HK1226953A1 - A topical composition containing sinomenine and a method for restraining carbonylation of protein - Google Patents

Description

Topical compositions comprising sinomenine and methods for inhibiting protein carbonylation

Technical Field

The present invention relates generally to topical compositions comprising sinomenine for use in inhibiting the carbonylation of skin proteins in a subject, and more particularly to topical compositions comprising sinomenine and methods of using the topical compositions to inhibit the carbonylation (or denaturation) of skin proteins in a subject.

Background

It is believed that a major contributor to aging-related skin yellowing is the process of protein denaturation in the dermis known as "carbonylation". Studies have shown that protein denaturation is the result of the binding between lipid degradation products produced by ultraviolet ("UV") radiation damage and dermal proteins. The resulting discoloration is a major factor contributing to yellowing of the skin with age of the individual and is particularly problematic for asians.

As an example of the foregoing process, lipids can interact with aldehydes that lead to carbonylation of dermal proteins, resulting in carbonylated proteins. In a similar manner, sugars can lead to the saccharification of dermal proteins, resulting in glycated proteins. Carbonylation is believed to produce more yellow skin relative to saccharification.

As a preventive measure, efforts have been made to prevent UV ray 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 use and require effective user compliance. In addition, some of these compositions can be irritating to the skin. Skin irritation or other application problems, such as undesirable greasiness, will cause the user to stop using the composition or to use the composition in a manner that is ineffective for its intended purpose.

In view of the above, there remains the need to provide other topical compositions that inhibit skin yellowing. There is also an opportunity to reverse, slow down or prevent skin carbonylation.

Disclosure of Invention

A topical composition is disclosed. The topical composition may be applied to the skin of a subject. The topical composition comprises sinomenine. The sinomenine is present in an amount effective to inhibit carbonylation of a skin protein in a 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 carbonylation of a skin protein in a subject is also disclosed. The cosmetic method comprises the step of applying sinomenine to the skin of the subject. The topical composition is generally applied in an amount and/or for a time sufficient to inhibit carbonylation of skin proteins. Unlike protein carbonylation inhibition, topical compositions are also useful for skin lightening (or whitening), particularly for inhibiting melanin synthesis, eliminating melanin, and/or inhibiting tyrosinase.

Drawings

Other advantages of the present disclosure will be readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings wherein:

FIG. 1 is a line graph illustrating cytotoxicity data ("data 1");

FIG. 2 is a line graph illustrating carbonylation inhibition data ("data 2");

FIG. 3 is another line graph illustrating cytotoxicity data ("data 3");

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

FIG. 5 is a line graph illustrating melanin elimination rate data ("data 5"); and

fig. 6 is a line graph illustrating tyrosinase inhibition rate data ("data 6").

Detailed Description

A topical composition ("composition") is disclosed. The composition is used for inhibiting skin protein carbonylation. More specifically, the compositions are useful for inhibiting the carbonylation of skin proteins in a subject. The skin layers include the epidermis and the dermis, and protein carbonylation generally occurs in at least the dermis. Typically, the composition is applied directly to the epidermis, e.g., the stratum corneum. The composition may also be applied indirectly to the epidermis, for example by a patch that carries/delivers the composition to the skin. At least a portion of the composition or an active ingredient thereof, such as sinomenine, migrates from the stratum corneum, through the epidermis and to the dermis to inhibit protein carbonylation therein.

The subject is typically a human, and may include men and women of various ages. The composition is not limited to a particular subject or location of the skin of a subject. For example, a person may apply the composition to their face, neck, arms, hands, chest, torso, legs, feet, and the like, or any combination thereof. Such skin areas may or may not be damaged. Dark spots, yellowing, sagging, and/or wrinkles generally indicate skin damage, such as UV radiation or photodamage. Methods of using the compositions are described further below.

It has surprisingly been found that the genus Toddalia (genus Sinomenium) can be used to inhibit protein carbonylation of the skin. Specifically, without being bound or limited to any particular theory, it is believed that the extract of the genus leptospira reduces and/or inhibits skin protein carbonylation. The compositions of the present disclosure generally comprise an extract from a plant of the genus leptospira, more typically a plant from the species leptospira (Sinomenium acutum). As described below, sinomenine is an active substance of the extract and is believed to be an effective compound for inhibiting protein carbonylation. As used herein, references to the genus tornado are used interchangeably with the dragon, and vice versa. The same is true for sinomenine and extract of the genus Toddalia (or Toddalia). Based on the findings herein, embodiments of the present disclosure generally relate to topical compositions comprising an extract of a plant of the genus chaenomeles, e.g., an extract of a plant of the species chaenomeles, wherein sinomenine is present in an amount sufficient to reduce/inhibit protein carbonylation in a subject following application.

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 may comprise plant material from the genus leptospira, including plant material from the species leptospira.

The active substances of aeolian dragon, which may also be called Sinomenium acutum (Thunb.) rehd. et Wils, aeolian dragon, include alkaloids such as sinomenine, isocratine, digoxin, L-tetrandrine and sinomenine, and β -phytosterol and stigmasterol, among which sinomenine is considered to be an effective compound inhibiting protein carbonylation (or denaturation), one or more of the other active substances may also be used for this effect, but sinomenine is considered to be most effective for protein carbonylation inhibition, the CAS number of sinomenine is 115-53-7, the molecular weight is 329g/mol, and the molecular formula C is C₁₉H₂₃NO₄. Sinomenine may be obtained commercially as a standard sample or may be obtained by extraction of plant material of the genus chaetomium. The standard sample 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 may also be obtained and used.

Any part of the plant of the genus chaenomeles may 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 parts. Sinomenine is generally present in the root of aeolian dragon, and thus, root extracts are generally most useful for the purposes of this disclosure. The genus Toddalia can be used in raw form, suspension form, dehydrated form, concentrated form, or extract form. Typically, the genera Toddalia are in dry or liquid form.

The extract of the genus salacia and/or other components of the composition may be obtained by conventional extraction methods known in the art, for example by water (e.g. steam) extraction or by solvent (e.g. alcohol) extraction. The compositions of the present disclosure are not limited to a particular extraction method, nor do they have to be extracted, as sinomenine is readily available from many commercial suppliers. Exemplary extraction methods are described below.

In many embodiments, the composition comprises an extract from a leptospira species. As used herein, reference to "an extract of the genus campylobacter" generally refers to a plant containing an extract from the genus campylobacter, including from the species campylobacter. Extracts of the genus leptospira are commercially available from a variety of sources. In addition, suitable extracts of the genus leptospira may be obtained by using any conventional extraction technique.

To obtain the extract of the genus leptospira, a polar solvent such as an alcohol (e.g., methanol, ethanol, butylene glycol), an ether (e.g., diethyl ether), a ketone (e.g., acetone), an ester (e.g., ethyl acetate), water, or a mixture thereof may be used as the solvent. The extract of the genus salacia may also be obtained by further extracting the extract with a non-polar solvent from a polar solvent. Suitable non-polar solvents include, but are not limited to, ethyl acetate, hexane, dichloromethane, chloroform or mixtures thereof.

There are many 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, which is hereby incorporated by reference in its entirety and is reproduced in part below with reference to some extraction methods. 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 alcohol solvents include, but are not limited to, C₁To C₄Alcohols such as methanol, ethanol, propanol, isopropanol and butanol; water-alcohol or a mixture of alcohol and water, including water-ethanol (hydro-ethanol); polyhydric alcohols such as propylene glycol and butylene glycol; and fatty alcohols. Any of these alcohol solvents may be used. Other solvents, such as but not limited to acetone, may also be used as the extraction solvent. Solvent-water blends of any ratio, such as alcohol-water and/or acetone-water blends, may also be used.

In one example, the extract of the genus leptospira can be obtained using organic solvent extraction techniques. In another example, solvent sequential fractionation may be used to obtain the extract of the genus leptospira. Total water-ethanol extraction techniques can also be used to obtain the extract of the genus Toddalia. In general, it is referred to as one-time extraction. The extract produced in this process will contain phytochemicals present in a wide range of extraction materials, including fat-soluble and water-soluble phytochemicals. After collecting the extraction solution, the solvent was evaporated to obtain an extract.

Total ethanol extraction may also be used. This technique uses ethanol as a solvent. The extraction techniques result in extracts that may include fat-soluble and/or lipophilic compounds in addition to water-soluble compounds. Total methanol extraction can also be used in a similar manner with similar results. In various embodiments, the arowana extract is obtained by alcohol extraction of plant material of the species arowana.

Another example of an extraction technique that may be used to obtain an extract of the genus leptospira is supercritical carbon dioxide supercritical fluid extraction ("SFE"). In this extraction procedure, the material to be extracted is not exposed to any organic solvent. Instead, the extraction solvent is carbon dioxide (CO) in a supercritical state (e.g., > 31.3 ℃ and > 73.8 bar)₂) With or without a modifier. One skilled in the art will appreciate that temperature and pressure conditions may be varied to obtain optimal extract yields. This technique produces an extract of fat-soluble and/or lipophilic compounds, which can also be used, similar to the total hexane and ethyl acetate extraction technique.

The extract of the genus leptospira 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 production of carbonyl derivatives from 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 μ g/mL of the topical composition. In specific embodiments, sinomenine is present in an amount of about 10, about 20, about 25, about 50, about 75, about 100, about 200, or about 300 μ g/mL of the composition. Also contemplated are various subranges and amounts from about 5 to about 500 μ g/mL of the composition, as well as amounts less than or greater than these amounts.

The amount of sinomenine present in the composition may depend on several factors, including the desired level of protein carbonylation inhibition, the level of protein carbonylation inhibition in a particular extract or composition, and other factors. In certain embodiments, sinomenine is present in an amount of from about 0.01 to about 20 parts by weight ("pbw") based on 100pbw of the composition. In a further embodiment, sinomenine is present in an amount of from about 0.05 to about 10pbw based on 100pbw of the composition. Also contemplated are various sub-ranges and amounts from about 0.01 to about 20pbw, as well as amounts less than or greater than these amounts. Other extracts or ingredients optionally useful in the compositions of the present disclosure are described in U.S. Pat. No. 5,747,006 to Dornoff et al, and U.S. Pat. Nos. 5,980,904, 6,994,874, 7,060,304, 7,247,321 and 7,364,759 to Leverett et al, the disclosures of which are incorporated herein by reference in their entirety.

In certain embodiments, the composition is free of other actives. By "other active" herein is generally meant that the composition does not contain other types of traditional Chinese medicines ("TCM"; or "Chinese medicines") other than Tornado. Other types of TCMs 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 by reference in their entirety. In certain embodiments, the compositions may comprise the following inactive materials. Inactive substances, if used, are different from other types of TCMs.

The compositions can be formulated to include a cosmetically acceptable carrier (or vehicle) and prepared and/or packaged and labeled to inhibit protein carbonylation, reduce skin yellowing, and/or lighten (or whiten) the skin. The composition may 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, an aerosol spray, gel, cream, dispersion, emulsion, foam, liquid, lotion, mousse, patch, pomade, powder, pump spray, solid, solution, stick, or towelette. 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 various methods known in the art. In one example of preparing the composition, the preparation method comprises the step of extracting plant material of the species chaulmoogra to obtain a chaulmoogra extract, i.e., to obtain sinomenine (with or without other active compounds). Typically, the method of preparation further comprises the step of combining sinomenine with a cosmetically acceptable carrier, such as one or more of the carriers described above. The components may be combined using conventional production methods and apparatus, such as mixers, blenders, and the like.

The compositions can be used to inhibit protein carbonylation in various ways. As an example, a cosmetic method for inhibiting carbonylation of a skin protein in a subject includes the step of applying sinomenine to the skin of the subject. Sinomenine can be applied in various ways, including applying a composition to the skin of a subject, wherein the composition comprises an extract from the species calophyllum. The composition may be applied to the skin directly or indirectly, for example, by hand, applicator, patch, and the like.

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

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

Examples

By way of background, oxidative stress is associated with the generation of reactive oxygen species by protein carbonylation, a process in which reactive aldehydes or ketones are introduced into proteins by oxidation. Protein carbonyls are the major products of protein oxidation and can be formed by oxidative cleavage of proteins, direct oxidation of amino acid residues, or covalent reaction with aldehydes derived from lipid peroxidation.

Protein carbonylation generally occurs by direct metal-catalyzed oxidation of amino acid side chains ("major protein carbonylation") or by addition of reactive aldehydes to amino acid side chains ("minor protein carbonylation"). Oxidative degradation of polyunsaturated fatty acids commonly found in the human body can lead to the formation of various aldehydes, such as acrolein and 4-hydroxynonanal ("4-HNE"). Studies have shown that the major protein carbonylation plays a role in the reactive oxygen species ("ROS") signaling mechanism. In particular, the major protein carbonylation mediates cell signaling, and the major protein carbonylation is reversible.

The major protein carbonylation involves direct metal-catalyzed oxidation of the amino acid side chain, particularly at the four susceptible amino acid residues: proline, arginine, lysine and threonine. Oxidation of the proline or arginine side chain results in the formation of glutamate semialdehyde, while lysine oxidizes to produce aminoadipate semialdehyde, thereby introducing carbonyl groups into the protein structure. Oxidation also converts the hydroxyl group of the threonine side chain to a carbonyl group, forming 2-amino-3-tetronic acid. The secondary protein carbonylation mechanism includes the michael addition reaction of reactive aldehydes to the side chains of lysine, histidine and cysteine residues, and the formation of schiff bases by reducing sugars with the amino groups on lysine and arginine, leading to an increase in the higher carbonylation end product.

Protein carbonyls are present in a variety of oxidative stress and disease conditions. In addition to the inactivation of the protein, the carbonylated cellular proteins undergo proteasome-dependent degradation. It is believed that the major protein carbonylation plays a role in labeling damaged proteins during oxidative stress to eliminate them from biological systems. The production of carbonyl proteins is a sign of oxidative stress, for example as an extrinsic factor, such as UV radiation-induced factors or external application of oxidative chemicals, or as an intrinsic factor, chemical attack by reactive carbonyl groups resulting from lipid peroxide degradation.

UV radiation or light damage skin is characterized by various features, such as dark spots, wrinkles and sagging. Older people, particularly asians, tend to show yellow skin color changes with photoaging. In china, a yellowish complexion generally means a lack of pink or a dull complexion. Common clinical signs of photodamage or aging include a characteristic pale or yellow skin appearance. Sallowness is often irreversible and thus prevention is desirable.

Various tests were performed and corresponding data sets were generated to determine the efficacy of sinomenine with respect to protein carbonylation. In a related section, cytotoxicity and inhibition of protein carbonylation are evaluated using potency assays and methods understood by those skilled in the art. The tabular data is shown below in data 1 and data 2 and in corresponding figures 1 and 2, respectively.

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

Data 1

Cytotoxicity of Sinomenine in HepG 2-CTG (Hydroquinone as reference)

From Promega Corporation("CTG") luminescence cell viability assay is a homogeneous method of determining the number of viable cells in culture, based on the quantification of ATP present as an indicator of metabolically active cells. According to the safety evaluation of CTG cell levels as illustrated by data 1 and fig. 1, sinomenine has no risk of cytotoxicity.

Data 2

Carbonylation inhibition of sinomenine in HepG2 (vitamin C as reference)

The gradient concentrations of sinomenine and vitamin C were 0, 33.3, 100 and 300. mu.g/ml. Two carbonylation inhibition rates were recorded for each concentration. The corresponding average carbonylation inhibition rates for sinomenine were 1.6%, 31.35%, 56.2% and 53.85%. The corresponding average carbonylation inhibition rates for vitamin C were-3.05%, 31.05%, 55.35% and 62.65%. In conclusion, sinomenine has an outstanding anti-carbonylation inhibitory potency in the cell HepG2 model compared to the positive control vitamin C without the risk of cytotoxicity. This data set is generally obtained by the assay methods listed below.

Carbonylation inhibition assay:

the purpose is as follows: the procedure describes a standard method for antioxidant assay of HepG2 cells.

Cell line: HepG2(ATCC #)

Materials:

·MEM(GIBCO#16600-082)

fetal bovine serum (GIBCO #10099-141)

Trypsin-EDTA (GIBCO #25200-072)

96 well cell plate (Costa)

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

The procedure is as follows:

day 1: plated cells

1. Trypsinize and determine cell density.

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

3. 90 μ l/well of cell suspension was dispensed onto assay plates (100,000 cells per well).

4. Under humidified conditions at 37 deg.C and 5% CO₂The assay plates were incubated for 24 hours.

Day 2: addition of test Compounds

1. Reference and test compound solutions (200 ×) were prepared according to plate diagrams.

2. Mu.l of the compound was transferred to 133. mu.l of the medium (final concentration: 10X).

3. Mu.l of the compound was transferred to an assay plate (final concentration: 1X).

4. Under humidified conditions at 37 deg.C and 5% CO₂The plates were incubated.

Day 4:

1. the medium was discarded and washed twice with hanks' balanced salt solution ("HBSS").

2. To each well 100 μ l HBSS (with mixture) was added.

3. The plate was placed at-80 ℃ for 30 min. And then unfreezing. Repeat at least 3 times.

4. The cell lysates were mixed.

5. 25 μ l of each lysate well was obtained and tested for protein concentration.

6. Regulating protein concentration.

7. 100 μ l of sample (using induced BSA dilution) was added to a 96-well protein binding plate. Incubate overnight at 4 ℃.

Day 5: ELISA assay-100 wells

1. Wells were washed 3 times with 250. mu.L of 1 XPBS per well. Excess wash solution was removed.

2. Add 100. mu.L of DNPH working solution and incubate for 45 min at room temperature in the dark.

3. Wash 5 times with 250. mu.L 1 XPBS/ethanol (1: 1, v/v) and incubate for 5 minutes on an orbital shaker.

4. Wash 2 times with 250 μ L1X PBS.

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

6. Wash 3 times with 250. mu.L of 1 XWash buffer, with sufficient aspiration between each wash.

7. 100 μ L of anti-DNP antibody was added and incubated on an orbital shaker for 1 hour at room temperature.

8. The strip wells were washed 3 times (1 × wash buffer).

9. To all wells 100 μ L of diluted HRP-conjugated secondary antibody was added and incubated on an orbital shaker for 1 hour at room temperature.

10. The strip wells were washed 5 times (1 × wash buffer).

11. The substrate solution was allowed to warm to room temperature. Add 100. mu.L of substrate solution to each well and incubate for 15 min.

12. The enzyme reaction was stopped by adding 100. mu.L of stop solution to each well. The result should be read immediately (color fading over time).

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

As background, human skin pigmentation is determined by the amount and location of melanin on the skin surface. Melanin is synthesized by the oxidation of the amino acid tyrosine to L-3, 4-dihydroxyphenylalanine ("L-DOPA") in cells present at the dermal-epidermal junction, commonly referred to as melanocytes. The oxidation process is catalyzed by tyrosinase. A series of cellular processes performed by melanocytes is commonly referred to as melanogenesis.

Skin pigmentation 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 UV radiation efficiently. As exposure of the skin to UV radiation increases, the amount and rate of melanin production generally increases, and can result in skin darkening, or "tan". Local or general pigmentation disorders, such as hyperpigmentation or hypopigmentation, can result from a number of factors, including in vivo hormone levels, diet, genetic disorders, and drug treatment. Common disorders of pigmentation disorders include chloasma (melisma), freckles and vitiligo.

Various compositions have been formulated to address pigmentation disorders, and have been contemplated for use in treating hyperpigmentation and/or hypopigmentation, for example. Such treatments are commonly referred to as "skin lightening," skin lightening, "or" skin lightening. There are several uses for skin lightening agents. For example, to lighten age spots (freckles or age spots), or to prevent darkening of the skin of caucasians and asians (i.e., maintaining a bright/white skin tone). Some of these formulations have included tyrosinase inhibitors such as hydroquinone, vitamin C, kojic acid, arbutin, glutathione, cysteine, lactic acid, ferulic acid, niacinamide, and plant extracts such as bearberry and mulberry extracts, and the like. However, of these compounds, hydroquinone and kojic acid, for example, have side effects including skin irritation, acute dermatitis, and cytotoxicity of skin cells.

Various tests were conducted and corresponding data sets were generated to determine the efficacy of sinomenine in lightening (or whitening) skin. In relevant part, cytotoxicity, melanin synthesis inhibition, melanin elimination, and tyrosinase inhibition are evaluated using potency assays and methods understood by those skilled in the art. The table data is shown below in data 3, data 4, data 5 and data 6 and in corresponding figures 3, 4, 5 and 6, respectively. Other extraction methods and analytical methods (e.g., assays) relevant to determining skin lightening efficacy are described in international publication nos. WO2011/019468 a2, WO 2011/109139 a2, and WO 2013/169634 a2 to Rana et al, the disclosures of which are incorporated herein by reference.

Data of3

Cytotoxicity of Sinomenine in B16F 10-CTG (Hydroquinone as reference)

According to the safety evaluation of cytotoxicity of CTG as illustrated by data 3 and fig. 3, sinomenine has no risk of cytotoxicity.

According to in vitro bioassay data, sinomenine was found to have skin lightening and depigmenting effects. Inhibition of melanin synthesis and the presence of melanin-eliminating activity, as well as inhibition of tyrosinase activity, was monitored by measurement of cell levels of model cells, B16F 10. This type of model cell-based assay for B16F10 cells is known to those skilled in the art.

Arbutin was considered a positive control for both melanin synthesis inhibition and tyrosinase activity inhibition, while hydroquinone was used as a positive control for the already existing melanin-eliminating activity. Summarizing the results described below, sinomenine has outstanding activity in vitro tests without the risk of cytotoxicity as a skin lightening or depigmenting agent.

Data 4

Melanin synthesis inhibition in B16F10 by sinomenine (arbutin as reference)

Sinomenine

Arbutin

In the melanin synthesis inhibitory activity in B16F10 cells, the inhibitory rate of sinomenine was 66.9% at 100 μ g/mL relative to the positive control arbutin. Thus, 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 outlined immediately below.

Melanin synthesis inhibition assay:

the purpose is as follows: this procedure describes a standard method for the determination of melanin synthesis inhibition in B16F10 cells.

Cell line: B16-F10(ATCC #)

Materials:

RPMI 1640 for B16 (GIBCO #22400-089 batch No. 1006397)

Fetal bovine serum (GIBCO #10099-141)

Trypsin-EDTA (GIBCO #25200-072)

·1M NaOH

24 orifice plate (Corning)

The procedure is as follows:

day 1: plated cells

5. Trypsinize and determine cell density.

6. The cell suspension was diluted to the desired volume at a density of 18,000 cells/ml.

7. 1 ml/well of cell suspension was dispensed onto assay plates.

8. Under humidified conditions at 37 deg.C and 5% CO₂The assay plates were incubated for 24 hours.

Day 2: addition of test Compounds

5. Reference and test compound solutions (200 ×) were prepared according to plate diagrams.

6. Mu.l of the compound was transferred to an assay plate (final concentration: 1X).

7. Under humidified conditions at 37 deg.C and 5% CO₂Plates were incubated for 72 hours.

Day 5: imaging a plate

14. The medium was removed.

15. Mu.l of 1M NaOH was added to the assay plate.

16. The plates were incubated at 80 ℃ for 30 minutes.

17. Transfer 140 μ l of the solution to a UV plate. The 400nm signal was tested.

18. Transfer 5. mu.l of the solution to a UV plate. To each well 200. mu.l BCA reagent was added and incubated for 20 minutes at 37 ℃. The 562nm signal was tested.

Data processing:

GraphPad was used.

Inhibition ═ maximum signal-compound signal)/(maximum signal-minimum signal) × 100.

The maximum signal is obtained from the effect of DMSO.

Minimal signal was obtained from 200 μ g/ml arbutin.

Data 5

Melanin elimination ratio of sinomenine in B16F10 (hydroquinone as reference)

Sinomenine

Hydroquinone

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

Determination of the elimination of melanin present:

the purpose is as follows: the procedure describes a standard method for melanin elimination assays in B16 cells.

Cell line: B16-F10(ATCC #)

Materials:

RPMI 1640 for B16 (GIBCO #22400-089 batch No. 1006397)

Fetal bovine serum (GIBCO #10099-141)

Trypsin-EDTA (GIBCO #25200-072)

·1M NaOH

24 orifice plate (Corning)

The procedure is as follows:

day 1: plated cells

1. Trypsinize and determine cell density.

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

3. 1 ml/well of cell suspension was dispensed onto assay plates.

4. Under humidified conditions at 37 deg.C and 5% CO₂The assay plates were incubated for 24 hours.

Day 2: cell processing

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

2. Under humidified conditions at 37 deg.C and 5% CO₂Assay plates were incubated for 72 hours.

Day 5: addition of test Compounds

1. Fresh medium was replaced, 1ml per well.

2. Reference and test compound solutions (200 ×) were prepared according to plate diagrams.

3. Mu.l of the compound was transferred to an assay plate (final concentration: 1X).

4. Under humidified conditions at 37 deg.C and 5% CO₂Plates were incubated for 72 hours.

Day 8: imaging a plate

1. The medium was removed.

2. Mu.l of 1M NaOH was added to the assay plate.

3. The plates were incubated at 80 ℃ for 30 minutes.

4. Transfer 140 μ l of the solution to a UV plate. The 400nm signal was tested.

5. Transfer 5. mu.l of the solution to a UV plate. To each well 200. mu.l BCA reagent was added and incubated for 20 minutes at 37 ℃. The 562nm signal was tested.

Data processing:

GraphPad was used.

Inhibition ═ maximum signal-compound signal)/(maximum signal-minimum signal) × 100.

Maximum signal was obtained from cells without compound effect.

The minimum signal was obtained from 10. mu.M forskolin and 50. mu.M 8-MOP.

Data 6

Tyrosinase inhibition by sinomenine in B16F10 (arbutin as reference)

Sinomenine

Arbutin

Sinomenine exerts excellent tyrosinase inhibitory activity in B16F10 cells, and the inhibition rate is 32.4% at a concentration of 100. mu.g/mL. Specifically, as shown in data 6 and fig. 6, sinomenine performed as well as or much better than arbutin in terms of tyrosinase inhibitory activity in B16F10 cells.

Briefly summarized, in addition to the good properties of tyrosinase inhibition and melanin synthesis inhibition, in particular, the inventors have found that sinomenine exhibits a great ability to eliminate existing melanin assay models. Thus, sinomenine has excellent activity as a skin whitening and depigmenting agent without the risk of cytotoxicity for skin care or topical use.

It is to be understood that the appended claims are not limited to the specific and specific compounds, compositions, or methods described in the detailed description, which may vary between specific embodiments within the scope of the appended claims. For any markush group on which particular features or aspects of various embodiments are described herein, it is to be understood that different, specific, and/or unexpected results can be obtained from each member of the respective markush group independently of all other markush members. Each member of the markush group may be relied upon individually or in combination and provide adequate support for specific embodiments within the scope of the appended claims.

It is also to be understood that any ranges and subranges relied upon in describing the various embodiments of the invention are independently and collectively within the scope of the appended claims, and are to be understood as describing and encompassing all ranges including all and/or some of the values therein, even if such values are not explicitly written herein. Those skilled in the art will readily recognize that the enumerated ranges and subranges sufficiently describe and enable various embodiments of the present invention, and that such ranges and subranges can be further delineated into relevant halves, thirds, quarters, fifths, and so on. As but one example, a range of "0.1 to 0.9" may be further delineated into a smaller third, i.e., 0.1 to 0.3, a middle third, i.e., 0.4 to 0.6, and a larger third, i.e., 0.7 to 0.9, which individually and generally fall within the scope of the appended claims, and may be relied upon and provide sufficient support for specific embodiments within the scope of the appended claims, individually and/or generally. Further, with respect to language that defines or modifies a range, such as "at least," "greater than," "less than," "no greater than," and the like, it is to be understood that such language includes subranges and/or an upper or lower limit. As another example, a range of "at least 10" inherently includes at least a sub-range of 10 to 35, a sub-range of at least 10 to 25, a sub-range of at least 25 to 35, and the like, and each sub-range can be relied upon individually and/or collectively and provides adequate support for specific embodiments within the scope of the appended claims. Finally, individual numerical values within the disclosed ranges may be relied upon and provide sufficient support for specific embodiments within the scope of the appended claims. For example, a range of "1 to 9" includes individual integers, such as 3, and individual values including decimal points (or fractions), such as 4.1, which may be relied upon and provide adequate support for specific embodiments within the scope of the appended claims.

The invention has been described herein by way of illustration and example, and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation. Many modifications and variations of the present invention are possible in light of the above teachings. The invention may be practiced otherwise than as specifically described within the scope of the appended claims. The subject matter of all combinations of independent and dependent claims (both single and multiple dependent) is expressly contemplated herein.

Claims (16)

1. A topical composition comprising sinomenine in an amount effective to inhibit carbonylation of a skin protein in a subject.

2. A topical composition for inhibiting carbonylation of a skin protein in a subject, the active ingredient of the topical composition consisting of sinomenine.

3. The topical composition of claim 1 or 2, wherein the topical composition comprises an extract from a leptospira fragrans.

4. The topical composition of claim 3, wherein the sinomenine is present in an amount effective to reduce the production of a carbonyl derivative in the skin of a subject, the carbonyl derivative comprising an aldehyde, a ketone, or a combination thereof.

5. The topical composition of claim 3, wherein the sinomenine is present in an amount from about 5 to about 500, or from about 25 to about 350 μ g/mL of the topical composition.

6. The topical composition of claim 3, wherein the sinomenine is obtained by solvent extraction, or by alcohol extraction of the plant material of the Fenglong species.

7. 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%.

8. The topical composition of claim 1 or 2, further comprising a cosmetically acceptable carrier.

9. 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, silicones, fatty esters, fatty alcohols, glycols, polyglycols, and combinations thereof.

10. A composition for inhibiting carbonylation of a skin protein in a subject, said composition consisting of sinomenine.

11. Use of the topical composition of any one of claims 1, 2, or 10 for inhibiting carbonylation of a skin protein in a subject.

12. A method of preparing a topical composition according to any one of claims 1, 2 or 10, the method comprising the step of extracting plant material of the plant species chaulmoogra to obtain sinomenine.

13. The method of claim 12, further comprising the step of combining said sinomenine with a cosmetically acceptable carrier.

14. A cosmetic method for inhibiting carbonylation of a skin protein in a subject, the method comprising the step of applying sinomenine to the skin of the subject.

15. A cosmetic method as set forth in claim 14 wherein the step of applying is further defined as applying a topical composition to the skin of the subject, wherein the topical composition comprises an extract from the leptospira.

16. The cosmetic method of claim 15, wherein the sinomenine is present in an amount effective to reduce the production of a carbonyl derivative in the skin of the subject, the carbonyl derivative comprising an aldehyde, a ketone, or a combination thereof.