Cosmetic compositions

Abstract

The present disclosure relates generally to cosmetic compositions comprising a Crocus sativus flower extract, and their use for skin treatment, particularly as anti-dandruff. The compositions provide very significant anti-dandruff, anti-fungal, anti-erythema, skin soothing, and antipruritic effects, and also improve skin barrier integrity.

Description

[0001] COSMETIC COMPOSITIONS

[0002] TECHNICAL FIELD

[0003] The present disclosure relates generally to cosmetic compositions comprising a Crocus sativus flower extract, and their use for skin care.

[0004] BACKGROUND

[0005] Currently, the main active molecules efficient on Malassezia spp. growth inhibition are mainly of synthetic origins, with significant regulatory and safety limitations. Thus, there is an unmet need for botanical natural active ingredients with 100% natural source according to ISO16128 guidelines, efficient as anti-Malassezia and anti -dandruff products, and efficient on seboregulation and anti-malodour.

[0006] In order to overcome the above-mentioned problem, the inventors have surprisingly discovered, and disclose herein, the use of a Crocus sativus flower extract as antifungal (especially anti-Malassezia). Notably, said extract, and skin care compositions prepared therefrom, are found to possess potent antidandruff effect when applied to scalp, as well as soothing activity, anti-erythema effect, and antipruritic effect, on human skin, and also improve skin barrier integrity.

[0007] SUMMARY OF THE INVENTION

[0008] In accordance with a first aspect of the present disclosure there is provided a cosmetic composition comprising a cosmetically acceptable excipient and at least one active cosmetic ingredient, wherein a first active cosmetic ingredient comprises or consists of a Crocus sativus flower extract.

[0009] In accordance with a second aspect of the present disclosure there is provided the use of the cosmetic composition according to the first aspect for anti-dandruff treatment, anti-fungal treatment, anti-erythema treatment, antipruritic treatment, skin soothing, and / or improvement of skin barrier integrity, comprising the step of applying the cosmetic composition to skin, e.g. to scalp.

[0010] In accordance with a third aspect of the present disclosure there is provided a method of cosmetic skin treatment, comprising the step of applying the cosmetic composition of the first aspect to the skin, preferably wherein the skin comprises the scalp.

[0011] In accordance with a fourth aspect of the present disclosure there is provided the use of a Crocus sativus flower extract in skin care, and in particular for anti-dandruff treatment, anti-fungal treatment, antierythema treatment, antipruritic treatment, skin soothing, and / or improvement of skin barrier integrity, preferably wherein the skin comprises the scalp. In accordance with a fifth aspect of the present disclosure there is provided a Crocus sativus flower extract, or a composition comprising thereof, for use in treating or preventing an inflammatory disease or a fungal infection.

[0012] These and other aspects of the invention will be better understood in view of the following detailed description of particular embodiments of the invention.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 shows Malassezia spp. growth inhibition. Cultures of Malassezia spp. were incubated for 24 h with a range of Crocus sativus flower extract concentrations from 100% to 0.02%. The results are presented in cells / mL.

[0015] Figure 2 shows Minimum Inhibitory Concentration of ketoconazole, piroctone olamine and climbazole at equivalent dry matter of Crocus sativus flower extract. Statistics determined by Mann Whitney test with **p<0.01.

[0016] Figure 3 shows Minimum Inhibitory Concentration of kaempferol-3-O-sophoroside (K3OS), kaempferol-3-O-glucoside (astragalin) (K3OG), and kinsenoside) and the mixture of the 3 tested in comparison with Crocus sativus flower extract at equivalent dose on M. globosa culture. Statistics determined by Mann Whitney test with *p<0.05.

[0017] Figure 4 shows bioguided inhibition of Malassezia growth. Cultures of Malassezia spp. were incubated for 24 h with Crocus sativus flower extract or the solvent alone (NADES Eutectys BLA) or Fractions (A, B, C, D) at a concentration range of 100% to 0.02% for each condition. The results are presented in cells / mL. Figure 5 shows fungistatic activity of Crocus sativus flower extract on Malassezia globosa. M. globosa (ATCC MYA 4612) was incubated for 36 h with Crocus sativus flower extract (3%), vehicle (3%) and Ketoconazole (50 pg / ml). Colony forming units (CFU) were quantified at different time points (TO, T+4h, T+8h, T+24h, T+32h and T+48h) in order to determine fungicidal activities.

[0018] Figure 6 shows membrane disruptive activity on Malassezia globosa membrane integrity. Cultures of M. globosa (MYA-4889) were treated with increasing concentrations of Crocus sativus flower extract for 4 h at 30 °C. Result are presented in MFI (mean fluorescent intensity). Statistical analysis was done by ANOVA.

[0019] Figure 7 shows depolarization of mitochondrial membrane of M. globosa. Cultures of M. globosa (MYA-4889) were treated with increasing concentrations of Crocus sativus flower extract and 10 mM sodium azide (NaN₃) as a positive control for 30 min at 30 °C. Result are presented in MFI (mean fluorescent intensity). Statistical analysis was done by ANOVA. Figure 8 shows anti-inflammatory activity under chemical stress. Human normal keratinocytes were pretreated with active or dexamethasone for 24 h then PMA (phorbol myristate acetate) was added in the culture media for inflammation induction. Cytokines were quantified by Multiplex method after 24 additional hours. Results are expressed using pro- and anti-inflammatory cytokines ratio for studying soothing response. Statistical analysis was done by Man Whitney test.

[0020] Figure 9 shows reduction of inflammation under inactivated Malassezia spp. topical application using RHE. Result are normalized relative to Stress condition and presented in percent. Student’s t-test was used for statistical analysis.

[0021] Figure 10 shows reduction of histamine release by itching receptor inhibition. Data was normalized relative to untreated and expressed in percent. Mann-Whitney test was used for statistical analysis.

[0022] Figure 11 shows measurement of viable epidermis thickness in pictures of PAS staining of RHE in presence or absence of M. globosa treated or not with Crocus sativus flower extract at 1%. Statistics analysis with Mann-Whitney t-test with *** p<0.001.

[0023] Figure 12 shows skin barrier integrity improvement on Malassezia-co\omzed RHE. Results are expressed in mean intensity of fluorescence and statistical analysis was done by Mann-Whitney test.

[0024] Figure 13 shows quantification of Lucifer yellow fluorescence intensity in RHE (modelling the skin permeability), in presence or absence of M. globosa treated or not with Crocus sativus flower extract at 1%. Statistics analysis was performed using Mann-Whitney test with *** p<0.001.

[0025] Figure 14 shows improvement of skin barrier on skin explant ex vivo model. Results are expressed in mean intensity of fluorescence and statistical analysis was done by Mann-Whitney test.

[0026] Figure 15 shows clinical reduction of dandruff area after 14 days of using shampoo comprising 3% Crocus sativus flower extract. Results are expressed in delta D0D14 expressed in percent. Statistical analysis was done using Wilcoxon test for paired data and Mann-Whitney for unpaired data.

[0027] Figure 16 shows soothing effect of shampoo comprising 3% Crocus sativus flower extract on human volunteers presenting Dandruff. Results are expressed in delta D0D14 expressed in percent. Statistical analysis was done using Student’s t-test.

[0028] Figure 17 shows reduction of Malassezia genus abundance from scalp swabbing after shampoo application with 3% Crocus sativus flower extract. Malassezia genus abundance was quantified by RT-QPCR method. Statistical analysis was done using Wilcoxon test.

[0029] Figure 18 shows the impact of switching Active shampoo to Neutral shampoo on dandruff reduction. Figure 19 shows the impact of switching active shampoo to Neutral shampoo on erythema reduction. Figure 20 shows summary of the effects of Placebo and Crocus sativus flower extract treatments on two features of microbial community stability: interdependence and forced collaboration, compared to healthy and dandruff-affected scalps. The blue lines represent the levels observed in healthy scalps, while the red lines indicate those in dandruff scalps. The triangles illustrate the changes between the compared conditions.

[0030] DETAILED DESCRIPTION

[0031] During biological evaluation of a Crocus sativus flower extract, the inventors have discovered, surprisingly, that cosmetic compositions comprising a Crocus sativus flower extract inhibit the growth of Malassezia spp. (M. restricta. M. furfur and M. globosa), which are involved in dandruff formation. We performed bioguided fractionation on anti-fungal activity on Malassezia in order to identify active compounds. We discovered that several molecules are efficient in Malassezia inhibition, but none of them is able to show the same performance and efficiency of the whole extract, even after blending them. We confirmed that the particularity and complexity of the extract is responsible for its anti-fungal activity, while isolated molecules only show part of the activity. We found that Crocus sativus flower extract had a fungistatic activity delivering its anti-fungal mode of action.

[0032] Without being bound by theory, we believe that Crocus sativus flower extract inhibits Malassezia growth by fungal membrane disruption activity and depolarization activity on mitochondrial membrane.

[0033] We also discovered that Crocus sativus flower extract has soothing activity, presumably by reducing pro-inflammatory cytokines under chemical stress or Malassezia-mediated inflammation induction. Interestingly, we showed that Crocus sativus flower extract can reduce instant itching, presumably by limiting histamine release by blocking MRGP2 receptor, reducing related pruritus. Crocus sativus flower extract also demonstrated additional biological activity like improvement of skin barrier integrity by controlling excessive keratinocytes migration and overexpression of Desmoglein-1 and Claudin-1. At clinical level, we showed that Crocus sativus flower extract significantly reduces dandruff count and area and limit erythema after 14 days.

[0034] Cosmetic compositions, and in particular skin care compositions, of the present invention comprise a cosmetically acceptable amount of a Crocus sativus flower extract and a cosmetically acceptable excipient (e.g. carrier), as disclosed herein.

[0035] In a first aspect, the present disclosure relates to a cosmetic composition comprising a cosmetically acceptable excipient and at least one active cosmetic ingredient, wherein a first active cosmetic ingredient comprises or consists of a Crocus sativus flower extract.

[0036] A particularly suitable Crocus sativus flower extract is an aqueous extract. In preferable embodiments, the Crocus sativus flowers do not comprise stigma (e.g. the stigma is removed before using the flowers for preparing the extract). As floral tissues (tepals and petals) are rich in kaempferol-3-O-sophoroside and also contain unique compounds such as kinsenoside and 3 -hydroxybutyrolactone, which are absent from stigmas, excluding the stigma form the material used for extraction would make the extract richer in said compounds, which have favourable effects on skin, e.g. as anti-erythema, antipruritic, skin soothing, and improvement of skin barrier integrity. In some embodiments, the flowers are sterilised, minced, dried and / or ground.

[0037] Exemplary extracts and production methods thereof are provided herein e.g. in Example 1.

[0038] A cosmetically effective amount of Crocus sativus flower extract is understood to be a non-toxic but sufficient quantity thereof to provide the desired effect. With regard to the total weight of the cosmetic composition, in particular the skin care composition, of the present invention the Crocus sativus flower extract may be present in amounts of about 0.01% w / w to about 5% w / w, particularly about 0.06% w / w to about 3% w / w. In some embodiments, the Crocus sativus flower extract is present in amounts of about 0.8% w / w to about 3% w / w of the total weight of the cosmetic composition.

[0039] In an embodiment, the cosmetically acceptable excipient comprises, e.g. consists of, a carrier. In a preferred embodiment, the carrier is a cosmetically acceptable carrier.

[0040] In an embodiment, the cosmetically acceptable excipient comprises or consists of a eutectic solvent, more preferably a natural deep eutectic solvent, and most preferably a eutectic solvent consisting of about 17% - 19% (e.g. about 18%) potassium lactate, about 7% - 9% (e.g. about 8%) lactic acid, about 26% - 35% (e.g. about 30%) betain, and about 37% - 50% (e.g. about 44%) water.

[0041] In an embodiment, the cosmetic composition further comprises one or more other active cosmetic ingredient(s), preferably one or more other active cosmetic ingredient(s) selected from the group consisting of lightening agents, dark spot erasers, skin tone agents, hydrating agents, moisturizers, UV protection actives, anti-ageing actives, and mixtures thereof.

[0042] In an embodiment, the cosmetic composition is a skin care composition, preferably a scalp care composition, e.g. an anti-dandruff composition.

[0043] Cosmetic preparations, e.g. skin care compositions, of the present invention comprise one or more cosmetically acceptable excipients. Any excipients commonly used in the preparation of cosmetic preparations for use on the human skin and / or hair may be employed in the present invention. Suitable excipients include, but are not limited to ingredients that can influence organoleptic properties, penetration of the skin, and the bioavailability of the Crocus sativus flower extract. More specifically, they include liquids, such as water, oils or surfactants, including those of petroleum, animal, plant or synthetic origin, such as and not restricted to, peanut oil, soybean oil, mineral oil, sesame oil, castor oil, polysorbates, sorbitan esters, ether sulfates, sulfates, betaines, glycosides, maltosides, fatty alcohols, nonoxynols, poloxamers, polyoxyethylenes, polyethylene glycols, dextrose, glycerol, digitonin, and the like.

[0044] The cosmetic preparation, and in particular the skin care composition, may be in the form of a liposome composition, mixed liposomes, oleosomes, niosomes, ethosomes, milliparticles, microparticles, nanoparticles and solid-lipid nanoparticles, vesicles, micelles, mixed micelles of surfactants, surfactantphospholipid mixed micelles, millispheres, microspheres and nanospheres, lipospheres, millicapsules, microcapsules and nanocapsules, as well as microemulsions and nanoemulsions, which can be added to achieve a greater penetration of the active cosmetic ingredient, e.g. the Crocus sativus flower extract. The cosmetic preparation, and in particular the skin care composition, may be produced in any solid, liquid, or semi-solid form useful for application to the skin topically or by transdermal application. Thus, these preparations of topical or transdermal application include, but are not restricted to, creams, multiple emulsions, such as and not restricted to, oil and / or silicone in water emulsions, water-in-oil and / or silicone emulsions, water / oil / water or water / silicone / water type emulsions, and oil / water / oil or silicone / water / silicone type emulsions, micro-emulsions, emulsions and / or solutions, liquid crystals, anhydrous compositions, aqueous dispersions, oils, milks, balsams, foams, aqueous or oily lotions, aqueous or oily gels, cream, hydro-alcoholic solutions, hydro-glycolic solutions, hydrogels, liniments, sera, soaps, shampoos, face masks, serums, polysaccharide films, ointments, mousses, pomades, pastes, powders, bars, pencils and sprays or aerosols (sprays), including leave-on and rinse-off formulations.

[0045] In an embodiment, the cosmetic composition is for anti-dandruff treatment, anti-fungal treatment, antierythema treatment, antipruritic treatment, skin soothing, and / or improvement of skin barrier integrity, comprising the step of applying the cosmetic composition to skin, e.g. to scalp.

[0046] In a second aspect, the present disclosure relates to the use of the cosmetic composition according to the first aspect for anti-dandruff treatment, anti-fungal treatment, anti-erythema treatment, antipruritic treatment, skin soothing, and / or improvement of skin barrier integrity, comprising the step of applying the cosmetic composition to skin, e.g. to scalp.

[0047] In a third aspect, the present disclosure relates to a method of cosmetic skin treatment, comprising the step of applying the cosmetic composition of the first aspect to the skin, preferably wherein the skin comprises the scalp.

[0048] In an embodiment, the method is for anti-dandruff treatment, anti-fungal treatment, anti-erythema treatment, antipruritic treatment, skin soothing, and / or improvement of skin barrier integrity.

[0049] In a fourth aspect, the present disclosure relates to the use of a Crocus sativus flower extract in skin care, and in particular for anti-dandruff treatment, anti-fungal treatment, anti-erythema treatment, antipruritic treatment, skin soothing, and / or improvement of skin barrier integrity, preferably wherein the skin comprises the scalp.

[0050] In a fifth aspect, the present disclosure relates to a Crocus sativus flower extract, or a composition comprising thereof, for use in treating or preventing an inflammatory disease or a fungal infection. In an embodiment, the fungal infection comprises infection with a Malassezia species, particularly with M. restricta M. furfur or M. globosa. In an embodiment, the fungal infection is atopical infection.

[0051] The present invention provides a formulation (e.g. a cosmetic formulation) comprising a Crocus sativus flower extract. The present invention also provides a product (e.g. a cosmetic product) comprising said composition. The cosmetic formulation / product may optionally further comprise cosmetic (including cosmetic active) ingredients, such as excipients, carriers (particularly, cosmetically acceptable carriers) and mixtures thereof as appropriate.

[0052] Throughout this disclosure, the terms “composition”, “preparation” and “formulation” are used interchangeably, unless otherwise noted.

[0053] A “cosmetic product” is intended to mean any substance or mixture intended to be placed in contact with the external parts of the human body (epidermis, hair system, nails, lips and external genital organs) or with the teeth and the mucous membranes of the oral cavity with a view exclusively or mainly to cleaning them, perfuming them, changing their appearance, protecting them, keeping them in good condition, correcting body odours and / or combinations thereof.

[0054] A “mixture” is intended to mean a mixture or solution composed of two or more substances.

[0055] “Cosmetic” or “cosmetic active ingredients” means any and all natural, naturally occurring, nature identical, synthetic synthetically produced, biosynthetically produced, sustainable, renewable and / or biodegradable compounds, ingredients, intermediates, molecules, substances, raw materials or products individually or as part of a mixture of compounds, ingredients, intermediates, molecules, substances, raw materials or products, blends, compositions, formulations (including but not limited to skin moisturizers, creams, balms, serums, oils, eye, facial makeup, wash off hair products, leave-on hair products, hair colorants (including but not limited to natural hair colorants) and / or combinations thereof), finished products and related technologies including, but not limited to components, incorporated, for example, into a cosmetic formulation (such as but not limited to natural colorants, preservatives, emulsifiers, antioxidants and the like which do not, for example, have an activity on the skin, hair, scalp and the like but play a role in the formulation of the finished product), delivery systems, marketing aids (such as, for example, coloured Unispheres™ applied to translucent formulations) and methods of making anything related thereto useful in / used for / intended for: - application by rubbing, pouring, sprinkling, spraying or otherwise directly on or to a human or animal body and / or by placing in contact with the various external and / or on surface parts of a human or animal body (including but not limited to the skin, hair, body hair, the hair system, scalp, nails, lips, external genitalia, teeth, oral and / or nasal mucosa and the like); and / or application indirectly on or to a human or animal body such as for example, application as part of a textile or application to a textile as part of a delivery device (such as a capsule) or a delivery system (such as blends or formulations) applied to a textile; and / or

[0056] - cleansing, caring, cooling, beautifying, conditioning, treating, soothing, texturizing, promoting attractiveness, protecting, maintaining, improving, enhancing, altering and / or changing an external part and / or surface of a human or animal body (such as, but not limited to the scalp) or the aesthetic appearance of a human or animal body; and / or with a view to mainly cleaning or perfuming, or protecting or maintaining in good condition or combating body odour or changing the appearance of or correcting or repairing a state of imbalance in skin, oral mucosa, scalp or hair by providing a calming, healing, repairing, or revitalization, hydration of the skin or in order to provide relief to, lubricate, moisten, tone, heal, sterilize, relieve, correct and / or remedy states of dryness, irritation, injury or fatigue, and / or with a view to correcting pigmentation disorders or providing a non-pharmaceutical prevention and / or treatment of dandruff, acne, irritation and / or inflammation and the like and / or rebalancing the bacterial flora (such as, for example, the microbiome) on the surface of the skin (such as, for example, by promoting the level of beneficial bacterial flora on the skin surface) and / or for the purposes of keeping a human or animal body in good condition for health and / or wellbeing purposes and / or for improving the appearance of a human or animal body by, for example, improving the appearance of a product applied to a human or body; and / or

[0057] - providing a cosmetic and / or dermatological function and / or benefit with a biological activity benefit (but without affecting a body’s structure or function). For the avoidance of doubt, a cosmetic or cosmetic active ingredient or any part thereof may also qualify as a functional ingredient.

[0058] As used herein, references to cosmetically acceptable excipients may refer to cosmetically acceptable adjuvants, diluents and / or carriers as known to those skilled in the art.

[0059] By “cosmetically acceptable” we mean that the additional components of the composition are generally safe, non-toxic, and neither biologically nor otherwise undesirable. For example, the additional components may be generally sterile and pyrogen free. Such components must be “acceptable” in the sense of being compatible with the composition of the invention and not deleterious to the recipients thereof. Thus, such excipients include any compound(s) used in forming a part of the formulation that is intended to act merely as an excipient, i.e. not intended to have biological activity itself.

[0060] The formulation or product (e.g. cosmetic formulation / product) may also contain one or more additional active ingredients, such as cosmetic active ingredients, such as lightening agents, dark spot erasers, skin tone agents, hydrating agents, moisturizers, UV protection actives, anti-ageing actives, hyaluronic acid, Centella asiatica extract, peptides such as Matrixyl® and Argireline®, and mixtures thereof.

[0061] Moreover, the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.

[0062] Suitable cosmetic carriers are typically those that are suitable for topical administration to the outer surface of the human body, such as the skin and / or hair and / or scalp.

[0063] Typically, such carriers are dermatologically acceptable.

[0064] The phrase "dermatologically acceptable carrier" means that the carrier is suitable for topical application to the keratinous tissue, has good aesthetic properties, is compatible with the actives in the composition, and will not cause any unreasonable safety or toxicity concerns.

[0065] The carrier can be in a wide variety of forms. In some instances, the solubility or dispersibility of the components (e.g. extracts, sunscreen active, additional components) may dictate the form and character of the carrier. Non-limiting examples include simple solutions (e.g. aqueous or anhydrous), dispersions, emulsions, and solid forms (e.g. gels, sticks, flowable solids, or amorphous materials).

[0066] The dermatologically acceptable carrier may be in the form of an emulsion. An emulsion may be generally classified as having a continuous aqueous phase (e.g. oil-in-water and water-in-oil-in-water) or a continuous oil phase (e.g. water-in-oil or oil-in-water). The oil phase of the present invention may comprise silicone oils, non-silicone oils such as hydrocarbon oils, esters, ethers, and the like, and mixtures thereof. The aqueous phase typically comprises water and water-soluble ingredients (e.g. water-soluble moisturizing agents, conditioning agents, antimicrobials, humectants and / or other skin care actives). However, in some instances, the aqueous phase may comprise components other than water, including but not limited to water-soluble moisturizing agents, conditioning agents, antimicrobials, humectants and / or other water-soluble skin care actives. In some instances, the non-water component of the composition comprises a humectant, such as glycerine and / or other polyol(s). Emulsions may also contain an emulsifier. Emulsifiers may be non-ionic, anionic or cationic.

[0067] The carrier may contain one or more dermatologically acceptable, hydrophilic diluents. As used herein, "diluent" includes materials in which the composition of the invention can be dispersed, dissolved, or otherwise incorporated. Hydrophilic diluents include water, organic hydrophilic diluents, such as lower monovalent alcohols (e.g., C1-C4), and low molecular weight glycols and polyols, including propylene glycol, polyethylene glycol, polypropylene glycol, glycerol, butylene glycol, 1,2,4-butanetriol, sorbitol esters, 1,2,6-hexanetriol, ethanol, isopropanol, sorbitol esters, butanediol, ether propanol, ethoxylated ethers, propoxylated ethers and combinations thereof. The cosmetic formulation / product may optionally include one or more additional ingredients commonly used in cosmetic compositions (e.g., colorants, preserving agents, skin tone agents, skin anti-aging agents, anti-inflammatory agents, sunscreen agents, combinations of these and the like), provided that the additional ingredients do not undesirably alter the benefits provided by the composition.

[0068] In some instances, it may be desirable to select skin tone agents that function via different biological pathways so that the actives do not interfere with one another, which could reduce the efficacy of both agents. The additional ingredients, when incorporated into the composition, should be suitable for use in contact with human skin tissue without undue toxicity, incompatibility, instability, allergic response, and the like.

[0069] As utilized in accordance with the methods, compounds, and compositions of the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0070] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or when the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” The use of the term “at least one” will be understood to include one as well as any quantity more than one, including but not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, or any integer inclusive therein. The term “at least one” may extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the quantities of 100 / 1000 are not to be considered limiting, as higher limits may also produce satisfactory results. In addition, the use of the term “at least one of X, Y, and Z” will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z.

[0071] The term “and / or” is understood to mean that all members of a group connected by the term “and / or” are represented both cumulatively with respect to each other in any combination, and alternatively with respect to each other. Exemplarily, for the expression “A, B and / or C”, the following disclosure is to be understood thereunder: i) (A or B or C), or ii) (A and B), or iii) (A and C), or iv) (B and C), or v) (A and B and C), or vi) (A and B or C), or vii) (A or B and C), or viii) (A and C or B).

[0072] As used herein, all numerical values or ranges include fractions of the values and integers within such ranges and fractions of the integers within such ranges unless the context clearly indicates otherwise. A range is intended to include any sub-range therein, although that sub-range may not be explicitly designated herein. Thus, to illustrate, reference to a numerical range, such as 1-10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., and so forth. Reference to a series of ranges includes ranges which combine the values of the boundaries of different ranges within the series. Thus, to illustrate reference to a series of ranges, for example, a range of 1-1,000 includes, for example, 1-10, 10-20, 20-30, 30-40, 40- 50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1,000, and includes ranges of 1-20, 10-50, 50-100, 100-500, and 500-1,000. Reference to an integer with more (greater) or less than includes any number greater or less than the reference number, respectively. Thus, for example, reference to less than 100 includes 99, 98, 97, etc. all the way down to the number one (1); and less than 10 includes 9, 8, 7, etc. all the way down to the number one (1).

[0073] The terms “increase,” “increasing,” "enhancing," or "enhancement" are defined as indicating a result that is greater in magnitude than a control number derived from analysis of a cohort, for example, the result can be a positive change of at least 5%, 10%, 20%, 30%, 40%, 50%, 80%, 100%, 200%, 300% or even more in comparison with the control number. Similarly, the terms “decrease,” “decreasing,” “lessening," or "reduction" are defined as indicating a result that is lesser in magnitude than a control number, for example, the result can be a negative change of at least 5%, 10%, 20%, 30%, 40%, 50%, 80%, 100%, 200%, 300% or even more in comparison with the control number.

[0074] As used in this specification and claims, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0075] When used herein “consisting of’ excludes any element, step, or ingredient not specified in the aspect, embodiment and / or claim element. When used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the aspect, embodiment and / or claim.

[0076] The term “or combinations thereof’ as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof’ is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0077] Throughout this application, the terms “about” or “approximately” are used to indicate that a value includes the inherent variation of error for the composition, the method used to administer the composition, or the variation that exists among the study subjects. As used herein the qualifiers “about” or “approximately” are intended to include not only the exact value, amount, degree, orientation, or other qualified characteristic or value, but are intended to include some slight variations due to measuring error, manufacturing tolerances, stress exerted on various parts or components, observer error, wear and tear, and combinations thereof, for example. The terms “about” or “approximately,” where used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass, for example, variations of ± 10%, or ± 5%, or ± 1%, or ± 0.1% (including all the values within this range) from the specified value, as such variations are appropriate to perform the disclosed methods and as understood by persons having ordinary skill in the art. In certain embodiments, the term about refers to ± 0.1, 0.2, or 0.3 logarithmic units, e.g. pH units. As used herein, the term “substantially” means that the subsequently described event or circumstance completely occurs or that the subsequently described event or circumstance occurs to a great extent or degree. For example, the term “substantially” means that the subsequently described event or circumstance occurs at least 90% of the time, or at least 95% of the time, or at least 98% of the time.

[0078] As used herein any reference to "one embodiment" or "an embodiment" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment and may be included in other embodiments. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment and are not necessarily limited to a single or particular embodiment. Further, all references to one or more embodiments or examples are to be construed as non-limiting to the claims.

[0079] Use of the word “we,” “us,” and / or “our” as a pronoun in the present disclosure refers generally to laboratory personnel, technicians, or other contributors who assisted in laboratory procedures and data collection and is not intended to represent an inventorship role by said laboratory personnel, technicians, or other contributors in any subject matter disclosed herein.

[0080] The term “carrier” as used herein, may also refer to a natural product or a product originating from nature that has been transformed or modified so that it is distinct from the natural product from which it originated, such as maltodextrin.

[0081] The amount of the cosmetic composition disclosed herein present in cosmetic products will vary depending on the application.

[0082] Typically, the amount of cosmetic composition disclosed herein that may be present in cosmetic products will be from about 0.001% to about 50% by weight, such as from about 0.01% to about 30% or from about 1% to about 20% of the cosmetic products, such as from about 0.01% to about 20%, or from about 0.1% to 10% or from about 1% to about 5% by weight of the product.

[0083] It is to be understood that this disclosure is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by the person skilled in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Where used herein, the specific term “single” is limited to only “one.” In accordance with the present disclosure there may be conventional molecular biology, microbiology, and recombinant DNA techniques employed which are within the skill of the art.

[0084] This disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. Preferably, the terms used herein are defined as described in " A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H. G. W, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

[0085] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, GenBank Accession Number sequence submissions etc.), whether supra or infra, is hereby incorporated by reference in its entirety.

[0086] EXAMPLES

[0087] The inventive concepts of the present disclosure will now be discussed in terms of several specific, nonlimiting, examples. The examples described below, which include particular embodiments, will serve to illustrate the practice of the present disclosure, it being understood that the particulars shown are by way of example and for purposes of illustrative discussion of particular embodiments of the present disclosure only and are presented in the cause of providing what is believed to be a useful and readily understood description of procedures as well as of the principles and conceptual aspects of the inventive concepts.

[0088] Example 1: Preparation of cosmetic agent: Crocus sativus extract

[0089] For evaluation of the cosmetic active agent of the invention, four specific Cosmetic Active Agents were prepared as below.

[0090] Cosmetic Active Agent A was prepared as follows at laboratory scale: 150 g of Crocus sativus flower was suspended in 3 litres of tap water and stirred for 2 hours at 60 °C. After a filtration on Fibrafix® filter plate AF6 (to remove the plant), a first filtrate was obtained. Then, the same biomass of Crocus sativus flower (150 g) was re-extracted with 3 litres of tap water and stirred for 2 hours at 60 °C and a second filtrate was obtained after filtration of Fibrafix® filter plate AF6 (to remove the plant). Both filtrates were combined and filtered again successively on Fibrafix® filter plates AF31H and AF140. Then a concentration (at 20 mBar, 50 °C) was performed to remove a part of the water to obtain 176.4 g of concentrated extract at 47.32% of dry matter. 10.56 g of this concentrated extract was mixed under stirring at 50 °C for 15 minutes with a mixture of 89.44 g of a liquid that contained 25% of tap water, 35% of TEGO® Natural betain and 40% of PURAC® BF P / 41 (comprising 45%-47% potassium lactate, 18.5%-20.5% lactic acid, and 32.5%-36.5% water).

[0091] Cosmetic Active Agent B was prepared as follows at laboratory scale: 150 g of Crocus sativus flower was suspended in 3 litres of tap water and stirred for 2 hours at 60 °C. After a filtration on Fibrafix® filter plate AF6 (to remove the plant), a first filtrate was obtained. Then, the same biomass of Crocus sativus flower (150 g) was re-extracted with 3 litres of tap water and stirred for 2 hours at 60 °C and a second filtrate was obtained after filtration of Fibrafix® filter plate AF6 (to remove the plant). Both filtrates were combined and filtered again successively on Fibrafix® filter plates AF31H and AF140. Then a concentration (at 20 mBar, 50 °C) was performed to remove a part of the water to obtain 92.4 g of concentrated extract at 67.28% of dry matter. 7.43 g of this concentrated extract was mixed under stirring at 50 °C for 15 minutes with a mixture of 92.57 g of a liquid that contain Eutectys™ buffered BLA (25% of tap water, 35% of TEGO® Natural betain and 40% of PURAC® BF P / 41).

[0092] Cosmetic Active Agent C was prepared as follows at laboratory scale: 80 g of Crocus sativus flower was suspended in 1.6 litres of tap water and stirred for 2 hours at 60 °C. After a filtration on Fibrafix® filter plate AF6 (to remove the plant), a first filtrate was obtained. Then, the same biomass of Crocus sativus flower (80 g) was re-extracted with 1.6 litres of tap water and stirred for 2 hours at 60 °C and a second filtrate was obtained after filtration of Fibrafix® filter plate AF6 (to remove the plant). Both filtrates were combined and filtered again successively on Fibrafix® filter plates AF31H. Then a concentration (at 20 mBar, 50 °C) was performed to remove a part of the water to obtain 83 g of concentrated extract at 56% of dry matter. 1.74 g of this concentrated extract was mixed under stirring at 50 °C during 15 minutes with a mixture of 18.26 g of a liquid that contain Eutectys™ buffered BLA (25% of tap water, 35% of TEGO® Natural betain and 40% of PURAC® BF P / 41).

[0093] Cosmetic Active Agent D was prepared as follows at pilot scale: 6 kg of Crocus sativus flower was suspended in 120 litres of tap water and stirred for 2 hours at 60 °C. After a filtration on Fibrafix® filter plate AF6 (to remove the plant), a first filtrate was obtained. Then, the same biomass of Crocus sativus flower (6 kg) was re-extracted with 120 litres of tap water and stirred during 2 hours at 60 °C and a second filtrate was obtained after filtration of Fibrafix® filter plate AF6 (to remove the plant). Both filtrates were combined and filtered again successively on Fibrafix® filter plates AF31H with perlite at 50 °C and finally on Fibrafix® filter plate AFST145. Then a concentration (at 60 mBar, 45-50 °C) was performed to remove a part of the water to obtain 6 kg of concentrated extract at 50.3% of dry matter. Eutectys™ buffered BLA was prepared by liquid blending at 50 °C about 30 min until complete dissolution of 13.5 kg of water, 21.6 kg of PURAC® BF P / 41 and 18.9 kg of TEGO® Natural betain. 54 kg of Eutectys™ buffered BLA is added to 6 kg of Crocus sativus flower extract concentrate by stirring at room temperature and finally a pasteurisation step is performed 1 hour at 85 °C. The final composition of the Cosmetic Active Agents samples is as in the below table.

[0094] INCI name CAS Extract wt % Range % Water 7732-18-5 44 25-49.99 Betaine 107-43-7 27 25-49.99 Potassium lactate 85895-78-9 / 996-31-6 17 10-24.99 Lactic acid 50-21-5 / 79-33-4 7 I 5-9.99

[0095]

[0096] Crocus sativus flower extract 84604-17-1 / 8022-19-3 5 5-9.99

[0097] Example 2: Inhibition of Malassezia spp. growth

[0098] During biological evaluation of the Crocus sativus flower extract of the invention, e.g. the Cosmetic Active Agents described in Example 1, we demonstrated that it inhibits growth of Malassezia spp. (re stricta, furfur and globosa). which are involved in dandruff formation.

[0099] 1- Material & method

[0100] Cultures of Malassezia resiricia. Malassezia furfur andMalassezia globosa were launched five days before contact on mLNA (Leeming & Notman Modified) agar at 32 °C. Cultures of Malassezia spp. were launched in modified Dixon medium at 32 °C until the exponential phase (Optical density ~ 12 corresponding to ~ 1 x 108CFU / ml) and were diluted in the modified Dixon medium to obtain a 1.106cell / ml solution.

[0101] To evaluate the fungicidal activity of the Cosmetic Active Agents of Example 1, ten different concentrations were tested from a range of 0.023% up to 6%. A dose at 100% was also tested. Targets of active ingredient content were 3% for vehicle Eutectys™ buffered BLA (25% of tap water, 35% of TEGO® Natural betain and 40% of PURAC® BF P / 41). Dilutions were performed using water as vehicle. Keteconazole was used as positive reference and tested from 1024 pg / ml to 2 pg / ml. Concentrations were expressed in % (v / v).

[0102] After 24 hours of incubation in presence of active ingredient and reference, the optical density (OD) was measured. Results were expressed in cells / mL and the percentage of inhibition was calculated as follows:

[0103] r. c. 1: c, •: • - i‘. 1. r-: <:

[0104] Inhibition value (%} = 100 - x: G'u

[0105]

[0106] i JW. lu.-t A'i-rj -N re cmtra:

[0107] 2- Results

[0108] Significant growth inhibition was observed at concentrations from 0.8% to 3% vs Eutectys™ buffered BLA. The Crocus sativus flower extract of the invention, e.g. the Cosmetic Active Agents described in Example 1, demonstrated fungicide activity toward Malassezia species. The results are also presented in Figure 1. Example 3: Minimum Inhibitory Concentration

[0109] The Minimum Inhibitory Concentration (MIC) was obtained in a culture of AT globosa. The Crocus sativus flower extract has been tested at equivalent dry matter concentration in comparison with a positive reference (ketoconazole) and benchmarks (Piroctone Olamine and Climbazole).

[0110] As presented in Figure 2, the results show that Crocus sativus flower extract inhibits the growth of M. globosa in a similar manner to the benchmarks, with a % superior to 90%.

[0111] Then, we tried to identify which molecule is responsible of this inhibiting activity. In a similar experiment to the previous one, the 3 molecules (kaempferol-3-O-sophoroside (K3OS), kaempferol-3-O-glucoside (astragalin) (K30G), and kinsenoside) and the mixture of the 3 were tested in comparison with Crocus sativus flower extract at equivalent dose. These molecules were identified by LC-MS.

[0112] The results, as presented in Figure 3, evidenced that none of the molecules identified was responsible of the growth inhibition in contrary to the bulk extract of Crocus sativus flower extract. It allows us to declare that the entire phytocomplex of Crocus sativus flower extract brings this activity. This efficacy suggests that with Crocus sativus flower extract, Malassezia species are less likely to develop antifungal resistance since it is not a single molecule.

[0113] Example 4: Selective growth inhibition

[0114] Crocus sativus flower extract demonstrated a significant activity in inhibiting Malassezia’s growth but to understand how it works and if it is a specific activity, other MIC were performed. The results are presented in the below table.

[0115] MIC (% growth in well) Tetracycline Crocus sativus flower Crocus sativus flower extract MIC=IC90 extract IC50 Staphylococcus epidermidis > 64 pg / mL > 8% > 8%

[0116] ATCC 12228

[0117] Staphylococcus aureus ATCC 0. 5 pg / mL > 8% > 8%

[0118] 25923

[0119] Escherichia coli ATCC 25922 0.5 pg / mL > 8% > 8%

[0120] Bacillus subtilis ATCC 11774 4 pg / mL > 8% > 8% Pseudomonas aeruginosa 8 pg / mL > 8% > 8%

[0121] ATCC 25853

[0122] Acinetobacter baumannii 64 pg / mL > 8% > 8%

[0123] ATCC BAA- 1605

[0124] Aspergillus brasiliensis ATCC - >12% >12%

[0125] 16404

[0126] Candida albicans ATCC >12% >12%

[0127]

[0128] 10231

[0129] The values of MIC for Tetracycline, used as a reference antibiotic, are consistent with those expected for the six strains tested. Of the 8 strains tested, there was no inhibitory activity in terms of MIC (Minimum Inhibitory Concentration), corresponding to the concentration that reduces growth in the well by at least 90%, i.e. 10% of the growth of the control without ingredient).

[0130] Based on these results, it is affirmed that Crocus sativus flower extract has a clear and specific inhibitory activity toward Malassezia.

[0131] Example 5: Bio-guided inhibition of Malassezia spp. growth

[0132] We performed bio-guided fractionation on anti-fungal activity on Malassezia in order to identify active compounds. We discovered that several molecules are efficient on Malassezia inhibition but no one is able to show same performance of the extract even by blending them. This confirms that the particularity and complexity of the extract is responsible for anti-fungal activity while isolated molecules only show part of the activity.

[0133] 1- Material and methods

[0134] Preparation of standards noticed K-3OG, K-3OS, KINS and MIX

[0135] “K-3OG” is a solution of Kaempferol-3-O-glucoside (supplier: extrasynthese, ref: 1243S) diluted at 0.01% in a mixture of DMSO / Water that is prepared as follows: 100 pL of DMSO and qsp water until 2 grams are added in 2.14 mg of Kaempferol-3-O-glucoside then a dilution of 1 / 10 in water is done.

[0136] “K-3OS” is a solution of Kaempferol-3-O-sophoroside (supplier: Achemblock, ref: AA00AKM8) diluted at 0.3% in a mixture of DMSO / Water that is prepared as follows: 200 pL of DMSO and qsp water until 2 grams are added in 5.65 mg of Kaempferol-3-O-sophoroside.

[0137] “KINS” is a solution of Kinsenoside (supplier: abcr, ref: AB493820) diluted at 0.6% in water that is prepared as follows: 500 pL of water is added in 12.11 mg of Kinsenoside.

[0138] “MIX” is a mixture of a solution of 0.6% Kinsenoside, 0.01% Kaempferol-3-O-glucoside and 0.3% of Kaempferol-3-O-sophoroside in a mixture of DMSO / Water.

[0139] Preparation of CPC (Centrifugal Partition Chromatography) fractions of Crocus sativus flower extract 300 g of Crocus sativus flower was suspended in 6000 litres of tap water and stirred for 2 hours at 60 °C. After a fdtration on Fibrafix® fdter plate AF6 (to remove the plant), a first filtrate was obtained. Then, the same biomass of Crocus sativus flower (300 g) was re-extracted with 6000 litres of tap water and stirred for 2 hours at 60 °C and a second filtrate was obtained after filtration of Fibrafix® filter plate AF6 (to remove the plant). Both filtrates were combined and filtered again successively on Fibrafix® filter plates AF31H. Then a concentration (at 20 mBar, 50 °C) was performed to remove a part of the water to obtain 303 g of concentrated extract at 56.0% of dry matter. A biphasic solvent system was prepared with a mixture of methyl tert-butyl ether / ethyl acetate / acetonitrile / water in proportion: (3 / 9 / 12 / 16, v / v / v / v).

[0140] The previous extract was submitted to a liquid-liquid fractionation described as follow: the 303 g were solubilized in 2 litres of lower phase of the biphasic solvent system ethyl acetate / acetonitrile / water (3 / 3 / 4, v / v / v) and extracted 3 times with 2 litres of fresh upper phase. The three upper phases were combined and evaporated to dryness to obtain a crude dry extract (CROC-UP, 13.8 g). This fraction was submitted to a CPC fractionation on a CPC instrument (CPC250-PRO (Gilson) column of 250 mb). The column was fdled with the lower phase of the above biphasic solvent system as the stationary phase and equilibrated with the upper phase of the above biphasic solvent system as the mobile phase at 20 mL / min and 1400 rpm. The extract was dissolved in 11 m of stationary phase + 11 mb of mobile phase and injected into the CPC column via a 20 mb sample loop. The mobile phase was pumped in the ascending mode for 60 min. The column was extruded by switching the mode from ascending to descending for 7 minutes. As a result, 4 final CPC fractions (A, B, C and D) were obtained, and their composition was determined through NMR13C analysis (Analytical Chemistry, 2014, Vol 86, Issue 6, p. 2955-2962) (see the table below).

[0141] Each CPC fraction (A, B, C and D) was separately mixed with a mixture of Eutectys™ buffered BLA (25% of tap water, 35% of TEGO® Natural betain and 40% of PURAC® BF P / 41) and standardised at 5% of dry matter, in analogy to the composition defined in Example 1.

[0142] % of

[0143] CPC- Mass (g ) crude Composition

[0144] Fractions

[0145] extract

[0146] Kaempferol 3-O-glucoside (Maj)* Kaempferol (Min); Dihydrokaempferol (Min); Mixture of faty acids, mainly linoleic acid (Maj); Tyrosol (Med); 3- A 1.0 10.9%

[0147] Hydroxybutyrolactone (Min); p-Hydroxybenzoic acid (Med); Phenylacetic acid (Med); Quercetin 3-O-glucoside (Min); Phenylethyl-glucoside (Min) 3-Hydroxybutyrolactone (Maj++); Isorhamnetin 3-O-sophoroside (Min); B 4.5 46.7%

[0148] Phenylethyl-glucoside (Min)

[0149] 3-Hydroxybutyrolactone (Min); Kaempferol: 3-O-sophoroside (Maj++); Quercetin 3-O-sophoroside (Min); Kinsenoside (Min); Goodyeroside A (Min); C 1.7 17.6%

[0150] Goodyeroside B (Min); Adenosine (Min); Uridine (Min); 3,4-Dihydroxybutyric acid (Med); Glycerol (Min)

[0151] 3-Hydroxybutyrolactone (Min); f-D-glucose (Maj); a-D-glucose (Maj); (3-D- fructofuranose (Med); a-D-fructofuranose (Min); p-D-fructopyranose (Min); Saccharose (Mih); Choline (Min); Kaempferol 3-O-sophoroside (Min); D 125.7 24.8%

[0152] Quercetin 3-O-sophoroside (Min-); Kinsenoside (Med); Goodyeroside A (Med); Goodyeroside B (Med); Uridine (Min); 3,4-Dihydroxybutyric acid

[0153]

[0154] (Min); Glycerol (Min) Identification Concentration to be tested

[0155] Crocus Flower 100 6 3 1.5 0.8 0.4 0.2 0.1 0.05 0.025 Fraction A

[0156] 11 0.66 0.33 0.165 0.088 0.44 0.22 0.11 0.055 0.003 (1156 of crude)

[0157] Fracton B

[0158] 47 2.82 1.41 0.71 0.38 0.19 0.09 0.05 0.024 0.011 (47% of crude)

[0159] Fraction C

[0160] 18 1.08 0.54 0.27 0.14 0.07 0.04 0,02 0.01 0.04 (18% of crude)

[0161] Fraction 0

[0162] 24 1.44 0.72 0.36 0.19 0.05 0.02 0.01 0.06

[0163]

[0164] (24% of crude) 0.01

[0165] Dilutions were performed using water as vehicle. Targets of active ingredient content were 6%, 3% and 1.5% of Crocus flower and its proportion of each crude.

[0166] 1 2 3 4 5 6 7 8 9 10 11 12

[0167] | I 100'4 6% 3% 1,5% 0,8% 0,4% 0,2% 0,1% 0,05% 0,03% HHH too* 0,66% 0,33% 0,17% 0,09% 0,04% 0,02% 0,01% 0,01% 0,003% | j IGCrfM 2,82% 1,41% 0,71% 0,36% 0,19% 0,10% 0,048% 0,024% 0,012%

[0168]

[0169] Ketoconazole? ><■ m Climbazote 0,2' - | Ven-de j Vi ' ce e

[0170]

[0171] 1 2 3 4 5 6 7 8 9 10 11 12

[0172] 100% 1,08% 0,54% 0,27% 0.014% 0,07% 0,04% 0,018% 0,009% 0,0045% 100% 1,44% 0,72% 0,36% 0,19% 0,1% 0,05% 0,023% 0,011% 0,0056% 100% 6% 3% 1,5% 0,8% 0.4% 0.2% 0,1% 0,05% 0,025% Ketoconazole Climbazote 0,2% e lOC% 1. •

[0173]

[0174] 0,5%

[0175] Isolated molecules or blend were also tested in the following condition.

[0176] Cosmetic 100% 6% 3% 1.5% 0.8% 0.4% 0.2% 0.1% 0.05% 0.025% Active Agent A 100% 6% 3% 1.5% 0.8% 0.4% 0.2% 0.1% 0.05% 0.025% K-3OG T- T+

[0177] 100% 6% 3% 1.5% 0.8% 0.4% 0.2% 0.1% 0.05% 0.025% K3-0S

[0178] Ketoconazole 0,5% Piroctone 1% Piroctone 0,5% Vehicle 100% Vehicle 3%

[0179]

[0180] 1 2 3 4 5 6 7 8 9 10 11 12 100% 6% 3% 1.5% 0.8% 0.4% 0.2% 0.1% 0.05% 0.025% KINS

[0181] 100% 6% 3% 1.5% 0.8% 0.4% 0.2% 0.1% 0.05% 0.025% MIX T- T+ Cosmetic 100% 6% 3% 1.5% 0.8% 0.4% 0.2% 0.1% 0.05% 0.025% Active Agent B Ketoconazole 0,5% Piroctone 1% Piroctone 0,5% Vehicle 100% Vehicle 3%

[0182]

[0183] In the above table, Crude crocus corresponds to the Cosmetic Active Agent C of Example 1.

[0184] 2- Results

[0185] Crocus sativus flower extract CPC fractions were tested at equivalent concentration to the Crocus sativus flower extracts of Example 1. Global data (curve data), and extract at 3% (optimal concentration, histogram data) for tested fractions showed that only CPC fractions A and D seem to carry the fungicidal activity. Results obtained on isolated molecules and blend are expressed in % of growth inhibition. Kaempferol-3-O-sophoroside (K3OS), kaempferol-3-O-glucoside (astragalin, K-30G) and kinsenoside (KINs) were all active but showed lower performance in comparison to the crude Crocus sativus extract. Even when we blended the three molecules in an equivalent manner to that of the full Crocus sativus flower extract we did not observe the same performance. This data demonstrated that it is the particularity and complexity of the Crocus sativus flower extract of the invention which is responsible for performance on wi-Malassezia activity.

[0186] Cosmetic 96 94 76 58 40 33 15 6 0 0 Active Agent A

[0187] 100 0 45 25 19 16 11 7 8 5 0 0 K-3OG

[0188] 15 12 11 9 6 2 3 0 0 0 K3-0S 95 92 94 85 33

[0189]

[0190] mean T- mean T+ 3 4 5 6 7 8 9 10 11 12

[0191] 28 17 8 3 0 0 0 0 0 0 KINS 31 19 14 12 9 4 0 0 0 0 MIX 100 0 Cosmetic 93 89 70 53 34 28 11 5 0 0 Active Agent B 94 92 91 86 30

[0192]

[0193] The results are also presented in Figure 4.

[0194] Example 6: Fungistatic activity measured by time-kill curve assay

[0195] We found that the Crocus sativus flower extract of the invention (Cosmetic Active Agent C in Example 1) has a fungistatic activity.

[0196] 1- Material and methods

[0197] The aim of the present study was to assess the potential fungicidal activity of an active ingredient on Malassezia globosa growth. A time-kill curve, also known as a time-kill assay, is an experimental method used to evaluate the efficacy of antimicrobial agents by studying their impact on microbial populations overtime.

[0198] Tested conditions:

[0199] The exploration on time-kill curve (TKC) was performed to assess the Crocus sativus flower extract (3%), vehicle (3%) and Ketoconazole (50 pg / ml) against Malassezia globosa ATCC MYA 4612 during growth.

[0200] Identification Comments

[0201] Crocus Flower Liquid, light brown, to be stored at 2-8 °C

[0202] Eutectys BLA Liquid, transparent, to be stored at 2-8 °C

[0203]

[0204] Ketoconazole Liquid, light brown, to be stored at 2-8 °C

[0205] The term “Crocus Flower” is used interchangeably with “crocus sativus flower extract” in the above table and throughout the document.

[0206] Study done on Malassezia globosa

[0207] Identification Comments

[0208] Crocus Flower Liquid, light brown, to be stored at 2-8 °C

[0209] Eutectys BLA Liquid, transparent, to be stored at 2-8 °C

[0210]

[0211] Ketoconazole Liquid, light brown, to be stored at 2-8 °C

[0212] Strain name Reference Supplier

[0213]

[0214] Malassezia globosa ATCC MYA 4612 American Type Culture Collection (ATCC) Malassezia yeast culture was realized in modified Dixon medium for which the composition is described hereunder:

[0215] Malt extract: 36 g

[0216] Oxidated Bile (Dehydrated): 20 g

[0217] Tween 40: 10 mL

[0218] Peptone: 6 g

[0219] Glycerol: 2 mL

[0220] Oleic Acid: 2 mL

[0221] Purified water: 1000 mL

[0222] The protocol used was described below:

[0223] Culture was launched five days before contact on mLNA (Leeming & Notman Mod-ified) agar at 32 °C, then in modified Dixon medium at 32 °C until the exponential phase (Optical density ~ 12 corresponding to ~ 1 x 108CFU / ml) and was diluted in the modified Dixon medium to obtain a 1 x 106cell / ml solution. At each time point of the kinetic (TO, T+4h, T+8h, T+24h, T+32h and T+48h) fungicidal activities were determined by yeast quantification. Colony forming units (CFU) were quantified for each condition.

[0224] 2- Results

[0225] Log 10 (CFU / ml) / Time Vehicle 3% \ Active 3% Ketoconazole 50 pg / ml

[0226] TO 6.3 6.3 6,3

[0227] T+4h 6.6 6.4 6.4

[0228] T+8h 7.3: 7.0 7.0

[0229] T+24h 7.8: 7.0 7.3

[0230]

[0231] T+30h 7.9: 7.3 7.2

[0232] Our results evidenced that the Crocus sativus flower extract of the invention only stops Malassezia globosa growth similar to tested reference Ketoconazole. We observed a significant fungistatic activity (but not a significant fungicidal activity) for Crocus sativus flower extract.

[0233] The results are also presented in Figure 5.

[0234] Example 7: Antifungal mode of action of Crocus sativus flower extract

[0235] We demonstrated that the Crocus sativus flower extract of the invention (Cosmetic Active Agent D in Example 1) inhibits Malassezia growth, presumably by fungal membrane disruption activity and depolarization activity on mitochondrial membrane.

[0236] 1- Material and methods To comprehensively assess the activity of the Crocus sativus flower extract on Malassezia globosa (MYA4889), we employed two different techniques: Mitochondrial Membrane Potential (MMP) Assay and Membrane-Disruptive Activity Assay. Each of these methods provides unique insights into the mechanisms by which the Crocus sativus flower extract interacts with and inhibits M. globosa.

[0237] Summary of methodologies:

[0238] - Membrane-Disruptive Activity Assay: This assay evaluates the ability of a test compound to disrupt the integrity of the cell membrane. By assessing membrane integrity, we can determine whether the compound causes direct damage to the fungal cell membrane, leading to cell lysis and death.

[0239] - Mitochondrial Membrane Potential (MMP) Assay: This technique measures changes in the mitochondrial membrane potential, an indicator of mitochondrial health and function. Disruption of MMP suggests that the compound interferes with energy production and induces cell death in the pathogen.

[0240] 2- Results

[0241] The Crocus sativus flower extract of the invention has a significant membrane disruptive activity (Figure 6). In addition, the Crocus sativus flower extract also has a significant depolarization activity on mitochondrial membrane (Figure 7).

[0242] Example 8: Anti-inflammatory property effect of Crocus sativus flower extract under chemical stress

[0243] We also discovered that the Crocus sativus flower extract of the invention (Cosmetic Active Agent D in Example 1) has skin soothing activity.

[0244] 1- Materials and methods

[0245] The aim of this study was to evaluate the anti-inflammatory property of the cosmetic active of the invention in keratinocytes. A pro-inflammatory stress was induced by a chemical agent (Phorbol Myristate Acetate) and the release of four cytokines was evaluated by multiplex assay: two of them are pro-inflammatory and two are anti-inflammatory.

[0246] Cell culture and treatment

[0247] The cell culture was done on primary cells isolated from fresh biopsies. Normal Human Epidermal Keratinocytes (NHEKs) were seeded in a type I collagen pre-coated 24 wells-plate at 30000 cells per well in triplicate. The cells were incubated for 24 h in complete medium (Dermalife medium supplemented with Life factors, Cell Systems) and 1% of antibiotics (Sigma- Aldrich) at 37 °C with 5% CO2.

[0248] At the end of the incubation, the cells were rinsed twice with PBS (Gibco) and pre-incubated with the test substances for 24 h in Dermalife complete medium without hydrocortisone and 1% of antibiotics at 37 °C with 5% CO2. The cells were treated in complete medium with the following conditions.

[0249] I Test substance I Concentration I

[0250] Dexamethasone (positive reference, from Sigma)! 1 pM

[0251]

[0252] Crocus sativus flower extract \ 0.06 wt%

[0253] The active was diluted in directly in medium.

[0254] After 24 h of pre -incubation, the cells were stressed with PMA (Phorbol 12-myristate 13-acetate, Sigma) at 1 ng / ml and incubated for 24 h at 37 °C with 5% CO2.

[0255] Skin cells untreated and cultivated with medium were used as a negative control. At the end of the culture, the cell media were collected and centrifuged at 2000 g for 10 min at 4 °C to eliminate dead cells. The media were stored at -20 °C.

[0256] An MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to evaluate treatments toxicity and for the normalisation of cytokines quantification.

[0257] MTT assay

[0258] MTT solution (Sigma) diluted at 1 mg / ml in basal medium was added to each well and incubated for 3 h at 37 °C, 5% CO2. Then, the medium was removed and 300 pL of dimethyl sulfoxide (DMSO, Sigma) was added to each well to dissolve the formazan crystals. Homogenization was done under orbital agitation for few minutes. The optical density was measured at a wavelength of 560 nm with mi-croplate reader (Spark®, TECAN).

[0259] Cytokines quantification

[0260] TNF-a, IL-6, IL- 10 and IL-IRa quantification were realized with a 4-plex assay (R& D Systems).

[0261] Briefly, the samples and the standard range were incubated for 2 h in 96-wells plate with specific beads with antibodies against TNF-a, IL-6, IL-10 and IL-IRa. After three washes with wash buffer using the magnetic device provided by the supplier, a Biotin-antibody cocktail was added to the wells for an incubation during 1 h under orbital agitation. Following three washes, a solution of Streptavidin-PE was added to the wells for 30 min. Finally, three more washes were conducted and the microparticles were resuspended in wash buffer. The read was then carried out by the Magpix® system (Luminex, Texas, U SA). The quantification was normalized with the optical density measured via the MTT assay. An inflammatory balance was calculated by comparing the modulation of anti-inflammatory and pro-inflammatory cytokines.

[0262] Statistical analysis

[0263] A Shapiro-Wilk normality test was performed to evaluate whether the data follow the Gaussian Law. The results did not follow the Gaussian Law. As a consequence, a non-parametric statistical analysis was performed by Kruskal-Wallis ANOVA followed by Mann Whitney U test. Results were considered significant with p<0.05 with *, p<0.01 with ** and p<0.001 with ***.

[0264] 2- Results

[0265] The Crocus sativus flower extract of the invention (Cosmetic Active Agent D in Example 1) significantly evidenced soothing activity by reducing inflammation under chemical stress condition (Figure 8).

[0266] Example 9: Anti-inflammatory property effect of Crocus sativus flower extract under inactivated Malassezia spp. stimulation

[0267] We also observed a soothing activity on Malassezia-mediated inflammation induction.

[0268] 1- Materials and methods

[0269] Reconstructed Human Epidermis (RHE; containing Langerhans cells, from Episkin) was topically pretreated overnight with the Crocus sativus flower extract of the invention (Cosmetic Active Agent D in Example 1) at 0.6%, then with an inactivated inoculum of M. Furfur, M. Globosa and M. Restricta (104CFU / mL) for 2 hours. The RHE were rinsed and the extract was re-applied for 24 hours. Culture medium was then collected and TNF-a was quantified using multiplex assay.

[0270] 2- Results

[0271] The Crocus sativus flower extract of the invention showed soothing activity on Malassezia-mediated inflammation by reducing TNFa (pro-inflammatory cytokine) (Figure 9).

[0272] Example 10: Reduction of itching by inhibition of histamine release from mastocytes Interestingly, we showed that the Crocus sativus flower extract of the invention (Cosmetic Active Agent D in Example 1) can reduce instant itching by limiting histamine release by blocking MRGP2 receptor.

[0273] 1- Materials and methods

[0274] Culture and treatment

[0275] Assay medium containing or not (controls) the test compound or the reference (cromoglycate, tested at 10 mM) was placed in a 96-well plate. After isolation, human mast cells were added and pre-incubated for 15 minutes. The cells were then stimulated or not (non-stimulated control) with Compound 48 / 80 (comprising poly-p-methoxyphenethylmethylamine; obtained from Sigma Aldrich, product Number C2313), tested at 5 pg / ml and the cells were incubated for 20 minutes.

[0276] Crocus sativus flower extract was tested at 0.6%. All experimental conditions were performed in n=3. Quantification of histamine release Histamine released in the culture supernatants was measured using a specific EIA kit according to the supplier’s instructions.

[0277] 2- Results

[0278] A basal release of histamine was detected in non-stimulated mast cell supernatants (83 nM). When stimulated with Compound 48 / 80 (comprising poly-p-methoxyphenethylmethylamine; obtained from Sigma Aldrich, product Number C2313), tested at 5 pg / ml, a strong degranulation of mast cells and histamine release was observed (767 nM). The reference cromoglycate, tested at 10 mM, highly inhibited the Compound 48 / 80-induced histamine release (5% of the stimulated control). These results were expected and validated the assay.

[0279] The Crocus sativus flower extract of the invention reduced histamine release by mast cells by *48%, suggesting a protective effect against mast cell degranulation (Figure 10).

[0280] Example 11: Normalisation of epidermis thickness after exposure to living M. globosa on RHE

[0281] A second model of Reconstructed Human Epidermis (RHE) was designed, reproducing the conditions and interactions with the skin by adding living culture of M. globosa on RHE.

[0282] In this context, we first evaluated the viable epidermis thickness in order to assess whether Crocus sativus flower extract was able to reduce the parakeratosis induced by the presence of M. globosa.

[0283] As presented in Figure 11, the presence of M. globosa on RHE significantly reduced the viable epidermis thickness by -52%***, evidencing its deleterious and pro-apoptotic effects. The RHE treated with Crocus sativus flower extract were significantly protected from this deleterious effect, display a +180%*** increase of viable epidermis thickness, with 164% rescue.

[0284] Example 12: Improvement of skin barrier integrity on RHE colonized by live Malassezia globosa

[0285] The Crocus sativus flower extract of the invention (Cosmetic Active Agent D in Example 1) also demonstrated additional biological activity, such as improvement of skin barrier by controlling excessive keratinocytes migration and overexpressed Desmoglein-1.

[0286] 1- Materials and methods

[0287] Culture treatment

[0288] Eight-day-old RHE were topically treated with 20 pl of artificial sebum for 20 minutes. Then, the RHE were topically treated with the test compound or water (control). After 30 minutes of incubation, Malassezia globosa was topically added (2* 105CFU / RHE). At day 9, the test compound or water (control) was topically applied for 30 minutes. Culture was stopped at day 10. For lucifer yellow analysis, RHE were incubated 5 hours with a 1 mM lucifer yellow solution. All experimental conditions were performed in n=6: 3 RHE for desmoglein analysis and 3 RHE for lucifer yellow analysis.

[0289] Histology

[0290] 1. Paraffin embedding

[0291] The RHE were fixed in formaldehyde solution. The fixed samples were then dehydrated in successive ethanol baths of increasing concentrations before being embedded in paraffin. The transversal sections were performed using a microtome (5 pm thickness) and kept at room temperature until analyses.

[0292] 2. Immunofluorescence labelling - Desmoglein expression

[0293] The sections were deparaffinised and the antigenic sites were retrieved using a specific buffer (pH 6) at 92 °C and then left at RT. After washes in a PBS-Tween 0.05% solution, the sections were saturated in PBS-Tween milk 5% and incubated at RT for 1 hour with the appropriate primary antibody (anti-desmoglein). After washes in a PBS-Tween 0.05% solution, the primary antibody was detected using the appropriate secondary fluorescent antibody (GAR- Alexa 488) and the cell nuclei were coloured with PI solution. The sections were washed with a PBS-Tween 0.05% solution and mounted in “Fluorescent Mounting medium”.

[0294] 2- Results

[0295] Malassezia globosa treatment induced a significant decrease (-54% ***) of Desmoglein intensity in the entire RHE. This result is aligned with the one observed with the PAS staining, showing a reduction of the viable epidermis thickness. Furthermore, DSG immunostaining is less cohesive into the tissues under Malassezia inoculum. The Crocus sativus flower extract of the invention (Cosmetic Active Agent D in Example 1) reduced the stress effect of Malassezia inoculum on the DSG staining, manifested by a significant increase (+135%***) in desmoglein-1 expression, which means a 117% rescue, evidencing a protective and restoring effect on the skin barrier (Figure 12).

[0296] These experiments, all together, demonstrated that Crocus sativus flower extract, by stimulating the desmoglein-1 and normalizing the skin epidermis, restores the barrier integrity.

[0297] Another experiment was performed to verify whether these beneficials effects, were able to reduce the skin permeability triggered by the presence of Malassezia spp..

[0298] The skin permeability was assessed using the Lucifer yellow probe, based on the principle that the more the liquid penetrates, the more the barrier is permissive. As presented in Figure 13, it was observed that the skin permeability is significantly increased by +92%* * * in presence of living M. globosa, showing the deleterious effect of its presence on skin integrity. In the same conditions, treatment with Crocus sativus flower extract prevented the skin barrier defect, as evidenced by the decreased of fluorescence by -14%*, for a 29% rescue.

[0299] Example 13: Improvement of epidermal cohesion in ex vivo model

[0300] We evaluated the impact of the Crocus sativus flower extract of the invention (Cosmetic Active Agent D in Example 1) on Claudin-1 expression on skin explants.

[0301] 1- Materials and methods

[0302] Skin explants were treated for 5 days with Crocus sativus flower extract at 1% and 3%. At the end of culture, immunostaining of claudin-1 was performed to visualize an effect in barrier function.

[0303] Human fresh skin explant coming from a donor were used in this study. Skin explants were topically treated for 5 days with Crocus sativus flower extract at 1% and 3%. Untreated condition was used a negative control condition. Culture medium was renewed every day. After sampling, skin explants were fixed in formalin before being dehydrated and embedded in paraffin for E-cadherin immunostaining. Sections of 4 pm thickness were dewaxed and then stained.

[0304] Claudine- 1 immunostaining was realized on paraffinized sections with a poly-clonal anti-claudine-1 antibody (abeam, ref ab211737; lot 1002148-1) diluted at 1:200 in PBS, BSA 0.3% and Tween 20 (0.05%) overnight at 4 °C, and revealed by secondary antibody anti rabbit diluted at 1 / 200 in same solution in presence of Hoechst au 1 / 5000 incubated Ih at room temperature. The staining was performed using a slide-processing system and stabilized using Eukitt media (Dako, AutostainerPlus). The immunostaining was assessed by microscopical observation.

[0305] 2- Results

[0306] We observed a significant increase of Claudine- 1 expression up to 31% induced by the Crocus sativus flower extract of the invention (Figure 14).

[0307] Example 14: Reduction of dandruff after shampoo application versus placebo control We evaluated the performance of the Crocus sativus flower extract of the invention (Cosmetic Active Agent D in Example 1) in shampoo during clinical evaluation.

[0308] 1- Materials and methods

[0309] Panel description

[0310] The clinical study was carried out on 20 volunteers (men and women) aged between 18 to 70 presenting dandruff, oily scalp and itching. This group was divided into two equal groups of volunteers, one applying active shampoo containing 3% of Crocus sativus flower extract and the other one applying placebo shampoo (same formula without active) for 14 days. Wash out of 15 days prior to start of the study was done for all volunteers to standardize the shampoo routine. Dandruff area and erythema were analysed after 14 days of application. Scalp microflora was collected to analyse the impact of the active on Malassezia abundance.

[0311] INCI formula

[0312] INCI Name Active Placebo AQUA / WATER 85.32 87.00 SODIUM LAURETH SULFATE 7.8 7.8 COCAMIDOPROPYLBETAINE 3.6 3.6 BETAINE 0.81 POTASSIUM LACTATE 0.51

[0313] LACTIC ACID 0.21

[0314] Crocus sativus flower extract 0.15

[0315] SODIUM BENZOATE 0.5 0.5 SODIUM CHLORIDE 0.5 0.5 HYDROXYPROPYL GUAR HYDROXYPROPYLTRIMONIUM CHLORIDE 0.2 0.2 CITRIC ACID 0.2 0.2 FRAGRANCE 0.2 0.2

[0316]

[0317] Erythema measurement

[0318] Dermatoscope C-Cube® (PIXIENCE) is a device which allows realizing high resolution skin pictures (10 million pixels 2D capture (UHD)). The probe is put directly on skin without any contact gel. Standardized photographs of the scalp with C-Cube®, Pixience were performed at DO and D 14.

[0319] In this study, erythema index was analysed. The inflammatory response is a way for the body to defend itself. Local irritations are initiated by the contact of every-day substances. Anti-inflammatory agents are being incorporated into skin care products to improve skin tone and texture while reducing the appearance of redness. The most common cause for inflammatory process is a disturbed and porous skin barrier. From the standard CIE L* a* b* measurements given by the C-Cube for every pixel, we define a specific severity parameter (based on a*) that can be uses to grade erythema. This index can be displayed with a blue / red colour map to localize and illustrate the erythema. Drawing ROI on images, it is possible to monitor changes across time or to analyse differences of evolution between zones. Scalp microflora swabbing

[0320] Sampling with a swab was performed at DO, D14 and D28 for the 2 non-healthy panels and at DO for 1 healthy panel (consisted of 10 healthy volunteers). All samples were collected by a single operator, using a single batch of sterile swabs and sodium chloride solution.

[0321] In addition, the swab (delivery by the promoter) was put 5 round trips x 8 hair parting. The sample was taken from rays and a length of 4 cm with a swab previously moister in physiological water.

[0322] One control sample was performed per day of sampling, which consists of unscrewing the buffer and immersing the cotton tip in the sodium chloride solution. It was left in contact for at least 1 minute in the open air to moisten the cotton perfectly.

[0323] Sampling:

[0324] 1. For each swab to be used, 500 pL of sterile 0.9% sodium chloride solution was transferred to a sterile 1.7 mL microtube.

[0325] 2. The pad was unscrewed and the cotton tip was immersed in the sodium chloride solution. It was left in contact for at least 1 minute to moisten the cotton perfectly.

[0326] 3. Sampling on the scalp 5 round trips x 8 hair parting was performed.

[0327] 4. Each swab was placed in separate container. These containers were placed in a zipped freezer bag and then kept at -80 °C prior to shipment and / or RNA / DNA extraction.

[0328] Microbiota analysis was performed by RT-qPCR.

[0329] Quantification of dandruffs with DandruffMeter DA 20®.

[0330] Dandruff collection:

[0331] The volunteer was seated in front of a desk covered by a black sheet of paper and was asked to rub the front of their head above the paper sheet. They must rub their head for 15 seconds and only with their fingertips in order to avoid a desquamation of the scalp with the nails. Then material on the paper sheet was collected in a Petri dish containing a black felt.

[0332] Dandruff sample analysis:

[0333] The analysis was made with DandruffMeter DA20 just after collecting because dandruff can alter over time. The system consists of a device in which the collected dandruff is inserted. A circularly arranged LED light source illuminates the sample homogeneously on a dark background. The high-resolution camera above the sample takes the image and the software detects all dandruff and categorizes it in 9 different size classes.

[0334] The software gives the following results: -Area: total area covered with green spots in mm2

[0335] For this study, measurement was performed on the scalp at DO and D14.

[0336] 2- Results

[0337] We demonstrated a significant reduction of dandruff area by up to -56.4% with the active shampoo, showing a significant difference in comparison with placebo by -46.2% after 14 days (Figure 15).

[0338] We also observed a significant reduction of erythema with active shampoo by -10.2% which is significantly better than placebo effect showing -14.1% of difference (Figure 16).

[0339] Scalp microflora analysis demonstrated that the Active significantly reduced the abundance of Malassezia while placebo did not modify it. This result confirmed the anti-fungal activity of the Crocus sativus flower extract of the invention at clinical level (Figure 17).

[0340] In conclusion, we demonstrated that the Crocus sativus flower extract of the invention has a strong antifungal activity specific to Malassezia spp., which contributes to significant reduction of dandruff and related erythema.

[0341] Example 15: Impact of stopping active shampoo application - maintenance of efficacy at long term

[0342] Most current anti-dandruff solutions are highly effective in reducing dandruff; however, there is often observed a rebound effect, with dandruff returning quickly after treatment cessation. We aimed to determine whether Crocus sativus flower extract could provide long-lasting benefits and prevent this rebound effect after discontinuation.

[0343] We instructed half of the volunteers to switch to a placebo after 14 days of using the active shampoo containing Crocus sativus flower extract at a concentration of 3% for an additional 14 days. Our results, presented in the below table and Figure 18, revealed impressive maintenance of dandruff reduction even after stopping the active shampoo.

[0344] Dandruff Active shampoo DO to D14

[0345] area Mean + / - SEM Average variation (%) vs DO Wilcoxon test versus DO (p)

[0346] (arbitrary unit)

[0347] DO 71.9 ± 50.9

[0348] D3 28.6 ± 20.6 - 60.5% ***

[0349]

[0350] D14 21.4 ± 18.2 - 70.2% #

[0351] Switch Active shampoo to Placebo shampoo (D14 to D28) DO 92.5 ± 62.0

[0352]

[0353] D28 23.1 ± 11.1 -75% These findings demonstrate that Crocus sativus flower extract is highly effective in reducing dandruff, and its benefits are sustained over the long term, even after discontinuation of use.

[0354] We also investigated the impact of switching from the active shampoo to a placebo on the erythema parameter. Our findings demonstrated that the soothing activity provided by Crocus sativus flower extract at a concentration of 3% is maintained even after discontinuing the application of the active shampoo. These results highlight the long-term benefits of Crocus sativus flower extract in reducing dandruff and promoting a healthier scalp condition. The results are presented in the below table and Figure 19.

[0355] Erythema average Crocus sativus flower extract at 3%

[0356] (maintenance phase) Mean + / - SEM Average variation Wilcoxon test versus (arbitrary unit) (%) vs DO DO (p) DO 27.7 ± 10.4

[0357] D3 20.6 ± 11.7 - 9.7%

[0358] D14 20.1 ± 11.3 -11.6%

[0359] Switch Active shampoo to Placebo shampoo (D14 to D28)

[0360] DO 27.7 ± 10.4

[0361] D28 24.4 ± 12.0 -11.8% #

[0362]

[0363] Example 16: Interabiome analysis after Crocus sativus flower extract at 3% versus placebo and healthy panel

[0364] A primary contributor to dandruff is the overgrowth of Malassezia. a lipid dependent yeast that thrives in sebaceous areas of the skin. While this yeast genus is normally part of the skin's microbiota, it can become problematic when environmental factors disrupt the microbial balance (Tao et al., 2021). Additionally, other microorganisms inhabiting the scalp, primarily bacteria, also appear to play a role in dandruff conditions, although discrepancies exist in various scientific publications (Tao et al., 2021).

[0365] Microbial absolute abundance evolution across treatment

[0366] o Impact on bacteria

[0367] Both treatments did not significantly impact composition of the scalp microbiota, considering the bacterial community. The below table shows the results of the statistical comparison of absolute abundances at DO versus D 14 for all bacterial targets.

[0368] Results of the Wilcoxon test (paired data) comparing absolute abundance at DO and D14 for each bacterial target, considering Placebo and Crocus sativus flower extract treatment. In bold Malassezia results. Significance = ns: p-value > 0.1, #: p-value <0.1. Active = 3% Crocus sativus flower extract shampoo. Target Placebo Active

[0369] I p-value Significance j p-value Significance

[0370] C. acnes \ 0.492 ns 0.652 ns

[0371] C. granulosum I 0.922 ns I 0.423 ns

[0372] C. modeslum I ns \ 0.500: ns

[0373] [ Corynebacterium spp. | 0.492 ns I 0.652 ns

[0374] | Cutibacterium spp. I 0.557 ns I 0.615 [ ns

[0375] Enterococcus spp. 0.411 ns > 0.500 ns

[0376] : L. clevelandensis i 0.232: ns! 0.903: ns

[0377] Lactobacillus spp. 1 0.193 ns j 0.688 | ns

[0378] P. aeruginosa 1 ns ■■■ 0.978 ns

[0379] Prevotella spp. 0.131 ns \ 0.161 ns

[0380] S. aureus ■■ 0.855 ns ■■. 0.860 \ ns

[0381] S. capitis: 0.846 > ns: 0.722 ns

[0382] S. caprae ■■. 0.789 ns ■■. 0.395 ns

[0383] S. epidermidis 1 0.160 | ns I 0.652 ns

[0384] Staphylococcus spp. I 0.375 ns j 0.754 | ns

[0385]

[0386] [ Streptococcus spp. | 0.232 ns 1 0.539 | ns

[0387] o Impact on Malassezia

[0388] After 14 days of treatment with Crocus sativus flower extract, the absolute abundances of the Malassezia genus, as well as M. restricta and M. globosa, were significantly reduced compared to pre-treatment levels. In contrast, treatment with placebo did not significantly impact the abundances of Malassezia. The below table shows the results of the statistical comparison of absolute abundances at DO versus D14 for all Malassezia targets.

[0389] Results of the Wilcoxon test (paired data) comparing absolute abundance at DO and D14 for each targeted Malassezia, considering placebo and Crocus sativus flower extract treatment. Significance = ns: p-value > 0.1, #: p-value <0.1. Active = 3% Crocus sativus flower extract shampoo.

[0390] Placebo Active

[0391] Target p-value Signifiance p-value Signifiance M. globosa 0.922 ns 0.096 #

[0392] M. restricta 0.432 ns 0.065 #

[0393] M. sympodialis 0.272 ns 0.990 ns

[0394] Malassezia spp. 0.232 ns 0.096 #

[0395]

[0396] In case of M. sympodalis. the low mean abundance (1.86 genomes / pl) combined with high variability (standard deviation = 5.69), may account for the lack of effect observed with the Active.

[0397] In conclusion, study on bacteria and fungi from scalps indicated that placebo did not impact the scalp microbiota composition. In contrast, shampoo with Crocus sativus flower extract specifically targeted Malassezia, leading to a significant decrease after 14 days of treatment. Microbial networks

[0398] o Healthy versus dandruff scalps

[0399] The clustering coefficient of a microbial network serves as a proxy for estimating the interdependence among the microorganisms within that network. A low to moderate level of interdependence among microorganisms enables the microbial community to be less sensitive to disturbances affecting only a single species; such communities are considered stable. In contrast, high interdependence reflects a community with low stability (Kajihara et al., 2024).

[0400] The positive edge percentage reflects the overall relationships among microorganisms within the community. Positive links are interpreted as collaboration, while negative links indicate competition. A community characterized by a predominance of competitive relationships is described as more stable (Coyte et al., 2015; Brandon-Mong et al., 2020); thus, a stable community is defined by a low to moderate proportion of collaborative interactions, represented by a low to moderate positive edge percentage. Therefore, we describe Positive edge percentage as forced collaborations. As also presented in the below table, we observed that both the clustering coefficient and positive edge percentage are significantly higher in the case of dandruff-affected scalps.

[0401] Network topological metrics of healthy and dandruff-affected scalps, representative of 10 volunteers for each condition. Significance:# = p-value < 0.1; * = p-value < 0.05.

[0402] Metric Healthy scalp Dandruff scalp Difference p-value significance ______ Clustering ~ ~ 24.2 ~ 74.4 (+)49.2 0.071

[0403] coefficient

[0404] Positive edge 76.47 84.55 (+)8.08 0.028

[0405]

[0406] percentage

[0407] This indicates that interdependence and forced collaboration are greater in dandruff-affected scalps community compared to healthy scalps community, suggesting that microbial communities are more stable on healthy scalps.

[0408] o Treatment with placebo

[0409] As presented in the below table, treatment with placebo does not impact the interdependence nor the forced collaboration. Thus, Placebo treatment did not improve the stability of dandruff-affected scalps.

[0410] Network topological metrics of dandruff-affected scalps before and after 14 days of treatment with placebo, representative of 10 volunteers for each condition. Significance: ns = p-value > 0.1.

[0411] Metric Placebo (DO) Placebo (D14) Difference p-value significance Clustering coefficient 63.8 64.2 (+)0.4 0.777 ns 66.68 63.50 (-)3.67 0.921

[0412]

[0413] Positive edge percentage ns o Treatment with shampoo with Crocus sativus flower extract

[0414] Treatment with shampoo with Crocus sativus flower extract resulted in a significant decrease in interdependence and forced collaboration. Consequently, said treatment enhanced the stability of the microbial community on dandruff-affected scalps after 14 days of treatment. The results are presented in the below table and Figure 18. An increase in interdependence and / or forced collaboration reflects a decrease in microbial community stability.

[0415] Network topological metrics of dandruff-affected scalps before and after 14 days of treatment with Crocus sativus flower extract (Active), representative of 10 volunteers for each condition. Significance: # = p-value < 0.1; ** = p-value < 0.01.

[0416] Metric Active (DO) Active (D14) Difference p-value significance Clustering coefficient 74.4 45.7 (-)28.7 0.089 #

[0417]

[0418] Positive edge percentage 84.48 52.17 (-)32.31 0.008 **

[0419] In conclusion, these results demonstrate that Crocus sativus flower extract, when used as a rinse-off solution for only 14 days, specifically targeted Malassezia fungi, results in a significant reduction in the absolute abundance of the predominant species within this genus. In contrast, Crocus sativus flower extract did not affect the abundance of bacteria on dandruff-affected scalps. Furthermore, this targeted action led to a significant increase in the stability of the microbial community on dandruff-affected scalps, approaching the stability observed in healthy scalps.

[0420] Example 17: Long term stability of Cosmetic Active Agent

[0421] The Active Cosmetic Agent C of Example 1 was stored for up to 12 months in the dark at 8 °C, at room temperature (RT; about 21 °C) and at 40 °C, respectively. During this time, the total flavonoid content % (measured by HPLC-UV method) and the color through measurement of dE2000 (measured with a spectrocolorimeter) were monitored. The results are shown in the following table. The results demonstrated a high stability of flavonoids even after 6 months at 40 °C and 12 months at 8 °C and 21 °C.

[0422] Date Temperature dE2000 Total Flavonoid content %

[0423] TO - - 0.48

[0424] RT 1.69 0.48

[0425] 3 months 40 °C 4.49 0.48

[0426] 8 °C 1.47 0.44

[0427] RT 1.85 0.40

[0428] 6 months 40 °C 4.78 0.40

[0429]

[0430] 8 °C 1.48 0.42 RT 9.29 0.40

[0431] 12 months

[0432]

[0433] 8 °C 6.56 0.42

[0434] Example 18: Stability testing of Cosmetic Active Agent in cosmetic formulations

[0435] The stability of the Cosmetic Active Agent D of Example 1 was further evaluated at concentrations of 3% in shampoo and conditioner formulations, as well as in a pH range, over 3 months at 3 °C, 21 °C, and 45 °C, and over 3 months at 3 °C and 21 °C. The following parameters were monitored:

[0436] - Viscosity mPa.s (Brookfield LVDV1M, Cylinder (S63), Speed (12 rpm), pH at 20 °C

[0437] Lab* Spectro measurements CM-25d

[0438] The following shampoo was prepared and tested:

[0439] Phase Material %

[0440] Aqua QSP 100

[0441] A Sodium Laureth sulfate 16

[0442] Coco betaine 10

[0443] Sodium benzoate 0.3

[0444] B

[0445] Potassium sorbate 0.2

[0446] C Citric acid 0.8

[0447]

[0448] D Cosmetic Active Agent D 3

[0449] The shampoo was prepared by weighing and mixing phase A until a homogenous mixture was obtained, then adding phase B, C and D. The results are summarized in the table below.

[0450] 1 Month 3 Months

[0451] Day 0

[0452] 3 °C 21 °C 45 °C 3 °C 21 °C 45 °C Limpid Limpid Limpid Limpid Limpid Limpid Limpid Aspect

[0453] Yellow Yellow Yellow Yellow Yellow Yellow Yellow Viscosity 780 480 480 360 480 480 480 pH 4.39 4.39 4.38 4.37 4.40 4.44 4.41

[0454]

[0455] dE2000 / 0.6 2.6 2.9 2.6 0.9 6

[0456] After 3 months at 4 °C, 21 °C and 45 °C, respectively, the shampoo containing the Cosmetic Active Agent C was slightly yellow, but the impact remains acceptable (dE2000 = 6 after 3 months at 45 °C) and the pH remains stable overtime.

[0457] The following conditioner was prepared and tested. Phase Material %

[0458] Aqua QSP 100

[0459] A

[0460] Disodium EDTA 0.15

[0461] B Cetrimonium chloride 2.6

[0462] C Lactic acid 80% 0.6

[0463] Cetearyl alcohol 6.5

[0464] Mineral oil 1

[0465] D

[0466] Vaseline 1

[0467] Guar hydroxypropyltrimonium chloride 0.5

[0468] Phenoxyethanol, ethylhexylglycerin 0.9

[0469] E

[0470]

[0471] Cosmetic Active Agent D 3

[0472] The conditioner was prepared by weighing phase A and heating to 75 °C while stirring slowly. Then at 75 °C, phase B was added to phase A and mixed for 15 minutes until homogeneous. Then, Phase C is added to AB and mixed for 15minutes until homogeneous. Then, phase D is added to phase A, B and C using a Polytron PT 3100 for 5 minutes until homogeneous. To finish, the formula was cooled down and at 40 °C, the phase E was added.

[0473] The results are summarized in the table below.

[0474] Day 0 1 Month 3 Months

[0475] 3 °C 21 °C 45 °C 3 °C 21 °C 45 °C Beige Beige Beige Beige Beige Beige Beige Aspect

[0476] emulsion emulsion emulsion emulsion emulsion emulsion emulsion Viscosity 29297 20000 14450 14100 24380 12460 14180 pH 4.00 3.98 3.98 3.98 3.97 3.98 3.97

[0477]

[0478] dE2000 / 2.9 3.3 4.3 2.8 3.3 5.25 The addition of 3% of the Cosmetic Active Agent D uniformly coloured the emulsion at the different tested temperatures, with no impact on the pH but decreases the initial viscosity by nearly 65%. The emulsion containing the Cosmetic Active Agent exhibited the same behaviour as the control emulsion over time.

[0479] Example 19: Determination of total flavonoid content

[0480] The Cosmetic Active Agent C was prepared as described in Example 1 above. The flavonoids content was determined by High Performance Liquid Chromatography coupled with UV detection at 266 nm using calibration curve of Kaempferol-3-O-glucoside (Sigma-Aldrich, Ref WE248703). The chromatographic conditions used could be those descried in the publication (Journal of pharmaceutical and biomedical analysis, 2024, 243, pl-15, “Plant Golden” C. sativus: Qualitative and quantitative analysis of major components in stigmas and petals and their biological activity in vitro). The results are summarized in the table below.

[0481] Total Flavonoids Kaempferol-3-O- Kaempferol-3-O- Quercetin-3-O- Sophoroside Glucoside Sophoroside

[0482]

[0483] 0.389 wt% 0.298 wt% 0.01 wt% 0.022 wt%

[0484] The main flavonoid is Kaempferol-3-O-sophoroside that represents 77% of the total flavonoids of the Cosmetic Active Agent C.

[0485] Example 20: Determination of Kinsenoside content

[0486] The Cosmetic Active Agent C was prepared as described in Example 1 above. The flavonoids content was determined by High Performance Liquid Chromatography coupled with ELSD detection using calibration curve of Kinsenoside (Aber, Ref AB493820). The chromatographic conditions used could be those described in the publication (Phytochemical analysis, 2020, 1-11).

[0487] The Kinsenoside content as measured was 0.42% (w / w).

[0488] Example 21: Identification of metabolites of Cosmetic Active Agent through CPC-NMR 1- Materials and Methods

[0489] Sample preparation

[0490] The Cosmetic Active Agent C was prepared as described in Example 1 above.

[0491] Liquid-Liquid Extraction

[0492] 50 mL of Cosmetic Active Agent C extract (corresponding to 58.6 g) were solubilized in a biphasic solvent system consisting of acetonitrile / ethyl acetate / water (3 / 3 / 4, v / v / v). Three successive extraction cycles were performed with 500 mL of fresh upper phase of the same biphasic solvent system. The upper phases were combined and evaporated to dryness (labelled as CRNA-UP; 3473 mg). The remaining aqueous lower phase was labelled CRNA-LOW.

[0493] Centrifugal Partition Chromatography

[0494] The column was filled with the stationary phase (lower phase of the two-phase solvent system) and equilibrated with the mobile phase at 20 mL / min and 1600 rpm. The CRNA-UP was dissolved in 8 mL of stationary phase + 8 mL of mobile phase and injected into the CPC column by a 20 mL loop. The mobile phase was pumped in the ascending mode for 60 min. The column was extruded by switching the mode from ascending to descending for 7 minutes. Tractions of 20 mL were collected over the whole experiment (elution and extrusion). As a result, six final fractions (from F01 to F06) were obtained.

[0495] NMR analyses and metabolites identification An aliquot of each fraction from F01 to F06 (up to 20 mg when possible in terms of quantity and solubility) and an aliquot of CRNA-LOW were dissolved in 600 pL of DMSO-d6 and analysed by 1H, 13C, HSQC, HMBC, and COSY NMR at 298 K on a Bruker Avance AVIII-600 spectrometer (Karlsruhe, Germany) equipped with a TCI cryoprobe. All spectra were processed using TopSpin 4.1.4. Structural elucidation of metabolites contained in the Cosmetic Active Agent C was performed by analysis of ID and 2D NMR spectra (including HSQC) of F01-F06.

[0496] 2- Results

[0497] The metabolites identified in fractions F01-F06 and CRNA-LOW are presented in the table below.

[0498] CPC Mass % of Composition

[0499] fractions (mg) CRNA- UP

[0500] 01 19 1.1% Kaempferol (Min); fatty acids (Maj); Tyrosol (Maj); p- Elution Hydroxybenzoic acid (Med); Phenylacetic acid (Min); Kaempferol 3- O-glucoside (Min)

[0501] 02 100 5.7% Eutectys™ BLA buffered constituents (Maj); Tyrosol (Min);

[0502] Elution Kaempferol 3-O-glucoside (Med); Quercetin 3-O-glucoside (Min) 03 918 52.3% Eutectys™ BLA buffered constituents (Maj); 3- Elution Hydroxybutyrolactone (Med); Kaempferol 3-O-sophoroside (Min);

[0503] 3,4-Dihydroxybutyric acid (Min)

[0504] 04 275 15.7% Eutectys™ BLA buffered constituents (Maj); 3- Elution Hydroxybutyrolactone (Min); Isorhamnetin 3-O-sophoroside (Min);

[0505] Kaempferol 3-O-sophoroside (Min)

[0506] 05 51 2.9% 3 -Hydroxybutyrolactone (Min); Kaempferol 3-O-sophoroside (Maj);

[0507] Elution Quercetin 3-O-sophoroside (Min); Choline (Min)

[0508] 06 391 22.3% Eutectys™ BLA buffered constituents (Maj); Kinsenoside (Min);

[0509] Extrusion Goodyeroside A (Min); P-D-glucose (Min); a-D-glucose (Min); P-D- fructofuranose (Min); a-D-fructofuranose (Min); P-D-fructopyranose (Min); Saccharose (Min); Glycerol (Min)

[0510] CRNA- Eutectys™ BLA buffered constituents (Maj); Kaempferol 3-O- LOW sophoroside (Min); Kinsenoside (Min); Goodyeroside A (Med); P-D- glucose (Med); a-D-glucose (Med); P-D-fructofuranose (Min); a-D- fructofuranose (Min); P-D-fructopyranose (Min); Saccharose (Min);

[0511]

[0512] Choline (Min)

[0513] The Cosmetic Active Agent C was partitioned in a liquid-liquid solvent system to attempt to concentrate a maximum of Crocus sativus L. metabolites in the organic upper phase while maintaining Eutectys™ BLA buffered components in the lower aqueous phase. A total of six fractions were then produced by CPC from the upper phase. All fractions were chemically profiled by NMR, resulting in the identification of 19 metabolites, including mainly glycosylated flavonoids (kaempferol 3-O-sophoroside as the major representative of this chemical class), 3 -hydroxybutyrolactone, kinsenoside, goodyeroside A, phenolic acids, choline, glycerol, and simple sugars.

Claims

Claims1- Cosmetic composition comprising a cosmetically acceptable excipient and at least one active cosmetic ingredient, wherein a first active cosmetic ingredient comprises or consists of a Crocus sativus flower extract.2- The cosmetic composition according to claim 1, wherein the Crocus sativus flower extract is present in an amount of about 0.01% to about 5%, preferably about 0.06% to about 3%, by weight of the cosmetic composition.3- The cosmetic composition according to claim 1 or 2, wherein the Crocus sativus flower extract is an aqueous extract.4- The cosmetic composition according to any one of claims 1 to 3, wherein the Crocus sativus flowers do not comprise stigma.5- The cosmetic composition according to any one of claims 1 to 4, wherein the Crocus sativus flowers are sterilised, minced, dried and / or ground.6- The cosmetic composition according to any one of claims 1 to 5, wherein the cosmetic composition is a skin care composition, preferably a scalp care composition, e.g. an anti -dandruff composition.7- The cosmetic composition according to any one of claims 1 to 5, wherein the cosmetically acceptable excipient comprises or consists of a eutectic solvent, more preferably a natural deep eutectic solvent, and most preferably a eutectic solvent consisting of about 18% potassium lactate, about 8% lactic acid, about 30% betain, and about 44% water.8- The cosmetic composition according to any one of claims 1 to 7, wherein the cosmetic composition further comprises one or more other active cosmetic ingredient(s), preferably one or more other active cosmetic ingredient(s) selected from the group consisting of lightening agents, dark spot erasers, skin tone agents, hydrating agents, moisturizers, UV protection actives, anti-ageing actives, and mixtures thereof.9- Use of the cosmetic composition according to any one of claims 1 to 8 for anti -dandruff treatment, antifungal treatment, anti-erythema treatment, antipruritic treatment, skin soothing, and / or improvement of skin barrier integrity, comprising the step of applying the cosmetic composition to skin, e.g. to scalp. 10- A method of cosmetic skin treatment, comprising the step of applying the cosmetic composition of any one of claims 1 to 8 to the skin, preferably wherein the skin comprises the scalp.11- The method of claim 10, for anti -dandruff treatment, anti-fungal treatment, anti-erythema treatment, antipruritic treatment, skin soothing, and / or improvement of skin barrier integrity.fungal treatment, anti-erythema treatment, antipruritic treatment, skin soothing, and / or improvement of skin barrier integrity, preferably wherein the skin comprises the scalp.13- A Crocus sativus flower extract, or a composition comprising thereof, for use in treating or preventing an inflammatory disease or a fungal infection.14- The Crocus sativus flower extract, or a composition comprising thereof, for use according to claim 13, wherein the fungal infection comprises infection with Malassezia species, particularly with M. restricta. M. furfur orM. globosa.15- The Crocus sativus flower extract, or a composition comprising thereof, for use according to claim 13 or 14, wherein the fungal infection is a topical infection.

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