WO2016169373A1 - 包含青藤碱的局部组合物及用于抑制蛋白羰基化的方法 - Google Patents

包含青藤碱的局部组合物及用于抑制蛋白羰基化的方法 Download PDF

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WO2016169373A1
WO2016169373A1 PCT/CN2016/076825 CN2016076825W WO2016169373A1 WO 2016169373 A1 WO2016169373 A1 WO 2016169373A1 CN 2016076825 W CN2016076825 W CN 2016076825W WO 2016169373 A1 WO2016169373 A1 WO 2016169373A1
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sinomenine
topical composition
skin
subject
carbonylation
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English (en)
French (fr)
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韩强
张翌
李廷钊
杜军
李波
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Access Business Group International LLC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/473—Quinolines; Isoquinolines ortho- or peri-condensed with carbocyclic ring systems, e.g. acridines, phenanthridines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/485—Morphinan derivatives, e.g. morphine, codeine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin

Definitions

  • the present invention generally relates to a sinomenine-containing topical composition for inhibiting skin protein carbonylation in a subject, and more particularly to a topical composition comprising sinomenine and inhibiting the skin protein carbonyl of a subject using the topical composition (or denaturation) method.
  • a major contributor to skin yellowing associated with aging is believed to be the protein denaturation process known as "carbonylation" in the dermis.
  • protein denaturation is the result of binding between lipid degradation products produced by ultraviolet ("UV)" ray damage and dermal proteins.
  • UV ultraviolet
  • the resulting discoloration is a major factor contributing to the yellowing of the skin with the age of the individual and is particularly problematic for Asians.
  • a lipid can interact with an aldehyde that causes carbonylation of the dermal protein, resulting in a carbonylated protein.
  • sugars can cause saccharification of dermal proteins, resulting in glycated proteins. It is believed that carbonylation produces a yellower skin relative to saccharification.
  • a topical composition is disclosed.
  • the topical composition can be used on the skin of a subject.
  • the topical composition comprises sinomenine.
  • the sinomenine is present in an amount effective to inhibit skin protein carbonylation in the subject after administration.
  • the active ingredient of the topical composition consists of sinomenine.
  • the topical composition consists of sinomenine.
  • a cosmetic method of inhibiting skin protein carbonylation in a subject includes the step of applying sinomenine to the skin of the subject.
  • the topical compositions are typically applied in an amount and/or time sufficient to inhibit skin protein carbonylation.
  • topical compositions are also used for skin lightening (or whitening), particularly for inhibiting melanin synthesis, eliminating melanin and/or inhibiting tyrosinase.
  • Figure 1 is a line graph illustrating cytotoxicity data ("Data 1");
  • Figure 2 is a line graph illustrating carbonylation inhibition data ("data 2");
  • Figure 3 is another line graph illustrating cytotoxicity data ("Data 3");
  • Figure 5 is a line graph illustrating melanin elimination rate data ("data 5");
  • Figure 6 is a line graph illustrating tyrosinase inhibition rate data ("data 6").
  • composition for inhibiting skin protein carbonylation. More specifically, the composition can be used to inhibit skin protein carbonyl in a subject Chemical.
  • the skin layer includes the epidermis and the dermis, and protein carbonylation generally occurs in at least the dermis.
  • the composition is applied directly to the epidermis, such as the stratum corneum.
  • the composition can also be applied indirectly to the epidermis, such as by a patch that carries/delivers the composition to the skin. At least a portion of the composition or active ingredient thereof, such as sinomenine, migrates from the stratum corneum through the epidermis and to the dermis to inhibit protein carbonylation therein.
  • Subjects are usually human and can include men and women of all ages.
  • the composition is not limited to the location of the skin of a particular subject or subject. For example, a person can apply the composition to his face, neck, arms, hands, chest, torso, legs, feet, and the like, or any combination thereof. Such areas of the skin may be damaged or not damaged. Black spots, yellowing, sagging, and/or wrinkles generally indicate skin damage, such as UV rays or light damage. Methods of using the compositions are further described below.
  • compositions of the present disclosure generally comprise extracts from plants of the genus Geochelone, more typically from plants of the species Sinomenium acutum.
  • sinomenine is the active substance of the extract and is believed to be an effective compound for inhibiting protein carbonylation.
  • reference to the genus Wind Dragon can be used interchangeably with Wind Dragon, and vice versa. The same is true for the mention of sinomenine and genus (or wind dragon) extract.
  • embodiments of the present disclosure generally relate to topical compositions comprising an extract of a plant of the genus Rhododendron, such as an extract of a plant of the genus Dragon, wherein the sinomenine is sufficient to reduce/inhibit protein carbonylation in the subject after administration.
  • the amount exists.
  • the composition comprises sinomenine.
  • the active ingredient of the composition consists of sinomenine.
  • the composition may further consist of one or more additional components and/or inactive components.
  • the composition consists of sinomenine.
  • the composition can include plant material from the genus Geochelone, including plant material from the genus.
  • the wind dragon is a genus of the family. It includes only one known species, Wind Dragon, produced in China, northern India, Nepal, Japan and northern Thailand.
  • the wind dragon can also be called the Sinomenium acutum (Thunb.) Rehd.et Wils.
  • the active substances of the wind dragon include alkaloids such as sinomenine, isoflavone, sinomenine, L-paraffin and chlorhexidine, and ⁇ -phytosterol and stigmasterol.
  • sinomenine is considered to be an effective compound for inhibiting protein carbonylation (or denaturation).
  • One or more other active substances may also be used for this effect, but it is considered that sinomenine is most effective for protein carbonylation inhibition.
  • Sinomenine CAS Number 115-53-7, molecular weight ⁇ 329g / mol, and the molecular formula C 19 H 23 NO 4.
  • Sinomenine can be obtained commercially as a standard sample or can be obtained by extracting a material of the genus Rhododendron.
  • Standard samples of sinomenine may have a purity of at least about 90%, at least about 95%, at least about 99%, and up to 99.99%. Other levels of purity are also available and used.
  • Genus plant can be used to obtain sinomenine for use in the composition, including but not limited to roots, stems, rhizomes, leaves, flowers, fruits, and/or extracts of these portions.
  • Sinomenine is generally present in the roots of the wind dragon, whereby root extracts are generally most useful for the purposes of the present disclosure.
  • the genus Rhododendron can be used in its original form, in a suspended form, in a dehydrated form, in a concentrated form or in the form of an extract.
  • the genus Dragon is in a dry or liquid form.
  • the other components of the extract and/or composition of the genus Dragon can be obtained by conventional extraction methods known in the art, such as extraction by water (e.g., steam) or by solvent (e.g., alcohol).
  • extraction methods known in the art, such as extraction by water (e.g., steam) or by solvent (e.g., alcohol).
  • solvent e.g., alcohol
  • the compositions of the present disclosure are not limited to a particular extraction method and are not necessarily extracted because sinomenine can be readily obtained from many commercial suppliers. An exemplary extraction method is described below.
  • the composition comprises an extract from the genus.
  • “Dragonfly extract” generally refers to extracts from the genus Rhododendron, including from the genus. Wind Dragon extracts are commercially available from a variety of sources.
  • suitable extracts of the genus Rhododendron can be obtained by using any conventional extraction technique.
  • a polar solvent such as an alcohol (for example methanol, ethanol, butylene glycol), an ether (for example diethyl ether), a ketone (for example acetone), an ester (for example ethyl acetate), water or a mixture thereof is available.
  • a polar solvent such as an alcohol (for example methanol, ethanol, butylene glycol), an ether (for example diethyl ether), a ketone (for example acetone), an ester (for example ethyl acetate), water or a mixture thereof.
  • Suitable non-polar solvents include, but are not limited to, ethyl acetate, hexane, dichloromethane, chloroform or mixtures thereof.
  • extraction methods that can be used to produce extracts suitable for the composition. These methods include, but are not limited to, the extraction methods disclosed in U.S. Patent No. 7,897,184 to Rana, et al. While the extraction solvent described specifically refers to ethanol, it should be understood that other alcohols such as, but not limited to, isopropanol, ethyl alcohol, and/or methyl alcohol may be used in addition to or in place of ethanol.
  • Exemplary alcoholic solvents include, but are not limited to, a C 1 to C 4 alcohols such as methanol, ethanol, propanol, isopropanol and butanol; water - alcohol or mixture of alcohols and water, including water - ethanol (hydro-ethanol); Multi Hydroxy alcohols such as propylene glycol and butylene glycol; and fatty alcohols. Any of these alcohol solvents can be used. Other solvents such as, but not limited to, acetone can also be used as the extraction solvent. Any ratio of solvent-water admixtures such as alcohol-water and/or acetone-water admixtures can also be used.
  • the extract of the genus Rhododendron can be obtained using an organic solvent extraction technique.
  • continuous extraction of the solvent can be used to obtain the extract of the genus.
  • the total water-ethanol extraction technique it is called a one-time extraction.
  • the extract produced in this process will contain a wide range of phytochemicals present in the extracted material, including fat soluble and water soluble phytochemicals. After collecting the extraction solution, the solvent was evaporated to obtain an extract.
  • Total ethanol extraction can also be used. This technique uses ethanol as a solvent.
  • the extract produced by the extraction technique may include a fat-soluble and/or lipophilic compound in addition to the water-soluble compound.
  • Total methanol extraction can also be used in a similar manner with similar results.
  • the extract of the genus Rhododendron is obtained by extracting the plant material of the genus.
  • SFE supercritical carbon dioxide supercritical fluid extraction
  • the material to be extracted is not exposed to any organic solvent. Rather, the extraction solvent is carbon dioxide (CO 2 ) in a supercritical state (eg, >31.3 ° C and >73.8 bar) with or without a conditioning agent.
  • CO 2 carbon dioxide
  • a supercritical state eg, >31.3 ° C and >73.8 bar
  • temperature and pressure conditions can be varied to achieve optimal extract yield.
  • This technique produces extracts of fat-soluble and/or lipophilic compounds similar to total hexane and ethyl acetate extraction techniques, which can also be used.
  • the extract of the genus Dioscorea can be added to the composition in any amount, provided that it is present in an amount effective to inhibit protein carbonylation.
  • sinomenine is present in an amount effective to reduce the formation of carbonyl derivatives of the skin of the subject.
  • carbonyl derivatives typically comprise an aldehyde, a ketone or a combination thereof.
  • sinomenine is present in an amount from about 5 to about 500, or from about 25 to about 350, g/mL of the topical composition. In a particular embodiment, the sinomenine is present in an amount of from about 10, about 20, about 25, about 50, about 75, about 100, about 200, or about 300 [mu]g/mL of the composition. Individual subranges and amounts of from about 5 to about 500 [mu]g/mL of composition, as well as amounts less than or greater than these amounts, are also contemplated.
  • sinomenine present in the composition can depend on several factors, including the level of protein carbonylation inhibition desired, the level of protein carbonylation inhibition in a particular extract or composition, and other factors.
  • sinomenine is present in an amount from about 0.01 to about 20 pbw based on 100 parts by weight ("pbw") of the composition.
  • the sinomenine is present in an amount of from about 0.05 to about 10 pbw based on the 100 pbw of the composition.
  • Other extracts or ingredients, which are optionally used in the compositions of the present disclosure, are described in U.S.
  • Patent No. 5,747,006 to Dornoff et al. and U.S. Patent Nos. 5,980,904, 6,994,874, 7, 060,304, 7, 247, 321 and 7, 364, 759 to Leverett et al. Into this article.
  • the composition is free of other actives.
  • "Other active substances” herein generally means that the composition does not contain other types of traditional Chinese medicines (“TCM”; or “Chinese medicine”) other than the genus Rhododendron.
  • TCM traditional Chinese medicines
  • Other types of TCM are understood in the art. Examples of other types of TCMs are generally described as "biologically active substances" in International Publication No. WO 01/22934 A2, the contents of which are incorporated herein in entirety by reference.
  • the composition may comprise the following inactive materials. If used, inactive materials are different from other types of TCM.
  • compositions can be formulated to contain a cosmetically acceptable carrier (or vehicle) and prepared and/or packaged and labeled to inhibit protein carbonylation, reduce skin yellowing, and/or skin lightening (or whitening).
  • a cosmetically acceptable carrier or vehicle
  • the composition can be administered topically.
  • cosmetically acceptable carriers include, but are not limited to, water, glycerin, waxes, various alcohols such as ethanol, propanol, vegetable oils, mineral oils, silicones such as silicone oils, fatty esters, fatty alcohols, glycols, polyglycols or Any combination thereof.
  • Such components are generally considered to be inactive components.
  • the final composition may be in any form suitable for topical application to the skin such as, but not limited to, aerosol sprays, gels, creams, dispersions, lotions, foams, liquids, lotions, mousses, patches, balms, Powder, pump spray, solid, solution, stick or towel.
  • the emulsions may include oil-in-water emulsions, water-in-oil emulsions, and water-in-silicone emulsions.
  • compositions of the present disclosure can be prepared using a variety of methods known in the art.
  • the method of preparation comprises the step of extracting the plant material of the genus Dioscorea zingiberensis to obtain the extract of fenglong, i.e., obtaining sinomenine (with or without other active compounds).
  • the method of preparation further comprises the step of combining one or more of sinomenine with a cosmetically acceptable carrier such as the above carrier.
  • the components can be combined using conventional production methods and devices such as mixers, blenders, and the like.
  • the composition can be used in various ways to inhibit protein carbonylation.
  • a cosmetic method for inhibiting skin protein carbonylation in a subject includes the step of applying sinomenine to the skin of the subject.
  • Sinomenine can be applied in a variety of ways, including applying the composition to the skin of a subject, wherein the composition comprises an extract from the species.
  • the composition can be applied directly or indirectly to the skin, such as by hand, applicator, patch, and the like.
  • the composition can be applied as needed, daily, several times a day or any suitable protocol to achieve the desired result.
  • the frequency of topical application can depend on several factors, including the level of protein carbonylation inhibition desired.
  • the regimen includes applying the composition to the skin once or twice daily to include morning application and/or evening application.
  • the amount of the composition applied to the skin in each application may depend on several factors, including the level of the desired result and the particular composition.
  • oxidative stress is associated with the production of reactive oxygen species by protein carbonylation, a process in which a reactive aldehyde or ketone is introduced into a protein by oxidation.
  • the protein carbonyl is the major product of protein oxidation and can be formed by oxidative cleavage of the protein, direct oxidation of the amino acid residue, or covalent reaction with an aldehyde derived from lipid peroxidation.
  • Protein carbonylation occurs generally by direct metal catalyzed oxidation of the amino acid side chain ("primary protein carbonylation") or by addition of a reactive aldehyde to the amino acid side chain (“secondary protein carbonylation”). Oxidative degradation of polyunsaturated fatty acids that are ubiquitous in the human body can lead to the formation of various aldehydes such as acrolein and 4-hydroxyfurfural (“4-HNE”). Studies have shown that major protein carbonylation plays a role in reactive oxygen species (“ROS”) signaling mechanisms. Specifically, major protein carbonylation mediates cellular signaling and major protein carbonylation is reversible.
  • ROS reactive oxygen species
  • Major protein carbonylation involves direct metal catalyzed oxidation of amino acid side chains, particularly on four susceptible amino acid residues: valine, arginine, lysine, and threonine.
  • Oxidation of the proline or arginine side chain results in the formation of a glutamic acid semialdehyde, while lysine oxidizes to produce an aminoadipate semialdehyde, thereby introducing a carbonyl group into the protein structure.
  • Oxidation also converts the hydroxyl group of the threonine side chain to a carbonyl group to form 2-amino-3-butanone acid.
  • the secondary protein carbonylation mechanism involves a Michael addition reaction of a reactive aldehyde with a side chain of lysine, histidine and a cysteine residue, and a substitution of a reducing sugar with an amino group on lysine and arginine.
  • Baseline leading to high carbonyl The increase in the final product of the base.
  • Protein carbonyls are found in a variety of oxidative stress and disease conditions. In addition to protein inactivation, carbonylated cellular proteins undergo proteasome-dependent degradation. It is believed that major protein carbonylation plays a role in labeling damaged proteins during oxidative stress to eliminate them from biological systems. The formation of carbonyl proteins is a hallmark of oxidative stress, for example as an external factor, such as UV radiation induced factors or external application of oxidizing chemicals, or as an intrinsic factor, a reactive carbonyl derived from lipid peroxide degradation. Chemical attack.
  • UV rays or light damaged skin are characterized by various features such as dark spots, wrinkles and sagging. Older people, especially Asians, tend to show changes in the color of yellow skin that age with light. In China, yellowing generally means lack of pink or dull complexion. Common clinical signs of photodamage or aging include the appearance of a characteristic pale or yellow skin. Face yellow is often irreversible, so prevention is ideal.
  • sinomenine has no cytotoxic risk based on safety assessment by CTG cell levels (see data / Figure 1). Moreover, the inventors have discovered that sinomenine has the efficacy and function of skin anti-carbonylation based on in vitro bioassay data (see data / Figure 2). Cell-based assays by model cell HepG2 were performed and inhibition of carbonylation activity was monitored. Vitamin C is considered to be a positive control for carbonylation inhibition. Those skilled in the art understand this type of model cell HepG2 cell-based assay.
  • Luminescent cell viability assay is a uniform method for determining the number of viable cells in culture, based on the quantification of ATP present as an indicator of metabolically active cells.
  • Sinomenine has no cytotoxic risk based on the safety evaluation of CTG cell levels as illustrated by Data 1 and Figure 1.
  • sinomenine has outstanding anti-carbonylation inhibitory potency compared to the positive control vitamin C in the cellular HepG2 model without the risk of cytotoxicity. This data set is generally obtained by the assay methods listed below.
  • This program describes a standard method for the anti-oxidation assay of HepG2 cells.
  • the assay plates were incubated for 24 hours at 37 ° C, 5% CO 2 under humidified conditions.
  • HBSS Hanks Balanced Salt Solution
  • human skin coloration is determined by the amount and location of melanin on the surface of the skin.
  • Melanin is synthesized by oxidation of amino acid tyrosine to L-3,4-dihydroxyphenylalanine ("L-DOPA") in cells, commonly referred to as melanocytes, present at the dermal-epidermal junction. The oxidation process is catalyzed by tyrosinase.
  • melanogenesis A series of cellular processes by melanocytes are commonly referred to as melanogenesis.
  • Skin coloration is regulated by the amount and type of melanin synthesized by melanocytes. Environmental factors can also affect skin color. A healthy amount of melanin in the skin effectively absorbs UV rays. When the skin is exposed to UV radiation, it generally increases the amount and rate of melanin production and can result in skin blackening or "brown”.
  • Local or general pigmentation disorders such as hyperpigmentation or hypopigmentation, can be derived from a number of factors, including hormone levels in the body, diet, genetic disorders, and medications. Common pigmentation disorders include melasma, freckles, and leukoplakia.
  • compositions have been formulated to address pigmentation disorders and have been contemplated, for example, for treating hyperpigmentation and/or hypopigmentation. Such treatments are often referred to as "skin brightening,” “skin whitening,” or “skin whitening.”
  • skin lightening agents For example, lightening age spots (freckles or aging spots), or preventing blackening of Caucasian and Asian skins (ie, maintaining a bright/white complexion).
  • Some of these formulations have included tyrosinase inhibitors such as hydroquinone, vitamin C, kojic acid, arbutin, glutathione, cysteine, lactic acid, ferulic acid, nicotinamide, and plant extracts.
  • tyrosinase inhibitors such as hydroquinone, vitamin C, kojic acid, arbutin, glutathione, cysteine, lactic acid, ferulic acid, nicotinamide, and plant extracts.
  • sinomenine was found to have efficacy in skin lightening and depigmentation.
  • Assays for this type of model cell-based B16F10 cells are known to those skilled in the art.
  • Arbutin is considered to be a positive control for both melanin synthesis inhibition and tyrosinase activity inhibition, while hydroquinone is used as a positive control for the existing melanin-eliminating activity.
  • sinomenine has the ability to strongly interrupt or reduce the melanin synthesis process in B16F10 cells. This data set is generally obtained by the assay methods listed below.
  • This program describes a standard method for the inhibition of melanin synthesis in B16 F10 cells.
  • the assay plates were incubated for 24 hours at 37 ° C, 5% CO 2 under humidified conditions.
  • % inhibition (maximum signal - compound signal) / (maximum signal - minimum signal) x 100.
  • This program describes a standard method for the determination of melanin in B16 cells.
  • the assay plates were incubated for 24 hours at 37 ° C, 5% CO 2 under humidified conditions.
  • the assay plates were incubated at 37 ° C, 5% CO 2 for 72 hours under humidified conditions.
  • % inhibition (maximum signal - compound signal) / (maximum signal - minimum signal) x 100.
  • Sinomenine exerts excellent tyrosinase inhibitory activity in B16F10 cells, and the inhibition rate is 32.4% at a concentration of 100 ⁇ g/mL. Specifically, as shown in Data 6 and FIG. 6, sinomenine exhibited much better or much better arbutin in tyrosinase inhibitory activity in B16F10 cells.
  • sinomenine has shown great ability to eliminate the presence of melanin assay models.
  • sinomenine has excellent activity as a skin whitening and depigmenting agent, and there is no risk of cytotoxicity for skin care or topical use.
  • the range of "0.1 to 0.9" can be further depicted as a smaller one-third, ie 0.1 to 0.3, the middle third, ie 0.4 to 0.6, and the larger one, ie 0.7 to 0.9, which is individual and generally within the scope of the appended claims, and may be relied upon and provided with sufficient support for the specific embodiments within the scope of the appended claims.
  • languages that define or modify a range such as “at least”, “greater than”, “less than”, “not greater than”, etc., it is to be understood that such language includes sub-ranges and/or upper or lower limits.
  • a range of "at least 10" inherently includes a subrange of at least 10 to 35, a subrange of at least 10 to 25, a subrange of at least 25 to 35, and the like, and each subrange may be in an individual and/or population Sufficient support is provided for the specific embodiments within the scope of the appended claims.
  • individual values within the scope of the disclosure may be relied upon and provide sufficient support for the specific embodiments within the scope of the appended claims.
  • a range of "1 to 9" includes each individual integer, such as 3, and an individual value including a decimal point (or fraction), such as 4.1, which may be relied upon and provide sufficient support for a particular embodiment within the scope of the appended claims. .

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Abstract

用于抑制皮肤蛋白羰基化的局部组合物、其制备方法和用途。所述局部组合物包含青藤碱作为活性成分。所述局部组合物的制备方法中包含从风龙种植物中获得青藤碱的步骤。

Description

包含青藤碱的局部组合物及用于抑制蛋白羰基化的方法 发明领域
本发明大体涉及用于抑制受试者皮肤蛋白羰基化的包含青藤碱的局部组合物，并且更具体涉及包含青藤碱的局部组合物以及使用所述局部组合物抑制受试者皮肤蛋白羰基化(或变性)的方法。
相关技术描述
认为与衰老相关的皮肤黄化的主要促成因素是真皮中称为“羰基化”的蛋白变性过程。研究显示，蛋白变性是紫外线(“UV”)射线损伤产生的脂质降解产物与真皮蛋白之间结合的结果。导致的脱色是促成皮肤随个人年龄而黄化的主要因素，并且对于亚洲人特别成问题。
作为前述过程的实例，脂质可以与导致真皮蛋白羰基化的醛相互作用，导致羰基化蛋白。以相似方式，糖类可以导致真皮蛋白的糖化，得到糖化的蛋白。认为羰基化相对于糖化产生更黄化的皮肤。
作为预防措施，已经进行努力以通过局部防晒组合物防止UV射线损伤。作为校正措施，已经进行努力以通过局部皮肤增亮(或增白)组合物逆转皮肤黄化。不幸的是，此类组合物要求进行中的应用，并且需要使用者顺从性有效。此外，这些组合物中的一些可以刺激皮肤。皮肤刺激或其他应用问题，例如不希望的油脂性，将引起使用者停止使用组合物或者以对于其预期目的无效的方式使用组合物。
鉴于上文，依然存在提供抑制皮肤黄化的其他局部组合物。还存在逆转、减缓或预防皮肤羰基化的机会。
发明概述
公开了一种局部组合物。所述局部组合物可在受试者皮肤上使用。所述局部组合物包含青藤碱。所述青藤碱以施用后有效抑制受试者皮肤蛋白羰基化的量存在。在某些实施方案中，所述局部组合物的活性成分由青藤碱组成。在具体实施方案中，所述局部组合物由青藤碱组成。
还公开了一种抑制受试者皮肤蛋白羰基化的美容方法。该美容方法包括应用青藤碱至受试者皮肤的步骤。所述局部组合物一般以足以抑制皮肤蛋白羰基化的量和/或时间应用。不同于蛋白羰基化抑制，局部组合物还用于皮肤增亮(或增白)，特别用于抑制黑色素合成、消除黑色素和/或抑制酪氨酸酶。
附图简述
本公开的其他优点将容易理解，因为通过参考以下详述并结合考虑附图而变得更好理解，附图中：
图1是示例说明细胞毒性数据(“数据1”)的线图；
图2是示例说明羰基化抑制数据(“数据2”)的线图；
图3是示例说明细胞毒性数据(“数据3”)的另一线图；
图4是示例说明黑色素合成抑制数据(“数据4”)的线图；
图5是示例说明黑色素消除率数据(“数据5”)的线图；和
图6是示例说明酪氨酸酶抑制率数据(“数据6”)的线图。
发明详述
公开了一种局部组合物(“组合物”)。所述组合物用于抑制皮肤蛋白羰基化。更具体地，所述组合物可用于抑制受试者皮肤蛋白羰基 化。皮肤层包括表皮和真皮，蛋白羰基化一般发生于至少真皮。通常，将所述组合物直接应用至表皮，例如角质层。还可以将所述组合物间接应用至表皮，例如通过携载/递送所述组合物至皮肤的贴片。至少一部分所述组合物或其活性成分，例如青藤碱，从角质层迁移穿过表皮并到达真皮以抑制其中的蛋白羰基化。
受试者通常是人，并且可以包括各年龄的男人和女人。所述组合物不限于特定受试者或受试者的皮肤位置。例如，一个人可以将所述组合物应用至其脸、颈、臂、手、胸、躯干、腿、脚等或其任意组合。此类皮肤区域可以受损或没有受损。黑斑、黄化、下垂和/或皱纹一般指示皮肤损伤，例如UV射线或光损伤。使用所述组合物的方法在下文进一步描述。
令人惊讶地发现风龙属(genus Sinomenium)可用于抑制皮肤的蛋白羰基化。具体地，不受束缚或受限于任何特定理论，认为风龙属的提取物减少和/或抑制皮肤蛋白羰基化。本公开的组合物一般包含来自风龙属的植物、更通常来自风龙(Sinomenium acutum)种的植物的提取物。如下文描述的，青藤碱是所述提取物的活性物质，并且据信是用于抑制蛋白羰基化的有效化合物。如本文使用的，对风龙属的提及可与风龙互换地使用，并且反之亦然。对青藤碱和风龙属(或风龙)提取物的提及也是一样。基于本文发现，本公开的实施方案一般涉及局部组合物，包含风龙属植物的提取物，例如风龙种植物的提取物，其中青藤碱以施用后足以减少/抑制受试者蛋白羰基化的量存在。
在各个实施方案中，所述组合物包含青藤碱。在进一步实施方案中，所述组合物的活性成分由青藤碱组成。在这些实施方案中，所述组合物可以进一步由一种或多种附加组分和/或非活性组分组成。在某些实施方案中，所述组合物由青藤碱组成。在所有这些实施方案中，所述组合物可以包括来自风龙属的植物材料，包括来自风龙种的植物材料。
风龙属是防己科植物属。其仅包括一种已知的种，风龙，产自中国、印度北部、尼泊尔、日本和泰国北部。风龙还可以被称为青藤(Sinomenium acutum(Thunb.)Rehd.et Wils)。风龙的活性物质包括生物碱例如青藤碱、异青藤碱、二青藤碱、L-防己碱和短防己碱、以及β-植物固醇和豆甾醇。这些活性物质中，认为青藤碱是抑制蛋白羰基化(或变性)的有效化合物。其他活性物质的一种或多种也可以用于这种作用，但是认为青藤碱用于蛋白羰基化抑制是最有效的。青藤碱的CAS号为115-53-7，分子量为～329g/mol，并且分子式为C19H23NO4。青藤碱可作为标准样品而商业途径获得，或者可以通过提取风龙属植物材料而获得。青藤碱的标准样品可以具有至少约90％、至少约95％、至少约99％和多达99.99％的纯度。还可以获得并使用其他纯度水平。
风龙属植物的任何部分可用于获得组合物中使用的青藤碱，包括但不限于根、茎、根茎、叶、花、果和/或这些部分的提取物。青藤碱一般存在于风龙的根中，由此，根提取物一般是对于本公开目的最有用的。风龙属可以原形式、混悬形式、脱水形式、浓缩形式或提取物形式使用。通常，风龙属是干或液体形式。
该风龙属提取物和/或组合物的其他组分可以通过本领域已知的常规提取方法获得，例如通过水(例如蒸汽)提取或通过溶剂(例如醇)提取。本公开的组合物不限于特定的提取方法，也不必须提取，因为青藤碱可以容易地获自许多商业供应商。示例性提取方法在下文描述。
在许多实施方案中，组合物包含来自风龙种的提取物。如本文使用的，对“风龙属提取物”的提及一般指含有来自风龙属的提取物，包括来自风龙种。风龙属提取物可商业获自各种来源。此外，适当的风龙属提取物可以通过使用任何常规提取技术获得。
为了获得风龙属提取物，极性溶剂例如醇(例如甲醇、乙醇、丁二醇)、醚(例如乙醚)、酮(例如丙酮)、酯(例如乙酸乙酯)、水或其混合物可用作溶剂。风龙属提取物还可以通过用非极性溶剂从极性溶剂进一步萃取提取物而获得。适合的非极性溶剂包括但不限于乙酸乙酯、己烷、二氯甲烷、氯仿或其混合物。
有许多可用于产生适合所述组合物的提取物的提取方法。这些方法包括但不限于Rana等人的美国专利号7,897,184中公开的提取方法，其在此通过引用整体并入本文，并且在下文参考一些提取方法而部分地复制。虽然描述的提取溶剂具体指乙醇，但是应该理解，其他醇，例如但不限于异丙醇、乙基醇和/或甲基醇可以补充或替代乙醇而使用。示例性醇溶剂包括但不限于C1至C4醇，例如甲醇、乙醇、丙醇、异丙醇和丁醇；水-醇或醇与水的混合物，包括水-乙醇(hydro-ethanol)；多羟基醇，例如丙二醇和丁二醇；和脂肪醇。可以使用这些醇溶剂的任何一个。其他溶剂，例如但不限于丙酮也可以作为提取溶剂使用。还可以使用任何比率的溶剂-水掺和物，例如醇-水和/或丙酮-水掺和物。
在一个实例中，可以使用有机溶剂提取技术获得风龙属提取物。在另一实例中，可以使用溶剂连续分级分离来获得风龙属提取物。还可以使用总的水-乙醇提取技术来获得风龙属提取物。一般而言，其称为一次性提取。在该过程中产生的提取物会含有宽范围的提取材料中存在的植物化学物，包括脂溶性和水溶性植物化学物。在收集提取溶液之后，蒸发溶剂，得到提取物。
还可以使用总的乙醇提取。该技术使用乙醇作为溶剂。该提取技术产生的提取物除了水溶性化合物以外还可以包括脂溶性和/或亲脂化合物。也可以相似方式使用总甲醇提取，具有相似的结果。在各个实施方案中，通过醇提取风龙种的植物材料而获得风龙属提取物。
可用于获得风龙属提取物的提取技术的另一实例是超临界二氧化碳超临界流体提取(“SFE”)。在该提取程序中，待提取的材料不暴露于任何有机溶剂。而是，提取溶剂是超临界状态(例如＞31.3℃和＞73.8巴)下的二氧化碳(CO2)，含有或没有调节剂。本领域技术人员会理解，温度和压力条件可以变化以获得最佳的提取物产率。该技术产生脂溶性和/或亲脂化合物的提取物，类似于总己烷和乙酸乙酯提取技术，其也可以使用。
风龙属提取物可以任何量加入组合物中，条件是其以有效抑制蛋白羰基化的量存在。通常，青藤碱以有效减少受试者皮肤羰基衍生物生成的量存在。此类羰基衍生物一般包含醛、酮或其组合。
在各个实施方案中，青藤碱以约5至约500、或者约25至约350μg/mL所述局部组合物的量存在。在具体实施方案中，青藤碱以约10、约20、约25、约50、约75、约100、约200或约300μg/mL组合物的量存在。还考虑约5至约500μg/mL组合物的各个子范围和量，以及小于或大于这些量的量。
组合物中存在的青藤碱的量可取决于几个因素，包括希望的蛋白羰基化抑制水平、特定提取物或组合物中的蛋白羰基化抑制水平、以及其他因素。在某些实施方案中，基于100重量份(“pbw”)组合物，青藤碱以约0.01至约20pbw的量存在。在进一步实施方案中，基于100pbw组合物，青藤碱以约0.05至约10pbw的量存在。还考虑约0.01至约20pbw的各个子范围和量，以及小于或大于这些量的量。任选用于本公开组合物的其他提取物或成分描述于Dornoff等人的美国专利号5,747,006，以及Leverett等人的美国专利号5,980,904、6,994,874、7,060,304、7,247,321和7,364,759，其公开内容通过引用整体并入本文。
在某些实施方案中，组合物不含其他活性物质。这里的“其他活性物质”一般表示组合物不含不同于风龙属的其他类型的传统中药(“TCM”；或“中药”)。其他类型的TCM是本领域所理解的。其他类型的TCM的实例一般在国际公开号WO 01/22934 A2中描述为“生物活性物质”，其内容通过引用整体并入本文。在某些实施方案中，组合物可以包含下述无活性物质。如果使用，无活性物质不同于其他类型的TCM。
可以配制组合物以包含美容上可接受的载体(或媒介物)并制备和/或包装并标示为抑制蛋白羰基化、减少皮肤黄化、和/或皮肤增亮(或增白)。组合物可以局部施用。美容上可接受的载体的实例包括但不限于水、甘油、蜡、各种醇例如乙醇、丙醇、植物油、矿物油、硅酮例如硅油、脂肪酯、脂肪醇、二醇、聚二醇或其任意组合。此类组分一般被认为是非活性组分。最终组合物可以是适合局部应用于皮肤的任何形式，例如但不限于气溶胶喷雾剂、凝胶、乳霜、分散液、乳液、泡沫、液体、洗液、摩丝、贴片、香膏剂、粉剂、泵式喷雾剂、固体、溶液、粘棒或小巾。乳液可以包括水包油乳液、油包水乳液和硅酮包水乳液。
可以使用本领域已知的各种方法制备本公开的组合物。在制备组合物的一个实例中，制备方法包括提取风龙种的植物材料以获得风龙提取物，即以获得青藤碱(含或不含其它活性化合物)的步骤。通常，制备方法还包括组合青藤碱与美容上可接受的载体例如上述载体的一种或多种的步骤。可以使用常规生产方法和装置例如混合器、掺和器等来组合组分。
组合物可以各种方式用于抑制蛋白羰基化。作为例子，用于抑制受试者皮肤蛋白羰基化的美容方法包括将青藤碱应用于受试者皮肤的步骤。青藤碱可以各种方式应用，包括将组合物应用于受试者皮肤，其中所述组合物包含来自风龙种的提取物。可以将组合物直接或间接应用于皮肤，例如通过手、涂布器、贴片等。
可以按需要、每天、一天几次或者任何适当方案施用组合物，以便实现所需结果。在美容方法中，局部应用频率可以取决于几个因素，包括所需的蛋白羰基化抑制水平。一般而言，方案包括每天一次或两次应用组合物于皮肤以包括早上应用和/或晚上应用。每次应用中应用于皮肤的组合物的量可以取决于几个因素，包括希望结果的水平和具体组合物。
示例说明本公开组合物和方法的以下实施例预期示例说明而非限制本发明。
实施例
作为背景，氧化应激通过蛋白羰基化与活性氧类的生成相关联，蛋白羰基化是其中反应性醛或酮通过氧化被引入蛋白的过程。蛋白羰基是蛋白氧化的主要产物，并且可以通过蛋白的氧化裂解、氨基酸残基的直接氧化或者与衍生自脂质过氧化的醛的共价反应而形成。
蛋白羰基化的发生一般通过直接金属催化的氨基酸侧链氧化(“主要蛋白羰基化”)或者将反应性醛添加至氨基酸侧链(“次要蛋白羰基化”)。普遍存在于人体的多不饱和脂肪酸的氧化降解可以导致各种醛的形成，例如丙烯醛和4-羟基壬醛(“4-HNE”)。研究显示，主要蛋白羰基化在活性氧类(“ROS”)信号传导机制中发挥作用。具体地，主要蛋白羰基化介导了细胞信号传导，并且主要蛋白羰基化是可逆的。
主要蛋白羰基化涉及直接金属催化的氨基酸侧链氧化，特别是在四个易感氨基酸残基上：脯氨酸、精氨酸、赖氨酸和苏氨酸。脯氨酸或精氨酸侧链的氧化导致谷氨酸半醛的形成，同时赖氨酸氧化产生氨基己二酸半醛，从而在蛋白结构中引入羰基。氧化还将苏氨酸侧链的羟基转化为羰基，形成2-氨基-3-丁酮酸。次要蛋白羰基化机制包括反应性醛与赖氨酸、组氨酸和半胱氨酸残基侧链的迈克尔加成反应，以及通过还原糖与赖氨酸和精氨酸上的氨基形成席夫碱，导致高级羰 基化终产物的增加。
蛋白羰基出现于各种氧化应激和疾病病症。除了蛋白被失活外，羰基化的细胞蛋白经历蛋白酶体依赖性降解。认为主要的蛋白羰基化在氧化应激期间标记受损蛋白中发挥作用，以从生物体系消除它们。羰基蛋白的生成是氧化应激的标志，例如作为外在因素，例如UV辐射诱导的因素或者氧化性化学品的外部应用，或者作为内在因素，源自脂质过氧化物降解的反应性羰基的化学攻击。
UV射线或光损伤皮肤由各种特征表征，例如黑斑、皱纹和下垂。老年人，特别是亚洲人，倾向于显示随光老化的黄色皮肤颜色变化。在中国，面黄一般意味着缺乏粉红色或者具有暗沉面色。光损伤或老化的常见临床体征包括特征性苍白或黄色皮肤外观。面黄常常是不可逆的，因此预防是理想的。
进行各种测试并产生相应的数据集以确定青藤碱相对于蛋白羰基化的效力。在相关部分，使用本领域技术人员理解的效力测定和方法来评价细胞毒性和蛋白羰基化抑制。表格数据在以下数据1和数据2以及分别相应的图1和2中显示。
如下文描述的，根据通过CTG细胞水平的安全性评价，青藤碱没有细胞毒性风险(参见数据/图1)。而且，本发明人已经发现，根据体外生物测定数据，青藤碱具有皮肤抗羰基化的效力和功能(参见数据/图2)。进行通过模型细胞HepG2的基于细胞的测定，并且监测了羰基化活性的抑制。认为维生素C是羰基化抑制的阳性对照。本领域技术人员理解该类型的模型细胞HepG2基于细胞的测定。
数据1
青藤碱在HepG2中的细胞毒性-CTG(氢醌作为参比)
Figure PCTCN2016076825-appb-000001
Promega Corporation的
Figure PCTCN2016076825-appb-000002
(“CTG”)发光细胞活力测定是测定培养物中存活细胞数目的均一方法，基于作为代谢活性细胞指示而存在的ATP的定量。根据通过数据1和图1中示例说明的CTG细胞水平的安全性评价，青藤碱没有细胞毒性风险。
数据2
青藤碱在HepG2中的羰基化抑制(维生素C作为参比)
Figure PCTCN2016076825-appb-000003
青藤碱和维生素C的梯度浓度都是0、33.3、100和300μg/ml。针对每个浓度记录了两个羰基化抑制率。青藤碱的相应的平均羰基化抑制率是1.6％、31.35％、56.2％和53.85％。维生素C的相应的平均羰基化抑制率是-3.05％、31.05％、55.35％和62.65％。总之，青藤碱在细胞HepG2模型中与阳性对照维生素C相比具有突出的抗羰基化抑制效力，而没有细胞毒性风险。该数据集一般通过下文列出的测定方法而获得。
羰基化抑制测定：
目的：该程序描述了HepG2细胞的抗氧化测定的标准方法。
细胞系：HepG2(ATCC#)
材料：
·MEM(GIBCO#16600-082)
·胎牛血清(GIBCO#10099-141)
·胰蛋白酶-EDTA(GIBCO#25200-072)
·96孔细胞板(Costa)
·OxiSelectTM蛋白羰基ELISA试剂盒(Cell Biology#STA-310)
程序：
第1天：铺板细胞
1.胰蛋白酶消化并测定细胞密度。
2.以1,100,000个细胞/ml的密度稀释细胞悬浆至所需体积。
3.将90μl/孔细胞悬浆分配至测定板上(100,000个细胞每孔)。
4.在加湿条件下以37℃、5％CO2孵育测定板24小时。
第2天：添加测试化合物
1.根据板图制备参比和测试化合物溶液(200×)。
2.将7μl化合物转移至133μl培养基(最终浓度：10×)。
3.将10μl化合物转移至测定板(最终浓度：1×)。
4.在加湿条件下以37℃、5％CO2孵育板。
第4天：
1.弃掉培养基并用汉克斯平衡盐溶液(“HBSS”)洗涤两次。
2.将100μl HBSS(含有混合物)添加至每个孔。
3.将板放在-80℃下30min。然后解冻。重复至少3次。
4.混合细胞裂解液。
5.获得25μl每孔裂解液并测试蛋白浓度。
6.调节蛋白浓度。
Figure PCTCN2016076825-appb-000004
7.将100μl样品(使用经诱导的BSA稀释液)添加至96孔蛋白结合平板中。在4℃孵育过夜。
第5天：ELISA测定-100孔
1.用250μL 1X PBS每孔洗涤孔3次。去除过量洗涤溶液。
2.添加100μL DNPH工作溶液并在黑暗中室温孵育45分钟。
Figure PCTCN2016076825-appb-000005
Figure PCTCN2016076825-appb-000006
3.用250μL 1X PBS/乙醇(1∶1，v/v)洗涤5次，在定轨摇床上孵育5分钟。
Figure PCTCN2016076825-appb-000007
4.用250μL 1X PBS洗涤2次。
5.每孔添加200μL封闭溶液并在定轨摇床上室温下孵育1至2小时。
Figure PCTCN2016076825-appb-000008
6.用250μL 1X洗涤缓冲液洗涤3次，每次洗涤之间充分抽吸。
Figure PCTCN2016076825-appb-000009
7.添加100μL抗DNP抗体并在定轨摇床上室温下孵育1小时。
Figure PCTCN2016076825-appb-000010
8.洗涤条形孔3次(1*洗涤缓冲液)。
9.向所有孔添加100μL稀释的HRP结合的二级抗体并在定轨摇床上室温下孵育1小时。
Figure PCTCN2016076825-appb-000011
10.洗涤条形孔5次(1*洗涤缓冲液)。
11.使底物溶液恒温至室温。向每孔添加100μL底物溶液，孵育15min。
12.通过向每孔添加100μL终止溶液而终止酶反应。结果应该立即读取(颜色随时间消褪)。
13.使用450nm作为主要波长在读板器上读取每个孔的吸光度。使用完全还原的BSA标准作为吸光度空白。
作为背景，人类皮肤着色由皮肤表面黑色素的量和位置决定。黑色素通过存在于真皮-表皮交界处的、通常称为黑素细胞的细胞中氨基酸酪氨酸氧化至L-3，4-二羟基苯丙氨酸(“L-DOPA”)而合成。所述氧化过程由酪氨酸酶催化。由黑素细胞进行的一系列细胞过程通常称为黑素生成。
皮肤着色通过黑素细胞合成的黑色素的量和类型来调控。环境因素也可影响皮肤颜色。皮肤中健康量的黑色素有效吸收UV射线。当皮肤在UV射线下暴露增加时，一般会提升黑色素生成的量和速率，并且可导致皮肤黑化，或者成“棕褐色”。局部或总体的色素沉着疾患，例如色素沉着过度或色素沉着不足，可以源自许多因素，包括体内激素水平、饮食、遗传疾患和药物治疗。常见的色素沉着失调疾患包括黄褐斑(melisma)、雀斑和白斑。
各种组合物已被配制以解决色素沉着疾患，并且例如已经预期用于治疗色素沉着过度和/或色素沉着不足。此类治疗通常被称为“皮肤增亮”、“皮肤美白”或“皮肤亮白”。存在皮肤美白剂的几种用途。例如，淡化老年斑(雀斑或衰老性色斑)，或者预防高加索人和亚洲人皮肤的黑化(即，维持亮/白肤色)。这些制剂中的一些已包括酪氨酸酶抑制剂例如氢醌、维生素C、曲酸、熊果苷、谷胱甘肽、半胱氨酸、乳酸、阿魏酸、烟酰胺，以及植物提取物例如熊果和桑树提取物等。然而，这些化合物中，例如氢醌和曲酸，具有副作用，包括皮肤刺激、急性皮炎和皮肤细胞的细胞毒性。
进行各种测试并产生相应数据集以测定青藤碱相对于皮肤增亮(或增白)的效力。在相关部分，使用本领域技术人员理解的效力测定和方法来评价细胞毒性、黑色素合成抑制、黑色素消除和酪氨酸酶抑制。表格数据在以下数据3、数据4、数据5和数据6以及分别相应的图3、4、5和6中显示。与确定皮肤增亮效力相关的其他提取方法和分析方法(例如测定方法)描述于Rana等人的国际公布号WO 2011/019468 A2、WO 2011/109139 A2和WO 2013/169634 A2，其公 开内容通过引用并入本文。
数据3
青藤碱在B16F10中的细胞毒性-CTG(氢醌作为参比)
Figure PCTCN2016076825-appb-000012
根据通过数据3和图3中示例说明的CTG的细胞毒性的安全性评价，青藤碱没有细胞毒性风险。
根据体外生物测定数据，发现青藤碱具有皮肤增亮和去色素沉着的效力。通过模型细胞B16F10细胞水平的测定，监测黑色素合成抑制和存在的黑色素消除活性，以及酪氨酸酶活性的抑制。该类型的基于模型细胞B16F10细胞的测定是本领域技术人员已知的。
熊果苷被视为黑色素合成抑制和酪氨酸酶活性抑制两者的阳性对照，而氢醌用作已存在的黑色素消除活性的阳性对照。总结以下描述的结果，青藤碱作为皮肤增亮或去色素沉着试剂在体外测试中具有突出活性而没有细胞毒性风险。
数据4
青藤碱在B16F10中的黑色素合成抑制(熊果苷作为参比)
青藤碱
Figure PCTCN2016076825-appb-000013
熊果苷
Figure PCTCN2016076825-appb-000014
在B16F10细胞中黑色素合成抑制活性中，相对于阳性对照熊果苷，青藤碱的抑制率在100μg/mL下是66.9％。因此，青藤碱有能力强烈中断或减少B16F10细胞中黑色素合成过程。该数据集一般通过下文即将列出的测定方法而获得。
黑色素合成抑制测定：
目的：该程序描述了B16 F10细胞中黑色素合成抑制测定的标准方法。
细胞系：B16-F10(ATCC#)
材料：
·用于B16的RPMI 1640(GIBCO#22400-089批号1006397)
·胎牛血清(GIBCO#10099-141)
·胰蛋白酶-EDTA(GIBCO#25200-072)
·1M NaOH
·24孔板(Corning)
程序：
第1天：铺板细胞
1.胰蛋白酶消化并测定细胞密度。
2.以18,000细胞/ml的密度稀释细胞悬浆至所需体积。
3.将1ml/孔细胞悬浆分配至测定板上。
4.在加湿条件下以37℃、5％CO2孵育测定板24小时。
第2天：添加测试化合物
1.根据板图制备参比和测试化合物溶液(200×)。
2.将5μl化合物转移至测定板(最终浓度：1×)。
3.在加湿条件下以37℃、5％CO2孵育板72小时。
第5天：使板成像
1.去除培养基。
2.将150μl 1M NaOH添加至测定板。
3.在80℃下孵育板30分钟。
4.将140μl溶液转移至UV板。测试400nm信号。
5.将5μl溶液转移至UV板。向每个孔添加200μl BCA试剂并在37℃孵育20分钟。测试562nm信号。
数据处理：
·使用GraphPad。
·％抑制＝(最大信号-化合物信号)/(最大信号-最小信号)×100。
·最大信号获自DMSO的作用。
·最小信号获自200μg/ml熊果苷。
数据5
青藤碱在B16F10中的黑色素消除率(氢醌作为参比)
青藤碱
Figure PCTCN2016076825-appb-000015
氢醌
Figure PCTCN2016076825-appb-000016
在存在的黑色素消除测定中，相对于阳性对照氢醌在100μg/mL时58.4％的消除率，发现B16F10培养细胞中39.4％色素在添加100μg/mL青藤碱72小时之后消除。这意味着青藤碱也用于黑色素消除。该数据集一般通过以下即将列出的测定方法而获得。
存在的黑色素消除测定：
目的：该程序描述了B16细胞中黑色素消除测定的标准方法。
细胞系：B16-F10(ATCC#)
材料：
·用于B16的RPMI 1640(GIBCO#22400-089批号1006397)
·胎牛血清(GIBCO#10099-141)
·胰蛋白酶-EDTA(GIBCO#25200-072)
·1M NaOH
·24孔板(Corning)
程序：
第1天：铺板细胞
1.胰蛋白酶消化并测定细胞密度。
2.以18,000个细胞/ml的密度稀释细胞悬浆至所需体积。
3.将1ml/孔细胞悬浆分配至测定板上。
4.在加湿条件下以37℃、5％CO2孵育测定板24小时。
第2天：细胞处理
1.向每孔添加10μM毛喉素和50μM 8-MOP。
2.在加湿条件下以37℃、5％CO2孵育测定板72小时。
第5天：添加测试化合物
1.更换新鲜培养基，每孔1ml。
2.根据板图制备参比和测试化合物溶液(200×)。
3.将5μl化合物转移至测定板(最终浓度：1×)。
4.在加湿条件下以37℃、5％CO2孵育板72小时。
第8天：使板成像
1.去除培养基。
2.将150μl 1M NaOH添加至测定板。
3.在80℃下孵育板30分钟。
4.将140μl溶液转移至UV板。测试400nm信号。
5.将5μl溶液转移至UV板。向每个孔添加200μl BCA试剂并在37℃孵育20分钟。测试562nm信号。
数据处理：
·使用GraphPad。
·％抑制＝(最大信号-化合物信号)/(最大信号-最小信号)×100。
·最大信号获自没有化合物作用细胞。
·最小信号获自10μM毛喉素和50μM 8-MOP。
数据6
青藤碱在B16F10中的酪氨酸酶抑制(熊果苷作为参比)
青藤碱
Figure PCTCN2016076825-appb-000017
熊果苷
Figure PCTCN2016076825-appb-000018
青藤碱在B16F10细胞中发挥优良的酪氨酸酶抑制活性，在100μg/mL浓度下抑制率为32.4％。具体地，如数据6和图6所示，在B16F10细胞中酪氨酸酶抑制活性方面，青藤碱表现与熊果苷相当或好得多。
简单概括，除了酪氨酸酶抑制和黑色素合成抑制的良好性能，特别地，本发明人还发现青藤碱显示了消除存在的黑色素测定模型的极大能力。由此，青藤碱具有优良的作为皮肤增白和脱色素试剂的活性，而对于护肤或局部使用而言没有细胞毒性风险。
要理解，所附权利要求不限于详述中描述的明确和特定的化合物、组合物或方法，其可以在落入所附权利要求范围内的特定实施方案之间变化。对于本文用于描述各个实施方案特定特征或方面所依据的任何马库什组而言，要理解，不同的、特殊的和/或未预料的结果 可以获自各个马库什组的每个成员而独立于所有其他马库什成员。马库什组的每个成员可以被单独或组合地依据并提供对所附权利要求范围内具体实施方案的足够支持。
还要理解，描述本发明各个实施方案中所依据的任何范围和子范围独立且总体落入所附权利要求范围，并且被理解为描述和涵盖包括其中全部和/或部分值在内的所有范围，即使此类值没有在本文明确书写。本领域技术人员容易发现，列举的范围和子范围充分描述并实现了本发明的各个实施方案，并且此类范围和子范围可以进一步描绘成相关的二分之一、三分之一、四分之一、五分之一等。仅作为一个实例，“0.1至0.9”的范围可以进一步描绘成较小的三分之一，即0.1至0.3，中间三分之一，即0.4至0.6，和较大的三分之一，即0.7至0.9，其个体且总体上在所附权利要求范围内，并且可以在个体和/或总体上被依据并提供对所附权利要求范围内具体实施方案的足够支持。此外，关于限定或修饰范围的语言，例如“至少”、“大于”、“小于”、“不大于”等，要理解此类语言包括子范围和/或上限或下限。作为另一实例，“至少10”的范围固有包括至少10至35的子范围，至少10至25的子范围，至少25至35的子范围等等，并且每个子范围可以在个体和/或总体上被依据并提供对所附权利要求范围内具体实施方案的足够支持。最后，所公开范围内个体数值可以被依据并提供对所附权利要求范围内具体实施方案的足够支持。例如，“1至9”的范围包括各个个体整数，例如3，以及包括小数点(或分数)的个体数值，例如4.1，其可以被依据并提供对所附权利要求范围内具体实施方案的足够支持。
本文已经以示例说明的方式描述了本发明，并且要理解，已经使用的术语预期在描述词语性质内而不是限制。鉴于以上教导，本发明的许多调整和变化是可能的。本发明可以在所附权利要求范围内以不同于具体描述的方式实施。在本文明确考虑独立权利要求和从属权利要求(单项和多项从属)的所有组合的主题。

Claims (16)

  1. 一种局部组合物，包含有效抑制受试者皮肤蛋白羰基化的量的青藤碱。
  2. 一种用于抑制受试者皮肤蛋白羰基化的局部组合物，所述局部组合物的活性成分由青藤碱组成。
  3. 如权利要求1或2所述的局部组合物，其中所述局部组合物包含来自风龙种的提取物。
  4. 如权利要求3所述的局部组合物，其中所述青藤碱以有效减少受试者皮肤中羰基衍生物生成的量存在，所述羰基衍生物包含醛、酮或其组合。
  5. 如权利要求3所述的局部组合物，其中所述青藤碱以约5至约500、或者约25至约350μg/mL所述局部组合物的量存在。
  6. 如权利要求3所述的局部组合物，其中所述青藤碱通过溶剂提取、或者通过醇提取所述风龙种的植物材料而获得。
  7. 如权利要求3所述的局部组合物，其中所述青藤碱具有至少1-99.99％纯度，较为推荐的是约90％、或者至少约90-99.9％的纯度。
  8. 如权利要求1或2所述的局部组合物，还包含美容上可接受的载体。
  9. 如权利要求8所述的局部组合物，其中所述美容上可接受的载体选自由以下组成的组：水、甘油、蜡、醇、植物油、矿物油、硅酮、脂肪酯、脂肪醇、二醇、聚二醇及其组合。
  10. 一种用于抑制受试者皮肤蛋白羰基化的组合物，所述组合物由青藤碱组成。
  11. 权利要求1、2或10任一项所述的局部组合物用于抑制受试者皮肤蛋白羰基化的用途。
  12. 一种制备权利要求1、2或10任一项所述的局部组合物的方法，所述方法包括提取所述风龙种的植物材料以获得青藤碱的步骤。
  13. 如权利要求12所述的方法，还包括组合所述青藤碱与美容上可接受的载体的步骤。
  14. 一种用于抑制受试者皮肤蛋白羰基化的美容方法，所述方法包括应用青藤碱至受试者皮肤的步骤。
  15. 如权利要求14所述的美容方法，其中所述应用步骤进一步限定为应用局部组合物至受试者皮肤，其中所述局部组合物包含来自所述风龙种的提取物。
  16. 如权利要求15所述的美容方法，其中所述青藤碱以有效减少受试者皮肤中羰基衍生物生成的量存在，所述羰基衍生物包含醛、酮或其组合。
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CN110381917B (zh) * 2017-03-06 2023-10-31 默克专利股份有限公司 相容性溶质的用途

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