a physiologically acceptable excipient.

2. The method according to claim 1, wherein the keratin structure is selected from the group consisting of human hair, beard, eyelashes, eyebrows and nails, or animal hair.

3. The use according to claim 1 or 2, wherein the Maslinic acid is of natural origin and exists in the form of an extract extracted from olive leaves and / or fruits, preferably from olive peels; and the extract of the Swertia plant, especially Swertia indica, is an extract obtained by extraction using a protonic polar solvent.

4. The method according to claim 3, wherein the protic polar solvent is selected from the group consisting of water, acetic acid, ethanol, ethyl acetate, n-butanol, isopropanol, n-propanol and mixtures thereof.

5. The use according to claim 3, wherein the Maslinic acid is present in the form of an extract extracted from olive leaves and / or fruits, and the extract further comprises oleanolic acid.

6. The use according to any one of claims 1 to 5, wherein the composition is used by oral administration or topical application, preferably on the scalp.

7. A composition comprising a combination of masleric acid and an extract of a Swertia plant, preferably Swertia indica, and a physiologically acceptable excipient for use in treating a growth disorder of keratinous structures in a mammal, the keratinous structures being selected from the group consisting of hair and skin appendages.

8. A composition for use according to claim 7, wherein the keratin structure is human hair.

9. A composition for use according to claim 7, wherein the keratinous structure is a skin appendage selected from the group consisting of human beard, eyelashes, nails and eyebrows.

10. A composition for use according to any one of claims 7 to 9, wherein the disorder of the growth of keratin structures is selected from the group consisting of alopecia, androgenic alopecia, alopecia areata, tinea, cicatricial alopecia and telogen effluvium.

11. The composition for use according to any one of claims 7 to 10, wherein the Maslinic acid is of natural origin and is present in the form of an extract obtained from olive leaves and / or fruits, preferably from olive peel; preferably, the composition comprises oleanolic acid and an extract of a plant of the genus Swertia, in particular an extract of Swertia indica; the extract of the plant of the genus Swertia is an extract obtained by extraction with a protic polar solvent; preferably, the protic polar solvent is selected from the group consisting of water, acetic acid, ethanol, ethyl acetate, n-butanol, isopropanol, n-propanol and mixtures thereof.

12. A composition for use according to any one of claims 7 to 11, wherein the composition is for oral administration or topical application.

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