TW201637662A - Topical composition comprising sinomenine and method for inhibiting protein carbonylation - 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 vine salt and method for inhibiting protein carbonylation

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 subject using the topical composition A method of carbonylation (or denaturation) of skin proteins.

A major contributor to skin yellowing associated with aging is the protein denaturation process known as "carbonylation" in the dermis. Studies have shown that protein denaturation is the result of the binding of lipid degradation products produced by ultraviolet ("UV") ray damage to 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. Similarly, saccharides can cause saccharification of dermal proteins to give glycated proteins. Salty carbonylation produces 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 implementation in action and require the user's willingness to use it in order to function. In addition, some of these compositions can irritate the skin. Skin irritation or other application problems such as nasty oil Greasy will cause the user to stop using the composition or use the composition in a manner that is ineffective for its intended purpose.

In view of the above, there are still opportunities to provide other skin lightening agents, especially high-efficiency skin lightening agents. Most specifically, there is still an opportunity to provide other topical compositions that inhibit skin yellowing. There is also a chance to reverse, slow or prevent skin carbonylation.

The present invention discloses a topical composition. 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 protein carbonylation. In certain embodiments, the active ingredient of the topical composition consists of sinomenine. In a particular embodiment, the topical composition consists of sinomenine.

The present invention also discloses a cosmetic method for inhibiting protein carbonylation in a subject. 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.

Other advantages of the present disclosure will be more readily understood by reference to the following detailed description taken in conjunction with the accompanying drawings in which: FIG. 1 is a line diagram illustrating cytotoxicity data ("Data 1"); the second figure is an illustration of tyrosine a line graph of enzyme inhibition rate data ("Data 2"); The third diagram is another line diagram illustrating melanin synthesis inhibition data ("Data 3"); and the fourth diagram is a line diagram illustrating melanin elimination rate data ("Data 4").

A topical composition ("composition") is disclosed. The composition is useful for inhibiting skin protein carbonylation in a subject. More specifically, the composition can be used to inhibit skin protein carbonylation in a subject. The skin layer includes the epidermis and the dermis, and protein carbonylation generally occurs in 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. Dark spots, yellowing, sagging, and/or wrinkles generally indicate skin 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, it is believed that the extract of the genus Rhododendron reduces and/or inhibits skin protein carbonylation. The compositions of the present disclosure generally comprise a plant extract from the genus Geochelone , more typically from 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. The dragons mentioned in this article can be used interchangeably with the wind dragons and vice versa. The same is true for the mention of sinomenine and genus (or wind dragon) extracts. 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 present in an amount sufficient to reduce/inhibit the test after administration. Protein carbonylation.

In various embodiments, the composition comprises sinomenine. In a further embodiment, the active ingredient of the topical 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 may comprise 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 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 an effective compound for inhibiting protein carbonylation (or denaturation). One or more other active substances may also be used for this effect, but 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 purity levels are also available and used.

Any portion of the genus Rhododendron 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, and therefore, the extract is generally most useful for the purposes of the present disclosure. The genus Rhododendron can be used in its original form, in suspended form, in dehydrated form, in concentrated form or as an extract. Usually, the larva is in a dry or liquid form.

The other components of the extract and/or composition of the genus 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 process and do not necessarily require extraction because sinomenine is readily available from a number of suppliers. Exemplary extraction methods are described below.

In many embodiments, the composition comprises an extract from the genus. As used herein, "Windragon Extract" generally refers to extracts from the genus Rhododendron, including from the wind dragon species. Wind Dragon extracts are commercially available from a variety of sources. In addition, a suitable extract of the genus Dragon 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 can be used. Solvent. The extract of the genus Rhododendron can also be obtained by further extraction from a polar solvent using 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 topical compositions. These methods include, but are not limited to, the extraction methods disclosed in U.S. Patent No. 7,897,184 to Rana, et al. Although 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 a mixture of alcohol and water, including aqueous 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 may 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, an 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 many of the phytochemicals present in the extract, including fat-soluble and water-soluble phytochemicals. After collecting the extraction solution, the solvent was evaporated to give an extract.

It is also possible to use total ethanol extraction completely. This technique uses ethanol as a solvent. The extract produced by the extraction technique may include, in addition to the water-soluble compound, a fat-soluble and/or lipophilic compound. Total methanol extraction can also be used in a similar manner with similar results. In various embodiments, the wind dragon extract is obtained by extracting the plant material of the wind dragon species with an alcohol.

Another example of an extraction technique that can be used to obtain a Wind Dragon extract is supercritical fluid carbon dioxide supercritical 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, and can also be used.

The extract of the genus Rhododendron added to the composition may be in any portion, provided that it is present in an amount effective to inhibit protein carbonylation. Generally, sinomenine is present in an amount effective to reduce the formation of carbonyl derivatives in 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 composition. In a particular embodiment, sinomenine is present in an amount of about 10, about 20, about 25, about 50, about 75, about 100, about 200, or about 300 [mu]g/mL of the composition. Also contemplated are individual subranges and portions of the composition from about 5 to about 500 [mu]g/mL, as well as smaller or larger portions.

The amount of sinomenine present in the composition can depend on several factors, including the desired protein carbonylation inhibition level, protein carbonylation inhibition levels in the 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 of the "(pbw") composition. In a further embodiment, sinomenine is present in an amount from about 0.05 to about 10 pbw based on the 100 pbw composition. Also contemplated are individual subranges and portions of from about 0.01 to about 20 pbw, as well as smaller or larger portions. Alternatively, other extracts or ingredients for use in the compositions of the present disclosure are described in Dornoff et al. U.S. Patent Nos. 5,747,006, and U.S. Patent Nos. 5,980,904, 6,994,874, 7, 060,304, 7, 247, 321 and 7, 364, 759, the disclosures of each of which are incorporated herein by reference.

In certain embodiments, the composition is free of other actives. The "other active substances" herein generally means that the composition does not contain other types of traditional Chinese medicines other than the genus Wind Dragon ("TCM"; or "Chinese medicine"). Other types of TCMs are contemplated 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 topical composition can comprise the following inactive materials. If used, inactive materials are different from other types of TCM.

The composition 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 (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, anthrone such as emu oil, fatty esters, fatty alcohols, glycols, polyglycols or random combination. Such components are generally considered to be inactive components. The final topical 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 emulsion may include an oil-in-water emulsion, a water-in-oil emulsion, and a fluorenone-in-water emulsion.

The compositions of the present disclosure can be prepared using a variety of methods known in the art. In one example of preparing a composition, the method of preparation comprises extracting plant material of the genus The step of obtaining the fenglong extract, ie 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 for skin lightening in a variety of ways. For example, a cosmetic method for inhibiting protein carbonylation includes the step of applying the original Lara vine to the skin of a subject. The original lara vine can be applied in a variety of ways, including applying a topical composition to the subject's skin. The topical composition can be applied directly or indirectly to the skin, such as by hand, applicator, patch, and the like.

The topical compositions can be administered as needed, daily, several times a day, or any suitable regimen to achieve the desired result. In cosmetic methods, the frequency of topical application can depend on several factors, including the desired protein carbonylation inhibition level. In general, the regimen includes applying the topical composition to the skin once or twice daily to include morning application and/or evening application. The amount of topical composition applied to the skin each time can depend on several factors, including the level of the desired result and the particular topical composition.

The following examples illustrate the topical compositions and methods of the present disclosure, which are intended to illustrate and not to limit the invention.

Example

As a background, oxidative stress is associated with the formation of reactive oxygen species by protein carbonylation, a process in which a reactive aldehyde or ketone is introduced into a protein by oxidation. Protein carbonyl is the major product of protein oxidation and can be oxidatively cleaved by a protein, directly oxidized by an amino acid residue, or covalently reacted with an aldehyde derived from lipid peroxidation. form.

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.

The main protein carbonylation involves direct metal catalyzed side chain oxidation of the amino acid, particularly on four susceptible amino acid residues: proline, arginine, lysine and threonine. Oxidation of the valine or arginine side chain results in the formation of glutamic acid semialdehyde, while lysine oxidizes to produce 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. Secondary protein carbonylation mechanisms include Michael addition reactions of reactive aldehydes with side chains of lysine, histidine, and cysteine residues, and by reducing sugars with amines on lysine and arginine The formation of a Schiff base results in an increase in the higher carbonylation end products.

Protein carbonyls occur in a variety of oxidative stress and disease conditions. In addition to protein deactivation, carbonylated cellular proteins undergo proteasome-dependent degradation. The major protein carbonylation acts to label 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, chemical attack from reactive carbonyls degraded by lipid peroxides. .

UV rays or light damaged skin manifest in a variety of features such as dark spots, wrinkles and sagging. Older people, especially Asians, tend to exhibit yellow skin color changes 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 it is best to prevent it in advance.

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 obtained data are shown in the following data 1 and data 2, and their respective corresponding Figs. 1 and 2.

As described below, sinomenine has no cytotoxic risk according to the safety evaluation by CTG cell level (see data 1/Fig. 1). Moreover, the inventors have found that sinomenine has the efficacy and function of skin anti-carbonylation according to in vitro bioassay data (see Data 2/Figure 2). Cell-based assays by model cell HepG2 were performed and inhibition of carbonylation activity was monitored. Vitamin C is a positive control for inhibition of carbonylation. Those skilled in the art will be able to understand this type of model cell HepG2 cell-based assay.

Data 1

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

Promega Corporation's CellTiter-Glo® (CTG) luminescence cell viability assay was used to determine the number of viable cells in culture as a reference for metabolically active cells and the quantification of existing ATP. According to the cell-based safety evaluation of CTG, as exemplified in Data 1 and the first figure, sinomenine has no risk of cytotoxicity.

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 average carbonylation inhibition rates of sinomenine were 1.6%, 31.35%, 56.2%, and 53.85%. The average carbonylation inhibition rate corresponding to vitamin C was -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 the standard side of anti-oxidation assay for HepG2 cells. law.

Cell line: HepG2 (ATCC #)

material:

̇MEM (GIBCO #16600-082)

Abortion bovine serum (GIBCO #10099-141)

̇Trypsin-EDTA (GIBCO #25200-072)

̇96-well cell culture 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 the cell suspension to the assay plate (100,000 cells per well) at 90 μl per well.

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

Day 2: Add test compound

1. Prepare the reference and test compound solution (200×) 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. The assay disk was incubated 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 assay plate at -80 ° C for 30 min. Then thaw. Repeat at least 3 times.

4. Mix the cell lysate.

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

6. Regulate protein concentration.

Reagent A 0.2ml

Reagent B 9.8ml

Total 10.0ml

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 each well 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. Dilution multiple DNPH stock solution (1mg/ml)

4. Wash twice with 250 μL of 1X PBS.

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

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 (1X 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 (1X wash buffer).

11. Allow the substrate solution to cool to room temperature. 100 μL of the substrate solution was added to each well and cultured 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 the disc 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 skin surface. Melanin is synthesized by oxidation of amino acid tyrosine to L-3,4-dihydroxyphenylalanine ("L-DOPA") by cells present at the dermal-epidermal junction, commonly referred to as melanocytes. 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 absorbs ultraviolet ("UV") rays. When the skin's exposure to UV rays increases, in general It will increase the amount and rate of melanin production and can cause the skin to darken or become "sepia". Local or general pigmentation disorders, such as hyperpigmentation or hypopigmentation, can result from a number of factors, including hormone levels in the body, diet, genetic disorders, and medications. Common pigmentation disorders include melasma, freckles and white spots.

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". Skin lightening agents have several uses. For example, lightening age spots (freckles or aging spots), diluting, or preventing blackening of the skin of Caucasians and Asians (ie, maintaining a bright/white complexion). Some of these preparations include tyrosinase inhibitors such as hydroquinone, vitamin C, citric acid, arbutin, glutathione, cysteine, lactic acid, ferulic acid, nicotinamide, and plant extracts. Such as bearberry and mulberry extracts, and the like. However, certain such compounds, such as hydroquinone and citric 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 in skin lightening (or whitening). In the relevant section, cytotoxicity, melanin synthesis inhibition, melanin elimination, and tyrosinase inhibition were evaluated using potency assays and methods as understood by those skilled in the art. The following data 3, data 4, data 5, and data 6 provide tabular data, each corresponding to the third, fourth, fifth, and sixth figures. 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 Incorporated herein by reference.

Data 3

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

Based on in vitro bioassay data, sinomenine was found to have efficacy in skin lightening and depigmentation. The cell-based assay was administered by model cell B16F10, monitoring of cell water was monitored, and melanin synthesis was monitored to inhibit existing ink stain elimination activity. The determination of this type of model-based cell B16F10 cells is well known to those skilled in the art.

Arbutin is considered to be a positive control for both inhibition of tyrosinase activity and inhibition of melanin synthesis, while hydroquinone is used as a positive control for deactivation of existing melanin. Summarizing the results described below, sinomenine has outstanding activity as an skin lightening or depigmenting agent in vitro tests 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 measurement methods to be listed below.

Melanin synthesis inhibition assay:

Purpose: This program describes a standard method for the inhibition of melanin synthesis in B16F10 cells.

Cell line: B16-F10 (ATCC #)

material:

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

Abortion bovine serum (GIBCO#10099-141)

̇Trypsin-EDTA (GIBCO # 25200-072)

̇1M NaOH

̇24-hole plate (Corning)

Program :

Day 1: Plating cells

1. Trypsinize and measure cell density.

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

3. Dispense the cell suspension to the assay plate at 1 ml/well.

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

Day 2: Add test compound

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

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

3. The assay disk was incubated at 37 ° C, 5% CO 2 for 72 hours under humidified conditions.

Day 5: Imaging the disc

1. Remove the medium.

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

3. Incubate the assay disk at 80 ° C for 30 minutes.

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

5. Transfer 5 μl of the solution to the UV disk. 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.

̇% suppression = (maximum signal - compound signal) / (maximum signal - minimum signal) × 100.

The maximal signal was 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 existing melanin elimination assay, 39.4% of the elimination rate of the positive control hydroquinone at 100 μg/mL was found to be eliminated after 3 hours of addition of 100 μg/mL sinomenine for 72 hours in the B16F10 cultured cells. This means that sinomenine is also used for melanin elimination. This data set is generally obtained by the following assay methods to be listed.

Existing melanin elimination assay:

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

Cell line: B16-F10 (ATCC #)

material:

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

Abortion bovine serum (GIBCO#10099-141)

̇Trypsin-EDTA (GIBCO # 25200-072)

̇1M NaOH

̇24-hole 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 the cell suspension to the assay plate at 1 ml per well.

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

Day 2: Cell Processing

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

2. The assay disk was 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 the reference and test compound solution (200×) according to the plate diagram.

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

4. The assay disk was incubated at 37 ° C, 5% CO 2 for 72 hours under humidified conditions.

Day 8: Imaging the disc

1. Remove the medium.

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

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

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

5. Transfer 5 μl of the solution to the UV disk. 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.

̇% suppression = (maximum signal - compound signal) / (maximum signal - minimum signal) × 100.

The maximal 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 inhibition activity in B16F10 cells The inhibition rate was 32.4% at a concentration of 100 μg/mL. Specifically, as exemplified in Data 6 and the sixth graph, sinomenine is equivalent to or better than arbutin in terms of 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 exhibits great ability in eliminating existing melanin assay models. Thus, it has excellent activity as a skin whitening and depigmenting agent, sinomenine, and no risk of cytotoxicity for skin care or topical use.

It is to be understood that the scope of the appended claims is not limited to the specific and specific embodiments of the invention and For any of the Markusi groups used herein to describe particular features or aspects of the various embodiments, it should be understood that different, specific, and/or unexpected results may be obtained from various Markusi groups. Each composition is composed independently of all other Markusi. Each of the components of the Marcuse group may be solely or in combination for the specific embodiments within the scope of the appended claims and may provide sufficient support.

It is also to be understood that any of the scope and sub-ranges that are described in the various embodiments of the present invention are intended to be Even these values are not explicitly written in this article. Those skilled in the art can readily appreciate that the various ranges and sub-ranges of the present invention fully describe and implement various embodiments of the present invention, and such ranges and sub-ranges can be further described as related one-half, one-third, and four-quarters One, one fifth, and so on. As an example only, 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, That is, 0.7 to 0.9, each of which is, and generally is, within the scope of the appended claims, and may be individually and/or generally based on the specific embodiments within the scope of the appended claims. In addition, with respect to languages that define or modify the scope, such as "at least", "greater than", "less than", "not greater than", etc., it is understood that such language includes subranges and/or upper or lower limits. As another example, the range of "at least 10" inherently includes at least a subrange of 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 a respective and/or overall It is based on and provides sufficient support for the specific embodiments within the scope of the appended claims. Finally, individual values within the scope of the disclosure may be individually and/or generally based on and provide sufficient support for the specific embodiments within the scope of the appended claims. For example, the range of "1 to 9" includes each individual integer, such as 3, and individual values including a decimal point (or fraction), such as 4.1, which may each and/or generally be a specific implementation within the scope of the appended claims. The examples are based on and provide sufficient support.

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