WO2016169373A1 - Topical composition containing sinomenine and method for restraining carbonylation of protein - Google Patents

Abstract

A topical composition for restraining carbonylation of skin protein, and a preparation method therefor and uses thereof. The topical composition contains sinomenine as an active component. The method for preparing the topical composition comprises a step of obtaining sinomenine from sinomenium acutum species plants.

Description

Topical composition comprising sinomenine and method for inhibiting protein carbonylation Field of invention

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.

Related technical description

A major contributor to skin yellowing associated with aging is believed to be the protein denaturation process known as "carbonylation" in the dermis. Studies have shown that protein denaturation is the result of binding between lipid degradation products produced by ultraviolet ("UV)" ray damage and dermal proteins. 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.

As an example of the foregoing process, a lipid can interact with an aldehyde that causes carbonylation of the dermal protein, resulting in a carbonylated protein. In a similar manner, sugars can cause saccharification of dermal proteins, resulting in glycated proteins. It is believed that carbonylation produces a yellower skin relative to saccharification.

As a precaution, efforts have been made to prevent UV rays damage by topical sunscreen compositions. As a corrective measure, efforts have been made to reverse skin yellowing by topical skin lightening (or whitening) compositions. Unfortunately, such compositions require ongoing applications and require user compliance. In addition, some of these compositions can irritate the skin. Skin irritation or other application problems, such as undesirable oleability, will cause the user to discontinue use of the composition or to use the composition in a manner that is ineffective for its intended purpose.

In view of the above, there are still other topical compositions that provide for the inhibition of skin yellowing. There are also opportunities to reverse, slow or prevent skin carbonylation.

Summary of invention

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. In certain embodiments, the active ingredient of the topical composition consists of sinomenine. In a particular embodiment, the topical composition consists of sinomenine.

A cosmetic method of inhibiting skin protein carbonylation in a subject is also disclosed. The cosmetic method 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. Unlike protein carbonylation inhibition, topical compositions are also used for skin lightening (or whitening), particularly for inhibiting melanin synthesis, eliminating melanin and/or inhibiting tyrosinase.

BRIEF DESCRIPTION OF THE DRAWINGS

Other advantages of the present disclosure will be readily understood, as it will become better understood by referring to

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");

4 is a line graph illustrating melanin synthesis inhibition data ("data 4");

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").

Detailed description of the invention

A topical composition ("composition") is disclosed. The composition is 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. Typically, 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.

Surprisingly, it has been found that genus Sinomenium can be used to inhibit protein carbonylation of the skin. In particular, without being bound or limited by any particular theory, extracts of the genus Rhododendron are believed to reduce and/or inhibit skin protein carbonylation. The compositions of the present disclosure generally comprise extracts from plants of the genus Geochelone, more typically from plants of the species Sinomenium acutum. As described below, sinomenine is the active substance of the extract and is believed to be an effective compound for inhibiting protein carbonylation. As used herein, 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. Based on the discovery herein, 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.

In various embodiments, the composition comprises sinomenine. In a further embodiment, the active ingredient of the composition consists of sinomenine. In these embodiments, the composition may further consist of one or more additional components and/or inactive components. In certain embodiments, the composition consists of sinomenine. In all of these embodiments, 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 β-phytosterol and stigmasterol. Among these active substances, 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.

Any portion of the 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. Usually, 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). 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.

In many embodiments, the composition comprises an extract from the genus. As used herein, reference to "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. In addition, suitable extracts of the genus Rhododendron can be obtained by using any conventional extraction technique.

In order to obtain the extract of the genus Dragon, 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. As a solvent. The extract of the genus Dioscorea can also be obtained by further extracting the extract from a polar solvent with a non-polar solvent. Suitable non-polar solvents include, but are not limited to, ethyl acetate, hexane, dichloromethane, chloroform or mixtures thereof.

There are a number of 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.

In one example, the extract of the genus Rhododendron can be obtained using an organic solvent extraction technique. In another example, continuous extraction of the solvent can be used to obtain the extract of the genus. It is also possible to use the total water-ethanol extraction technique to obtain the extract of the genus. In general, 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. In various embodiments, the extract of the genus Rhododendron is obtained by extracting the plant material of the genus.

Another example of an extraction technique that can be used to obtain the extract of the genus Rhododendron is supercritical carbon dioxide supercritical fluid extraction ("SFE"). In the extraction procedure, 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. Those skilled in the art will appreciate that 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. Typically, sinomenine is present in an amount effective to reduce the formation of carbonyl derivatives of the skin of the subject. Such carbonyl derivatives typically comprise an aldehyde, a ketone or a combination thereof.

In various embodiments, 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.

The amount of 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. In certain embodiments, sinomenine is present in an amount from about 0.01 to about 20 pbw based on 100 parts by weight ("pbw") of the composition. In a further embodiment, the sinomenine is present in an amount of from about 0.05 to about 10 pbw based on the 100 pbw of the composition. Also contemplated are individual subranges and amounts of from about 0.01 to about 20 pbw, and amounts less than or greater than these amounts. 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.

In certain embodiments, 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. 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. In certain embodiments, the composition may comprise the following inactive materials. If used, inactive materials are different from other types of TCM.

The 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). The composition can be administered topically. Examples of 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.

The compositions of the present disclosure can be prepared using a variety of methods known in the art. In one example of the preparation of the composition, 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). In general, 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. As an example, 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. In cosmetic methods, the frequency of topical application can depend on several factors, including the level of protein carbonylation inhibition desired. In general, 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.

The following examples of the disclosed compositions and methods are intended to illustrate and not to limit the invention.

Example

As a background, 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.

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.

Various tests were performed and corresponding data sets were generated to determine the efficacy of sinomenine relative to protein carbonylation. In the relevant section, cytotoxicity and inhibition of protein carbonylation are evaluated using potency assays and methods as understood by those skilled in the art. The tabular data is shown in the following data 1 and data 2 and in respective corresponding Figures 1 and 2.

As described below, 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.

Data 1

Cytotoxicity of sinomenine in HepG2 - CTG (hydroquinone as a reference)

(“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。Promega Corporation

("CTG") 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.

Data 2

Inhibition of carbonylation of sinomenine in HepG2 (vitamin C as a reference)

The gradient concentrations of sinomenine and vitamin C were 0, 33.3, 100 and 300 μg/ml. Two carbonylation inhibition rates were recorded for each concentration. The corresponding average carbonylation inhibition rates of sinomenine were 1.6%, 31.35%, 56.2% and 53.85%. The corresponding average carbonylation inhibition rates of vitamin C were -3.05%, 31.05%, 55.35%, and 62.65%. In conclusion, 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.

Carbonylation inhibition assay:

Purpose: This program describes a standard method for the anti-oxidation assay of HepG2 cells.

Cell line: HepG2 (ATCC#)

material:

·MEM (GIBCO#16600-082)

·Fetal bovine serum (GIBCO#10099-141)

·Trypsin-EDTA (GIBCO#25200-072)

·96-well cell plate (Costa)

· OxiSelect ^TM protein carbonyl ELISA kit (Cell Biology # STA-310)

program:

Day 1: Plating cells

1. Trypsinize and measure cell density.

2. Dilute the cell suspension to the desired volume at a density of 1,100,000 cells/ml.

3. Dispense 90 μl/well of cell suspension onto the assay plate (100,000 cells per well).

4. The assay plates were incubated for 24 hours at 37 ° C, 5% CO ₂ under humidified conditions.

Day 2: Add test compound

1. Prepare a reference and test compound solution (200x) according to the plate diagram.

2. Transfer 7 μl of the compound to 133 μl of medium (final concentration: 10×).

3. Transfer 10 μl of the compound to the assay plate (final concentration: 1×).

4. Incubate the plates at 37 ° C, 5% CO ₂ under humidified conditions.

Day 4:

1. Discard the medium and wash twice with Hanks Balanced Salt Solution ("HBSS").

2. Add 100 μl of HBSS (containing the mixture) to each well.

3. Place the plate at -80 ° C for 30 min. Then thaw. Repeat at least 3 times.

4. Mix the cell lysate.

5. Obtain 25 μl per well lysate and test for protein concentration.

6. Regulate protein concentration.

7. Add 100 μl of sample (using the induced BSA dilution) to the 96-well protein-binding plate. Incubate overnight at 4 °C.

Day 5: ELISA assay - 100 wells

1. Wash the wells 3 times with 250 μL of 1X PBS per well. Excess wash solution is removed.

2. Add 100 μL of DNPH working solution and incubate for 45 minutes at room temperature in the dark.

3. Wash 5 times with 250 μL IX PBS/ethanol (1:1, v/v) and incubate for 5 minutes on an orbital shaker.

4. Wash twice with 250 μL of 1X PBS.

5. Add 200 μL of blocking solution to each well and incubate for 1 to 2 hours at room temperature on an orbital shaker.

6. Wash 3 times with 250 μL of 1X Wash Buffer and draw well between each wash.

7. Add 100 μL of anti-DNP antibody and incubate for 1 hour at room temperature on an orbital shaker.

8. Wash the strip holes 3 times (1* wash buffer).

9. Add 100 μL of diluted HRP-conjugated secondary antibody to all wells and incubate for 1 hour at room temperature on an orbital shaker.

10. Wash the strip holes 5 times (1* wash buffer).

11. Allow the substrate solution to be tempered to room temperature. 100 μL of the substrate solution was added to each well and incubated for 15 min.

12. Stop the enzymatic reaction by adding 100 μL of stop solution to each well. The result should be read immediately (the color fades over time).

13. Read the absorbance of each well on a plate reader using 450 nm as the primary wavelength. The fully reduced BSA standard was used as the absorbance blank.

As a background, 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. 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.

Various 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." There are several uses for 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. For example, bearberry and mulberry extracts. However, among these compounds, such as hydroquinone and kojic acid, have side effects including skin irritation, acute dermatitis, and cytotoxicity of skin cells.

Various tests were performed and corresponding data sets were generated to determine the efficacy of sinomenine relative to skin lightening (or whitening). In the relevant section, cytotoxicity, melanin synthesis inhibition, melanin elimination, and tyrosinase inhibition are evaluated using potency assays and methods as understood by those skilled in the art. The tabular data is shown in the following data 3, data 4, data 5 and data 6 and corresponding Figures 3, 4, 5 and 6, respectively. Other extraction methods and analytical methods (e.g., assay methods) associated with determining skin lightening efficacy are described in Rana et al., International Publication No. WO 2011/019468 A2, WO 2011/109139 A2, and WO 2013/169634 A2, the disclosure of which is incorporated herein by reference. The contents are incorporated herein by reference.

Data 3

Cytotoxicity of sinomenine in B16F10-CTG (hydroquinone as a reference)

Sinomenine has no cytotoxic risk based on the safety evaluation of cytotoxicity of CTG as illustrated by Data 3 and Figure 3.

Based on in vitro bioassay data, sinomenine was found to have efficacy in skin lightening and depigmentation. The inhibition of melanin synthesis and the presence of melanin-eliminating activity, as well as inhibition of tyrosinase activity, were monitored by assay of model cell B16F10 cell levels. 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. Summarizing the results described below, sinomenine has outstanding activity as an skin lightening or depigmentation reagent in an in vitro test without the risk of cytotoxicity.

Data 4

Inhibition of melanin synthesis by sinomenine in B16F10 (arbutin as a reference)

Sinomenine

Arbutin

Among the melanin synthesis inhibitory activities in B16F10 cells, the inhibition rate of sinomenine was 66.9% at 100 μg/mL with respect to the positive control arbutin. Therefore, 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.

Melanin synthesis inhibition assay:

Purpose: This program describes a standard method for the inhibition of melanin synthesis in B16 F10 cells.

Cell line: B16-F10 (ATCC#)

material:

· RPMI 1640 for B16 (GIBCO #22400-089 lot number 1006397)

·Fetal bovine serum (GIBCO#10099-141)

·Trypsin-EDTA (GIBCO#25200-072)

·1M NaOH

·24-well plate (Corning)

program:

Day 1: Plating cells

1. Trypsinize and measure cell density.

2. Dilute the cell suspension to the desired volume at a density of 18,000 cells/ml.

3. Dispense 1 ml/well of cell suspension onto the assay plate.

4. The assay plates were incubated for 24 hours at 37 ° C, 5% CO ₂ under humidified conditions.

Day 2: Add test compound

1. Prepare a reference and test compound solution (200x) according to the plate diagram.

2. Transfer 5 μl of the compound to the assay plate (final concentration: 1×).

3. Incubate the plates for 72 hours at 37 ° C, 5% CO ₂ under humidified conditions.

Day 5: Imaging the board

1. Remove the medium.

2. Add 150 μl of 1 M NaOH to the assay plate.

3. Incubate the plate for 30 minutes at 80 °C.

4. Transfer 140 μl of the solution to the UV plate. Test the 400 nm signal.

5. Transfer 5 μl of the solution to the UV plate. 200 μl of BCA reagent was added to each well and incubated at 37 ° C for 20 minutes. The 562 nm signal was tested.

data processing:

· Use GraphPad.

% inhibition = (maximum signal - compound signal) / (maximum signal - minimum signal) x 100.

• The maximum signal is obtained from the action of DMSO.

• The minimum signal was obtained from 200 μg/ml arbutin.

Data 5

Melanin elimination rate of sinomenine in B16F10 (hydroquinone as a reference)

Sinomenine

Hydroquinone

In the presence of the melanin-eliminating assay, 39.4% of the pigment in the B16F10 cultured cells was found to be eliminated after 72 hours of addition of 100 μg/mL sinomenine relative to the 58.4% elimination rate of the positive control hydroquinone at 100 μg/mL. This means that sinomenine is also used for melanin elimination. This data set is generally obtained by the assay methods listed below.

The presence of melanin elimination assay:

Purpose: This program describes a standard method for the determination of melanin in B16 cells.

Cell line: B16-F10 (ATCC#)

material:

· RPMI 1640 for B16 (GIBCO #22400-089 lot number 1006397)

·Fetal bovine serum (GIBCO#10099-141)

·Trypsin-EDTA (GIBCO#25200-072)

·1M NaOH

·24-well plate (Corning)

program:

Day 1: Plating cells

1. Trypsinize and measure cell density.

2. Dilute the cell suspension to the desired volume at a density of 18,000 cells/ml.

3. Dispense 1 ml/well of cell suspension onto the assay plate.

4. The assay plates were incubated for 24 hours at 37 ° C, 5% CO ₂ under humidified conditions.

Day 2: Cell Processing

1. Add 10 μM forskolin and 50 μM 8-MOP to each well.

2. The assay plates were incubated at 37 ° C, 5% CO ₂ for 72 hours under humidified conditions.

Day 5: Add test compound

1. Replace fresh medium with 1 ml per well.

2. Prepare a reference and test compound solution (200x) according to the plate diagram.

3. Transfer 5 μl of the compound to the assay plate (final concentration: 1×).

4. Incubate the plates for 72 hours at 37 ° C, 5% CO ₂ under humidified conditions.

Day 8: Imaging the board

1. Remove the medium.

2. Add 150 μl of 1 M NaOH to the assay plate.

3. Incubate the plate for 30 minutes at 80 °C.

4. Transfer 140 μl of the solution to the UV plate. Test the 400 nm signal.

5. Transfer 5 μl of the solution to the UV plate. 200 μl of BCA reagent was added to each well and incubated at 37 ° C for 20 minutes. The 562 nm signal was tested.

data processing:

· Use GraphPad.

% inhibition = (maximum signal - compound signal) / (maximum signal - minimum signal) x 100.

• The maximum signal is obtained from cells that have no compound action.

• The minimum signal was obtained from 10 μM forskolin and 50 μM 8-MOP.

Data 6

Tyrosinase inhibition of sinomenine in B16F10 (arbutin as a reference)

Sinomenine

Arbutin

Sinomenine exerts excellent tyrosinase inhibitory activity in B16F10 cells, and the inhibition rate is 32.4% at a concentration of 100 μg/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.

Briefly summarized, in addition to the good performance of tyrosinase inhibition and melanin synthesis inhibition, in particular, the inventors have also discovered that sinomenine has shown great ability to eliminate the presence of melanin assay models. Thus, sinomenine has excellent activity as a skin whitening and depigmenting agent, and there is no risk of cytotoxicity for skin care or topical use.

It is to be understood that the appended claims are not limited to the specific embodiments of the invention, and the invention may be varied between the specific embodiments falling within the scope of the appended claims. For any Markush group that is used herein to describe a particular feature or aspect of each embodiment, it is understood that different, specific, and/or unexpected results are obtained. It can be obtained from each member of each Markush group and is independent of all other Markush members. Each member of the Markush group may rely on and provide sufficient support for the specific embodiments within the scope of the appended claims, either individually or in combination.

It is also to be understood that any range and sub-ranges that are described in the various embodiments of the invention are intended to be Even such values are not explicitly written in this article. Those skilled in the art will readily recognize that the scope and sub-ranges of the present invention fully describe and implement various embodiments of the present invention, and such scope and sub-ranges can be further described as related one-half, one-third, one-quarter One-fifth, etc. As just one example, 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. Further, with respect to 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. As another example, 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. Finally, 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. For example, 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. .

The present invention has been described herein by way of illustrative example, and it is understood that Many modifications and variations of the present invention are possible in light of the above teaching. The invention may be practiced otherwise than as specifically described within the scope of the appended claims. The subject matter of all combinations of the independent claims and the dependent claims (single and multiple subordinates) is explicitly considered herein.

Claims (16)

A topical composition comprising sinomenine in an amount effective to inhibit skin protein carbonylation in a subject. A topical composition for inhibiting skin protein carbonylation in a subject, the active ingredient of which is comprised of sinomenine. The topical composition of claim 1 or 2, wherein the topical composition comprises an extract from the genus. The topical composition of claim 3 wherein said sinomenine is present in an amount effective to reduce the formation of a carbonyl derivative in the skin of the subject, said carbonyl derivative comprising an aldehyde, a ketone or a combination thereof. The topical composition of claim 3 wherein said sinomenine is present in an amount of from about 5 to about 500, or from about 25 to about 350, g/mL of said topical composition. The topical composition according to claim 3, wherein the sinomenine is obtained by solvent extraction or by alcohol extraction of the plant material of the genus. The topical composition of claim 3 wherein said sinomenine has a purity of at least 1-99.99%, more preferably about 90%, or at least about 90-99.9%. A topical composition according to claim 1 or 2 further comprising a cosmetically acceptable carrier. The topical composition of claim 8 wherein said cosmetically acceptable carrier is selected from the group consisting of water, glycerin, waxes, alcohols, vegetable oils, mineral oils, silicones, fatty esters, fatty alcohols, Alcohols, polyglycols, and combinations thereof. A composition for inhibiting skin protein carbonylation in a subject, the composition consisting of sinomenine. Use of a topical composition according to any one of claims 1, 2 or 10 for inhibiting skin protein carbonylation in a subject. A method of preparing the topical composition of any one of claims 1, 2 or 10, the method comprising the step of extracting plant material of the genus of the genus to obtain sinomenine. The method of claim 12 further comprising the step of combining said sinomenine with a cosmetically acceptable carrier. A cosmetic method for inhibiting skin protein carbonylation in a subject, the method comprising the step of applying sinomenine to the skin of the subject. A cosmetic method according to claim 14 wherein said applying step is further defined as applying a topical composition to the skin of the subject, wherein said topical composition comprises an extract from said species. The cosmetic method of claim 15 wherein said sinomenine is present in an amount effective to reduce formation of a carbonyl derivative in the skin of the subject, said carbonyl derivative comprising an aldehyde, a ketone or a combination thereof.

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