Cosmetic compositions and uses thereof

Abstract

A method of applying a topical skin composition to skin is disclosed. The method can include applying the composition to the skin. The composition can include Saussurea involucrate extract, Rhododendron anthopogon extract, and Oenothera biennis oil.

Description

[0001] Cross-reference related applications

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 540,182, filed September 25, 2023, which is incorporated herein by reference in its entirety. Background of the Invention

[0004] A. Field of Invention

[0005] This invention generally relates to cosmetic compositions that can be used for moisturizing and protecting the skin barrier. In a particular embodiment, the composition is an oil for facial use.

[0006] B. Background

[0007] The epidermis is the outermost layer of the skin, comprising a cellular continuum of four layers: the stratum corneum, the stratum granulosum, the stratum spinosum, and the stratum basale. Each cellular layer in the epidermis represents a different stage in a process in which basal epidermal keratinocytes undergo a continuous cycle of proliferation, differentiation, and apoptosis, moving upwards from the stratum basale to eventually produce keratinocytes. These keratinocytes form the stratum corneum, also known as the stratum corneum.

[0008] Keratinocytes, which form the stratum corneum, are linked by proteins and lipids, creating a protective barrier against the external environment commonly known as the skin barrier. This tightly regulated epidermal permeability barrier acts as both a physical and selective barrier against chemical and biological damage. Key functions of the skin barrier include reducing the penetration of free radicals and preventing harmful radiation, including ultraviolet radiation, from penetrating deeper layers. The skin barrier also functions as a permeability barrier, preventing the loss of body moisture to the external environment. Summary of the Invention

[0009] This invention overcomes the deficiencies in the art by providing stable skincare compositions that also effectively moisturize and protect the skin barrier. These compositions can effectively protect skin proteins from oxidative stress-induced damage, reduce uneven skin tone, and / or support collagen production.

[0010] In some aspects, a topical skin composition is disclosed, comprising a combination of Rhododendron ferrugineum (alpine rose) extract, Oenothera biennis (evening primrose) oil, and Saussurea involucrata (snow lotus) extract, and a combination of structuring agents. The amounts of the components in the composition can vary (e.g., amounts can be as low as 0.000001% w / w or as high as 80% w / w or any range thereof). In one example, for instance, the following ranges / amounts have been found to be effective: 0.0001% by weight to 0.1% by weight of snow lotus extract; 0.0001% by weight to 0.1% by weight of Rhododendron ferrugineum extract; and 0.01% by weight to 5.0% by weight of evening primrose oil. This combination can be used for all skin types (e.g., dry skin, normal skin, oily skin, and combination skin).

[0011] In some aspects, the composition contains at least 75% by weight squalene. In some aspects, the cosmetic vehicle may also include any one, any combination of, or at least one, two, three, four, five, six, seven, eight, or all nine of the following ingredients: caprylic / capric triglyceride, hexyldecyl alcohol, bisabolol, N-palmitoylhydroxyproline cetyl ester, stearic acid, brassica campestris (rapeseed) sterols, olea europaea (olive) fruit oil, tocopheryl acetate, and BHT. The amounts of the ingredients in the composition may vary (e.g., amounts may be as low as 0.000001% w / w or as high as 80% w / w or any range thereof). In some aspects, the composition comprises 75.0% to 95.0% by weight of squalene, 5.0% to 10.0% by weight of caprylic / capric triglycerides, 0.1% to 5.0% by weight of hexyldecyl alcohol, 0.001% to 1.0% by weight of bisabolol, 0.001% to 1.0% by weight of N-palmitoylhydroxyproline cetyl ester, 0.0001% to 0.1% by weight of stearic acid, 0.0001% to 0.1% by weight of brassicosterols, 0.01% to 5.0% by weight of olive (Olea europaea) fruit oil, 0.01% to 5.0% by weight of tocopheryl acetate, and 0.0001% to 0.1% by weight of BHT. The dosage of ingredients in cosmetic carriers, or the addition of excipients and other components, can also be used to adjust the rheological properties of cosmetic carriers, change the combination of plant extracts, or add additional skincare benefits. In some cases, the composition is structured into an oily substance for facial use.

[0012] Methods for applying any of the topical skin compositions of the present invention to the skin are also disclosed. The skin may be facial skin, body skin (e.g., arms, hands, legs, feet, neck, back, chest, abdomen, or scalp). The composition may be applied to the skin within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes after cleansing the skin with a cleansing composition (e.g., soap, cleansing product, wipes, etc.). Furthermore, a skin moisturizing product may be applied to the skin within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes after applying any of the topical skin compositions. In this sense, the topical skin compositions of the present invention can be used in other product application regimens. For example, a person may begin by cleansing the skin, followed by topical application of the composition of the present invention, then topical application of a moisturizing product, and in some cases, followed by cleansing the skin to apply a face mask (typically during the evening hours).

[0013] In other embodiments, the inventors envision using the compositions of the present invention to treat specific skin conditions. Skin conditions may include fine lines or wrinkles, uneven skin tone, loose or sagging skin, erythematous skin, sensitive skin, dry skin, flaky skin, itchy skin, cracked skin, pruritus, spider angiomas, freckle-like nevi, age spots, senile purpura, keratosis, melasma, spots, nodules, photodamaged skin, dermatitis (including but not limited to seborrheic dermatitis, nummular dermatitis, contact dermatitis, atopic dermatitis, exfoliative dermatitis, perioral dermatitis, and stasis dermatitis), psoriasis, folliculitis, rosacea, acne, impetigo, erysipelas, tinea corporis, eczema, sunburn, burned skin, open wounds, and / or inflammatory skin conditions.

[0014] In certain aspects, the compositions of the present invention are formulated as topical skin compositions. The compositions disclosed herein may be in the form of sprays, foams, toners, creams, ointments, gels, lotions, emulsions, solutions, aerosols, or powders. The compositions may also be formulated for topical skin application at least once, twice, three times, four times, five times, six times, seven times, or more than seven times daily during use. In other aspects of the invention, the compositions may be storage-stable or color-stable, or both storage-stable and color-stable. It is also envisioned that the viscosity of the composition can be selected to achieve the desired results. For example, depending on the desired type of composition, the viscosity of the composition can be from about 1 cps to well over 1 million cps or any range or integer derived therefrom (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). The viscosities were measured using a TC rotor at 2.5 rpm at 25°C (s, 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, 600000 cps, 700000 cps, 800000 cps, 900000 cps, 1000000 cps, etc., measured with a Brookfield viscometer at 25°C using a TC rotor). In a non-limiting aspect, the pH of the composition can be from about 6 to about 9. In other respects, the pH can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.

[0015] The compositions of the present invention can also be modified to have a desired oxygen radical scavenging capacity (ORAC) value. In some non-limiting aspects, the compositions of the present invention, or their components or extracts, as defined in this specification, can be modified to have an ORAC value of 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, or 60 per milligram. 70, 80, 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 derived therefrom.

[0016] In a non-limiting aspect, the pH of the composition can be from 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 may 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 may also include preservatives. Non-limiting examples of preservatives include phenoxyethanol, methylparaben, propylparaben, or any mixture thereof. In some embodiments, the composition does not contain parabens.

[0017] The compositions of the present invention can have UVA and UVB absorption properties. The compositions can 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 higher, or any integer or range of SPF derived therefrom. The compositions can be sunscreen lotions, sprays, or creams.

[0018] The compositions of the present invention may further comprise any one, any combination of, or all of the following additional ingredients: water, chelating agents, humectants, preservatives, thickeners, polysiloxane-containing compounds, essential oils, structuring agents, vitamins, pharmaceutical ingredients, or antioxidants, or any combination or mixture of such ingredients. In some aspects, the composition may comprise at least two, three, four, five, six, seven, eight, nine, ten, or all of these additional ingredients identified in the preceding sentence. Non-limiting examples of these additional ingredients are identified throughout this specification and are incorporated herein by reference. The amount of such ingredients may be from 0.0001% to 99.9% by weight or volume of the composition, or any integer or range as disclosed in other parts of this specification, which are incorporated herein by reference.

[0019] Kits comprising the compositions of the present invention are also envisioned. In some embodiments, the composition is contained in a container. The container may be a bottle, dispenser, or package. The container may dispense a predetermined amount of the composition. In some aspects, the composition is dispensed in the form of a spray, clump, or liquid. The container may include markings on its surface. The markings may be words, abbreviations, pictures, or symbols.

[0020] It is conceivable 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.

[0021] In the context of this invention, at least 20 aspects are described. Aspect 1 includes a method of treating skin in need of treatment, comprising topically applying a combination of snow lotus extract, alpine rose rhododendron extract, and evening primrose oil to the skin, wherein the skin is treated. Aspect 2 pertains to aspect 1, wherein the composition further comprises squalene. Aspect 3 pertains to either aspect 1 or aspect 2, wherein the composition further comprises caprylic / capric triglyceride, hexyldecyl alcohol, bisabolol, N-palmitoylhydroxyproline cetyl ester, stearic acid, BHT, and brassicosterols. Aspect 4 pertains to any one of aspects 1 to 3, wherein the composition comprises 0.0001% to 0.1% by weight of snow lotus extract, 0.0001% to 0.1% by weight of alpine rose rhododendron extract, and 0.01% to 5.0% by weight of evening primrose oil. Aspect 5 pertains to aspect 2, wherein the composition further comprises 75.0% to 95.0% by weight of squalene. Aspect 6 is subordinate to aspect 3, wherein the composition further comprises 5.0% to 10.0% by weight of caprylic / capric triglyceride, 0.1% to 5.0% by weight of hexyldecyl alcohol, 0.001% to 1.0% by weight of bisabolol, 0.001% to 1.0% by weight of N-palmitoylhydroxyproline cetyl ester, 0.0001% to 0.1% by weight of stearic acid, 0.0001% to 0.1% by weight of BHT, and / or 0.0001% to 0.1% by weight of brassicosterols. Aspect 7 is subordinate to any one of aspects 1 to 6, wherein the composition further comprises olive fruit oil, tocopheryl acetate, or tocopherol. Aspect 8 is subordinate to aspect 7, wherein the composition comprises 0.01% to 5.0% by weight of olive fruit oil and 0.01% to 5.0% by weight of tocopheryl acetate or tocopherol. Aspect 9 belongs to any one of Aspects 1 to 8, wherein the composition moisturizes and protects the skin barrier. Aspect 10 belongs to any one of Aspects 1 to 9, wherein the composition protects skin proteins from oxidative stress-induced damage. Aspect 11 belongs to any one of Aspects 1 to 10, wherein the composition reduces uneven skin tone. Aspect 12 belongs to any one of Aspects 1 to 11, wherein the composition supports collagen production. Aspect 13 belongs to any one of Aspects 1 to 12, wherein the composition remains stable for 12 weeks when stored at a maximum temperature of 45°C. Aspect 14 belongs to any one of Aspects 1 to 13, wherein the composition is an oil. Aspect 15 comprises a topical skin care composition containing effective amounts of snow lotus extract, alpine rose rhododendron extract, and evening primrose oil. Aspect 16 is subordinate to aspect 15, wherein the composition further comprises squalene, caprylic / capric triglyceride, hexyldecyl alcohol, bisabolol, N-palmitoylhydroxyproline cetyl ester, stearic acid, BHT and brassicosterols.Aspect 17 belongs to any one of aspects 15 to 16, wherein the composition further comprises olive fruit oil, tocopheryl acetate, or tocopherol. Aspect 18 belongs to any one of aspects 15 to 17, wherein the composition comprises 0.0001% to 0.1% by weight of snow lotus extract, 0.0001% to 0.1% by weight of alpine rose rhododendron extract, and 0.01% to 5.0% by weight of evening primrose oil. Aspect 19 is subordinate to aspect 16, wherein the composition comprises 75.0% to 95.0% by weight of squalene, 5.0% to 10.0% by weight of caprylic / capric triglyceride, 0.1% to 5.0% by weight of hexyldecyl alcohol, 0.001% to 1.0% by weight of bisabolol, 0.001% to 1.0% by weight of N-palmitoylhydroxyproline cetyl ester, 0.0001% to 0.1% by weight of stearic acid, 0.0001% to 0.1% by weight of BHT, and / or 0.0001% to 0.1% by weight of brassicosterols. Aspect 20 is subordinate to aspect 17, wherein the composition comprises 0.01% to 5.0% by weight of olive fruit oil and 0.01% to 5.0% by weight of tocopheryl acetate or tocopherol.

[0022] In some embodiments, the compositions of the present invention may be pharmaceutically elegant or cosmetically elegant, or may have pleasant tactile properties. "Pharmaceutically elegant," "cosmetically elegant," and / or "pleasant tactile properties" describe compositions having specific tactile properties that feel comfortable on the skin (e.g., compositions that are not too thin or too oily, compositions with a silky texture, non-sticky or non-viscous compositions, etc.). Pharmaceutically elegant or cosmetically elegant may also refer to the creamy or lubricating properties of the composition, or the moisturizing properties of the composition.

[0023] The composition and its application may “contain / component,” “substantially constitute,” or “consist of” any of the ingredients disclosed in full in this specification. “Substantially constitute” means that the inclusion of additional ingredients in the composition will not materially affect the properties of the aforementioned combination of plant extracts and cosmetic carrier. An example of such an inclusion would be an ingredient that has an adverse effect (e.g., reduces efficacy or stability) on any of the ingredients mentioned in the composition.

[0024] Acne includes pimples, blackheads, whiteheads, papules, nodules, pustules, inflammatory lesions, or cysts.

[0025] "Topical application" refers to applying or spreading the composition onto the surface of the lips or keratinized tissue. "Topical skin compositions" include compositions suitable for topical application to the lips or keratinized tissue. Such compositions are generally dermatologically acceptable, meaning they do not exhibit excessive toxicity, incompatibility, instability, allergic reactions, etc., when applied to the lips or skin. The topical skin care compositions of the present invention may have a selected viscosity to avoid significant dripping or accumulation after application to the skin.

[0026] "Keratin tissue" includes the keratin-containing layer that forms the outermost protective layer of mammals, and includes, but is not limited to, the lips, skin, hair, and nails.

[0027] The term “about” or “approximately” is defined as close to what a person skilled in the art would understand, and in a non-limiting embodiment, the term is defined as within ±10%, preferably within ±5%, more preferably within ±1%, and most preferably within ±0.5%.

[0028] The term “substantially” and its variations are defined primarily, but not necessarily entirely, as understood by one of ordinary skill in the art, and in a non-limiting embodiment, substantially means within ±10%, ±5%, ±1%, or ±0.5%.

[0029] When used in the claims and / or description, the terms “inhibition” or “reduction”, “or treatment”, or any variations thereof include any measurable reduction or complete inhibition made to achieve the desired result.

[0030] When used in the specification and / or claims, the term “effective” means sufficient to achieve the desired, anticipated, or predetermined result.

[0031] When used in conjunction with the term "comprising / including" in the claims and / or description, the absence of a quantifier before the element may mean "one / type," but it is also consistent with the meaning of "one / type or more / types," "at least one / type," and "one / type or more than one / type."

[0032] As used in this specification and claims, the terms “comprising,” “having,” “including,” or “containing” are inclusive or open-ended and do not exclude additional, unlisted elements or method steps.

[0033] Other objects, features, and advantages of the invention will become apparent from the following detailed description. However, it should be understood that while the detailed description and examples point to specific embodiments of the invention, they are given by way of illustration only. Furthermore, it is contemplated that changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art based on this detailed description. Detailed Implementation

[0034] The cosmetic compositions of the present invention can be used to moisturize and protect the skin barrier. For example, facial oils offer numerous advantages, including: removing impurities such as sebum from the skin; unclogging skin pores; correcting or balancing imbalanced skin pH levels; refreshing and revitalizing the skin; smoothing and tightening skin pores; and preparing the skin for subsequent application of moisturizing products.

[0035] In one aspect, the present invention provides compositions and methods for moisturizing and protecting the skin barrier. In a non-limiting embodiment, the compositions of the present invention can be used for moisturizing and protecting the skin barrier by topically applying the compositions of the present invention to skin areas requiring such treatment. As described throughout this specification, the compositions may comprise at least one or any combination of the following ingredients: alpine rose rhododendron extract, evening primrose oil, and snow lotus extract, and a combination of structuring agents. The amounts of these plant extracts in a given composition can vary. In one example, for instance, the following ranges / amounts have been found to be effective: 0.0001% by weight to 0.1% by weight of snow lotus extract; 0.0001% by weight to 0.1% by weight of alpine rose rhododendron extract; and 0.01% by weight to 5.0% by weight of evening primrose oil. However, it is also contemplated that the amounts of the plant extracts may be lower or higher than these ranges. In this regard, the amount of any of the plant extracts in a given composition may range from 0.00001% to 10%, 0.0001% to 5%, 0.001% to 2%, 0.01% to 1%, 0.1% to 0.5%, etc. The inclusion of alpine rose rhododendron extract can protect skin proteins from oxidative stress-induced damage, while evening primrose oil can reduce uneven skin tone, and snow lotus extract can support collagen production. The non-limiting aspects of the invention are described in more detail in the following subsections.

[0036] Some of the compositions disclosed herein can be applied to the skin or hair and remain 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 longer). Afterward, if necessary, the composition can be rinsed off or peeled off the skin. Some of the compositions disclosed herein can be applied to the skin and immediately rinsed off. Some of the compositions disclosed herein can be applied to the skin and at least partially absorbed by the skin.

[0037] These and other non-limiting aspects of the invention are described in the following sections.

[0038] A. Active ingredients

[0039] This invention is based on the discovery that a combination of one or more active ingredients—alpine rose rhododendron extract, evening primrose oil, and snow lotus extract—can be used to moisturize and protect the skin barrier.

[0040] 1. Alpine rose and rhododendron extract

[0041] Alpine rose (Rhododendron simsii), also known as the Alpine rose, is a flowering plant native to remote mountainous regions such as the Alps and Pyrenees. Alpine rose leaf extract is commercially available from a variety of sources. The leaves and flowers of the Alpine rose are known sources of flavonoids. An exemplary source is produced by Mibelle Group Biochemistry (Buchs, Switzerland, USA) under the trade name AlpRose O, and is available from Bronson & Jacobs (Shanghai) International Trading Co., Ltd. or Mibelle Biochemistry. The extracted compounds can be used as powders and / or solutions in water and organic solvents (e.g., water, glycerol, or combinations thereof) in the compositions / formulations of this invention. In some embodiments, the extract includes stem cells from Alpine rose leaves. In vitro testing has shown that this component has antioxidant activity and inhibits interleukin-6 expression.

[0042] 2. Evening Primrose Oil

[0043] Evening primrose (Oenothera biennis, also known as evening primrose) is a flowering plant native to eastern and central North America. The extract from evening primrose seeds is an oil that can be used as a skin conditioner. This oil can be encapsulated in polymer materials to help deliver the oil to deeper tissues. The encapsulated form of the oil can be a powder containing at least 23% evening primrose seed extract. This ingredient is commercially available from a variety of sources. In some cases, evening primrose seed extract is commercially available from Carrubba, Inc., which supplies it under the trade names Evening Primrose Oil N23776 and Evening Primrose Oil N23776 Plant Extract. Antioxidant activity has been observed in vitro.

[0044] 3. Snow lotus extract

[0045] Snow lotus extract is an extract from the whole plant, the snow lotus, which is native to the mountainous regions of Central Asia. In some cases, snow lotus extract is commercially available from Carrubba, Inc., which supplies snow lotus extract under the name N62744-OS plant extract. This extract is a glycerol extract. In vitro testing has shown that this extract increases the production of type I collagen.

[0046] B. Amount of components

[0047] It is envisioned that the compositions of the present invention may contain any amount of the ingredients discussed in this specification. The compositions may also contain any combination of additional ingredients (e.g., pigments, or additional cosmetic or pharmaceutical ingredients) described throughout this specification. The concentration of any ingredient within the composition may vary. For example, in a non-limiting embodiment, the composition in its final form may contain, substantially constitute, 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%, and 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%, 0.0040%, 0.0041%, 0.0042%, 0.0043%, 0.0044%, 0.0045%, 0.0046%, 0.0047%, 0.0048%, 0.0049%, 0.0050%, 0 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 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%、0.5500%、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 thereof, at least one of the ingredients mentioned throughout this specification and claims. In a non-limiting respect, percentages may be calculated by weight or volume of the total composition. Those skilled in the art will understand that concentrations can vary depending on the addition, substitution, and / or reduction of ingredients in a given composition.

[0048] C. Carrier

[0049] The compositions of the present invention can be contained in or incorporated into all types of carriers and supports. The carrier or support can be pharmaceutically or dermatologically acceptable. Non-limiting examples of carriers or supports include water, glycerin, alcohols, oils, silicon-containing compounds, silicone compounds, and waxes. Variations and other suitable carriers will be apparent to those skilled in the art and are applicable to the present invention. In some aspects, the concentrations and combinations of compounds, ingredients, and agents can be selected in such a way that these combinations are chemically compatible and do not form complexes that precipitate from the final product.

[0050] D. Structure

[0051] The compositions of the present invention can be structured or formulated into a variety of different forms. Non-limiting examples include emulsions (e.g., water-in-oil, water-in-oil, water-in-oil, water-in-polysiloxane, polysiloxane-in-water, oil-in-water-in-oil, polysiloxane-in-water-in-oil), creams, lotions, solutions (aqueous solutions and water-alcohol solutions), anhydrous bases (such as lipsticks and powders), gels, masks, scrubs, body lotions, exfoliants, and ointments. Variations and other structures will be apparent to those skilled in the art and are applicable to the present invention.

[0052] E. Additional ingredients

[0053] In addition to the combinations of ingredients disclosed by the inventors, the composition may also include additional ingredients, such as cosmetic ingredients and pharmaceutical active ingredients. Non-limiting examples of such additional ingredients are described in the following subsections.

[0054] 1. Cosmetic ingredients

[0055] The CTFA International Dictionary and Handbook of Cosmetic Ingredients (2004 and 2008) describes a variety of non-limiting cosmetic ingredients that can be used in the context of this invention. Examples of these ingredient categories include: fragrances (artificial and natural; e.g., gluconic acid, phenoxyethanol, and triethanolamine), dyes and coloring agents (e.g., Blue 1, Blue 1 Lake, Red 40, titanium dioxide, D&C Blue 4, D&C Green 5, D&C Orange 4, D&C Red 17, D&C Red 33, D&C Violet 2, D&C Yellow 10, D&C Yellow 11), flavorings / fragrances (e.g., stevia). Rebaudiana extract and menthol), adsorbents, lubricants, solvents, moisturizers (including, for example, emollients, humectants, film-forming agents, occlusive agents, and agents that affect the skin's natural moisturizing mechanisms), waterproofing agents, UV absorbers (physical and chemical absorbers, such as para-aminobenzoic acid ("PABA") and corresponding PABA derivatives, titanium dioxide, zinc oxide, etc.), essential oils, vitamins (e.g., vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, and vitamin K), trace metals (e.g., zinc, calcium, and selenium), anti-irritants (e.g., steroidal and nonsteroidal anti-inflammatory drugs), plant extracts (e.g., aloe vera, chamomile, cucumber extract, ginkgo biloba extract). 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); absorbents (e.g., aluminum octenyl succinate, kaolin, corn starch, oat starch, cyclodextrin, talc, and zeolite); skin whitening agents (bleach). (e.g., hydroquinone, nicotinamide lactate), humectants (e.g., sorbitol, urea, methyl gluceth-20, saccharide isomers and mannitol), exfoliants, waterproofing agents (e.g., magnesium hydroxide / aluminum stearate), skin conditioning agents (e.g., aloe vera extract, allantoin, bisabolol, ceramide, dimethicone, hyaluronic acid, biosaccharide gum-1, ethylhexylglycerin, pentylene glycol, hydrogenated polydecene, octyl dodecyl oleate and dipotassium glycyrrhizate). Non-limiting examples of some of these ingredients are provided in the following subsections.

[0056] a. Ultraviolet absorbers and / or ultraviolet reflectors

[0057] UV absorbers and / or UV reflectors that can be used in combination with the compositions of the present invention include chemical sunscreens and physical sunscreens. Non-limiting examples of chemical sunscreens that can be used include para-aminobenzoic acid (PABA), PABA esters (glyceryl PABA ester, dimethyl PABA pentyl ester, and dimethyl PABA octyl ester), butyl PABA, ethyl PABA, ethyl dihydroxypropyl PABA, benzophenones (hydroxybenzophenone, sulphone, benzophenone, and benzophenone-1 to benzophenone-12), cinnamates (octinoxate, isoamyl p-methoxycinnamate, octyl methoxycinnamate, cinoxate, diisopropyl methyl cinnamate, DEA salt of methoxycinnamate, ethyl diisopropyl cinnamate, glyceryl octyl ester dimethoxycinnamate, and ethyl methoxycinnamate), cinnamates, and salicylates (homomethyl cinnamate). (salicylate), benzyl salicylate, ethylene glycol salicylate, isopropyl benzyl salicylate, etc.), anthranilates, ethyl urocarbamate, homosalate, octyl salicylate, dibenzoylmethane derivatives (e.g., avobenzone), octocrylene, octyl triazine, gallyl gallate trioleate, glyceryl aminobenzoate, 2-hydroxy-1,4-naphthoquinone and dihydroxyacetone, ethylhexyl triazine, dioctylbutamidotriazine, benzyl malonic acid polysiloxane, terephthalomethyl dicamphor sulfonic acid, disodium phenyl dibenzimidazole tetrasulfonate, diethylaminohydroxybenzoyl benzoate, bis(diethylaminohydroxybenzoyl)benzoate, bis(benzoxazolylphenylethylhexylimino)triazine. Examples of physical sunscreens include ethylhexyliminotriazine, cresoltrazol trisiloxane, methylene bis(benzotriazolyltetramethylbutylphenol) and bis(ethylhexyloxyphenol)methoxyphenyltriazine, 4-methylbenzyl camphor, and isopentyl-4-methoxycinnamate. Non-limiting examples of physical sunscreens include kaolin, talc, petrolatum, and metal oxides (e.g., titanium dioxide and zinc oxide).

[0058] b. Moisturizer

[0059] Non-limiting examples of moisturizers that can be used with the compositions of the present invention include amino acids, chondroitin sulfate, diglycerides, 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 factors, PEG-15 butylene glycol, polyglycerol sorbitol, pyrrolidone carboxylic acid salts, potassium PCA, propylene glycol, sugar isomers, sodium glucuronide, sodium PCA, sorbitol, sucrose, trehalose, urea, and xylitol.

[0060] Other examples include acetylated lanolin, acetylated lanolin alcohol, alanine, algae extracts, Aloe barbadensis, Aloe barbadensis extract, Aloe barbadensis gel, Althea officinalis extract, Prunus armeniaca kernel oil, arginine, arginine aspartate, Arnica montana extract, aspartic acid, Persea gratissima oil, barrier sphingolipids, butanol, beeswax, behenol, β-sitosterol, Betula alba bark extract, Borago officinalis extract, Ruscus aculeatus extract, butylene glycol, Calendula officinalis extract, calendula oil, Euphorbia cerifera wax, rapeseed oil, caprylic / capric triglycerides, and Elettaria cardamom. Cardamomum oil, Copernicia cerifera wax, Daucus carota sativa oil, Ricinus communis oil, ceramide, ceresin, cetearyl alcohol polyether-5, cetearyl alcohol polyether-12, cetearyl alcohol polyether-20, cetearyl alcohol caprylate, cetearyl alcohol polyether-20, cetearyl alcohol polyether-24, cetearyl acetate, cetearyl caprylate, cetearyl palmitate, Anthemis nobilis oil, cholesterol, cholesterol ester, cholesterol hydroxystearate, citric acid, clary sage (Southern European sage),Salvia sclarea oil, cocoa butter (Theobroma cacao), cocoyl caprylate / caprylate, coconut oil (Cocos nucifera), collagen, collagen amino acids, corn oil (Zea mays), fatty acids, decyl oleate, polydimethylsiloxane copolyol, polydimethylsiloxane alcohol, dioctyl adipate, dioctyl succinate, dimeric pentaerythritol hexacaprylate / hexacaprylate, DNA, erythritol, ethoxydiethylene glycol, ethyl linoleate, Eucalyptus globulus oil, evening primrose oil (Oenothera biennis), fatty acids, Geranium maculatum oil, glucosamine, glucosamine glutamate (glucose) glutamate, glutamic acid, glyceryl polyether-26, glycerin, glycerol, glyceryl distearate, glyceryl hydroxystearate, glyceryl laurate, glyceryl linoleate, glyceryl myristate, glyceryl oleate, glyceryl stearate, glyceryl stearate SE, glycine, ethylene glycol stearate, ethylene glycol stearate SE, glycosaminoglycans, grape (Vitis vinifera) seed oil, hazelnut (American hazelnut, Corylus americana) nut oil, hazelnut (European hazelnut, Corylus avellana) nut oil, hexanediol, hyaluronic acid, hybrid safflower (Carthamus tinctorius) Hydrogenated castor oil, hydrogenated coconut oil glycerides, hydrogenated coconut oil, hydrogenated lanolin, hydrogenated lecithin, hydrogenated palm oil glycerides, hydrogenated palm kernel oil, hydrogenated soybean oil, hydrogenated tallow glycerides, hydrogenated vegetable oil, hydrolyzed collagen, hydrolyzed elastin, hydrolyzed glycosaminoglycans, hydrolyzed keratin, hydrolyzed soybean protein, hydroxylated lanolin, hydroxyproline, isocetyl stearate, isocetyl stearyloxy stearate, isodecanol oleate, isopropanol isostearate, isopropanol lanolinate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, isostearamide DEA, isostearic acid, isostearyl lactate, isostearyl neopentanoate, jasmine (Jasminum officinale) oil, jojoba (Buxus) Ingredients include: chinensis oil, kelp, kukui (Aleurites moluccana) nut oil, lactamide MEA, lanolin alcohol polyether-16, lanolin alcohol polyether-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, macadamia ternifolia nut oil, maltitol, chamomile (motherwort).Chamomilla recutita oil, methyl glucosesquistearate, methylsilanol PCA, mineral oil, mink oil, *Morchella esculenta* oil, myristyl lactate, myristyl myristate, myristyl propionate, neopentyl glycol dicaprylate / dicaprylate, octyl dodecyl alcohol, octyl dodecyl myristate, octyl dodecyl stearyl stearate, octyl hydroxy stearate, octyl palmitate, octyl salicylate, octyl stearate, oleic acid, olive oil, orange oil, palm oil, palmitic acid, panthenyl thioethylamine, panthenol, panthenol ethyl ether, paraffin wax, PCA, peach kernel oil, peanut oil, PEG-8 C12-18 ester, 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 methylglucose sesquistearate, PEG-40 sorbitan peroleate, PEG-5 soybean sterol, PEG-10 soybean sterol, PEG-2 stearate, PEG-8 stearate, PEG-20 stearate, PEG-32 stearate, PEG-40 stearate, PEG-50 stearate, PEG-100 stearate, PEG-150 stearate, pentadecyl lactone, peppermint (Mentha) Piperrita oil, petrolatum, phospholipids, plankton extract, polyamino acid polysaccharide condensate, polyglycerol-3 diisostearate, polyquaternium-24, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, polysorbate 85, potassium myristate, potassium palmitate, propylene glycol, propylene glycol dicaprylate / dicaprylate, propylene glycol dicaprylate, propylene glycol dinonanoate, propylene glycol laurate, propylene glycol stearate, propylene glycol stearate SE, PVP, pyridoxine dipalmitate, retinol, retinyl palmitate, rice bran oil, RNA, rosemary oil, rose oil, safflower oil, sage oil, sandalwood oil, serine, serum albumin, sesame oil. Indicum oil, shea butter (avocado oil),Butyrospermum parkii lipids, 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, stearoxydimethylsiloxane, stearoxytrimethylsilane, stearyl alcohol, stearyl glycyrrhetinic acid, stearyl heptanoate, stearyl stearate, sunflower (Helianthus annuus) seed oil, sweet almond (Prunus amygdalus) Dulcis oil, synthetic beeswax, tocopherol, tocopheryl acetate, tocopheryl linoleate, triterpenoid, tridecyl stearate, triethanolamine, triglyceride stearate, urea, vegetable oil, water, wax, wheat (Triticum vulgare) germ oil, and ylang-ylang (Cananga odorata) oil.

[0061] c. Antioxidants

[0062] Non-limiting examples of antioxidants that can be used with the compositions of the present invention include acetylcysteine, ascorbic acid peptides, ascorbate dipalmitate, ascorbate methylsilanol pectate, ascorbate palmitate, ascorbate stearate, BHA, BHT, tert-butylhydroquinone, cysteine, cysteine ​​HCl, dipentylhydroquinone, ditert-butylhydroquinone, diceryl thiodipropionate, dioleotocopherol methylsilanol, disodium ascorbate sulfate, distearate thiodipropionate, ditridecyl thiodipropionate, dodecyl gallate, isoascorbic acid, ascorbate ester, ethyl ferulic acid, ferulic acid, gallate, and hydroquinone. Isooctyl mercaptoacetate, kojic acid, magnesium ascorbate, magnesium ascorbate phosphate, methylsilanol ascorbic acid, natural plant antioxidants such as green tea extract or grape seed extract, nordihydroguaiac acid, octyl gallate, phenyl mercaptoacetic acid, potassium ascorbate tocopheryl phosphate, potassium sulfite, propyl gallate, quinones, rosmarinic acid, sodium ascorbate, sodium bisulfite, sodium isoascorbate, sodium metabisulfite, sodium sulfite, superoxide dismutase, sodium mercaptoacetate, sorbitol furfural, thiodiglycol, thiodiglycolamide, thiodiglycolic acid (acid), mercaptoacetic acid, thiolactic acid, thiosalicylic acid, tocopherol polyether-5, tocopherol polyether-10, tocopherol polyether-12, tocopherol polyether-18, tocopherol polyether-50, tocopherol, tocophersolan, tocopherol acetate, tocopherol linoleate, tocopherol nicotinate, tocopherol succinate and tris(nonylphenyl) phosphite.

[0063] d. Structuring agents

[0064] In other non-limiting aspects, the compositions of the present invention may include a structuring agent. In some aspects, the structuring agent helps to provide rheological properties to the composition to promote its stability. In other aspects, the structuring agent may also be used as an emulsifier or a surfactant. Non-limiting examples of structuring agents include stearic acid, palmitic acid, stearyl alcohol, cetyl alcohol, behenyl alcohol, stearic acid, palmitic acid, polyethylene glycol ethers of stearyl alcohol having an average of about 1 to about 21 ethylene oxide units, polyethylene glycol ethers of cetyl alcohol having an average of about 1 to about 5 ethylene oxide units, and mixtures thereof.

[0065] e. Emulsifiers

[0066] In some aspects of the invention, the composition does not include an emulsifier. However, in other aspects, the composition may include one or more emulsifiers. Emulsifiers can reduce interfacial tension between phases and improve the formulation and stability of the emulsion. Emulsifiers may be nonionic, cationic, anionic, and zwitterionic emulsifiers (see U.S. Patent Nos. 5,011,681, 4,421,769, and 3,755,560). Non-limiting examples include esters of glycerol, esters of propylene glycol, fatty acid esters of polyethylene glycol, fatty acid esters of polypropylene glycol, esters of sorbitol, esters of dehydrated sorbitan anhydride, carboxylic acid copolymers, esters and ethers of glucose, ethoxylated ethers, ethoxylated alcohols, alkyl phosphates, polyoxyethylene fatty ether phosphates, fatty acid amides, acyl lactates, soaps, TEA salts of stearic acid, DEA salts of oleyl alcohol polyether-3 phosphate, polyethylene glycol 20 dehydrated sorbitan monolaurate (polysorbate 20), polyethylene glycol 5 soybean sterol, stearyl alcohol polyether-2, stearyl alcohol polyether-20, stearyl... Cetearyl alcohol polyether-21, cetearyl alcohol polyether-20, cetearyl alcohol glucoside, cetearyl alcohol, C12 to C13 alkyl alcohol polyether-3, PPG-2 methyl gluconate distearate, PPG-5-cetearyl alcohol polyether-20, bis-PEG / PPG-20 / 20 polydimethylsiloxane, cetearyl alcohol polyether-10, polysorbate 80, cetearyl phosphate, cetearyl phosphate potassium, diethanolamine cetearyl phosphate, polysorbate 60, glyceryl stearate, PEG-100 stearate, arachidyl alcohol, arachidyl alcohol glucoside, and mixtures thereof.

[0067] f. Compounds containing polysiloxanes

[0068] In a non-limiting sense, polysiloxane compounds include any member of the polymer product family whose molecular backbone consists of alternating silicon and oxygen atoms, with side groups attached to silicon atoms. By varying the length of the -Si-O- chains, the side groups, and the crosslinking, polysiloxanes can synthesize a wide variety of materials. Their consistency can vary from liquid to gel to solid.

[0069] Polysiloxane-containing compounds that may be used in the context of this invention include those described herein or those known to those skilled in the art. Non-limiting examples include silicone oils (e.g., volatile and non-volatile oils), gels, and solids. In some aspects, polysiloxane-containing compounds include silicone oils, such as polyorganosiloxanes. Non-limiting examples of polyorganosiloxanes include polydimethylsiloxane, cyclomethicone, polysiloxane-11, phenyltrimethylsiloxane, trimethylsilyl-terminated trimethylsiloxane, stearoxytrimethylsilane, or mixtures of these with other organosiloxane materials in any given proportion to achieve a desired consistency and application properties depending on the intended use (e.g., application to a specific area such as skin, hair, or eyes). "Volatile silicone oil" includes silicone oils with low heat of vaporization, i.e., silicone oils with a heat of vaporization typically less than about 50 calories per gram. Non-limiting examples of volatile silicone oils include: cyclomethyl polysiloxanes, 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.); and low-viscosity polydimethylsiloxanes, i.e., polydimethylsiloxanes having a viscosity of about 50 cst or less (e.g., polydimethylsiloxanes such as Dow Corning 200-0.5 cst fluid). Dow Corning fluids are available from Dow Corning Corporation, Midland, Michigan. Cyclomethyl polysiloxanes and polydimethylsiloxanes are described in the CTFA Cosmetic Ingredient Dictionary, Third Edition (incorporated by reference), as mixtures of cyclic dimethyl polysiloxane compounds and fully methylated linear siloxane polymers capped with trimethylsiloxy units, respectively. Other non-limiting volatile polysiloxanes that may be used in the context of this invention include those available from General Electric Co., Silicone Products Div., Waterford, NY, and Stauffer Chemical Co., Adrian, Michigan, SWS Silicones Div.

[0070] g. Exfoliating agent

[0071] Exfoliants comprise ingredients that remove dead skin cells from the outer surface of the skin. These agents can act by mechanical, chemical, and / or other means. Non-limiting examples of mechanical exfoliants include abrasives such as pumice, silica, fabrics, paper, shells, beads, solid crystals, solid polymers, etc. Non-limiting examples of chemical exfoliants include acid exfoliants and enzyme exfoliants. Acids that can be used as exfoliants include, but are not limited to, glycolic acid, lactic acid, citric acid, α-hydroxy acids, β-hydroxy acids, etc. Other exfoliants known to those skilled in the art are also envisioned to be useful within the context of this invention.

[0072] h. Essential oils

[0073] Essential oils include oils derived from herbs, flowers, trees, and other plants. These oils typically exist as tiny droplets between plant cells and can be extracted using several methods known to those skilled in the art (e.g., steam distillation, enfleurage (i.e., by using fat extraction), 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 darken over time. Essential oils are insoluble in water but soluble in alcohols, ethers, non-volatile oils (vegetable oils), and other organic solvents. Typical physical properties found in essential oils include a boiling point of about 160°C to about 240°C and a density of about 0.759 to about 1.096.

[0074] Essential oils are typically named after the plant from which they are found. For example, rose oil or peppermint oil is derived from the rose or mint plant, respectively. Non-limiting examples of essential oils that may be used in the context of this invention include sesame oil, macadamia nut oil, tea tree oil, evening primrose oil, Spanish sage oil, Spanish rosemary oil, coriander oil, thyme oil, allspice oil, rose oil, fennel oil, balsam oil, bergamot oil, rosewood oil, cypress 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, citrus oil, marjoram oil, myrrh oil, neroli oil, orange oil, patchouli oil, pepper oil, black pepper oil, petitgrain oil, pine oil, and rose of Otto. Oils such as ott-Oil, rosemary oil, sandalwood oil, spearmint oil, spikenard oil, vetiver oil, wintergreen oil, or ylang-ylang oil are also permitted. Other essential oils known to those skilled in the art are also envisioned to be useful within the context of this invention.

[0075] i. Thickener

[0076] Thickening agents, including thickeners or gelling agents, comprise substances that can increase the viscosity of a composition. Thickeners include those substances that can increase the viscosity of a composition without significantly altering the efficacy of the active ingredient within the composition. Thickeners can also increase the stability of the compositions of the present invention. In some aspects of the invention, thickeners include hydrogenated polyisobutylene, trihydroxystearin, ammonium acryloyldimethyl taurate / VP copolymer, or mixtures thereof.

[0077] Non-limiting examples of additional thickeners that may be used in the context of this invention include carboxylic acid polymers, crosslinked polyacrylate polymers, polyacrylamide polymers, polysaccharides, and gums. Examples of carboxylic acid polymers include crosslinked compounds containing one or more monomers derived from acrylic acid, substituted acrylic acid, and salts and esters of such acrylic acid and substituted acrylic acid, wherein the crosslinking agent contains two or more carbon-carbon double bonds and is derived from polyols (see U.S. Patent Nos. 5,087,445, 4,509,949, and 2,798,053; CTFA International Cosmetic Ingredient Dictionary, Fourth edition, 1991, pp. 12 and 80). Examples of commercially available carboxylic acid polymers include carbomer, which is a homopolymer of acrylic acid crosslinked with an allyl ether of sucrose or pentaerythritol (e.g., CARBOPOL from BFGOODRICH). TM 900 series).

[0078] Non-limiting examples of crosslinked 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, and 4,599,379.

[0079] Non-limiting examples of polyacrylamide polymers (including nonionic polyacrylamide polymers, including substituted branched polymers or unbranched polymers) include polyacrylamide, isoparaffins and lauryl ether-7, acrylamide and substituted acrylamide multiblock copolymers with acrylic acid and substituted acrylic acid.

[0080] 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. Another example is alkyl-substituted cellulose, wherein the hydroxyl groups of the cellulose polymer are hydroxyalkylated (preferably hydroxyethylated or hydroxypropylated) to form hydroxyalkylated cellulose, and then further modified by C10 to C30 straight-chain or branched alkyl groups via ether bonds. Typically, these polymers are ethers of C10-C30 straight-chain or branched alcohols with hydroxyalkyl cellulose. Other useful polysaccharides include stearyl glucans, which comprise a straight chain containing (1 to 3) linked glucose units, with one (1 to 6) linked glucose units for every three units.

[0081] Non-limiting examples of gums that can be used with the present invention include gum arabic, agar, alginic acid, alginate, ammonium alginate, amylopectin, calcium alginate, carrageenan calcium, carnitine, carrageenan, dextrin, gelatin, gellan gum, guar gum, guar hydroxypropyltrimethylammonium chloride, lithium montmorillonite, hyaluronic acid, hydrated silica, hydroxypropyl chitosan, hydroxypropyl guar gum, black privet gum, kelp, black 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.

[0082] j. Preservatives

[0083] Non-limiting examples of preservatives that may be used in the context of this invention include quaternary ammonium salt preservatives such as polyquaternary ammonium salt-1 and benzalkonium halide (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.

[0084] 2. Drug components

[0085] It is also envisioned that pharmaceutically active agents can be used with the compositions of the present invention. Non-limiting examples of pharmaceutically active agents include anti-acne agents, agents for treating rosacea, analgesics, anesthetics, anorectal medications, antihistamines, anti-inflammatory agents including nonsteroidal anti-inflammatory drugs, antibiotics, antifungal agents, antiviral agents, antimicrobial agents, anticancer actives, antiscabicides, antilice agents, antitumor agents, antiperspirants, antipruritics, antipsoriatic agents, and antiseborrheic agents. Agents), bioactive proteins and peptides, burn treatment agents, cauterizing agents, depigmenting agents, depilatory agents, diaper rash treatment agents, enzymes, hair growth stimulants, hair growth depressants including DFMO and its salts and analogues, hemostatic agents, keratolytic agents, oral ulcer treatment agents, cold sore treatment agents, dental and periodontal treatment agents, photosensitizing agents, skin protectants / barrier agents, steroids including hormones and corticosteroids, sunburn treatment agents, sunscreens, transdermal agents, nasal agents, vaginal agents, wart treatment agents, wound treatment agents, wound healing agents, etc.

[0086] F. Reagent Kit

[0087] The use of a kit is also contemplated in certain aspects of the invention. For example, the compositions of the invention may be contained in a kit. The kit may include a container. The container may include a bottle, metal tube, laminated tube, plastic tube, dispenser, pressurized container, isolating container, packaging, compartment, lipstick container, cosmetic tray for containing cosmetic compositions, or other types of containers, such as injection-molded or blow-molded plastic containers in which a dispersion or composition or desired bottle, dispenser, or packaging is retained. The kit and / or container may include markings on its surface. For example, the markings may be words, phrases, abbreviations, pictures, or symbols.

[0088] The container can dispense a predetermined amount of the composition. In other embodiments, the container (e.g., a metal, laminated, or plastic tube) can be squeezed to dispense the desired amount of composition. The composition can be dispensed as a spray, aerosol, liquid, fluid, or semi-solid form. The container can have a spray, pump, or squeeze mechanism. The kit may also include instructions for using the kit components and any other compositions contained in the container. The instructions may include explanations of how to apply, use, and maintain the composition.

[0089] Example

[0090] The following embodiments are included to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques that the inventors have discovered that function well in the practice of the invention and can therefore be considered as constituting a preferred mode of practice. However, based on this disclosure, those skilled in the art will understand that many changes can be made to the specific embodiments disclosed and similar or analogous results can still be obtained without departing from the spirit and scope of the invention.

[0091] According to this disclosure, all compositions disclosed and claimed herein can be prepared and carried out without excessive experimentation. While the compositions and methods of the invention have been described according to preferred embodiments, it will be apparent to those skilled in the art that changes can be made to the compositions and the steps or sequence of steps of the methods described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically relevant reagents can be substituted for the reagents described herein while achieving the same or similar results. All such similar substitutions and modifications that are apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0092] A. Example 1 (Materials used)

[0093] The combinations of active ingredients disclosed herein can be included in a wide range of topical skin product formulations. Non-limiting examples of combinations of active ingredients include those listed in Table 1.

[0094] Table 1

[0095]

[0096] B. Example 2 (Exemplary Formulation)

[0097] Formulations having the ingredients from Example 1 were prepared as topical skin compositions. The formulations in Table 2 are examples of topical skin compositions that can be used for moisturizing and protecting the skin barrier.

[0098] Table 2*

[0099]

[0100] *The formulation can be prepared by mixing the components in a beaker at a heating temperature of 70°C to 75°C until homogeneous. The formulation can then be cooled to normal room temperature (20°C to 25°C). Furthermore, if desired, additional components can be added, for example, to modify the rheological properties of the composition.

[0101] **Excipients may be added, for example, to modify the rheological properties of the composition.

[0102] C. Example 3 (Stability)

[0103] Stability testing: The ability of the composition to maintain stable appearance, color, consistency, odor, pH, light stability, durability, texture, and viscosity over time under different conditions was analyzed. The composition remained stable for 12 weeks when stored at 45°C.

[0104] D. Example 4 (In vitro assay)

[0105] ORAC assay: The oxygen radical uptake (or absorbance) capacity (ORAC) of alpine rose rhododendron extract and evening primrose oil is determined by measuring their antioxidant activity. Antioxidant activity indicates the ability to reduce oxidizing agents (oxidants). This assay quantifies the extent and time required to inhibit the effects of oxidants (such as oxygen radicals) known to cause damage to cells (e.g., skin cells). The ORAC value of any of the active ingredients, combinations of ingredients, or compositions having said combinations disclosed in this specification can be determined by methods known to those skilled in the art (see U.S. Publications 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 luciferin fluorescence over time due to peroxy radicals generated from the decomposition of AAPH (2,2'-azobis(2-methylpropanediamine) dihydrochloride). The water-soluble vitamin E analog Trolox, used as a positive control, inhibited luciferin decay in a dose-dependent manner. Alpine rose and rhododendron extract was found to increase antioxidant activity by 106%. Evening primrose oil was found to increase antioxidant activity by 108%.

[0106] Collagen Stimulation Assay: Collagen is an extracellular matrix protein essential for skin structure. Increased collagen synthesis contributes to improved skin firmness and elasticity. This bioassay was used to examine the effect of snow lotus extract on the production of procollagen peptides (collagen precursors) by human epidermal fibroblasts. The endpoint of this assay was a spectrophotometric measurement reflecting the presence of procollagen peptides and cell viability. The assay employed a quantitative sandwich enzyme immunoassay technique, in which monoclonal antibodies specific to procollagen peptides were pre-coated onto microplates. Standards and samples were transferred to the wells, and any present procollagen peptides bound to the immobilized antibody. After washing away any unbound material, an enzyme-linked polyclonal antibody specific to procollagen peptides was added to the wells. After washing to remove any unbound antibody-enzyme reagent, substrate solution was added to the wells, and color development was performed, with the degree of color development proportional to the amount of procollagen peptides bound in the initial step, detected at 450 nm using a microplate reader. Color development was stopped, and the color intensity was measured. It was found that snow lotus extract can induce and increase the expression of type I collagen by 42%.

[0107] Cytokine array: Human epidermal keratinocytes were cultured to 70%–80% confluence. The culture medium was aspirated from the plate, and 0.025% trypsin / EDTA was added. When the cells became rounded, the culture dish was gently tapped to release the cells. The cells containing trypsin / EDTA were removed from the dish and neutralized. The cells were centrifuged at 180 × g for 5 minutes to form a cell pellet. The supernatant was aspirated. The resulting pellet was resuspended in EPILIFE™ medium (Cascade Biologics). The cells were seeded in 6-well plates at approximately 10%–20% confluence. Once the cells reached approximately 80% confluence, the culture medium was aspirated, and 1.0 ml of EPILIFE™, along with phorbol 13-myristate 12-acetate (PMA) (a known inflammatory inducer) and test composition dilution buffer (i.e., diluting 1.0% (100 µl of 100X stock solution) and 0.1% (10 µl of 100X stock solution) of the test composition to a final volume of 1 ml of EPILIFE™ growth medium) were added to two replicate wells. The medium was gently stirred to ensure thorough mixing. Additionally, 1.0 ml of EPILIFE™ was added to the control wells. TM With or without adding additional PMA. The plates were then incubated at 37±1℃ and 5.0±1% CO2 for approximately 5 hours. After this 5-hour incubation, all culture media were collected in conical tubes and frozen at -70℃.

[0108] For analysis, 16-well hybridization chambers were attached to 16-well FAST slides, with triplicate of the 16-well FAST slides containing 16 anti-cytokine antibodies and experimental controls (WHATMAN BIOSCIENCES). The slides were then placed in FASTFrames (4 slides per frame) for processing. The arrays were blocked for 15 minutes at room temperature using 70 ml of S&S protein array blocking buffer (WHATMAN SCHLEICHER AND SCHEULL). The blocking buffer was removed, and 70 ml of each supernatant sample was added to each array. The arrays were incubated with gentle agitation at room temperature for 3 hours. The arrays were washed three times with TBS-T. The arrays were then treated with 70 ml of an antibody mixture containing a biotinylated antibody corresponding to the capture antibody for each arrangement. The arrays were incubated with gentle agitation at room temperature for 1 hour. The arrays were washed three times with TBS-T. The arrays were then incubated with 70 ml of a solution containing streptavidin-Cy5 conjugates with gentle agitation at room temperature for 1 hour. The arrays were washed three times with TBS-T, rapidly rinsed in deionized water, and dried.

[0109] The slides were imaged using the PERKIN-ELMER SCANARRAY 4000 confocal fluorescence imaging system. The array images can be saved and analyzed using the IMAGING RESEARCH ARRAYVISION software. In short, spot intensity is determined by subtracting the background signal. The average value of spots from each sample condition can be repeatedly taken and then compared with an appropriate control.

[0110] Cells treated with alpine rose rhododendron extract showed that interleukin-6 expression was suppressed by 83% compared to the appropriate control.

[0111] E. Example 5 (In vivo testing)

[0112] Determinations that can be used to determine the efficacy of any single ingredient or any combination of ingredients, or a composition having a combination of said ingredients, disclosed in the entirety of the specification and claims, can be determined by methods known to those skilled in the art. The following are non-limiting determinations that may be used in the context of this invention. It should be appreciated that other testing procedures, including, for example, objective and subjective procedures, may be used.

[0113] It has been determined that different formulations containing snow lotus extract, alpine rose rhododendron extract, and evening primrose oil can reduce the appearance of fine lines and wrinkles, improve texture / smoothness, and increase firmness. The results are shown in Table 3, and the methods used to analyze the effectiveness of the formulations are provided below.

[0114] Table 3*

[0115]

[0116] * Bold values ​​are significantly different from the baseline (p<0.05)

[0117] The results shown in Table 3 were determined through a trial involving 29 female participants, one-third of whom were Asian. All participants exhibited mild to moderate fine lines and wrinkles. Participants applied the test product twice daily, once in the morning and once in the evening. The trial evaluated the product's efficacy at three time points: 4 weeks, 8 weeks, and 12 weeks. Assessments were conducted using an expert clinical scoring method, including visual and tactile assessments. Visual assessments focused on fine lines and wrinkles, while tactile assessments measured improvements in skin firmness and texture / smoothness.

[0118] F. Example 6 (Additional Detection)

[0119] Determinations that can be used to determine the efficacy of any ingredient or combination of ingredients disclosed throughout the specification and claims, or a composition having a combination of said ingredients, can be determined by methods known to those skilled in the art. The following are non-limiting determinations that may be used in the context of this invention. It should be appreciated that other testing procedures, including, for example, objective and subjective procedures, may be used.

[0120] B16 Pigmentation Assay: Melanogenesis is the process by which melanocytes produce melanin, a naturally occurring pigment that gives skin, hair, and eyes color. Inhibiting melanin production is beneficial in preventing skin darkening and reducing age-related dark spots. This bioassay utilizes B16-F1 melanocytes (ATCC), an immortalized mouse melanoma cell line, to analyze the effects of compounds on melanogenesis. The endpoint of this assay was spectrophotometric measurement of melanin production and cell viability. B16-F1 melanocytes were cultured in standard DMEM growth medium containing 10% fetal bovine serum (MEDIATECH) at 37°C in 10% CO2, and then treated for 6 days with any of the active ingredients, combinations of ingredients, or compositions containing said combinations disclosed in the instructions. After incubation, melanin secretion was measured by absorbance at 405 nm, and cell viability was quantified.

[0121] For the preparation of samples and controls, nearly confluent normal adult epidermal fibroblasts (Cascade Biologics) cultured at 37°C in 10% CO2 in standard DMEM growth medium containing 10% fetal bovine serum (MEDIATECH) were treated for 3 days with a combination of each component disclosed in the instructions or a composition having said combinations. After incubation, the cell culture medium was collected, and the amount of procollagen peptide secretion was quantified using a sandwich enzyme-linked immunosorbent assay (ELISA) from TAKARA (#MK101).

[0122] Elastin Stimulation Assay: Elastin is a connective tissue protein that helps the skin regain its shape after stretching or contraction. Elastin is also an important load-bearing protein, used in areas where mechanical energy needs to be stored. Elastin is produced by linking numerous soluble proelastin molecules in a reaction catalyzed by lysyl oxidase. Elastin secretion and elastin fibers can be monitored in cultured human fibroblasts by staining them with an immunofluorescent antibody targeting elastin.

[0123] Laminin and Fibronectin Stimulation Assay: Laminin and fibronectin are major proteins at the dermal-epidermal junction (DEJ), also known as the basement membrane. The DEJ lies between the dermis and epidermis, forming finger-like projections called reteridges. Epidermal cells obtain nutrients from blood vessels in the dermis. The reteridges increase the surface area of ​​the epidermis exposed to these vessels and the necessary 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 within the DEJ. They are considered adhesives that hold cells together and are secreted by dermal fibroblasts, contributing to intracellular and intercellular adhesion of epidermal cells to the DEJ. The secretion of laminin and fibronectin can be monitored by quantifying laminin and fibronectin in the supernatant of cultured human fibroblasts treated for 3 days with or without one or more test components at a final concentration of 1.0%. Following incubation, the levels of laminin and fibronectin can be measured using enzyme-linked immunosorbent assay (ELISA) using immunofluorescent antibodies targeting laminin and antibodies targeting fibronectin directly. Measurements of cellular metabolic activity are standardized, for example, by biotransformation with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazole (MTS).

[0124] Tumor necrosis factor-α (TNF-α) assay: TNF-α, the proto-ligand of the TNF superfamily, is a pleiotropic cytokine that plays a central role in inflammation. Increased expression of TNF-α is associated with upregulation of pro-inflammatory activity. This bioassay can be used to analyze the effect of any of the active ingredients, combinations of ingredients, or compositions having said combinations disclosed in this specification on the production of TNF-α by human epidermal keratinocytes. The endpoint of this assay can be a spectrophotometric measurement reflecting TNF-α and cell viability. This assay employs a quantitative sandwich enzyme immunoassay technique, wherein a monoclonal antibody specific to TNF-α is pre-coated on a microplate. Standards and samples can be transferred into the wells, and any TNF-α present binds to the immobilized antibody. After washing away any unbound material, an enzyme-linked polyclonal antibody specific to TNF-α can be added to the wells. After washing to remove any unbound antibody-enzyme reagent, a substrate solution can be added to the wells, and color development can be performed proportionally to the amount of TNF-α bound in the initial step, and detection can be performed using a microplate reader at 450 nm. The color development can be terminated, and the color intensity can be measured. Submerged normal adult keratinocytes (Cascade Biologics) were cultured in EPILIFE™ standard growth medium (Cascade Biologics) at 37°C and 5% CO2, and treated for 6 hours with phorbol 12-myristate 13-acetate (PMA, 10 ng / ml, SIGMACHEMICAL, #P1585-1MG) and any active ingredient, combination of ingredients, or composition having said combination disclosed in the instructions. PMA has been shown to induce a significant increase in TNF-α secretion, peaking at 6 hours post-treatment. After incubation, the cell culture medium was collected, and the amount of TNF-α secreted was quantified using a sandwich enzyme-linked immunosorbent assay (ELISA) from R&D Systems (#DTA00C).

[0125] Antioxidant (AO) assay: An in vitro bioassay used to measure the total antioxidant capacity of any one of the ingredients, combinations of ingredients, or compositions having said combinations disclosed in this specification. This assay relies on the antioxidants in the sample inhibiting metmyoglobin from releasing ABTS. ® (2,2'-Azido-bis-[3-ethylbenzothiazoline sulfonate]) oxidized to ABTS ®•+ capacity. An organism's antioxidant system includes enzymes such as superoxide dismutase, catalase, and glutathione peroxidase; macromolecules such as albumin, ceruloplasmin, and ferritin; and a range of small molecules, including ascorbic acid, α-tocopherol, β-carotene, reduced glutathione, uric acid, and bilirubin. The sum of endogenous and dietary antioxidants represents the total antioxidant activity in extracellular fluid. The synergistic effect of all the different antioxidants provides stronger protection against reactive oxygen species or nitrogen free radical attacks than any single compound acting alone. Therefore, total antioxidant capacity may provide more relevant biological information than information obtained by measuring individual components, as it takes into account the cumulative effect of all antioxidants present in plasma and body fluids. The ability of antioxidants in a sample to inhibit ABTS® oxidation was compared to Trolox (a water-soluble tocopherol analog) and quantified as molar equivalents of Trolox. The antioxidant capacity kit #709001 from CAYMAN CHEMICAL (Ann Arbor, Michigan, USA) can be used as an in vitro bioassay to measure the total antioxidant capacity of each of any active ingredient, combination of ingredients, or composition having said combination disclosed in this specification. The protocol can be performed according to the manufacturer's recommendations.

[0126] Mushroom Tyrosinase Activity Assay: In mammalian cells, tyrosinase catalyzes two steps in the multi-step biosynthesis of melanin, from tyrosine (and from dopachrome polymerization). Located in melanocytes, tyrosinase produces melanin (aromatic quinone compounds), which imparts color to skin, hair, and eyes. Purified mushroom tyrosinase (from SIGMA) can be incubated with its substrate L-Dopa (from FISHER) in the presence or absence of each active ingredient, any combination of ingredients, or a composition having said combinations disclosed in this specification. Pigment formation can be assessed by a microplate reader reading at 490 nm. The percentage inhibition of mushroom tyrosinase activity compared to an untreated control can be calculated to determine the ability of the test ingredient or its combination to inhibit the activity of the purified enzyme. The inhibition of the test extract is compared with the inhibition of kojic acid (SIGMA).

[0127] Matrix metalloproteinase 3 and matrix metalloproteinase 9 (MMP3; MMP9) activity assays: In vitro matrix metalloproteinase (MMP) inhibition assays. MMPs are extracellular proteases that function in many normal and disease states due to their broad substrate specificity. MMP3 substrates include collagen, fibronectin, and laminin; while MMP9 substrates include type VII collagen, fibronectin, and laminin. Using the Colorimetric Drug Discovery kits for MMP3 (AK-400) and MMP-9 (AK-410) from BioMol International, this assay aims to measure the protease activity of MMPs using a thiopeptide as a chromogenic substrate (Ac-PLG-[2-mercapto-4-methyl-valeryl]-LG-OC2H5)5,6. The peptide bonds at the MMP cleavage site are replaced by thioester bonds in the thiopeptide. MMP hydrolyzes this bond to generate a thiol group, which reacts with DTNB [5,5'-dithiobis(2-nitrobenzoic acid), Ellman's reagent] to form 2-nitro-5-thiobenzoic acid, which can be determined by its absorbance at 412 nm (ε=13600 M). -1 cm -1 The test is performed at pH 6.0 and above 7. It can determine the active ingredients disclosed in this specification, any combination of ingredients, or compositions having said combinations.

[0128] Matrix Metalloproteinase 1 (MMP) Activity Assay: In vitro matrix metalloproteinase (MMP) inhibition assay. MMPs are extracellular proteases that function in many normal and disease states due to their broad substrate specificity. MMP 1 substrates include type IV collagen. The ENZ / CHEK Gelatinase / Collagen Assay Kit (#E12055) utilizes a fluorescent gelatin substrate to detect MMP 1 protease activity. Following protein hydrolysis, a bright green fluorescence is observed, which can be monitored using a fluorescent microplate reader to measure enzyme activity.

[0129] The ENZ / CHEK Gelatinase / Collagenase Assay Kit (#E12055) from Invitrogen is designed for the in vitro assay of MMP1 enzyme activity. It can determine the active ingredients disclosed in this specification, any combination of ingredients, or compositions containing said combinations. The assay relies on the ability of purified MMP1 enzyme to degrade fluorescent gelatin substrates. Once the substrate is specifically cleaved by MMP1, bright green fluorescence appears and can be monitored using a fluorescent microplate reader. Test materials are incubated in the presence or absence of purified enzyme and substrate to determine their protease inhibitory activity.

[0130] Cyclooxygenase (COX) Assay: In vitro inhibition assays of cyclooxygenase-1 and cyclooxygenase-2 (COX-1, COX-2). COX is a bifunctional enzyme with both cyclooxygenase and peroxidase activities. Cyclooxygenase activity converts arachidonic acid into hydroperoxymonoperoxide (prostaglandin G2; PGG2), and the peroxidase component reduces the peroxidase (prostaglandin H2; PGH2) to the corresponding alcohols, namely precursors of prostaglandins, thromboxanes, and prostacyclin. This COX inhibitor screening assay measures the peroxidase component of cyclooxygenase. Peroxidase activity is determined colorimetrically by monitoring the presence of oxidized N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). This inhibitor screening assay includes both COX-1 and COX-2 enzymes to screen for isoenzyme-specific inhibitors. The effect of each active ingredient, any combination of ingredients, or a combination having said combinations disclosed in this specification on the activity of purified cyclooxygenase (COX-1 or COX-2) can be analyzed using a colorimetric COX (sheep) inhibitor screening assay (#760111, CAYMAN CHEMICAL). Following the manufacturer's instructions, the purified enzyme, heme, and test extract are mixed in assay buffer and incubated with shaking at room temperature for 15 minutes. After incubation, arachidonic acid and the colorimetric substrate are added to initiate the reaction. Colorimetric development can be assessed using a microplate reader at 590 nm. The percentage of COX-1 or COX-2 activity inhibition can be calculated and compared to an untreated control to determine the ability of the test extract to inhibit the activity of the purified enzyme.

[0131] Lipoxygenase (LO) Assay: In vitro lipoxygenase (LO) inhibition assay. LO is a non-heme iron dioxygenase that catalyzes the addition of molecular oxygen to fatty acids. Linoleic acid and arachidonic acid are the main substrates of LO in plants and animals. Arachidonic acid can then be converted into hydroxyeicosatetrienoic acid (HETE) derivatives, which are subsequently converted into leukotrienes, potent inflammatory mediators. This assay provides an accurate and convenient method for screening lipoxygenase inhibitors by measuring the hydroperoxides produced when lipoxygenases (5-LO, 12-LO, or 15-LO) are incubated with arachidonic acid. The ability of each active ingredient, any combination of ingredients, or a combination of said combinations to inhibit enzyme activity can be determined using a colorimetric LO inhibitor screening kit (#760700, CAYMAN CHEMICAL). The purified 15-lipoxygenase and the test ingredient are mixed in assay buffer and incubated with shaking at room temperature for 10 minutes. After incubation, arachidonic acid is added to initiate the reaction, and the mixture is incubated at room temperature for another 10 minutes. A colorimetric substrate can be added to terminate the catalysis, and color development can be assessed by fluorescence microplate reading at 490 nm. The percentage of inhibition of lipoxygenase activity can be calculated compared to the untreated control to determine the ability of each active ingredient, any combination of ingredients, or a composition having the combinations disclosed in the specification to inhibit the activity of the purified enzyme.

[0132] Elastase Assay: The ENZCHEK® Elastase Assay Kit (#E-12056) from MOLECULAR PROBES (Eugene, Oregon USA) can be used as an in vitro enzyme inhibition assay to measure the inhibition of elastase activity by each active ingredient, any combination of ingredients, or a composition having said combinations disclosed in this specification. The ENZCHEK 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 resulting increase in fluorescence can be monitored using a fluorescence microplate reader. The digestion product of the elastin substrate has maximum absorption at approximately 505 nm and maximum fluorescence emission at approximately 515 nm. When screening for elastase inhibitors using the ENZCHEK Elastase Assay Kit, the peptide N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone can be used as a selective, aggregate inhibitor of elastase.

[0133] Ceramide production: Ceramide in cell or tissue samples can be incubated at room temperature with a 1 / 50 dilution of mouse monoclonal antibody against ceramide (ENZO LIFE SCIENCE, catalog number ALX-804-196, clone MID15B4) for 2 hours and labeled using a biotin / streptavidin amplification system. Video microscopy can be used to observe the ceramide (pink staining).

[0134] Oil Control Assay: Measurements of reduced sebum secretion and / or reduced sebum production can be performed using standard techniques known to those skilled in the art. In one case, the forehead can be used. Each active ingredient disclosed in the instructions, any combination of ingredients, or a composition having said combinations is applied once or twice daily to a portion of the forehead for a set number of days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more than 14 days), while another portion of the forehead is left untreated with the composition. After the set number of days has elapsed, sebum secretion can be measured by applying fine blotting paper to the treated and untreated forehead skin. This is done by first removing any sebum from the treated and untreated areas with a damp and dry cloth. Blotting paper can then be applied to the treated and untreated areas of the forehead, and an elastic band can be placed around the forehead to gently press the blotting paper onto the skin. After 2 hours, remove the blotting paper, let it dry, and then examine it through the light. Darker blotting paper is associated with more sebum production (or lighter blotting paper is associated with less sebum production).

[0135] Erythema assay: A reduction in skin redness can be assessed using a MINOLTA colorimeter. Applying a 0.2% sodium lauryl sulfate solution to the subject's forearm induces skin erythema. 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 within the red area. Immediately after reading, the area is treated with the active ingredient disclosed in this specification, any combination of ingredients, or a composition having said combination. Measurements can be repeated periodically to determine the formulation's ability to reduce redness and irritation.

[0136] Skin Moisture / Hydration Assay: Skin moisture / hydration can be measured using impedance measurement with the Nova dermal phase meter. The impedance meter measures changes in skin moisture content. The outer layer of skin has unique electrical properties. When skin is dry, its conductivity is poor. As skin becomes more hydrated, its conductivity increases. Therefore, changes in skin impedance (correlation with conductivity) can be used to assess changes in skin hydration. The device can be calibrated according to the instrument's instructions on each test day. Temperature and relative humidity should also be recorded. Subjects can be assessed as follows: Before measurement, they can equilibrate in a room with specified humidity (e.g., 30% to 50%) and temperature (e.g., 68°C to 72°C). Three independent impedance readings can be taken on each side of the face, recorded, and averaged. A T5 setting can be used for the impedance meter, which applies an average impedance value to the face every five seconds. Changes can be reported using statistical variance and significance. Each active ingredient, any combination of ingredients, or composition having said combinations can be determined according to this procedure.

[0137] Determination of Skin Transparency and Reduction of Freckles and Age Spots: Skin transparency and the 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 the Minolta colorimeter to determine the potential for irritation caused by product treatment. The a* value measures the change in skin color in the red zone. This is used to determine whether each active ingredient, any combination of ingredients, or composition having said combinations disclosed in this specification causes irritation. Measurements can be taken on each side of the face and averaged as values ​​for the left and right sides. Skin transparency can also be measured using a Minolta colorimeter. This measurement is a combination of the a*, b, and L values ​​of the Minolta colorimeter, which are related to skin brightness and closely related to skin smoothness and hydration. Skin readings are performed as described above. In a non-limiting aspect, skin transparency can be described as L / C, where C is chromaticity, defined as (a... 2 +b 2 ) 1 / 2 .

[0138] Skin dryness, fine lines, skin smoothness, and skin color assessment: Skin dryness, fine lines, skin smoothness, and skin color can be assessed using clinical grading techniques. For example, clinical grading of skin dryness can be determined using the five-point Kligman scale: (0) Skin is soft and moist; (1) Skin appears normal with no obvious dryness; (2) Skin feels slightly dry with no obvious scaling; (3) Skin feels dry and rough, appears whitish, and has some scaling; (4) Skin feels very dry and rough, appears whitish, and has scaling. Assessments can be performed independently by two clinicians, and the average value can be taken.

[0139] Clinical grading of skin color: Clinical grading of skin color can be performed using a ten-point analog number scale: (10) Uniform pinkish-brown skin. No dullness, redness, or flaking patches when examined with a handheld magnifying glass. The skin texture is very uniform to the touch; (7) Uniform skin color to the naked eye. No flaking areas, but slight discoloration caused by pigmentation or erythema. No discoloration exceeding 1 cm in diameter; (4) Skin discoloration and uneven texture are easily noticeable. Slight flaking. Some areas of skin feel rough to the touch; (1) Uneven skin color and texture. Many areas have flaking and discoloration, whether hypopigmentation, erythema, or melasma. Large areas of uneven color exceeding 1 cm in diameter. The assessment is performed independently by two clinicians and the average value is taken.

[0140] Clinical grading of skin smoothness: Clinical grading of skin smoothness can be analyzed using a ten-point analog number scale: (10) Smooth, moist and shiny skin, no resistance when fingers run across the surface; (7) Relatively smooth, slight resistance; (4) Rough, obvious changes, frictional sensation when rubbed; (1) Rough, flaking, uneven surface. The assessment was conducted independently by two clinicians and the average value was taken.

[0141] Skin smoothness and wrinkle reduction were assessed using the method described by Packman et al. (1978): Skin smoothness and wrinkle reduction can also be visually assessed using the method described by Packman et al. (1978). For example, at each subject visit, the depth, shallowness, and total number of superficial facial lines (SFLs) for each subject can be carefully scored and recorded. Numerical scores are obtained by multiplying a numerical factor by a depth / width / length factor. Scores are given for the eye and mouth areas (left and right sides) and summed together as the total wrinkle score.

[0142] Skin firmness measured 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 ball onto the skin and recording its first two rebound peaks. The ballistometry uses a small, lightweight probe with a relatively blunt probe (4 square millimeters of contact area). The probe penetrates the skin slightly, and the measurement results depend on the characteristics of the outer layers of the skin, which include the stratum corneum, epidermis, and part of the dermis.

[0143] Skin softness / flexibility measurement using the Gas Bearing Electrodynamometer: Skin softness / flexibility can be assessed using the Gas Bearing Electrodynamometer, which measures the stress / strain properties of the skin. The viscoelastic properties of the skin are related to skin hydration. Measurements can be taken by fixing the probe to a predetermined location on the cheek area using double-sided tape. A force of approximately 3.5 gm can be applied parallel to the skin surface, and skin displacement can be accurately measured. Skin flexibility can then be calculated and expressed as DSR (Dynamic Elastic Coefficient in gm / mm).

[0144] Appearance assessment of lines and wrinkles using replicas: Replicas can be used to assess the appearance of fine lines and wrinkles on the skin; the replica is an impression of the skin surface. Silicone rubber-based materials can be used. Replicas can be analyzed through image analysis. Silicone replicas can be taken from the subject's face, and the images of the replicas can be analyzed using a computer image analysis system to objectively quantify changes in the visibility of fine lines and wrinkles. Replicas can be taken from the eye and neck areas, and photographed with a digital camera using low-angle incident light. Image processing programs can be used to analyze the digital images and determine the area of ​​the replica covered by wrinkles or fine lines.

[0145] Skin surface profile determination using a surface photometer / stylus method: The surface profile of the skin can be measured using a surface photometer / stylus method. This involves shining light onto the surface of a replica or tracing a probe across the surface of the replica. The vertical displacement of the probe is fed into a computer via a distance sensor, and after scanning a replica of a fixed length, a cross-sectional analysis of the skin profile can be generated as a two-dimensional curve. This scan can be repeated any number of times along a fixed axis to generate a simulated 3-D image of the skin. Ten random portions of the replica can be obtained using the probe technique and combined to generate 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 average profile height. Rt is the maximum vertical distance between the highest and lowest peaks, and Rz is the average peak amplitude minus the average peak height. These values ​​are calibration values ​​in mm. Before each use, the equipment should be standardized by scanning metal standards with known values. The Ra value can be calculated using the following formula: R a =Standardized roughness; l m = Horizontal (scanning) length; y = Absolute value of the profile position relative to the average profile height (x-axis).

[0146] MELANODERM TM Assay: In other non-limiting respects, it can be achieved by using skin analogues, such as MELANODERM. TMThis is used to evaluate the efficacy of each active ingredient, any combination of ingredients, or composition having said combinations disclosed in this specification. Melanocytes are a type of cell found in skin analogs that stain positively when exposed to the melanin precursor L-dihydroxyphenylalanine (L-DOPA). Skin analog MELANODERM TM Various matrices containing each active ingredient, any combination of ingredients, or compositions having the combinations disclosed in the specification, or the matrix alone, can be used as controls. Alternatively, an untreated sample of the skin mimic can be used as a control.

[0147] Polyfilament production: Changes in polyfilament production in keratinocytes can be measured as a result of each active ingredient, any combination of ingredients, or a composition having said combinations disclosed in this specification. Polyfilament is a precursor to natural moisturizing factor (NMF) in the skin. Increased NMF increases skin hydration. Polyfilament production in treated and untreated keratinocytes can be determined using bioassays that analyze polyfilament concentrations in keratinocyte lysates. A non-limiting example of a bioassay that can be used to quantify polyfilament production is PROTEINSIMPLE® SIMON. TM Western blot protocol. For each sample, normal human epidermal keratinocytes (NHEK) were grown in calcium-containing EPI-200–MATTEK EPILIFE® growth medium from Life Technologies (M-EP-500-CA). Prior to treatment, NHEK was incubated overnight at 37°C in growth medium with 5% CO2. Then, NHEK was incubated for 24 to 36 hours in growth medium containing 1% test compound / extract or without the compound / extract (negative control). NHEK was then washed, collected, and placed on ice or colder until lysed on ice using lysis buffer and sonication. Protein concentrations in the samples can be determined and used to standardize the samples. Lysates can be stored at -80°C until used for quantification.

[0148] PROTEINSIMPLE® SIMON TMWestern blot bioassay employs quantitative Western blot immunoassay, using a primary antibody specific to fimbriaein to quantitatively detect fimbriaein in the test sample. Cell samples are lysed and protein concentrations are normalized. Then, the normalized sample and molecular weight standards are loaded onto a denatured protein separation gel using capillary electrophoresis and run. The proteins in the gel are then immobilized and detected using an immunoprobe with a fimbriae-specific primary antibody. The immobilized proteins can then be immunodetected using an enzyme-linked immunosorbent assay (ELISA) antibody bound to the primary antibody. A chemiluminescent substrate solution can then be added to the immobilized proteins to allow chemiluminescence development, with the intensity of the color development proportional to the amount of fimbriaein bound during immobilization. Chemiluminescence development is stopped at specific times, and the intensity of the chemiluminescent signal can be measured and compared with positive and negative controls.

[0149] Closure protein production: Changes in closure protein production in keratinocytes due to each active ingredient, any combination of ingredients, or a composition having said combinations disclosed in this specification can be measured. Closure proteins are key proteins in the formation of tight junctions and the skin's moisture barrier function. A non-limiting example that can determine closure protein production in treated and untreated keratinocytes is the use of a bioassay analyzing the concentration of closure proteins in keratinocyte lysates. This bioassay can be performed using PROTEINSIMPLE® SIMON. TM Western blotting was performed. For the sample, adult epidermal keratinocytes (HEKa) from Life Technologies (C-005-5C) could be blotted using EPILIFE. TM EPILIFE was grown in the growth medium at 37°C in 5% CO2 for 24 hours. TM The growth medium was supplemented with calcium (M-EP-500-CA) from Life Technologies and keratinocyte growth supplement (HKGS) (S-101-5) from Life Technologies. HEKa samples were then incubated for 24 to 48 hours in growth medium containing the test compound / extract, without the compound / extract (negative control), or containing 1 mM CaCl2 (positive control). The HEKa samples were then washed, collected, and placed on ice or colder until lysed on ice using lysis buffer and sonication. Protein concentrations in the samples can be determined and used for sample standardization. Lysates were stored at -80°C until used for bioassays.

[0150] PROTEINSIMPLE® SIMON TMWestern blot bioassay employs quantitative Western blot immunoassay, using a primary antibody specific to the closure protein to quantitatively detect the closure protein in the test sample. Cell samples are lysed and protein concentrations are normalized. Then, the normalized sample and molecular weight standards are loaded onto a denaturing protein separation gel using capillary electrophoresis and run. The proteins in the gel are then immobilized, and immunoprobes are used to detect them using a primary antibody specific to the closure protein. The immobilized proteins are then immunodetected using an enzyme-linked immunosorbent assay (ELISA) antibody bound to the primary antibody. A chemiluminescent substrate solution is then added to the immobilized proteins so that the chemiluminescence development is proportional to the amount of closure protein bound in the immobilization. Chemiluminescence development can be stopped at specific times, and the intensity of the chemiluminescent signal can be measured and compared with positive and negative controls.

[0151] Keratinocyte monolayer permeability: Changes in keratinocyte monolayer permeability due to each active ingredient, any combination of ingredients, or a composition having said combinations disclosed in this specification can be measured. Keratinocyte monolayer permeability is a measure of skin barrier integrity. As a non-limiting example, MILLIPORE's in vitro vascular permeability assay (ECM642) can be used to determine keratinocyte monolayer permeability in treated and untreated keratinocytes. This assay analyzes endothelial cell adsorption, transport, and permeability. In short, adult epidermal keratinocytes (C-005-5C) from Life Technologies can be seeded onto a porous collagen-coated membrane within collection wells. The keratinocytes are then subjected to EPILIFE... TM The culture medium was incubated at 37°C in 5% CO2 for 24 hours, containing EPILIFE. TMThe growth medium was supplemented with calcium (M-EP-500-CA) from LIFE TECHNOLOGIES and keratinocyte growth supplement (HKGS) (S-101-5) from LIFE TECHNOLOGIES. This culture time allowed cells to form a monolayer and close 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 another 48 hours at 37°C and 5% CO2. To determine the permeability of the keratinocyte monolayer after incubation with / without the test compound / extract, the medium was replaced with fresh medium containing high molecular weight fluorescein isothiocyanate (FITC)-glucan, and the keratinocytes were incubated for 4 hours at 37°C and 5% CO2. During the 4-hour incubation, FITC could pass through the keratinocyte monolayer and porous membrane into the collection wells at a rate proportional to the monolayer permeability. After the 4-hour incubation, cell viability and the FITC content in the collection wells could be determined. For FITC content, the culture medium in the collection wells was collected, and the fluorescence of the culture medium at 480 nm was measured when excited at 520 nm. The percentage of permeability and percentage change compared to the untreated control can be determined by the following formulas: Permeability percentage = ((average Ex / Em of the test sample) / average Ex / Em of the untreated control) * 100; Permeability change percentage = Permeability percentage of the test sample – Permeability percentage of the untreated control.

[0152] Hyaluronic acid production: Changes in hyaluronic acid (HA) production in human dermal fibroblasts can be measured as a result of each active ingredient disclosed in this specification, any combination of ingredients, or a composition having said combinations. HA is a polysaccharide involved in matrix structural stability and in providing turgor pressure to tissues and cells.

[0153] As a non-limiting example, the production of HA in treated and untreated adult dermal fibroblasts (HDFa) can be determined using the R&DSystems Hyaluronic Acid DuoSet ELISA Kit (DY3614). In this assay, to produce samples, nearly confluent HDFa cells (C-13-5C) from Cascade Biologics were cultured for 72 hours at 37°C in 10% CO2 in starvation medium (Dulbecco modified Eagle medium containing 0.15% fetal bovine serum and 1% penicillin-streptomycin solution) prior to treatment. The cells were then incubated for 24 hours with fresh starvation medium and the test compounds, positive controls (phorbol 12-myristate 13-acetate from SIGMA-ALDRICH and platelet-derived growth factor from SIGMA-ALDRICH (P3201)), or without any added substances. The medium was then collected and frozen at -80°C until used for the ELISA assay.

[0154] In short, the ELISA assay employs a quantitative sandwich enzyme immunoassay technique, in which a capture antibody specific for HA is pre-coated onto a microplate. Standards and culture medium from treated and untreated cells are transferred into the wells of the microplate to allow any HA present to be bound by the immobilized antibody. After washing away any unbound material, an HA-specific enzyme-linked detection antibody is added to the wells. After washing to remove any unbound antibody-enzyme reagent, substrate solution is added to the wells to allow color development, the intensity of which is 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 can be measured using a microplate reader.

[0155] As another non-limiting example, human skin explants can be cultured in viable explant medium at 37°C under a humid atmosphere supplemented with 5% CO2. The explants can be treated by topical application of the sample product (n=3) on days D0, D2, D3, D6, D8, and D9. Control explants (n=3) are treated without any changes except for replacement of the viable explant medium. Half the volume of viable medium can be replaced on days D3, D6, and D8. On day D9, three explants from each condition are harvested and cut in half. One half of the explants is fixed in buffered formalin, and the other half is frozen at -80°C.

[0156] After fixation in plain Bouin for 48 hours and in formalin for 24 hours, the samples were dried and paraffin-embedded using an automated tissue processor, Leica TP 1020. Five-micron sections were prepared using a microtome (Leica RM2125 Minotaur) and mounted on a SUPERFROST. TMOn histological slides. Microscopic observation can be performed using an optical microscope, employing a LEICA ORTHOPLAN or LEICA DM LB microscope. Images can be captured using an OLYMPUS DP72 camera and CELL^D software. General morphology can be examined on paraffin sections stained with Masson's trichrome Goldner variant. Hyaluronic acid staining can be performed at room temperature with 1 / 100 diluted anti-hyaluronic acid biotinylated protein (HABP) (SEIKAGAKU, catalog number 400763-1A) and a magnification system biotin / streptavidin (VECTOR, VECTASTAIN PK-7200) for 1 hour.

[0157] Inhibition of hyaluronidase activity: Changes in hyaluronidase activity due to each active ingredient, any combination of ingredients, or a composition having said combinations disclosed in this specification can be measured. Hyaluronidase is an enzyme that degrades HA. HA is a polysaccharide involved in matrix structural stability and in providing turgor pressure for 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, 1-S type hyaluronidase from SIGMA-ALDRICH (H3506) is added to microplate reaction wells containing the test compound or control. Tannic acid can be used as a positive control inhibitor, the control enzyme is not added to the test compound, and wells containing the test compound or positive control but not hyaluronidase serve as background negative controls. The wells are incubated at 37°C for 10 minutes before adding the substrate (HA). The substrate is added, and the reaction is incubated at 37°C for 45 minutes. A portion of each reaction solution was then transferred to a solution of sodium acetate and acetic acid at pH 3.75 and gently mixed to stop that portion of the reaction (terminus well). The terminus well and reaction well should contain the same volume of solution after a portion of the reaction solution was added to the terminus well. Both reaction wells and terminus wells were incubated at room temperature for 10 minutes. The absorbance of the reaction wells and terminus wells at 600 nm was then measured. The inhibition rate can be calculated using the following formulas: Inhibitor (or control) activity = (Absorbance of inhibitor terminus well at 600 nm – Absorbance of inhibitor reaction well at 600 nm); Initial activity = Absorbance of control enzyme at 600 nm; Inhibition percentage = [(Initial activity / Inhibitor activity) * 100] - 100.

[0158] Peroxisome proliferator-activated receptor γ (PPAR-γ) activity: Changes in PPAR-γ activity due to each active ingredient, any combination of ingredients, or a composition having said combinations disclosed in this specification can be measured. PPAR-γ is a key receptor for sebum production. As a non-limiting example, PPAR-γ activity can be determined using a bioassay analyzing the ability of a compound or composition to inhibit ligand binding. In short, fluorescent small molecule pan-PPAR ligand, FLUORMONE. TM Pan-PPAR Green, available from Life Technologies (PV4894), can be used to determine whether a test compound or composition can inhibit the binding of a ligand to PPAR-γ. The sample wells contain PPAR-γ and the fluorescent ligand, as well as: the test compound or composition (test); a reference inhibitor, rosiglitazone (positive control); or no test compound (negative control). The wells are incubated for a predetermined time to allow the ligand to bind to PPAR-γ. The fluorescence polarization of each sample well can then be measured and compared to the negative control wells to determine the percentage of inhibition by the test compound or composition.

[0159] Endothelial tube formation: Endothelial tube formation is involved in angiogenesis and microvascular capillary formation. Capillary formation and angiogenesis may contribute to skin redness and rosacea. In the presence or absence of test extracts and compounds, the ability of endothelial cells to form tubes can be determined using a capillary disruption assay, which utilizes pre-formed primary human umbilical vein endothelial cells (HUVECs) in a cell culture system.

[0160] In short, culturing HUVECs in vitro on the extracellular matrix stimulates endothelial cell attachment and tubular morphogenesis to form capillary-like lumen structures. These in vitro-formed capillary tubes resemble human vascular capillaries in many ways. Capillary tube assays are based on this phenomenon and are used to evaluate potential agents targeting the vascular system.

[0161] HUVEC cultures were grown in a 5% CO2, 37°C cell culture incubator. The complete growth medium for HUVECs was endothelial cell 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-amphotericidal). All assays were performed using HUVEC cultures from passage 3 to passage 8.

[0162] HUVECs were pre-labeled with the fluorescent agent calcein AM and seeded in complete growth medium into extracellular matrix-coated 96-well culture plates. Endothelial capillaries should form approximately four hours after morphogenesis. Then, a 50 µl volume of the designed dose of the test agent was administered to the formed capillary culture as a treatment. An untreated control could be added with a carrier of the test agent. The FDA-approved anti-angiogenic drug SUTENT... ® The concentration of the sample can be used as a performance control for the assay. Approximately six hours after treatment, the morphology of the endothelial tubules in each well is examined under a microscope for imaging, and the capillary disruptive activity under the treatment conditions is quantitatively analyzed. Each test condition can be performed in duplicate wells, including controls.

[0163] * * * * * * * * * * * * * *

[0164] According to this disclosure, all compositions and / or methods disclosed and claimed herein can be prepared and performed without excessive experimentation. While the compositions and methods of the present invention have been described according to preferred embodiments, it will be apparent to those skilled in the art that changes can be made to the said compositions and / or methods, and to the steps or sequence of steps of the methods described herein, without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically relevant reagents can be substituted for the reagents described herein while achieving the same or similar results. All such similar substitutions and modifications that are apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the invention as defined by the appended claims.

Claims

1. A method for treating skin in need of treatment, the method comprising topically applying a combination of snow lotus extract, alpine rose rhododendron extract, and evening primrose oil to the skin, wherein the skin is treated.

2. The method according to claim 1, wherein the composition further comprises squalene.

3. The method of claim 1, wherein the composition further comprises: Caprylic / capric triglycerides; Hexyldecyl alcohol; Bisabolol; N-palmitoylhydroxyproline cetyl ester; Stearic acid; BHT; and Brassic sterols.

4. The method of claim 1, wherein the composition further comprises: 0.0001% to 0.1% by weight of snow lotus extract; 0.0001% to 0.1% by weight of alpine rose rhododendron extract; and Evening primrose oil, 0.01% to 5.0% by weight.

5. The method of claim 2, wherein the composition further comprises 75.0% to 95.0% by weight of squalene.

6. The method of claim 3, wherein the composition further comprises: Caprylic / capric triglycerides, ranging from 5.0% to 10.0% by weight; 0.1% to 5.0% by weight of hexyldecyl alcohol; 0.001% to 1.0% by weight of bisabolol; 0.001% to 1.0% by weight of N-palmitoylhydroxyproline cetyl ester; 0.0001% to 0.1% by weight of stearic acid; 0.0001% to 0.1% by weight of BHT; and Brassic sterols, ranging from 0.0001% to 0.1% by weight.

7. The method according to claim 1, wherein the composition further comprises olive fruit oil, tocopheryl acetate, or tocopherol.

8. The method of claim 7, wherein the composition further comprises: Olive fruit oil from 0.01% to 5.0% by weight; and 0.01% to 5.0% by weight of tocopherol acetate or tocopherol.

9. The method of claim 1, wherein the composition moisturizes and protects the skin barrier.

10. The method of claim 1, wherein the composition protects skin proteins from oxidative stress-induced damage.

11. The method of claim 1, wherein the composition reduces uneven skin tone.

12. The method of claim 1, wherein the composition supports collagen production.

13. The method of claim 1, wherein the composition remains stable for 12 weeks when stored at a maximum temperature of 45°C.

14. The method of claim 1, wherein the composition is an oil.

15. A topical skin care composition, said composition comprising: Snow lotus extract; Alpine rose rhododendron extract; and Evening primrose oil.

16. The composition of claim 15, wherein the composition further comprises: Squalene; Caprylic / capric triglycerides; Hexyldecyl alcohol; Bisabolol; N-palmitoylhydroxyproline cetyl ester; Stearic acid; BHT; and Brassic sterols.

17. The composition according to claim 15, further comprising olive fruit oil, tocopheryl acetate, or tocopherol.

18. The composition according to claim 15, further comprising: 0.0001% to 0.1% by weight of snow lotus extract; 0.0001% to 0.1% by weight of alpine rose rhododendron extract; and Evening primrose oil, 0.01% to 5.0% by weight.

19. The composition of claim 16, further comprising: 75.0% to 95.0% by weight of squalene; Caprylic / capric triglycerides, ranging from 5.0% to 10.0% by weight; 0.1% to 5.0% by weight of hexyldecyl alcohol; 0.001% to 1.0% by weight of bisabolol; 0.001% to 1.0% by weight of N-palmitoylhydroxyproline cetyl ester; 0.0001% to 0.1% by weight of stearic acid; 0.0001% to 0.1% by weight of BHT; and Brassic sterols, ranging from 0.0001% to 0.1% by weight.

20. The composition of claim 17, further comprising: Olive fruit oil from 0.01% to 5.0% by weight; and 0.01% to 5.0% by weight of tocopherol acetate or tocopherol.

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