Cosmetic compositions and methods of use thereof

Abstract

The present invention relates generally to methods and compositions useful for treating skin, and in particular, improving skin gloss, improving overall complexion, reducing skin discoloration, and / or reducing the appearance of dark spots. In some aspects, methods are disclosed that include topically applying an effective amount of a topical composition to the skin, the topical composition comprising a silybum marianum extract and / or a white water lily extract.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 435,838, filed on December 29, 2022, which is incorporated herein by reference in its entirety. Background of the Invention A. Technical field

[0003] The present invention generally relates to cosmetic compositions that can be used to improve the visual appearance of skin by accelerating skin desquamation and nighttime skin pigmentation. The compositions can include milk thistle (Silybum marianum) extract and / or white water lily (Nymphaea alba) extract. This combination is chemically compatible and can be incorporated into a wide range of product formulations, such as masks, serums, creams, cleansers, toners, gels, emulsions, gel emulsions, gel serums, and the like.

[0004] B. Related technical description

[0005] Aging, chronic exposure to adverse environmental factors, malnutrition, fatigue, etc. can alter the visual appearance, physical properties, or physiological functions of the skin in visually undesirable ways. The most significant and noticeable changes include the appearance of fine lines and wrinkles, loss of elasticity, increased sagging, decreased firmness, loss of color uniformity or tone, rough surface texture, and mottled pigmentation. As the skin ages or undergoes chronic environmental insults, the skin undergoes less obvious but measurable changes, including a general decrease in cell and tissue viability, decreased cell replication rates, decreased skin blood flow, decreased moisture content, accumulation of structural and functional errors, alterations in the normal regulation of common biochemical pathways, and a decrease in the skin's ability to remodel and repair itself. Many changes in the skin's appearance and function are caused by changes in the outer epidermal layer of the skin, while others are caused by changes in the dermis.

[0006] Ethnic background has been reported to be associated with differences in structural and functional skin properties. Muizzuddin et al., Structural and functional differences in barrier properties of African American, Caucasian and East Asian skin. J Dermatol Sci. 2010 Aug;59(2):123-8; Rawlings, Ethnic skin types: are there differences in skin structure and function? Int J Cosmet Sci. 2006 Apr;28(2):79-93.

[0007] The diverse expression or activation levels of proteases in different ethnic backgrounds may play a role in shaping distinct skin characteristics. For example, a member of the kallikrein (KLK) family of serine proteases, KLK7 (also known as stratum corneum chymotryptic enzyme (SCCE)), is involved in the skin desquamation process by degrading adhesion proteins of the corneodesmosome. Previous studies have shown that the expression of KLK7 / SCCE proteins is significantly reduced in certain ethnic groups or races. For example, low levels of active KLK7 / SCCE in black skin may contribute to higher corneocyte maturation, stronger cohesion, and overall stronger barrier function compared to other ethnic groups. However, low protease activity may also lead to undesirable skin desquamation, which raises concerns in skin care, such as uneven skin tone and dark spots.

[0008] Historically, skin discoloration has been addressed by modulating the skin's melanin production pathway, for example by inhibiting tyrosinase. This approach is hypothesized to be less effective in people of color due to inefficient desquamation. For example, keratinocytes, which form dark spots in the stratum corneum, contain excess melanin, which must be removed to reduce the appearance of discoloration. Therefore, without addressing the underlying issue of inefficient desquamation, existing solutions may not effectively remove keratinocytes and, consequently, may not effectively improve skin appearance. Summary of the Invention

[0009] The inventors have identified a solution to problems associated with undesirable skin desquamation, such as uneven skin tone and dark spots. This solution is based on a combination of plant-based ingredients, including milk thistle extract and / or white water lily extract. This combination can be used to create a topical skin composition that improves skin radiance, improves overall skin tone, reduces skin discoloration, and / or reduces the appearance of dark spots.

[0010] In some aspects, topical compositions are disclosed. In some aspects, the topical composition comprises any one, any combination, or all of milk thistle extract and / or white water lily extract.

[0011] In some examples, the topical composition comprises an effective amount of any one, any combination, or all of milk thistle extract and / or white water lily extract to increase gene expression of KLK family serine proteases, including KLK5, KLK7, and / or KLK14. In some examples, the topical composition comprises an effective amount of any one, any combination, or all of milk thistle extract and / or white water lily extract to stimulate the skin's natural desquamation process, more effectively shedding the top layer of keratinocytes, thereby improving skin radiance and even skin tone. In some examples, the topical composition comprises an effective amount of any one, any combination, or all of milk thistle extract and / or white water lily extract to accelerate skin desquamation. In some examples, the topical composition comprises an effective amount of any one, any combination, or all of milk thistle extract and / or white water lily extract to improve skin radiance, improve overall skin tone, reduce skin discoloration, and / or reduce the appearance of dark spots.

[0012] The amounts of the ingredients in the composition can vary (e.g., the amounts can be as low as 0.000001 weight / weight % to as high as 80 weight / weight % or any range therebetween). In one example, the composition comprises any one, any combination, or all of milk thistle extract and / or white water lily extract. The composition may also comprise one or more ingredients as described herein. For example, the composition may comprise one or more additional ingredients selected from one or more of a conditioning agent, a humectant, a pH adjuster, a structuring agent, an inorganic salt, and a preservative.

[0013] In some embodiments, the milk thistle extract is milk thistle seed extract and / or the white water lily extract is white water lily flower extract.

[0014] Also disclosed are methods of using the compositions disclosed herein. In some examples, the methods comprise topically applying any of the compositions disclosed herein to an area of ​​skin and / or facial area and / or eye area in need thereof. In one aspect, any of the compositions disclosed herein is topically applied and the composition is left on the application area and removed from the application area after a period of time, and / or the composition is removed directly after application.

[0015] In particular, the composition of the present invention is formulated as a topical skin composition. The composition can have a dermatologically acceptable carrier or vehicle for compounds, compositions, and extracts. The composition can also include a moisturizer or wetting agent, a surfactant, a silicone-containing compound, a UV agent, an oil, and / or other ingredients described in this specification sheet or other ingredients known in the art. The composition can be a lotion, a cream, a body milk, a facial mask, a scrub, a lotion, a gel, an essence, an emulsion (e.g., oil-in-water, water-in-oil, silicone-in-water, water-in-water-in-water, oil-in-water-in-oil, silicone-in-water-in-oil, etc.), a solution (e.g., an aqueous solution or a water-alcohol solution), an anhydrous matrix (e.g., a lipstick or powder), an ointment, an emulsion, a paste, an aerosol, a solid form, an eye gel, etc. The composition can be in a powder form (e.g., dried, freeze-dried, granular, etc.). The composition can be formulated for topical skin application at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or more than 7 times per day during use. In some aspects of the invention, the composition can be storage stable or color stable, or both storage stable and color stable. It is also contemplated that the viscosity of the composition can be selected to achieve the desired result. For example, the viscosity of such a composition can range from about 1 cps to well over 1 million cps, or any range or integer derivable therein, depending on the type of composition desired (e.g., 2 cps, 3 cps, 4 cps, 5 cps, 6 cps, 7 cps, 8 cps, 9 cps, 10 cps, 20 cps, 30 cps, 40 cps, 50 cps, 60 cps, 70 cps, 80 cps, 90 cps, 100 cps, 200 cps, 300 cps, 400 cps, 500 cps, 600 cps, 700 cps, 800 cps, 900 cps, 1000 cps, 2000 cps, 3000 cps, 4000 cps, 5000 cps, 6000 cps, 7000 cps, 8000 cps, 9000 cps, 10000 cps, 20000 cps, 30000 cps, 40000 cps, 50000 cps, 60000 cps, 70000 cps, 80000 cps, 90000 cps, 100000 cps, 200000 cps, 300000 cps, 400000 cps, 500000 cps, 500000 cps, 600000 cps, 600000 s, 4000cps, 5000cps, 6000cps, 7000cps, 8000cps, 9000cps, 10000cps, 20000cps, 3000 0cps, 40000cps, 50000cps, 60000cps, 70000cps, 80000cps, 90000cps, 100000cps, 2000 00cps, 300000cps, 400000cps, 500000cps, 600000cps, 700000cps, 800000cps, 900000 cps, 1000000cps, 2000000cps, 3000000cps, 4000000cps, 5000000cps, 10000000cps, etc.).

[0016] The compositions of the present invention can also be modified to have a desired oxygen radical absorbance capacity (ORAC) value. In certain non-limiting aspects, the compositions or components of the present invention identified throughout this specification, or extracts thereof, can be modified to have at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410 0, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 30000, 50000, 100000 or more than 100000, or any range of ORAC values ​​derivable therein.

[0017] In non-limiting aspects, the composition can have a pH value of about 6 to about 9. In some aspects, the pH can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. The composition can include triglycerides. Non-limiting examples include short-chain triglycerides, medium-chain triglycerides and long-chain triglycerides. In some aspects, the triglyceride is a medium-chain triglyceride (e.g., caprylic capric triglyceride). The composition can also include a preservative. Non-limiting examples of a preservative include phenoxyethanol, methylparaben, propylparaben or any mixture thereof. In some embodiments, the composition does not contain parabens.

[0018] The composition of the present invention may have UVA and UVB absorbing properties. The composition may have a sun protection factor (SPF) of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or greater, or any integer or derivative thereof. The composition may be a sunscreen lotion, spray, or cream.

[0019] The composition of the present invention can also include any one, any combination or all of the following additional ingredients: water, conditioning agent, chelating agent, wetting agent, pH adjusting agent, inorganic salt, preservative, thickening agent, silicone-containing compound, essential oil, structurant, vitamin, pharmaceutical ingredient or antioxidant, or any combination of these ingredients or the mixture of these ingredients. In some aspects, said composition can include at least two, three, four, five, six, seven, eight, nine, ten or more than ten or all of these additional ingredients determined in the aforementioned sentence. The non-limiting examples of these additional ingredients run through this specification sheet and are incorporated into this section by reference. The amount of these ingredients can be 0.0001 weight % to 99.9 weight % or 0.0001 volume % to 99.9 volume % of the composition, or any integer or scope as disclosed in other parts of this specification sheet, which are incorporated into this section by reference.

[0020] Also contemplated are kits comprising the compositions of the present invention. In certain embodiments, the composition is contained in a container. The container can be a bottle, dispenser, or bag. The container can dispense a predetermined amount of the composition. In certain aspects, the composition is dispensed in the form of a spray, mist, mass, or liquid. The container can carry a marking on its surface. The marking can be a word, abbreviation, picture, or symbol.

[0021] It is also contemplated that the compositions disclosed in this specification can be used as leave-in compositions or rinse-out compositions. For example, a leave-in composition can be a composition that is topically applied to the skin and retained on the skin for a period of time (e.g., at least 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 20 minutes, or 30 minutes, or at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours, or all night or all day). Alternatively, a rinse-out composition can be a product that is applied to the skin and then removed or rinsed (e.g., with water) from the skin within a period of time, such as less than 5 minutes, 4 minutes, 3 minutes, 2 minutes, or 1 minute. The example of a rinse-out composition can be a skin cleanser, shampoo, conditioner, or soap. Examples of leave-on compositions may be skin moisturizers, sunscreens, face masks, night creams or day creams.

[0022] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present invention, and vice versa.Furthermore, the compositions of the present invention can be used to implement the methods of the present invention.

[0023] In some embodiments, the compositions of the present invention may be pharmaceutically or cosmetically suitable, or may have pleasant tactile properties. "Pharmaceutically suitable," "cosmetically suitable," and / or "pleasant tactile properties" describe compositions that have specific tactile properties that make the skin feel pleasant (e.g., a composition that is not too watery or too oily, a composition with a silky texture, a non-sticky or non-adhesive composition, etc.). Pharmaceutically or cosmetically suitable may also refer to the creamy or lubricating properties of the composition, or the moisturizing properties of the composition.

[0024] Products comprising the compositions of the present invention are also contemplated. In non-limiting aspects, the product can be a cosmetic. The cosmetic can be those described elsewhere in this specification or known to those skilled in the art. Non-limiting examples of products include moisturizers, creams, lotions, skin softeners, serums, gels, lotions, body milks, scrubs, foundations, night creams, lipsticks, cleansers, toners, sunscreens, facial masks, anti-aging products, deodorants, antiperspirants, perfumes, colognes, and the like.

[0025] Aspects 1 to 20 are also disclosed in the context of the present invention. Aspect 1 is a method of treating skin comprising topical application of a topical skin composition, wherein the topical skin composition comprises milk thistle extract and white water lily extract. Aspect 2 is the method of aspect 1, wherein topical application of the topical skin composition results in improved skin radiance, improved overall skin tone, reduced skin discoloration, and / or reduced appearance of dark spots. Aspect 3 is the method of aspect 1, wherein the topical skin composition increases gene expression of kallikrein-5 (KLK5), kallikrein-7 (KLK7), and / or kallikrein-14 (KLK14). Aspect 4 is the method of aspect 1, wherein the topical skin composition stimulates shedding of skin keratinocytes. Aspect 5 is the method of aspect 1, wherein the topical skin composition accelerates skin desquamation. Aspect 6 is the method of aspect 1, wherein the topical skin composition is a serum, cream, gel, lotion, gel-emulsion, gel serum, mask, cleanser, and / or toner. Aspect 7 is the method of aspect 1, wherein the milk thistle extract is milk thistle seed extract and / or the white water lily extract is white water lily flower extract. Aspect 8 is the method of aspect 7, wherein the topical skin composition comprises an effective amount of milk thistle seed extract and white water lily flower extract. Aspect 9 is the method of aspect 7, wherein the topical skin composition comprises 0.0001% to 10% by weight of milk thistle seed extract and 0.0001% to 5% by weight of white water lily flower extract. Aspect 10 is the method of aspect 9, wherein the topical skin composition is free of parabens. Aspect 11 is the topical skin composition, wherein the topical skin composition comprises milk thistle extract and white water lily flower extract. Aspect 12 is the topical skin composition of aspect 11, wherein topical application of the topical skin composition results in improved skin radiance, improved overall skin tone, reduced skin discoloration, and / or reduced appearance of dark spots. Aspect 13 is the topical skin composition of aspect 11, wherein the topical skin composition increases gene expression of kallikrein-5 (KLK5), kallikrein-7 (KLK7), and / or kallikrein-14 (KLK14). Aspect 14 is the topical skin composition of aspect 11, wherein the topical skin composition stimulates shedding of skin keratinocytes. Aspect 15 is the topical skin composition of aspect 11, wherein the topical skin composition accelerates skin desquamation. Aspect 16 is the topical skin composition of aspect 11, wherein the topical skin composition is a serum, cream, gel, emulsion, gel emulsion, gel serum, mask, cleanser, and / or toner. Aspect 17 is the topical skin composition of aspect 11, wherein the milk thistle extract is milk thistle seed extract and / or the white water lily extract is white water lily flower extract. Aspect 18 is the topical skin composition of aspect 17, wherein the topical skin composition comprises an effective amount of milk thistle seed extract and white water lily flower extract.Aspect 19 is a topical skin composition according to aspect 17, wherein the topical skin composition comprises 0.0001% to 10% by weight of milk thistle seed extract and 0.0001% to 5% by weight of white water lily extract. Aspect 20 is a topical skin composition according to aspect 19, wherein the topical skin composition is free of parabens.

[0026] "Topical application" means applying or coating a composition onto the surface of the lips or keratinous tissue. "Topical skin composition" includes compositions suitable for topical application to the skin, lips, and / or keratinous tissue. Such compositions are generally dermatologically acceptable in that they do not exhibit excessive toxicity, incompatibility, instability, allergic reactions, etc., when applied to the lips, skin, and / or keratinous tissue. The topical skin care compositions of the present invention may have a selected viscosity to avoid noticeable dripping or pooling after application to the lips, skin, and / or keratinous tissue.

[0027] "Keratinous tissue" includes the keratin-containing layer that is the outermost protective covering of mammals and includes, but is not limited to, lips, skin, hair, and nails.

[0028] The term "about" or "approximately" is defined as close as understood by one of ordinary skill in the art. In one non-limiting embodiment, the term is defined as within ±10%, preferably within ±5%, more preferably within ±1%, and most preferably within ±0.5%.

[0029] The term "substantially" and variations thereof are defined as being understood by one of ordinary skill in the art to be largely, but not necessarily completely, and in one non-limiting embodiment, substantially means within ±10%, within ±5%, within ±1%, or within ±0.5%.

[0030] The terms "inhibit" or "reduce" or any variations of these terms include any measurable reduction or complete inhibition that achieves a desired result. The terms "promote" or "increase" or any variations of these terms include any measurable increase, such as a measurable increase in a protein or molecule (e.g., a matrix protein such as fibronectin, laminin, collagen or elastin or a molecule such as hyaluronic acid), to achieve a desired result.

[0031] The term "effective" as used in the specification and / or claims means sufficient to achieve a desired, expected, or intended result.

[0032] When used in conjunction with the terms "comprises," "comprising," "having," or "containing" or any variations of these terms in the claims and / or description, an element may mean "a," but it also means "one or more," "at least one," and "one or more than one."

[0033] The phrase "and / or" means "and" or "or." For example, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.

[0034] As used in this specification and in one or more claims, the words "comprising," "having," "including," or "containing" are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0035] The compositions and methods of use thereof may "comprise," "consist essentially of," or "consist of" any ingredient or step disclosed in the specification. With respect to the phrase "consisting essentially of," a fundamental and novel property of the compositions and methods of the present invention is the ability to accelerate skin desquamation.

[0036] Other objects, features and advantages of the present invention will become apparent from the detailed description that follows. However, it should be understood that the detailed description and examples, while indicating specific embodiments of the present invention, are given by way of illustration only. In addition, it is expected that changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. DETAILED DESCRIPTION

[0037] As mentioned above, several unique aspects of the present invention are the combination of milk thistle extract and white water lily extract in a topical cosmetic composition. This allows for the benefits of improved skin radiance, improved overall skin tone, reduced skin discoloration, and / or reduced appearance of dark spots to be realized.

[0038] In some aspects, the composition is applied to the skin to improve skin radiance, improve overall skin tone, reduce skin discoloration, and / or reduce the appearance of dark spots by accelerating skin desquamation and / or by increasing gene expression of KLK family serine proteases, including KLK5, KLK7, and / or KLK14.

[0039] The following subsections describe non-limiting aspects of the invention in greater detail.

[0040] Some compositions disclosed herein can be applied to the skin and left on the skin or hair for a period of time (e.g., at least 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, or 60 minutes or more). Thereafter, the composition can be washed off or peeled off from the skin if desired. Some compositions disclosed herein can be applied to the skin and immediately rinsed off from the skin. Some compositions disclosed herein can be applied to the skin and at least partially absorbed by the skin.

[0041] These and other non-limiting aspects of the present invention are described in the following sections.

[0042] A.Active ingredients

[0043] The present invention is based on the identification of one or a combination of more than one active ingredients - milk thistle extract and white water lily extract - that can be used to accelerate skin desquamation, increase gene expression of KLK family serine proteases, including KLK5, KLK7 and / or KLK14, and improve skin radiance, improve overall skin tone, reduce skin discoloration and / or reduce the appearance of dark spots.

[0044] Milk thistle, commonly known as milk thistle, is a plant native to southern Europe and Asia. It is known for its red to purple flowers, shiny light green leaves with white veins, and fruit. In some instances, the milk thistle extract is a milk thistle fruit extract. In some embodiments, the milk thistle fruit extract is a hydroalcoholic extract containing silymarin as the active ingredient (silymarin is a mixture of flavonoid derivatives, including silibine, silicristine, silidianin, isosilibine, and isosiliicristine). In some embodiments, the milk thistle extract is a milk thistle seed extract. In some embodiments, the milk thistle seed extract contains a mixture of esters from milk thistle seeds. In some instances, the milk thistle seed extract contains ethyl silymarin. In some instances, the milk thistle seed extract does not contain silymarin. In some instances, such ingredients are commercially available, for example from Alban Muller, which provides silymarin ethyl ester (and) optionally tocopherol, under the trade name ER.

[0045] Nymphaea alba, commonly known as white water lily, is a plant native to North Africa, Asia, and Europe. It is known for its white flowers and green leaves. In some embodiments, water and / or propylene glycol extracts from white water lilies are used. In some instances, such ingredients are commercially available, for example from Solabia, which provides propylene glycol (and) water (and) white water lily extracts under the trade name PROV.

[0046] This combination of ingredients can be used in different product forms to treat various skin conditions. As non-limiting examples, the combination of ingredients can be formulated as an emulsion (e.g., oil-in-water, water-in-oil), a gel, a serum, a gel emulsion, a gel serum, a toner, a facial mask, a scrub, a lotion, a cream, or a body lotion.

[0047] The extracts described herein may be extracts prepared by extraction methods known in the art and combinations thereof. Non-limiting examples of extraction methods include liquid-liquid extraction, solid phase extraction, aqueous phase extraction, ethyl acetate extraction, ethanol extraction, acetone extraction, oil extraction, supercritical carbon dioxide extraction, heat extraction, pressure extraction, pressure drop extraction, ultrasonic extraction, etc. The extract may be liquid, solid, dried liquid, resuspended solid, etc.

[0048] B. Amount of ingredients

[0049] It is contemplated that the compositions of the present invention may include any amount of the ingredients discussed herein. The compositions may also include any number of combinations of additional ingredients (e.g., pigments, or additional cosmetic ingredients or additional pharmaceutical ingredients) described herein. The concentration of any ingredient in the composition may vary. For example, in non-limiting embodiments, the composition in its final form may comprise, consist essentially of, or consist of, for example, at least about 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.0010%, 0.0011%, 0.0012%, 0.0013%, 0.0014%, 0.0015%, 0.0016%, 0.0017%, 0.0018%, 0.0019%, 0.0020%, 0.0021%, 0.0022%, 0.0023%, 0.0024%, 0.0025%, 0.0026%, 0.0027%, 0.0028%, 0.0029%, 0.0030%, 0.0031%, 0.0032%, 0.0033%, 0.0034%, 0.0035%, 0.0036%, 0.0037%, 0.0038%, 0.0039%, 0.010 3%, 0.0024%, 0.0025%, 0.0026%, 0.0027%, 0.0028%, 0.0029%, 0.0030%, 0.0031%, 0.0032%, 0.0033%, 0.0034%, 0.0035%, 0.0036%, 0.0037%, 0.0038%, 0.0039%, 0.0040%, 0.0041%, 0.0042%, 0.0043%, 0.0044%, 0.0045%, 0.0046%, 0.0047%, 0.0048%, 0.0049%, 0.0050%, 0.0051% %, 0.0052%, 0.0053%, 0.0054%, 0.0055%, 0.0056%, 0.0057%, 0.0058%, 0.0059%, 0.0060%, 0.0061%, 0.0062%, 0.0063%, 0.0064%, 0.0065%, 0.0066%, 0.0067%, 0.0068%, 0.0069%, 0.0070%, 0.0071%, 0.0072%, 0.0073%, 0.0074%, 0.0075%, 0.0076%, 0.0077%, 0.0078%, 0.0079%, 0.0080%, 0.0081%, 0.0082%, 0.0083%, 0.0084%, 0.0085%, 0.0086%, 0.0087%, 0.0088%, 0.0089%, 0.0090%, 0.0091%, 0.0092%, 0.0093%, 0.0094%, 0.0095%, 0.0096%, 0.0097%, 0.0098%, 0.0099%, 0.0100%, 0.0200%, 0.0250%, 0.0275%, 0.0300%, 0.0325%, 0.0350%, 0.0375%, 0.0400％、0.0425％、0.0450％、0.0475％、0.0500％、0.0525％、0.0550％、0.0575％、0.0600％、0.0625％、0.0650％、0.0675％、0.0700％、0.0725％、0.0750％、0.0775％、0.0800％、0.0825％、0.0850％、0.0875％、0.0900％、0.0925％、0.0950％、0.0975％、0.1000％、0.1250％、0.1500％、0.1750％、0.2000％、0.2250％、0.2500％、0.2750％、0.3000％、0.3250％、0.3500％、0.3750％、0.4000％、0.4250％、0.4500％、0.4750％、0.5000％、0.5250％、0.0550％、0.5750％、0.6000％、0.6250％、0.6500％、0.6750％、0.7000％、0.7250％、0.7500％、0.7750％、0.8000％、0.8250％、0.8500％、0.8750％、0.9000％、0.9250％、0.9500％、0.9750％、1.0％、1.1％、1.2％、1.3％、1.4％、1.5％、1.6％、1.7％、1.8％、1.9％、2.0％、2.1％、2.2％、2.3％、2.4％、2.5％、2.6％、2.7％、2.8％、2.9％、3.0％、3.1％、3.2％、3.3％、3.4％、3.5％、3.6％、3.7％、3.8％、3.9％、4.0％、4.1％、4.2％、4.3％、4.4％、4.5％、4.6％、4.7％、4.8％、4.9％、5.0％、5.1％、5.2％、5.3％、5.4％、5.5％、5.6％、5.7％、5.8％、5.9％、6.0％、6.1％、6.2％、6.3％、6.4％、6.5％、6.6％、6.7％、6.8％、6.9％、7.0％、7.1％、7.2％、7.3％、7.4％、7.5％、7.6％、7.7％、7.8％、7.9％、8.0％、8.1％、8.2％、8.3％、8.4％、8.5％、8.6％、8.7％、8.8％、8.9％、9.0％、9.1％、9.2％、9.3％、9.4％、9.5％、9.6％、9.7％、9.8％、9.9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% or any range derivable therein of at least one ingredient recited throughout the present specification and claims. In non-limiting aspects, the percentages may be calculated by weight or volume of the entire composition. One of ordinary skill in the art will appreciate that the concentrations in a given composition may vary depending on the addition, substitution and / or removal of ingredients.

[0050] C. Carrier

[0051] The compositions of the present invention can include or be incorporated into all types of carriers and vehicles. The carrier or vehicle can be a pharmaceutically or dermatologically acceptable carrier or vehicle. Non-limiting examples of carriers or vehicles include water, glycerol, alcohol, oil, silicon-containing compounds, silicone compounds, and waxes. Variants and other suitable vehicles will be apparent to those skilled in the art and are suitable for use in the present invention. In some aspects, the concentrations and combinations of the compounds, ingredients, and reagents are selected so that the combination is chemically compatible and does not form complexes that precipitate from the final product.

[0052] D.Structure

[0053] The compositions of the present invention can be constructed or formulated into a variety of different forms. Non-limiting examples include emulsions (e.g., water-in-oil, water-in-oil-in-water, oil-in-water, silicone-in-water, silicone-in-water, oil-in-oil-in-water, silicone-in-water-in-oil emulsions), creams, lotions, solutions (aqueous or hydro-alcoholic), anhydrous bases (e.g., lipsticks and powders), gels, facial masks, scrubs, body lotions, peels, and ointments. Variants and other structures will be apparent to those skilled in the art and are suitable for use with the present invention.

[0054] E. Additional ingredients

[0055] In addition to the combination of ingredients disclosed herein, the compositions may also include additional ingredients, such as cosmetic ingredients and pharmaceutical active ingredients. Non-limiting examples of these ingredients are described in the following subsections.

[0056] 1. Cosmetic ingredients

[0057] The CTFA International Cosmetic Ingredient Dictionary and Handbook (2004 and 2008) describes a variety of non-limiting cosmetic ingredients that can be used in the context of the present invention. Examples of these ingredient categories include: fragrances (artificial and natural; for example, gluconic acid, phenoxyethanol, and triethanolamine), dyes and coloring ingredients (for example, Blue 1, Blue 1 Lake, Red 40, titanium dioxide, D&C Blue No. 4, D&C Green No. 5, D&C Orange No. 4, D&C Red No. 17, D&C Red No. 33, D&C Violet No. 2, D&C Yellow No. 10, and D&C Yellow No. 11), flavorings / fragrances (for example, Stevia), and rebaudiana, sweetleaf) extract and menthol), adsorbents, lubricants, solvents, humectants (including, for example, emollients, humectants, film formers, occlusives and agents that affect the skin's natural moisturizing mechanisms), water repellents, UV absorbers (physical and chemical absorbers, such as para-aminobenzoic acid ("PABA") and corresponding PABA derivatives, titanium dioxide, zinc oxide, etc.), essential oils, vitamins (such as A, B, C, D, E and K), trace metals (such as zinc, calcium and selenium), anti-irritants (such as steroids and nonsteroidal anti-inflammatory drugs), plant extracts (such as aloe vera, chamomile, cucumber extract, Ginkgo biloba), biloba), ginseng, and rosemary), antimicrobial agents, antioxidants (e.g., BHT and tocopherol), chelating agents (e.g., disodium EDTA and tetrasodium EDTA), preservatives (e.g., methylparaben and propylparaben), pH adjusters (e.g., sodium hydroxide and citric acid), adsorbents (e.g., aluminum starch octenylsuccinate, kaolin, corn starch, oat starch, cyclodextrin, talc, and zeolite), skin bleaching and whitening agents (e.g., hydroquinone and niacinamide lactate / ester), humectants (e.g., sorbitol, urea, methyl gluceth-20, saccharide isomers, and mannitol), exfoliants, water-resistant agents (e.g., magnesium hydroxide / aluminum stearate), skin conditioning agents (e.g., aloe vera extract, allantoin, bisabolol, ceramides, dimethicone, hyaluronic acid, biosaccharide gum-1, ethylhexylglycerin, pentylene glycol, hydrogenated polydecene, octyldodecyl oleate, and dipotassium glycyrrhizate). Non-limiting examples of some of these ingredients are provided in the following subsections.

[0058] a. UV absorbers and / or reflectors

[0059] UV absorbers and / or reflectors that can be used in combination with the compositions of the present invention include chemical and physical sunscreens. Non-limiting examples of chemical sunscreens that can be used include para-aminobenzoic acid (PABA), PABA esters (glyceryl PABA, amyl p-dimethylaminobenzoate, and octyl p-dimethylaminobenzoate), butyl PABA, ethyl PABA, dihydroxypropyl PABA ethyl ester, benzophenones (oxybenzone, sulfisobenzone, benzophenone, and benzophenone-1 to benzophenone-12), cinnamates (octyl methoxycinnamate (octyl methoxycinnamate), isopentyl p-methoxycinnamate, octyl methoxycinnamate, cinoxate, methyl diisopropyl cinnamate, DEA methoxycinnamate, ethyl diisopropyl cinnamate, glyceryl caprylate dimethoxycinnamate, and ethyl methoxycinnamate), cinnamate esters, salicylates (homosalate, benzyl salicylate, glycol salicylate, isopropyl salicylate), Benzyl esters, etc.), anthranilates / esters, ethyl urocanate, benzoyl salicylate, octyl salicylate, dibenzoylmethane derivatives (such as avobenzone), octyl triazone, galloyl gallate trioleate, glyceryl aminobenzoate, 2-hydroxy-1,4-naphthoquinone and dihydroxyacetone, ethylhexyl triazone, dioctyl butyramido triazone, benzylmalonate polysiloxane, terephthalylidene dicamphor sulfonic acid, disodium phenyl dibenzimidazole tetrasulfonate, diethylamino hydroxybenzoyl hexyl benzoate, bis-diethylamino hydroxybenzoyl benzoate, bis-benzoxazolylphenyl ethylhexyliminotriazine, drometrizole trisiloxane, methylene bis-benzotriazolyl tetramethylbutylphenol and bis-ethylhexyloxyphenol methoxyphenyl triazine, 4-methylbenzylidene camphor and isoamyl 4-methoxycinnamate. Non-limiting examples of physical sunscreens include kaolin, talc, petrolatum, and metal oxides (eg, titanium dioxide and zinc oxide).

[0060] b. Moisturizer

[0061] Non-limiting examples of moisturizers that can be used with the compositions of the present invention include amino acids, chondroitin sulfate, diglycerin, erythritol, fructose, glucose, glycerin, glycerol polymers, ethylene glycol, 1,2,6-hexanetriol, honey, hyaluronic acid, hydrogenated honey, hydrogenated starch hydrolysate, inositol, lactitol, maltitol, maltose, mannitol, natural moisturizing factor, PEG-15 butylene glycol, polyglyceryl sorbitol, salts of pyrrolidone carboxylic acid, potassium PCA, propylene glycol, saccharide isomers, sodium glucuronate, sodium PCA, sorbitol, sucrose, trehalose, urea, and xylitol.

[0062] Other examples include acetylated lanolin, acetylated lanolin alcohol, alanine, algae extract, aloe barbadensis, aloe vera extract, aloe vera gel, Althea officinalis extract, apricot (Prunus armeniaca) kernel oil, arginine, arginine aspartate, arnica montana extract, aspartic acid, avocado (Persea gratissima) oil, barrier sphingolipids, butanol, beeswax, behenyl alcohol, beta-sitosterol, Betula alba bark extract, Borago officinalis extract, Ruscus aculeatus extract, butylene glycol, Calendula officinalis extract, calendula oil, Euphorbia cerifera (Candelilla) seed extract, butylene glycol, maltose ... cerifera wax, rapeseed oil, caprylic / capric triglyceride, elettaria cardamomum oil, copernicia erifera wax, daucus carota sativa (carrot) oil, ricinus communis (castor) oil, ceramides, ozokerite, ceteareth-5, ceteareth-12, ceteareth-20, cetearyl octanoate, ceteth-20, ceteth-24, cetyl acetate, cetyl octanoate, cetyl palmitate, anthemis nobilis (chamomile) oil, cholesterol, cholesterol esters, cholesterol hydroxystearate, citric acid, clary sage (salvia sclarea) oil, theobroma cacao (cocoa) cacao butter, coco-caprylate / caprate, coconut (Cocosnucifera) oil, collagen, collagen amino acids, corn (Zea mays) oil, fatty acids, decyl oleate, dimethicone copolyol, dimethiconol, dioctyl adipate, dioctyl succinate, dipentaerythrityl hexacaprylate / hexacaprate, DNA, erythritol, ethoxydiglycol, ethyl linoleate, eucalyptus globulus oil, evening primrose (Oenothera biennis) oil, fatty acids, geranium scabra (Geranium)maculatum) oil, glucosamine, glucosamine glutamate / ester, glutamic acid, glycereth-26, glycerin, glyceryl distearate, glyceryl hydroxystearate, glyceryl laurate, glyceryl linoleate, glyceryl myristate, glyceryl oleate, glyceryl stearate, glyceryl stearate SE, glycine, glycol stearate, glycol stearate SE, glycosaminoglycans, grape (Vitis vinifera) seed oil, hazelnut (Corylus americana) nut oil, hazelnut (Corylus avellana) nut oil, hexylene glycol, hyaluronic acid, hybrid safflower (Carthamus tinctorius) oil, hydrogenated castor oil, hydrogenated coco-glycerides, hydrogenated coconut oil, hydrogenated lanolin, hydrogenated lecithin, hydrogenated palm glycerides, hydrogenated palm kernel oil, hydrogenated soybean oil, hydrogenated tallow glycerides, hydrogenated vegetable oil, hydrolyzed collagen, hydrolyzed elastin, hydrolyzed glycosaminoglycans, hydrolyzed keratin, hydrolyzed soy protein, hydroxylated lanolin, hydroxyproline, isocetyl stearate, isocetyl stearoyl stearate, isodecyl oleate, isopropyl isostearate, isopropyl lanolinate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, isostearamide DEA, isostearic acid, isostearyl lactylate, isostearyl neopentanoate, jasmine (Jasminum officinale) oil, jojoba (Buxus chinensis) oil, macroalgae, Aleurites aurantifolia (Aleurites moschata) seed oil moluccana) Nut Oil, Lactamide MEA, Lanolin Eth-16, Lanolin Eth-10 Acetate, Lanolin, Lanolin Acid, Lanolin Alcohol, Lanolin Oil, Lanolin Wax, Lavender (Lavandula angustifolia) Oil, Lecithin, Lemon (Citrus medica limonum) Oil, Linoleic Acid, Linolenic Acid, Macadamiaternifolia Nut Oil, Maltitol, Chamomilla recutita (Chamomilla) recutita) oil, methyl glucose sesquistearate, methylsilanol PCA esters, mineral oil, mink oil, Mortierella fuchsia oil, myristyl lactate, myristyl myristate, myristyl propionate, neopentyl glycol dicaprylate / dicaprate, octyldodecanol, octyldodecanol myristate, octyldodecanol stearoyl stearate, octyl hydroxystearate, octyl palmitate, octyl salicylate, octyl stearate, oleic acid, olive (Olea europaea) oil, orange (Citrus aurantium dulcis) oil, palm (Elaeis guineensis) oil, palmitic acid, panthenol, panthenyl ethyl ether, paraffin, PCA, peach (Prunus persica) kernel oil, peanut (ArachisHypogaea) Oil, PEG-8 C12-18 Esters, PEG-15 Cocoamine, PEG-150 Distearate, PEG-60 Glyceryl Isostearate, PEG-5 Glyceryl Stearate, PEG-30 Glyceryl Stearate, PEG-7 Hydrogenated Castor Oil, PEG-40 Hydrogenated Castor Oil, PEG-60 Hydrogenated Castor Oil, PEG-20 Methyl Glucose Sesquistearate, PEG40 Sorbitan Full Oleate, PEG-5 Soybean Sterols, PEG-10 Soybean Sterols, PEG-2 Stearate, PEG-8 Stearate, PEG-20 Stearate, PEG-32 Stearate, PEG-40 Stearate, PEG-50 Stearate, PEG-100 Stearate, PEG-150 Stearate, Pentadecanoate, Peppermint (Peppermint, Mentha piperita oil, petrolatum, phospholipids, plankton extract, polyamino acid polysaccharide condensate, polyglyceryl-3 diisostearate, polyquaternium-24, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, polysorbate 85, potassium myristate, potassium palmitate, propylene glycol, propylene glycol dicaprylate / dicaprate, propylene glycol dicaprylate, propylene glycol dipelargonate, propylene glycol laurate, propylene glycol stearate, propylene glycol stearate SE, PVP, pyridoxine dipalmitate, retinol, retinyl palmitate, rice (Oryza sativa) bran oil, RNA, rosemary (Rosmarinus officinalis) oil, rose oil, safflower (Carthamus tinctorius) oil, sage (Salvia officinalis) oil, sandalwood (Santalum) album) oil, serine, serum albumin, sesame (Sesamumindicum) oil, shea butter (Butyrospermum parkii), silk powder, sodium chondroitin sulfate, sodium hyaluronate, sodium lactate, sodium palmitate, sodium PCA, sodium polyglutamate, soluble collagen, sorbitan laurate, sorbitan oleate, sorbitan palmitate, sorbitan sesquioleate, sorbitan stearate, sorbitol, soybean (Glycine soja) oil, sphingolipids, squalane, squalene, stearamide MEA-stearate, stearic acid, stearoxydimethicone, stearoxytrimethylsilane, stearyl alcohol, stearyl glycyrrhetinate, stearyl heptanoate, stearyl stearate, sunflower (Helianthus annuus) seed oil, sweet almond (Prunus amygdalus dulcis) seed oil dulcis) oil, synthetic beeswax, tocopherol, tocopheryl acetate, tocopheryl linoleate, tribehenin, tridecyl neopentanoate, tridecyl stearate, triethanolamine, tristearin, urea, vegetable oil, water, wax, wheat (Triticum)vulgare) germ oil and Ylang-ylang (Canangaodorata) oil.

[0063] c. Antioxidants

[0064] Non-limiting examples of antioxidants that can be used with the compositions of the present invention include acetylcysteine, ascorbic acid polypeptide, ascorbyl dipalmitate, ascorbyl methylsilanol pectinate, ascorbyl palmitate, ascorbyl stearate, BHA, BHT, t-butylhydroquinone, cysteine, cysteine ​​hydrochloride, diamylhydroquinone, di-t-butylhydroquinone, dicetylthiodipropionate, dioleoyl tocopheryl methylsilanol, disodium ascorbyl sulfate, distearylthiodipropionate, ditridecylthiodipropionate, lauryl gallate, isoascorbic acid, ascorbyl esters, ethyl ferulate, ferulic acid, gallic esters, hydroquinone, 2-ethylhexyl thioglycolate, kojic acid, magnesium ascorbate, magnesium ascorbyl phosphate, methylsilanol Ascorbyl esters, natural plant antioxidants such as green tea or grape seed extract, nordihydroguaiaretic acid, octyl gallate, phenylthioglycolic acid, potassium ascorbyl tocopheryl phosphate, potassium sulfite, propyl gallate, quinone, rosmarinic acid, sodium ascorbate, sodium bisulfite, sodium erythorbate, sodium metabisulfite, sodium sulfite, superoxide dismutase, sodium thioglycolate, sorbitol furfural, thiodiglycol, thiodiethylamide, thiodiacetic acid, thioglycolic acid, thiolactic acid, thiosalicylic acid, tocophereth-5, tocophereth-10, tocophereth-12, tocophereth-18, tocophereth-50, tocopherol, tocopherol acetate, tocopheryl linoleate, tocopheryl nicotinate, tocopheryl succinate, and tris(nonylphenol) phosphite.

[0065] d. Structuring agent

[0066] In other non-limiting aspects, the composition of the present invention may include a structuring agent. In some aspects, the structuring agent helps provide rheological characteristics to the composition to contribute to the stability of the composition. In other aspects, the structuring agent can also play the role of an emulsifier or surfactant. The limiting examples of structuring agents include stearic acid, palmitic acid, stearyl alcohol, cetyl alcohol, behenyl alcohol, stearic acid, palmitic acid, a polyethylene glycol ether with an average of about 1 to about 21 ethylene oxide units of stearyl alcohol, a polyethylene glycol ether with an average of about 1 to about 5 ethylene oxide units of cetyl alcohol, and mixtures thereof.

[0067] e. Emulsifier

[0068] In certain aspects of the invention, the composition does not include an emulsifier. However, in other aspects, the composition may include one or more than one emulsifier. Emulsifiers can reduce interphase surface tension and improve the formulation and stability of the emulsion. Emulsifiers can be nonionic, cationic, anionic, and zwitterionic emulsifiers (see U.S. Patents Nos. 5,011,681; 4,421,769; 3,755,560). Non-limiting examples include glycerides, propylene glycol esters, fatty acid esters of polyethylene glycol, fatty acid esters of polypropylene glycol, esters of sorbitol, sorbitan esters, carboxylic acid copolymers, esters and ethers of glucose, ethoxylated esters, ethoxylated alcohols, alkyl phosphates, polyoxyethylene fatty ether phosphates, fatty acid amides, acyl lactylates, fatty acid salts, TEA stearate, oleth-3 phosphate DEA salt, polyethylene glycol 20 sorbitan monolaurate (polysorbate 20), polyethylene glycol 5 soya sterol, steareth-2, steareth-20, steareth ... Ceteareth-21, Ceteareth-20, Cetearyl Glucoside, Cetearyl Alcohol, C12-13 Pareth-3, PPG-2 Methyl Glucose Ether Distearate, PPG-5-Ceteth-20, Bis-PEG / PPG-20 / 20 Dimethicone, Ceteth-10, Polysorbate 80, Cetyl Phosphate, Potassium Cetyl Phosphate, Diethanolamine Cetyl Phosphate, Polysorbate 60, Glyceryl Stearate, PEG-100 Stearate, Arachidyl Alcohol, Arachidyl Glucoside, and mixtures thereof.

[0069] f. Silicone-containing compounds

[0070] In non-limiting terms, silicone-containing compounds include any member of a family of polymeric products whose molecular backbone consists of alternating silicon and oxygen atoms, with pendant groups attached to the silicon atoms. By varying the length of the -Si-O- chains, the pendant groups, and the crosslinking, silicones can be synthesized into a wide variety of materials. Their consistency can vary from liquid to gel to solid.

[0071] The silicone-containing compound that can be used under the environment of the present invention includes those described in this specification or known to those of ordinary skill in the art. Non-limiting examples include silicone oils (such as volatile and non-volatile oils), silica gel and silicone solids. In some aspects, the silicone-containing compound includes silicone oils, such as polyorganosiloxanes. The non-limiting examples of polyorganosiloxanes include dimethicone, cyclomethicone, polysilicone-11, phenyl trimethicone, trimethylsilyl amino dimethicone, stearoxy trimethyl silane or the organosiloxane material of its mixture and other any given ratio, to reach the desired consistency and application characteristics according to expected application (such as, applied to specific areas such as skin, hair or eyes). " Volatile silicone oil " includes silicone oil with low heat of vaporization, i.e., usually lower than about 50 calories per gram of silicone oil. Non-limiting examples of volatile silicone oils include: cyclomethicones, such as Dow Corning 344 Fluid, Dow Corning 345 Fluid, Dow Corning 244 Fluid, and Dow Corning 245 Fluid, Volatile Silicon 7207 (Union Carbide Corp., Danbury, Conn.); low viscosity dimethicones, i.e., dimethicones having a viscosity of about 50 cSt or less (e.g., dimethicones such as Dow Corning 200-0.5 cSt Fluid). Dow Corning Fluid is commercially available from Dow Corning Corporation of Midland, Michigan. In the third edition of the CTFA Cosmetic Ingredient Dictionary (incorporated by reference), cyclomethicone and dimethicone are described as mixtures of cyclic dimethylpolysiloxane compounds and fully methylated linear siloxane polymers end-capped with trimethylsiloxy units, respectively. Other non-limiting volatile silicone oils that can be used in the context of the present invention include those available from General Electric Co., Silicone Products Div., Waterford, NY and SWS Silicones Div., Stauffer Chemical Co., Adrian, MI.

[0072] g. Keratolytics

[0073] Exfoliants include ingredients that remove dead skin cells from the outer surface of the skin. These agents can act mechanically, chemically, or in other ways. Non-limiting examples of mechanical exfoliants include abrasives such as pumice, silica, cloth, paper, shells, beads, solid crystals, solid polymers, and the like. Non-limiting examples of chemical exfoliants include acids and enzyme exfoliants. Acids that can be used as exfoliants include, but are not limited to, glycolic acid, lactic acid, citric acid, alpha-hydroxy acids, beta-hydroxy acids, and the like. Other exfoliants known to those skilled in the art are also contemplated for use within the scope of the present invention.

[0074] h. Essential oils

[0075] Essential oils include oils from herbs, flowers, trees, and other plants. Such oils typically exist as tiny droplets between plant cells and can be extracted by several methods known to those skilled in the art (e.g., steam distillation, enfleurage (i.e., extraction using fat), maceration, solvent extraction, or mechanical pressing). When these types of oils are exposed to air, they tend to evaporate (i.e., they are volatile oils). Therefore, although many essential oils are colorless, they oxidize and become darker in color over time. Essential oils are insoluble in water but soluble in alcohols, ethers, fixed oils (of plants), and other organic solvents. Typical physical properties of essential oils include a boiling point of about 160°C to 240°C and a density of about 0.759 to about 1.096.

[0076] Essential oils are generally named after the plant in which they are found. For example, rose oil or peppermint oil come from the rose or mint plant, respectively. Non-limiting examples of essential oils that can be used in the context of the present invention include sesame oil, macadamia oil, tea tree oil, evening primrose oil, Spanish sage oil, Spanish rosemary oil, cilantro oil, thyme oil, pimentoberries oil, rose oil, anise oil, balsam oil, bergamotoil, rosewood oil, cedar oil, chamomile oil, sage oil, clary sage oil, clove oil, cypress oil, eucalyptus oil, fennel oil, sea fennel oil, frankincense oil, geranium oil, ginger oil, grapefruit oil, jasmine oil, juniper oil, lavender oil, lemon oil, lemongrass oil, lime oil, mandarin oil,

[0013] The present invention also includes, but is not limited to, essential oils such as citrus oil, thyme oil, thyme oil, thyme oil, thyme leaf oil, thyme oil, thyme leaf oil, thyme leaf oil, thyme leaf oil, thyme leaf oil, thyme leaf oil, thyme leaf oil, thyme leaf oil, thyme leaf oil, thyme leaf oil, thyme leaf oil, thyme leaf oil, thyme leaf oil, thyme leaf oil, thyme leaf oil, thyme leaf oil, thyme leaf oil, thyme leaf oil, thyme leaf oil, thyme leaf oil, thyme leaf oil, thyme leaf oil, thyme leaf oil, thyme leaf oil, thyme leaf oil, thyme leaf oil, thyme leaf oil, thyme leaf oil, thyme leaf oil, thyme leaf oil

[0077] i.Thickener

[0078] Thickeners, including thickeners or gelling agents, include substances that increase the viscosity of a composition. Thickeners include agents that increase the viscosity of a composition without substantially altering the efficacy of the active ingredients within the composition. Thickeners can also increase the stability of the compositions of the present invention. In some aspects of the present invention, the thickener includes hydrogenated polyisobutylene, trihydroxystearin, ammonium acryloyldimethyltaurate / VP copolymer, or mixtures thereof.

[0079] Non-limiting examples of additional thickening agents that can be used in the context of the present invention include carboxylic acid polymers, cross-linked polyacrylate polymers, polyacrylamide polymers, polysaccharides, and gums. Examples of carboxylic acid polymers include cross-linked compounds containing one or more monomers derived from acrylic acid, substituted acrylic acid, and salts and esters of these acrylic acid and substituted acrylic acid, wherein the cross-linking agent contains two or more carbon-carbon double bonds and is derived from a polyol (see U.S. Patent Nos. 5,087,445; 4,509,949; 2,798,053; CTFA International Cosmetic Ingredient Dictionary, Fourth Edition, 1991, pages 12 and 80). Examples of commercially available carboxylic acid polymers include carbomers, which are homopolymers of acrylic acid cross-linked with allyl ethers of sucrose or pentaerythritol (e.g., CARBOPOL from BF GOODRICH). TM 900 series).

[0080] Non-limiting examples of cross-linked polyacrylate polymers include cationic and nonionic polymers. Examples are described in U.S. Patent Nos. 5,100,660; 4,849,484; 4,835,206; 4,628,078; 4,599,379).

[0081] Non-limiting examples of polyacrylamide polymers (including nonionic polyacrylamide polymers, including substituted branched or unbranched polymers) include polyacrylamide, isoparaffins and laureth-7, multi-block copolymers of acrylamide and substituted acrylamides with acrylic acid and substituted acrylic acid.

[0082] Non-limiting examples of polysaccharides include cellulose, carboxymethyl hydroxyethyl cellulose, cellulose acetate propionate carboxylate, hydroxyethyl cellulose, hydroxyethyl ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methyl hydroxyethyl cellulose, microcrystalline cellulose, sodium cellulose sulfate, and mixtures thereof. Other examples are alkyl-substituted celluloses, wherein the hydroxyl groups of the cellulose polymer are hydroxyalkylated (preferably hydroxyethylated or hydroxypropylated) to form hydroxyalkylated cellulose, which is then further modified by ether linkages with C10 to C30 linear or branched alkyl groups. Typically, these polymers are ethers of C10 to C30 linear or branched alcohols and hydroxyalkyl celluloses. Other useful polysaccharides include scleroglucans, which comprise linear chains of (1-3) linked glucose units with one (1-6) linked glucose per three units.

[0083] Non-limiting examples of gums that can be used in the present invention include gum arabic, agar, algin, alginic acid, ammonium alginate, amylopectin, calcium alginate, calcium carrageenan, carnitine, carrageenan, dextrin, gelatin, gellan gum, guar gum, guar hydroxypropyltrimonium chloride, hectorite, hyaluronic acid, hydrated silica, hydroxypropyl chitosan, hydroxypropyl guar, karaya gum, kelp, locust bean gum, natto gum, potassium alginate, potassium carrageenan, propylene glycol alginate, sclerotium gum, sodium carboxymethyl dextran, sodium carrageenan, tragacanth gum, xanthan gum, and mixtures thereof.

[0084] j. Preservatives

[0085] Non-limiting examples of preservatives that can be used in the context of the present invention include quaternary ammonium preservatives (e.g., polyquaternium-1 and benzalkonium halides (e.g., benzalkonium chloride ("BAC") and benzalkonium bromide)), parabens (e.g., methylparaben and propylparaben), phenoxyethanol, benzyl alcohol, chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

[0086] 2. Drug ingredients

[0087] Pharmaceutically active agents are also contemplated as useful in the compositions of the present invention. Non-limiting examples of pharmaceutical active agents include anti-acne agents, agents for treating rosacea, analgesics, anesthetics, anorectal medications, antihistamines, anti-inflammatory agents including nonsteroidal anti-inflammatory drugs, antibiotics, antifungals, antivirals, antimicrobials, anticancer actives, scabicides, pediculicides, anti-tumor agents, antiperspirants, antipruritics, antipsoriatic agents, antiseborrheic agents, biologically active proteins and peptides, burn treatments, cautery agents, depigmenting agents, depilatories, diaper rash treatments, enzymes, hair growth stimulants, hair growth retardants including DFMO and its salts and analogs, hemostats, keratolytics, mouth ulcer treatments, cold sore treatments, dental and periodontal treatments, photosensitizing actives, skin protectants / barrier agents, steroids including hormones and corticosteroids, sunburn treatments, sunscreens, transdermal actives, nasal actives, vaginal actives, wart treatments, wound treatments, wound healing agents, and the like.

[0088] F. Kit

[0089] Also contemplated are kits for use in certain aspects of the present invention. For example, the compositions of the present invention may be included in a kit. The kit may include a container. The container may include a bottle, metal tube, laminated tube, plastic tube, dispenser, high-pressure vessel, barrier container, packaging, compartment, lipstick container, compacted container, cosmetic tray or other type of container capable of storing a cosmetic composition, such as an injection or blow molded plastic container, in which the dispersion or composition or the desired bottle, dispenser or packaging is stored. The kit and / or container may include indicia on its surface. For example, the indicia may be a word, phrase, abbreviation, picture or symbol.

[0090] The container can distribute a predetermined amount of composition. In other embodiments, a squeeze container (e.g., metal, laminate, or plastic tube) can be used to distribute the composition of a desired amount. The composition can be distributed in the form of a spray, an aerosol, a liquid, a fluid, or a semisolid. The container can have a spray mechanism, a pump mechanism, or an extrusion mechanism. The test kit can also include instructions for using the test kit components and for using any composition contained in the container. Instructions can include instructions for how to use, use, and preserve the composition.

[0091] Example

[0092] The following examples are used to illustrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the technology disclosed in the following examples can represent the technology that the inventors have found to play a good role in the practice of the present invention, and therefore it can be considered that it constitutes a preferred embodiment for its practice. However, based on this disclosure, it will be appreciated by those skilled in the art that many changes can be made in the disclosed specific embodiments without departing from the spirit and scope of the present invention, and still obtain the same or similar results.

[0093] All compositions and / or methods disclosed and claimed herein can be made and implemented according to the present disclosure without excessive experimentation. Although the compositions and methods of the present invention have been described in accordance with preferred embodiments, it will be apparent to those skilled in the art that variations can be implemented in the compositions and / or methods and in the steps or order of steps of the methods described herein without departing from the concept, spirit, and scope of the present invention. More specifically, it will be apparent that certain chemically and physiologically related reagents can replace the reagents described herein while achieving the same or similar results. All such similar substitutions and changes apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the present invention as defined by the appended claims.

[0094] Example 1

[0095] (Materials used)

[0096] The combinations of active ingredients disclosed herein can be included in a wide range of topical product formulations for use on the skin and / or hair. Non-limiting examples of active ingredient combinations include those found in Table 1.

[0097] Table 1

[0098]

[0099] Example 2

[0100] (Additional determination)

[0101] Assays that can be used to determine the efficacy of any ingredient, or any combination of ingredients or compositions comprising a combination of ingredients disclosed throughout this specification and claims can be determined by methods known to those of ordinary skill in the art. The following are non-limiting assays that can be used in the context of the present invention. It should be appreciated that other testing procedures, including, for example, objective and subjective procedures, can be used.

[0102] B16 Pigmentation Assay: Melanogenesis is the process by which melanocytes produce melanin, the naturally occurring pigment that gives skin, hair, and eyes its color. Inhibiting melanogenesis is beneficial in preventing skin darkening and reducing dark spots associated with aging. This bioassay uses the immortalized mouse melanoma cell line B16-F1 melanocytes (ATCC) to analyze the effects of compounds on melanin production. The endpoints of this assay are spectrophotometric measurements of melanin production and cell viability. B16-F1 melanocytes can be cultured in standard DMEM growth medium with 10% fetal bovine serum (MEDIATECH) at 37°C in 10% CO2 and then treated with any of the active ingredients, combinations of ingredients, or compositions having said combinations disclosed in this specification. Following incubation, melanin secretion is measured by absorbance at 405nm and cell activity is quantified.

[0103] Collagen Stimulation Assay: Collagen is an extracellular matrix protein that is crucial to skin structure. Increased collagen synthesis contributes to improved skin firmness and elasticity. This bioassay can be used to examine the effects of any of the active ingredients, combinations of ingredients, or compositions comprising such combinations disclosed herein on the production of procollagen peptides (collagen precursors) by human dermal fibroblasts. The endpoint of this test is a spectrophotometric measurement reflecting the presence of procollagen peptides and cell viability. This assay utilizes a quantitative sandwich enzyme immunoassay technique, whereby a monoclonal antibody specific for procollagen peptides is pre-coated on a microplate. Standards and samples are pipetted into the wells, and any procollagen peptide present is bound by the immobilized antibody. After washing away any unbound material, an enzyme-linked polyclonal antibody specific for procollagen peptides is added to the wells. After rinsing to remove any unbound antibody-enzyme reactants, a substrate solution can be added to the wells and detected using a microplate reader at 450 nm, with color development proportional to the amount of procollagen peptide bound in the initial step. The color development can be stopped and the intensity of the color measured.

[0104] To generate samples and controls, subconfluent normal human mature dermal fibroblasts (Cascade Biologics) cultured in standard DMEM growth medium with 10% fetal bovine serum (MEDIATECH) at 37° C. in 10% CO 2 can be treated with each of the combinations of components disclosed in the present specification or compositions having said combinations for 3 days. After incubation, the cell culture medium can be collected, and the amount of procollagen peptide secretion can be quantified using TAKARA's sandwich enzyme-linked immunosorbent assay (ELISA) (#MK101).

[0105] Elastin stimulation assay: Elastin is a connective tissue protein that helps the skin regain its shape after stretching or contracting. Elastin is also an important load-bearing protein used where mechanical energy storage is required. Elastin is made by linking many soluble tropoelastin molecules in a reaction catalyzed by the enzyme lysyl oxidase. Elastin secretion and elastin fibers can be monitored in cultured human fibroblasts by staining the cells with an immunofluorescent antibody against elastin.

[0106] Laminin and Fibronectin Stimulation Assay: Laminin and fibronectin are the major proteins in the dermal-epidermal junction (DEJ), also known as the basement membrane. The DEJ is located between the dermis and epidermis and interlocks to form finger-like projections called ridges. Epidermal cells receive their nutrients from blood vessels in the dermis. The ridges increase the surface area of ​​the epidermis exposed to these blood vessels and required nutrients. The DEJ provides adhesion between the two tissue compartments and controls the structural integrity of the skin. Laminin and fibronectin are two structural glycoproteins located in the DEJ. Laminin and fibronectin are believed to be the adhesives that bind cells together. They are secreted by dermal fibroblasts and help promote intracellular and intercellular adhesion of epidermal cells and the DEJ. The secretion of laminin and fibronectin can be monitored by quantifying laminin and fibronectin in the cell supernatant of cultured human fibroblasts, where the fibroblasts are treated for 3 days with culture medium containing or not containing a final concentration of 1.0% of the test component. After incubation, the levels of laminin and fibronectin can be measured in an enzyme-linked immunosorbent assay (ELISA) using immunofluorescent antibodies against laminin and antibodies against fibronectin. Measurements of cellular metabolic activity are normalized, as determined by the bioconversion of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazole (MTS).

[0107] Tumor Necrosis Factor Alpha (TNF-α) Assay: TNF-α, the prototypic ligand of the TNF superfamily, is a pleiotropic cytokine that plays a major role in inflammation. Increased expression of TNF-α is associated with upregulation of proinflammatory activity. This bioassay can be used to examine the effects of any of the active ingredients, combinations of ingredients, or compositions comprising such combinations disclosed herein on TNF-α production by human epidermal keratinocytes. The assay endpoint can be a spectrophotometric measurement reflecting the presence of TNF-α and cell viability. The assay utilizes a quantitative sandwich enzyme immunoassay technique, whereby a monoclonal antibody specific for TNF-α is pre-coated on a microplate. Standards and samples can be pipetted into the wells, and any TNF-α present is bound by the immobilized antibody. After washing away any unbound material, an enzyme-linked polyclonal antibody specific for TNF-α can be added to the wells. After rinsing to remove any unbound antibody-enzyme reactants, a substrate solution can be added to the wells and detected using a microplate reader at 450 nm, with color development proportional to the amount of TNF-α bound in the initial step. The color development can be stopped and the intensity of the color can be measured. EPILIFE at 37°C in 5% CO2 can be treated with phorbol 12-myristate 13-acetate (PMA, 10 ng / mL, SIGMACHEMICAL, #P1585-1MG) and any one of the active ingredients, ingredient combinations, or compositions having said combinations disclosed in the instructions. TM Subconfluent normal adult human keratinocytes (Cascade Biologics) were cultured in standard growth medium (Cascade Biologics) for 6 hours. PMA has been shown to cause a significant increase in TNF-α secretion, which peaked 6 hours after treatment. Following incubation, cell culture medium was collected and the amount of TNF-α secretion was quantified using a sandwich enzyme-linked immunosorbent assay (ELISA) from R&D Systems (#DTA00C).

[0108] Antioxidant (AO) Assay: An in vitro bioassay that measures the total antioxidant capacity of any of the ingredients, combinations of ingredients, or compositions comprising said combinations disclosed herein. This assay relies on the ability of antioxidants in a sample to inhibit (2,2'-Azo-bis-[3-ethylbenzthiazoline sulfonate]) is oxidized by metmyoglobin to The antioxidant system of living organisms includes enzymes such as superoxide dismutase, catalase and glutathione peroxidase; macromolecules such as albumin, ceruloplasmin and ferritin; and a series of small molecules, including ascorbic acid, α-tocopherol, β-carotene, reduced glutathione, uric acid and bilirubin. The total number of endogenous and food-derived antioxidants represents the total antioxidant capacity of the extracellular fluid. The collaboration of all different antioxidants provides stronger protection against the invasion of reactive oxygen or nitrogen free radicals than any single compound alone. Therefore, the total antioxidant capacity can give more relevant biological information than that obtained by measuring the individual components because it takes into account the cumulative effect of all antioxidants present in plasma and body fluids. The ability of the antioxidants in the sample to prevent ABTS oxidation was compared with Trolox, a water-soluble tocopherol analogue, and quantified as the molar equivalent of Trolox. As an in vitro bioassay for measuring the total antioxidant capacity of any of the ingredients, combinations of ingredients, or compositions having said combinations disclosed in this specification, the Antioxidant Capacity Kit #709001 from CAYMAN CHEMICAL (Ann Arbor, Michigan USA) can be used. The protocol can be performed according to the manufacturer's recommendations.

[0109] ORAC Assay: The oxygen radical absorbance (or absorbance) capacity (ORAC) of any of the active ingredients disclosed herein, combinations of active ingredients, or compositions containing such combinations can also be tested by measuring the antioxidant activity of such ingredients or compositions. Antioxidant activity indicates the ability to reduce oxidants (oxidizing substances). This assay quantifies the time and extent required to inhibit the action of oxidants, such as oxygen free radicals, which are known to cause damage to cells (e.g., skin cells). The ORAC value of any of the active ingredients disclosed herein, combinations of active ingredients, or compositions containing such combinations can be determined by methods known to those skilled in the art (see US Publication Nos. 2004 / 0109905 and 2005 / 0163880; and commercially available kits such as the Zen-Bio ORAC Antioxidant Assay Kit (#AOX-2)). The Zen-Bio ORAC Antioxidant Assay Kit measures the loss of fluorescein fluorescence over time due to the formation of hydroperoxide radicals by the decomposition of AAPH (2,2'-azobis-2-methylpropylamidine dihydrochloride). Trolox is a water-soluble vitamin E analog used as a positive control that inhibits fluorescein decay in a dose-dependent manner.

[0110] Mushroom Tyrosinase Activity Assay: In mammalian cells, tyrosinase catalyzes two steps in melanin biosynthesis from tyrosine (and from dopachrome polymerization). Tyrosinase is located in melanocytes and produces melanin (an aromatic quinone compound) that gives skin, hair, and eyes their color. Mushroom tyrosinase (SIGMA) can be incubated with its substrate, L-Dopa (from FISHER), in the presence or absence of any of the active ingredients disclosed herein, combinations of ingredients, or combinations comprising such combinations. Pigment formation can be assessed by colorimetric plate reading at 490 nm. The percent inhibition of mushroom tyrosinase activity can be calculated by comparison to an untreated control to determine the ability of the test ingredient or combination to inhibit the activity of the purified enzyme. The inhibitory effect of the test extracts was compared to that of kojic acid (SIGMA).

[0111] Matrix metalloproteinase 3 and matrix metalloproteinase 9 activity (MMP3; MMP9) assay: In vitro matrix metalloproteinase (MMP) inhibition assay. MMPs are extracellular proteases that play a role in many normal and disease states due to their broad substrate specificity. MMP3 substrates include collagen, fibronectin, and laminin; while MMP9 substrates include collagen VII, fibronectin, and laminin. Colorimetric drug discovery kits for MMP3 (AK-400) and MMP-9 (AK-410) from BioMol International were used. The assay was designed to measure the protease activity of MMPs using a sulfur-containing peptide as a chromogenic substrate (Ac-PLG-[2-mercapto-4-methyl-pentanoyl]-LG-OC2H5)5,6. The peptide bond at the MMP cleavage site is replaced by a thioester bond in the sulfur-containing peptide. This bond is hydrolyzed by MMP to produce a sulfhydryl group, which reacts with DTNB [5,5'-dithiobis(2-nitrobenzoic acid), Ellman's reagent] to produce 2-nitro-5-thiobenzoic acid, which can be detected by its absorbance at 412 nm (ε = 13600 M-1 cm-1 at pH 6.0 and above). Any of the active ingredients, ingredient combinations disclosed in this specification, or compositions containing such combinations can be tested.

[0112] Matrix Metalloproteinase 1 (MMP1) Enzyme Activity Assay: This in vitro matrix metalloproteinase (MMP) inhibition assay is performed. MMPs are extracellular proteases that play a role in many normal and disease states due to their broad substrate specificity. MMP1 substrates include collagen IV. The Molecular Probes ENZ / CHEK Gelatinase / Collagenase Assay Kit (#E12055) utilizes a fluorescent gelatin substrate to detect MMP1 protease activity. After proteolytic cleavage, bright green fluorescence is displayed and can be observed using a fluorescence microplate reader to measure enzyme activity.

[0113] The ENZ / CHEK Gelatinase / Collagenase Assay Kit (#E12055) from Invitrogen is designed as an in vitro assay for measuring MMP1 enzyme activity. Any of the active ingredients, combinations of ingredients, or compositions containing such combinations disclosed herein can be tested. The assay relies on the ability of pure MMP1 enzyme to degrade a fluorescent gelatin substrate. Once the substrate is specifically cleaved by MMP1, bright green fluorescence is displayed, which can be observed using a fluorescence microplate reader. The test material is incubated in the presence or absence of pure enzyme and substrate to determine its protease inhibitory ability.

[0114] Cyclooxygenase (COX) Assay: In vitro cyclooxygenase-1 and cyclooxygenase-2 (COX-1, COX-2) inhibition assay. COX is a bifunctional enzyme with both cyclooxygenase and peroxidase activity. The cyclooxygenase activity converts arachidonic acid to hydroperoxide endoperoxides (prostaglandin G2; PGG2), and the peroxidase component reduces the endoperoxides (prostaglandin H2; PGH2) to the corresponding alcohols, precursors of prostaglandins, thromboxanes, and prostacyclins. This COX inhibition screening assay measures the peroxidase component of cyclooxygenase. Peroxidase activity is measured colorimetrically by monitoring the appearance of oxidized N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). This inhibition screening assay includes both COX-1 and COX-2 enzymes to screen for isozyme-specific inhibitors. The colorimetric COX (sheep) inhibitor screening test (#760111, CAYMAN CHEMICAL) can be used to analyze the effect of each active ingredient disclosed in the instructions, any combination of ingredients, or a composition having said combination on the activity of purified cyclooxygenase (COX-1 or COX-2). According to the manufacturer's instructions, the purified enzyme, heme, and test extract can be mixed in assay buffer and incubated with shaking at room temperature for 15 minutes. After incubation, arachidonic acid and a colorimetric substrate can be added to start the reaction. The color change can be assessed by a colorimetric plate measured at 590 nm. The percentage of inhibition of COX-1 or COX-2 activity can be calculated by comparison with an untreated control to determine the ability of the test extract to inhibit the activity of the pure enzyme.

[0115] Lipoxygenase (LO) Assay: In vitro lipoxygenase inhibition assay. LO is a non-heme, iron-containing dioxygenase that catalyzes the addition of molecular oxygen to fatty acids. Linoleate and arachidonate are the primary substrates for LO in plants and animals. Arachidonic acid can then be converted to hydroxyeicosatetraenoic (HETE) acid derivatives, which are subsequently converted to leukotrienes, potent inflammatory mediators. This assay provides an accurate and convenient method for screening lipoxygenase inhibitors by measuring the hydroperoxides produced by lipoxygenases (5-LO, 12-LO, or 15-LO) incubated with arachidonic acid. The ability of each of the active ingredients disclosed herein, combinations of ingredients, or compositions comprising such combinations to inhibit enzyme activity can be determined using the Colorimetric LO Inhibitor Screening Kit (#760700, Cayman Chemical). Purified 15-lipoxygenase and the test ingredients can be mixed in assay buffer and incubated with shaking at room temperature for 10 minutes. After incubation, arachidonic acid can be added to initiate the reaction, and the mixture can be incubated for an additional 10 minutes at room temperature. Catalysis can be terminated by adding a colorimetric substrate, and the color change can be assessed by fluorescence plate measurement at 490 nm. The percent inhibition of lipoxygenase activity can be assessed compared to an untreated control to determine the ability of each of the active ingredients disclosed herein, the combination of ingredients, or any of the compositions comprising such combinations to inhibit the activity of the purified enzyme.

[0116] Elastase assay: MOLECULAR PROBES (Eugene, Oregon USA) can be used. Elastase assay (kit # E-12056) is an in vitro enzyme inhibition assay for inhibiting the activity of any of the elastase enzymes disclosed in this specification sheets, combinations of ingredients, or compositions with the combination. The ENZCHEK test kit contains soluble bovine cervical ligament elastin, which can be labeled with a dye to quench the fluorescence of the conjugate. Non-fluorescent substrates can be digested by elastase or other proteases to produce highly fluorescent fragments. The fluorescence enhancement produced can be monitored with a fluorescence microplate reader. The digestion product from the elastin substrate has an absorption maximum at approximately 505nm and a fluorescence emission maximum at approximately 515nm. When using the ENZCHEK elastase assay kit to screen for elastase inhibitors, peptides, N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone, can be used as selective, common elastase inhibitors.

[0117] Ceramide production: Ceramide in cells or tissue samples can be labeled with a mouse monoclonal antibody anti-ceramide (ENZO LIFE SCIENCE, reference ALX-804-196 clone MID15B4) diluted 1 / 50 using the amplification system biotin / streptavidin for 2 hours at room temperature. Ceramide can be visualized by video microscopy (pink staining).

[0118] Oil Control Assay: An assay for measuring a reduction in sebum secretion and / or a reduction in sebum production in the sebaceous glands can be performed using standard techniques known to those of ordinary skill in the art. In a specific example, the forehead can be used. Each of the active ingredients disclosed herein, a combination of ingredients, or a composition comprising such combinations can be applied once or twice daily to a portion of the forehead for a fixed number of days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more), while the rest of the forehead is not treated with the composition. After the fixed number of days has expired, sebum secretion can be analyzed by applying fine blotting paper to the treated and untreated forehead skin. This is accomplished by first removing all sebum from the treated and untreated areas with wet and dry cloths. Blotting paper is then applied to the treated and untreated forehead areas, and an elastic band can be placed around the forehead to gently press the blotting paper against the skin. After 2 hours, the blotter can be removed, allowed to dry, and then transilluminated. Darker blotters correspond to more sebum secretion (or lighter blotters correspond to reduced sebum secretion).

[0119] Erythema Assay: An assay measuring the reduction of skin redness can be evaluated using a MINOLTA colorimeter. Skin erythema can be induced by applying a 0.2% sodium lauryl sulfate solution to the subject's forearm. The area is protected with an occlusive patch for 24 hours. After 24 hours, the patch is removed and the irritation-induced redness can be assessed using the a* value of the MINOLTA colorimeter. The a* value measures the change in skin color in the red area. Immediately following the reading, the area is treated with any of the active ingredients, combinations of active ingredients, or compositions containing such combinations disclosed herein. Repeated measurements can be performed periodically to determine the formulation's ability to reduce redness and irritation.

[0120] Determination of Skin Moisture / Hydration: Skin moisture / hydration benefits can be measured using impedance measurements performed with a Nova DermalPhase Meter. Impedance meters measure changes in skin moisture content. The outer layer of skin has different electrical properties. When skin is dry, it conducts electricity poorly. As it becomes more hydrated, it increases in conductivity. Therefore, changes in skin impedance (which is related to conductivity) can be used to assess changes in skin hydration. The device can be calibrated for each test day according to the instrument's instructions. Temperature and relative humidity can also be recorded. Subjects can be assessed as follows: they can equilibrate in a room with a defined humidity (e.g., 30% to 50%) and temperature (e.g., 68°C to 72°C) before measurement. Three independent impedance measurements are taken on each side of the face, recorded, and averaged. The impedance meter can be set to T5, which applies impedance to the face every five seconds and averages the measured values. Changes can be reported with statistical variance and significance. This method can be used to test each of the active ingredients disclosed in this specification, any combination of ingredients, or compositions containing such combinations.

[0121] Determination of Skin Clarity and Reduction of Freckles and Age Spots: Skin clarity and reduction of freckles and age spots can be assessed using a Minolta colorimeter. Changes in skin color can be assessed using the a* value of a Minolta colorimeter to determine potential irritation due to product treatment. The a* value measures the change in skin color in the red area. This is used to determine whether each of the active ingredients disclosed in this specification, any of the combinations of ingredients, or a composition containing said combination reduces irritation. The measurement can be made on each side of the face and averaged as the values ​​for the left and right sides of the face. Skin clarity can also be measured using a Minolta Meter. The measurement is a combination of the a*, b, and L values ​​of the Minolta Meter and is related to the brightness of the skin, and corresponds very well to the smoothness and hydration of the skin. Skin measurements are performed as above. In one non-limiting aspect, skin clarity can be described as L / C, where C is chromaticity and is defined as (a 2 +b 2 ) 1 / 2 .

[0122] Skin dryness, surface wrinkles, skin smoothness, and skin color assessment: Skin dryness, surface wrinkles, skin smoothness, and skin color can be assessed using clinical scoring techniques. For example, the clinical score for skin dryness can be determined using a five-point standard Kligman Scale: (0) skin is soft and moist; (1) skin appears normal with no visible dryness; (2) skin feels slightly dry to the touch without visible flaking; (3) skin feels dry, tough, and has a whitish appearance with some scaling; and (4) skin feels very dry, rough, and has a whitish appearance with scaling. The assessment can be performed independently by two clinicians and averaged.

[0123] Skin color clinical scoring: Skin color was clinically scored using a ten-point analog numerical scale: (10) Smooth, even skin, pink-brown color. No dark, red, or scaly patches when examined with a handheld magnifier. The microtexture of the skin felt very even; (7) Even skin color when examined without a magnifier. No scaly areas, but discoloration due to pigmentation or erythema. No discoloration greater than 1 cm in diameter; (4) Easily noticeable skin discoloration and uneven texture. Minor scales. Skin that feels rough to the touch in some areas; and (1) Uneven skin coloration and texture. Multiple areas of scales and discoloration, hypopigmented, red, or black spots. Large areas of uneven color greater than 1 cm in diameter. The assessments were performed independently by two clinicians and averaged.

[0124] Clinical Score Measurement of Skin Smoothness: The clinical score of skin smoothness can be analyzed using a ten-point analog numerical scale: (10) smooth, the skin is moist and shiny, and there is no resistance when a finger is passed over the surface; (7) somewhat smooth, with little resistance; (4) rough, visibly changed, with friction when rubbed; and (1) rough, flaky, uneven surface. The evaluation was performed independently by two clinicians and the average was calculated.

[0125] Skin smoothness and wrinkle reduction measurements using the method disclosed in Packman et al. (1978): Skin smoothness and wrinkle reduction can also be measured visually using the method disclosed in Packman et al. (1978). For example, at each visit, the depth, shallowness, and total number of surface face lines (SFL) of each subject are carefully scored and recorded. A numerical score is obtained by multiplying the number factor by the depth / width / length factor. Scores are obtained for the eye area and the mouth area (left and right) and added together to form a total wrinkle score.

[0126] Skin firmness measurement using the Hargens Ballistometer: Skin firmness can be measured using the Hargens Ballistometer, a device that assesses skin elasticity and firmness by dropping a small object on the skin and recording the first two rebound peaks. Ballistometry uses a small, lightweight probe with a relatively blunt tip (4 square millimeters contact area). The probe gently penetrates the skin, resulting in measurements that rely on the properties of the skin's outer layers, which include the stratum corneum and epidermis, as well as part of the dermis.

[0127] Skin softness / pliability measurement using a Gas Bearing Electrodynamometer: Skin softness / pliability can be assessed using a Gas Bearing Electrodynamometer, an instrument that measures the pressure / tension properties of the skin. The viscoelasticity of the skin is related to skin moisturization. Measurements can be made at specific locations in the cheek area by attaching the probe to the skin surface with double-sided tape. A force of approximately 3.5 gm is applied parallel to the skin surface, accurately measuring skin displacement. Skin flexibility can then be calculated and expressed as DSR (dynamic spring rate, in gm / mm).

[0128] Line and Wrinkle Visibility Assay Using Replicas: The appearance of lines and wrinkles on the skin can be assessed using replicas, which are impressions of the skin surface. Materials such as silicone rubber can be used. The replicas can be analyzed using image analysis. Changes in line and wrinkle visibility can be objectively quantified by forming a subject's face using a silicon replica and analyzing the replica image using a computer image analysis system. Replicas can be obtained from the eye and neck areas and photographed using a digital camera using low-angle incident light. The digital images can be analyzed using an image processing program to determine the areas of the replica covered by wrinkles and fine lines.

[0129] Skin surface profile determination using the profilometer / stylus method: The surface profile of the skin can be measured using the profilometer / stylus method. This involves illuminating a light or dragging a stylus across the surface of a replica. The vertical displacement of the stylus can be recorded in a computer using a distance sensor and after scanning a certain distance of the replica, an analysis of the skin profile can be generated as a two-dimensional surface. This scan can be repeated any number of times along a fixed axis to produce a simulated 3-D image of the skin. Ten random sections of the replica can be obtained using the stylus technique and combined to produce an average value. Values ​​of interest include Ra, which is the arithmetic mean of all roughness (height) values ​​calculated by integrating the profile height relative to the mean profile height. Rt, which is the maximum vertical distance between the highest peak and the lowest valley, and Rz, which is the average peak amplitude minus the average peak height. Values ​​are given as values ​​calibrated in mm. The equipment should be standardized before each use by scanning a metal standard of known value. The Ra value can be calculated using the following formula: R a = normalized roughness; lm = lateral (scan) length; and y = absolute value of profile position relative to the mean profile height (x-axis).

[0130] MELANODERM TM Assay: In other non-limiting aspects, the assay can be performed using a skin mimetic such as MELANODERM TM To evaluate the efficacy of each active ingredient, any combination of ingredients, or a composition comprising such a combination disclosed in this specification. Melanocytes, a type of cell in skin analogs, stain positively when exposed to L-dihydroxyphenylalanine (L-DOPA), a precursor of melanin. Skin analogs, MELANODERM TM Treatments can be performed using various matrices containing each active ingredient, any combination of ingredients, or compositions containing combinations disclosed herein, or using the matrices alone as controls. Alternatively, untreated skin analog samples can be used as controls.

[0131] Production of filaggrin: Changes in the production of filaggrin in keratinocytes due to each of the active ingredients disclosed herein, any one of the combinations of ingredients, or compositions containing said combinations can be measured. Filaggrin is a precursor to the natural moisturizing factor (NMF) in the skin. Increased NMF increases the moisture content of the skin. Using a bioassay that analyzes the concentration of filaggrin in keratinocyte lysates, the production of filaggrin in treated and untreated keratinocytes can be determined. Non-limiting examples of bioassays that can be used to quantify the production of intermediate filament-related proteins are SIMON TMWestern blotting protocol. For each sample, normal human epidermal keratinocytes (NHEKs) were grown in EPI-200-MATTEK EPI Life growth medium (M-EP-500-CA) containing calcium from Life Technologies. Before treatment, NHEKs were incubated overnight at 37°C in growth medium with 5% CO2. NHEKs were then incubated in growth medium containing 1% test compound / extract or no compound / extract (negative control) for 24 to 36 hours. NHEKs can then be washed, collected, and stored on ice or in a cooler place until lysed on ice using lysis buffer and sonication. The protein concentration of the sample can be determined and used for sample standardization. The lysate can be stored at -80°C until used for quantitative analysis.

[0132] SIMON TM The Western blot bioassay utilizes a quantitative Western blot immunoassay technique to quantify filaggrin in a test sample using a filaggrin-specific antibody. Cell samples are lysed and normalized for protein concentration. Normalized samples and molecular weight standards can then be loaded and run on a denaturing protein separation gel using capillary electrophoresis. The proteins in the gel are immobilized and immunoprobed using a primary antibody specific for filaggrin. The immobilized proteins can then be immunoprobed using an enzyme-linked detection antibody that is conjugated to the primary antibody. A chemiluminescent substrate solution can then be added to the immobilized proteins, resulting in chemiluminescence development proportional to the amount of filaggrin bound to the fixative. Chemiluminescence development is stopped at a specific time, and the intensity of the chemiluminescent signal can be measured and compared to positive and negative controls.

[0133] Production of occludin: Changes in the production of occludin in keratinocytes caused by each of the active ingredients disclosed herein, any of the combinations of ingredients, or compositions containing said combinations can be measured. Ocludin is a protein that is essential for the formation of tight junctions and the moisture barrier function of the skin. A non-limiting example of how to determine the production of occludin in treated and untreated keratinocytes is to use a bioassay that analyzes the concentration of occludin in keratinocyte lysates. SIMON TM Western blot protocol for bioassays. For samples, adult human epidermal keratinocytes (HEKa) from Life Technologies (C-005-5C) were cultured in EPILIFE® (Calcium-containing) medium from Life Technologies supplemented with Keratinocyte Growth Supplement (HKGS) from Life Technologies (S-101-5).TM Grow in growth medium (M-EP-500-CA) at 37°C and 5% CO2 for 24 hours. HEKa cells are then incubated in growth medium containing the test compound / extract, no compound / extract as a negative control, or 1 mM CaCl2 as a positive control for 24 to 48 hours. The HEKa cells are then washed, collected, and stored on ice or in a colder place until lysed on ice using lysis buffer and sonication. The protein concentration of the sample can be determined and used for sample normalization. The lysate is stored at -80°C until used in the bioassay.

[0134] SIMON TM The Western Blot Bioassay utilizes a quantitative Western Blot immunoassay technique that uses an antibody specific for occludin to quantitatively detect occludin in a test sample. Cell samples are lysed and the protein concentration is standardized. The standardized sample and molecular weight standards are then loaded and run on a denaturing protein separation gel using capillary electrophoresis. The proteins in the gel are then immobilized and immunoprobed using a primary antibody against occludin. The immobilized proteins are immunoprobed using an enzyme-linked detection antibody that is conjugated to the primary antibody. A chemiluminescent substrate solution is then added to the immobilized proteins, resulting in chemiluminescence development proportional to the amount of occludin bound to the fixative. The development of chemiluminescence can be stopped at a specific time, and the intensity of the chemiluminescent signal can be measured and compared to positive and negative controls.

[0135] Keratinocyte monolayer permeability: Changes in the permeability of the keratinocyte monolayer caused by each of the active ingredients disclosed in this specification, any one of the ingredient combinations, or a composition containing the combination can be measured. Keratinocyte monolayer permeability is a measure of skin barrier integrity. As a non-limiting example, the in vitro vascular permeability test of MILLIPORE (ECM642) can be used to determine the permeability of the keratinocyte monolayer in treated and untreated keratinocytes. This test analyzes the adsorption, transport and permeability of endothelial cells. Briefly, adult epidermal keratinocytes (C-005-5C) from Life Technologies can be seeded onto a porous collagen-coated membrane in a collection well. The keratinocytes are then grown in EPILIFE® from LIFE TECHNOLOGIES containing calcium supplemented with Keratinocyte Growth Supplement (HKGS) (S-101-5) from LIFE TECHNOLOGIES. TMThe cells were incubated in growth medium (M-EP-500-CA) at 37°C and 5% CO for 24 hours. This incubation period allowed the cells to form a monolayer and seal the membrane pores. The medium was then replaced with fresh medium containing (test sample) or without (untreated control) the test compound / extract, and the keratinocytes were incubated for an additional 48 hours at 37°C and 5% CO. To determine the permeability of the keratinocyte monolayer after incubation with or without the test compound / extract, the medium was replaced with fresh medium containing high molecular weight fluorescein isothiocyanate (FITC)-dextran, and the keratinocytes were incubated at 37°C and 5% CO for 4 hours. During the 4-hour incubation, FITC can pass through the keratinocyte monolayer and the porous membrane, entering the collection wells at a rate proportional to the monolayer permeability. After 4 hours of incubation, cell viability and FITC content in the collection wells can be determined. For FITC content, the medium in the collection wells was measured for fluorescence at 480 nm (Em) when excited at 520 nm. Permeability percentage and percent change compared to the untreated control can be determined by the following formulas: Permeability percentage = ((average Ex / Em of test sample) / average Ex / Em of untreated control) * 100; Percent change = percent permeability of test sample - percent permeability of untreated control.

[0136] Hyaluronic Acid Production: Changes in hyaluronic acid production in human dermal fibroblasts induced by any of the active ingredients disclosed herein, any combination of ingredients, or compositions containing such combinations can be measured. HA is a polysaccharide that participates in stabilizing matrix structure and provides turgor pressure to tissues and cells.

[0137] As a non-limiting example, the HA production in treated and untreated adult human dermal fibroblasts (HDFa) can be determined using the Hyaluronic Acid DuoSet ELISA Kit (DY3614) from R&D Systems. In this analysis, sub-confluent HDFa cells (C-13-5C) from Cascade Biologics were incubated in starvation medium (0.15% fetal bovine serum and 1% penicillin-streptomycin solution in Dulbecco's Modified Eagle medium) for 72 hours at 37°C and 10% CO2 before treatment. The cells were then incubated for 24 hours with fresh starvation medium containing the test compound, a positive control (phorbol 12-myristate 13-acetate (P1585) from SIGMA-ALDRICH and platelet-derived growth factor (P3201) from SIGMA-ALDRICH) or without additives. The culture medium was then collected and frozen at -80°C until used for ELISA determination.

[0138] In short, the ELISA assay uses a quantitative sandwich enzyme immunoassay technique, whereby a HA-specific capture antibody can be pre-coated onto a microplate. Standards and culture medium from treated and untreated cells are pipetted into the microplate wells to allow any HA present to be bound by the immobilized antibody. After washing away all unbound material, an enzyme-linked detection antibody specific for HA is added to the wells. After washing to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells to allow color development proportional to the amount of HA bound in the initial step. Color development is stopped at a specific time, and the color intensity at 450 nm is measured using a microplate reader.

[0139] As another non-limiting example, human skin explants can be cultured in a survival explant culture medium in a humidified atmosphere at 37°C supplemented with 5% CO2. The treatment of the explants can be carried out by topical application of the sample product (n=3) on day 0, day 2, day 3, day 6, day 8, and day 9. Control explants (n=3) do not receive any treatment except that the survival explant culture medium is renewed. On day 3, day 6, and day 8, half the volume of the survival culture medium can be renewed. On day 9, three explants under each condition can be taken out and cut in half. Half the explants are fixed in buffered formalin and the other half are frozen at -80°C.

[0140] After fixation for 48 hours in plain Bouin and 24 hours in formalin, the samples can be dried and immersed in paraffin using the automated tissue processor Leica TP 1020. 5-μm sections can be made using a microtome (Minot model LEICA RM2125) and mounted on SUPERFROST TM On histological slides. Microscopic observation can be carried out by using the optical microscope of LEICAORTHOPLAN or LEICA DM LB microscope. Images can be taken with OLYMPUS DP72 camera and CELL 24D software. General morphology can be examined on paraffin sections stained with Masson's tricolor Goldner variant. The staining of hyaluronic acid can be diluted to 1 / 100 1 hour at room temperature with anti-hyaluronic acid biotinylated protein (HABP) (SEIKAGAKU ref400763-1A), carried out with amplification system biotin / streptavidin (VECTOR, VECTASTAIN PK-7200).

[0141] Inhibition of Hyaluronidase Activity: Changes in the activity of hyaluronidase due to each of the active ingredients disclosed herein, any combination of ingredients, or a composition containing such a combination can be measured. Hyaluronidase is an enzyme that degrades hyaluronic acid. HA is a polysaccharide that participates in the stabilization of matrix structure and provides turgor pressure to tissues and cells. As a non-limiting example, hyaluronidase activity can be determined using an in vitro protocol modified from SIGMA-ALDRICH protocol #EC 3.2.1.35. Briefly, type 1-S hyaluronidase (H3506) from SIGMA-ALDRICH is added to microplate reaction wells containing a test compound or control. Tannic acid can be used as a positive control inhibitor, and the control enzyme can be treated without the addition of the test compound. Wells containing the test compound or positive control but without hyaluronidase can be used as background negative controls. Prior to the addition of the substrate (HA), the wells are incubated at 37°C for 10 minutes. The substrate is added and the reaction is incubated at 37°C for 45 minutes. A portion of each reaction solution is then transferred to a solution of sodium acetate and acetic acid and gently mixed at a pH of 3.75 to stop the partial reaction (stopped well). After adding a portion of the reaction solution to the stopped well, the stopped well and the reaction well should both contain the same volume of solution. Both the reaction well and the stopped well are incubated at room temperature for 10 minutes. The absorbance at 600 nm of the reaction well and the stopped well is then measured. The inhibition can be calculated using the following formula: inhibitor (or control) activity = (absorbance at 600 nm of the well where the inhibitor is stopped - absorbance at 600 nm of the inhibitor reaction well); initial activity = absorbance of the control enzyme at 600 nm; inhibition percentage = [(initial activity / inhibitor activity) * 100] - 100.

[0142] Peroxisome proliferator-activated receptor gamma (PPAR-γ) activity: Changes in PPAR-γ activity caused by each active ingredient disclosed in the specification, any combination of ingredients, or compositions having such combinations can be measured. PPAR-γ is a key receptor for sebum production. As a non-limiting example, the activity of PPAR-γ can be determined using a bioassay that analyzes the ability of a test compound or composition to inhibit ligand binding. In short, the fluorescent small molecule pan-PPAR ligand, FLUORMONE TMPan-PPAR Green, available from Life Technologies (PV4894), can be used to determine whether a test compound or composition is able to inhibit the binding of a ligand to PPAR-γ. Sample wells contain PPAR-γ and a fluorescent ligand along with: a test compound or composition (test); a reference inhibitor, rosiglitazone (positive control); or no test compound (negative control). The wells are incubated for a set period of time to allow the ligand an opportunity to bind to PPAR-γ. The fluorescence polarization of each sample well can then be measured and compared to the negative control well to determine the percentage of inhibition by the test compound or composition.

[0143] Cytokine Array: Human epidermal keratinocytes were cultured to 70% to 80% confluency. The culture medium was aspirated from the plate, and 0.025% trypsin / EDTA was added. When the cells became rounded, the dish was tapped to release the cells. The cells containing trypsin / EDTA were removed from the dish and equilibrated. The cells were centrifuged for 5 minutes to form a cell pellet. The supernatant was aspirated. The resulting pellet was resuspended in EPILIFE. TM Culture medium (Cascade Biologics). Cells were seeded into 6-well plates at approximately 10% to 20% confluency. When the cells became approximately 80% confluent, the culture medium was aspirated and 1.0 mL of EPILIFETM was added along with phorbol 13-myristate 12-acetate ("PMA") (a known inflammatory inducer) and a test composition dilution to duplicate wells (i.e., 1.0% (100 μL of 100× stock solution) and 0.1% (10 μL of 100× stock solution). The test composition was diluted to a final volume of 1 mL of EPILIFETM. TM Gently agitate the medium to ensure thorough mixing. In addition, add 1.0 mL of EPILIFE TM , with or without the addition of PMA. After preparation, the plates were then incubated at 37 ± 1 °C and 5.0 ± 1% CO2 for approximately 5 hours. After the 5-hour incubation, all the culture medium was collected in conical tubes and frozen at -70 °C.

[0144] In order to analyze, 16 lattice hybridization boxes are attached to three 16 lattice FAST slides of repeated arrangement with 16 kinds of anti-cytokine antibodies plus experimental control (WHATMAN BIOSCIENCES), and slides are placed in FAST frames (4 sheets for each frame) for processing. Arrays are sealed at room temperature using 70mL S&S protein array blocking buffer (WHATMAN SCHLEICHER and SCHEULL) for 15 minutes. Remove blocking buffer and add 70mL every kind of supernatant sample to each array. Arrays are incubated at room temperature with gentle stirring for 3 hours. Wash arrays 3 times with TBS-T. Arrays are processed with 70mL antibody mixture, which contains a biotinylated antibody, corresponding to every kind of array capture antibody. Arrays are incubated with gentle stirring for 1 hour at room temperature. Wash arrays 3 times with TBS-T. Arrays are incubated with gentle stirring for 1 hour at room temperature with 70mL of solution containing streptavidin-Cy5 conjugate. Wash arrays 3 times with TBS-T, quickly rinsed in deionized water, and dried.

[0145] Slides can be imaged in a PERKIN-ELMER SCANARRAY 4000 confocal fluorescence imaging system. Array images can be saved and analyzed using imaging research ARRAYVISION software. In short, dot intensity is determined by subtracting background signal. The dot replicates from each sample condition can be averaged and then compared with a suitable control.

[0146] Endothelial Tube Formation: Endothelial tube formation is associated with angiogenesis and microvascular capillary formation. Capillary formation and angiogenesis may contribute to skin redness and rosacea. The ability of endothelial cells to form tubes can be determined using a capillary tube burst assay with preformed primary human umbilical vein endothelial cells (HUVEC) in a cell culture system in the presence or absence of test extracts and compounds.

[0147] Briefly, HUVECs cultured in vitro on an extracellular matrix stimulate the attachment and tubular morphogenesis of endothelial cells, forming capillary-like luminal structures. These in vitro-formed capillaries resemble human vascular capillaries in many respects. The capillary assay builds on this phenomenon and is used to evaluate potential vascular targeting agents.

[0148] HUVEC cultures were grown in a 5% CO2, 37°C cell culture incubator. The complete growth medium for HUVEC was endothelial basal medium (EBM) supplemented with 2% fetal bovine serum (FBS), 12 μg / mL bovine brain extract, 1 μg / mL hydrocortisone, and 1 μg / mL GA-1000 (gentamicin-amphotericin). HUVEC cultures between passages 3 and 8 were used for all analytical experiments.

[0149] HUVECs were pre-labeled with the fluorescent agent Calcein AM and seeded in 96-well culture plates coated with extracellular matrix containing their complete growth medium. After approximately four hours of morphogenesis, endothelial capillaries should have formed. A 50 μL volume of the designed dose of the test agent was then applied to the formed capillary cultures as a treatment condition. Untreated controls were treated with the test agent's vehicle. The concentration of FDA-approved anti-angiogenic drug α-glucose (α-glucose) serves as a performance control for the assay. Approximately six hours after treatment, endothelial tubule morphology in each well is examined microscopically, allowing for imaging and quantitative analysis of capillary disruption activity under the treatment conditions. Each test condition can be run in duplicate, including a control.

[0150] **************

[0151] All compositions and / or methods disclosed and claimed herein can be made and implemented according to the present disclosure without excessive experimentation. Although the compositions and methods of the present invention have been described in accordance with preferred embodiments, it will be apparent to those skilled in the art that variations can be implemented in the compositions and / or methods and in the steps or order of steps of the methods described herein without departing from the concept, spirit, and scope of the present invention. More specifically, it will be apparent that certain chemically and physiologically related reagents can replace the reagents described herein while achieving the same or similar results. All such similar substitutions and changes apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the present invention as defined by the appended claims.

Claims

1. A method of treating skin comprising topical application of a topical skin composition, wherein the topical skin composition comprises milk thistle extract and white water lily extract.

2. The method of claim 1, wherein topical application of the topical skin composition results in improved skin radiance, improved overall skin tone, reduced skin discoloration, and / or reduced appearance of dark spots.

3. The method of claim 1, wherein the topical skin composition increases gene expression of kallikrein-5 (KLK5), kallikrein-7 (KLK7), and / or kallikrein-14 (KLK14).

4. The method of claim 1, wherein the topical skin composition stimulates exfoliation of skin keratinocytes.

5. The method of claim 1, wherein the topical skin composition accelerates skin desquamation.

6. The method of claim 1, wherein the topical skin composition is a serum, a cream, a gel, an emulsion, a gel-emulsion, a gel serum, a mask, a cleanser, and / or a toner.

7. The method of claim 1, wherein the milk thistle extract is a milk thistle seed extract and / or the white water lily extract is a white water lily flower extract.

8. The method of claim 7, wherein the topical skin composition comprises an effective amount of: Milk thistle seed extract; and White water lily extract.

9. The method of claim 7, wherein the topical skin composition comprises: 0.0001% to 10% by weight of milk thistle seed extract; and 0.0001% to 5% by weight of white water lily flower extract.

10. The method of claim 9, wherein the topical skin composition is free of parabens.

11. A topical skin composition, wherein the topical skin composition comprises milk thistle extract and white water lily extract.

12. The topical skin composition of claim 11, wherein topical application of the topical skin composition results in improved skin radiance, improved overall skin tone, reduced skin discoloration, and / or reduced appearance of dark spots.

13. The topical skin composition of claim 11, wherein the topical skin composition increases gene expression of kallikrein-5 (KLK5), kallikrein-7 (KLK7), and / or kallikrein-14 (KLK14).

14. The topical skin composition of claim 11, wherein the topical skin composition stimulates exfoliation of skin keratinocytes.

15. The topical skin composition of claim 11, wherein the topical skin composition accelerates skin desquamation.

16. The topical skin composition of claim 11, wherein the topical skin composition is a serum, a cream, a gel, an emulsion, a gel-emulsion, a gel serum, a mask, a cleanser, and / or a toner.

17. The topical skin composition of claim 11, wherein the milk thistle extract is milk thistle seed extract and / or the white water lily extract is white water lily flower extract.

18. The topical skin composition of claim 17, wherein the topical skin composition comprises an effective amount of: Milk thistle seed extract; and White water lily extract.

19. The topical skin composition of claim 17, wherein the topical skin composition comprises: 0.0001% to 10% by weight of milk thistle seed extract; and 0.0001% to 5% by weight of white water lily flower extract.

20. The topical skin composition of claim 19, wherein the topical skin composition is free of parabens.

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