CN106137807B

CN106137807B - Compositions comprising thalictrum foeniculaceum and methods for skin lightening

Info

Publication number: CN106137807B
Application number: CN201510128115.3A
Authority: CN
Inventors: 韩强; 张翌; 李廷钊; 杜军; 李波; 申晓娟
Original assignee: Jietong International Co ltd
Current assignee: Jietong International Co ltd
Priority date: 2015-03-23
Filing date: 2015-03-23
Publication date: 2020-11-06
Anticipated expiration: 2035-03-23
Also published as: TW201634044A; WO2016150309A1; CN106137807A

Abstract

A composition comprises plant material from the genus thalictrum. The composition may be used topically to lighten (or whiten) the skin of a subject. Typically, the composition comprises plant material, such as an extract, from the thalictrum petersonii species. In certain embodiments, the active ingredient of the composition consists of thalictrum petersonii. In a particular embodiment, the composition consists of thalictrum petersonii. A cosmetic method for lightening a subject's skin includes the step of applying thalictrum petitkii to the subject's skin. The composition is generally applied in an amount and/or for a time sufficient to lighten the skin.

Description

Compositions comprising thalictrum foeniculaceum and methods for skin lightening

Technical Field

The present invention relates generally to compositions comprising Thalictrum (Thalictrum) for lightening the skin of a subject, and more particularly to compositions comprising Thalictrum petaloideum (Thalictrum petaloideum) and methods of lightening the skin of a subject using the compositions.

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. In the other mode, melanin is produced by melanocytes existing at the dermal-epidermal junction, and the chemical nature is that tyrosine in the melanocytes is catalyzed and oxidized by tyrosinase to produce L-DOPA, and then melanin is produced through a series of enzymatic reactions. This series of cellular processes is commonly referred to as melanogenesis. The oxidation process is catalyzed by an enzyme called 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. Healthy amounts of melanin in the skin absorb ultraviolet ("UV") radiation effectively. In said another way, in healthy skin, melanin is present in an amount effective to absorb the ultraviolet radiation from the external environment. Increased exposure of the skin to UV radiation generally increases the amount and rate of melanin production (with a corresponding increase in the amount and rate of skin melanin production as the skin is exposed to UV radiation for an increased period of time and intensity), and may result in darker skin (darker skin color), 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 pigmentation disorders include chloasma (melasma), 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, lightening age spots (freckles or age spots), lightening, or preventing darkening of human skin (i.e., maintaining a bright/white skin tone) in caucasians and asians. 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, some of these compounds, such as hydroquinone and kojic acid, have side effects, including skin irritation, acute dermatitis, and cytotoxicity of skin cells.

In view of the above, there remains an opportunity to provide other skin lightening agents, in particular high efficacy skin lightening agents. Most particularly, there remains an opportunity to provide effective skin lightening agents and to add natural materials to the compositions to address the problems associated with conventional compounds such as hydroquinone.

Disclosure of Invention

A composition is disclosed. The composition may be used topically to lighten (or whiten) the skin. The composition comprises thalictrum petitchii. In certain embodiments, the active ingredient of the composition consists of thalictrum petersonii. In a particular embodiment, the composition consists of thalictrum petersonii.

A cosmetic method of lightening a subject's skin is also disclosed. The method includes the step of applying Thalictrum petaloides to the skin of the subject. The composition is generally applied in an amount and/or for a time sufficient to lighten the skin.

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 tyrosinase inhibition rate data ("data 2");

fig. 3 is a line graph illustrating melanin synthesis inhibition data ("data 3"); and

fig. 4 is a line graph illustrating melanin elimination rate data ("data 4").

Detailed Description

A composition is disclosed. The composition is typically a topical composition. The composition is used for skin lightening (or whitening). More specifically, the compositions may be used to lighten the skin of a subject. 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 areas of skin may be hyperpigmented or hypopigmented. Methods of using the compositions are described further below.

It has surprisingly been found that Thalictrum (genus Thalictrum) can be used for skin lightening. Specifically, without being bound or limited to any particular theory, it is believed that the extract of thalictrum reduces and/or inhibits melanin production. Alternatively or additionally to reduction/inhibition, an extract of thalictrum is believed to eliminate melanin. The compositions of the present disclosure generally comprise an extract from a plant of the genus thalictrum, more typically from the species thalictrum amboinense. As used herein, reference to thalictrum may be used interchangeably with thalictrum petitkii, and vice versa. Based on the findings herein, embodiments of the present disclosure generally relate to topical compositions comprising an extract of a thalictrum plant, e.g., an extract of a thalictrum plant, in an amount sufficient to reduce/inhibit melanin production in a subject in need thereof and/or in an amount sufficient to eliminate melanin in a subject in need thereof.

In various embodiments, the composition comprises thalictrum, or thalictrum. In a further embodiment, the active ingredient of the composition consists of thalictrum aquifolium, or consists of thalictrum petersonii. 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 thalictrum aquifolium, or consists of thalictrum petersonii. In all of these embodiments, the composition can include plant material from the genus Thalictrum, including plant material from one or more species such as Thalictrum petersonii (also known as Thalictrum petaloideum L.).

Thalictrum aquilegifolium is a genus of 120 to 200 perennial flowering plants of Ranunculaceae (buttercup) which mainly grow in temperate regions. Although commonly named "grass rue," the thalictrum species is generally unrelated to true rue (rutaceae), but has two or three petioles similar to its members. Grassland rue is common in shady or damp places, spread throughout most of the northern hemisphere and sub-world in south to south africa and tropical south america, but is generally not present in australia. Thalictrum is most common in the world's temperature regions, with at least 22 being present in north america. The leaves are alternately feathered, compound leaves, and the staining is usually blue-green. Flowers are small and without petals, but have many long stamens, usually bright white, yellow, pink or light purple, and are produced in prominent dense inflorescences. In some species, sepals are large, bright and petaloid, but in most cases they are small and flagging when or shortly after the flower is blooming. Typical natural products present in thalictrum are benzylisoquinoline alkaloids, such as magnoflorine and the structurally related alkaloid berberine.

The scientific classification of this component of the composition is generally as follows-mesh: ranunculus (Ranunculus); family: ranunculaceae (Ranunculaceae); and belongs to: thalictrum aquilegifolium (Merr.) Kuntze. Exemplary species of thalictrum foeniculaceum (T.) include, but are not limited to: down pine (t.alpinum), t.aquilegifolia, t.calabroccoli, thalictrum arborescens (t.clifolium), t.coelenti, thalictrum foeniculiforme (t.delavayi), thalictrum citriodorum (t.delvanum), thalictrum citrifolia (t.diferum), thalictrum foeniculatum (t.dioecium), thalictrum yunnanense (t.diptercarpum), t.fendleri, thalictrum foeniculatum (t.flavum), thalictrum serpentium (t.fonticulatum), thalictrum glandulosum (t.foeniculum), thalictrum crepidum (t.foenium), thalictrum crepidum (t.melo), thalium, thalictrum crepidum, thalictrum atratum (t.e), thalictrum citrinum, thalictrum, thalium, and a, or a variety t.e, or a. In many embodiments, the thalictrum includes or is thalictrum petersonii.

Any part of the thalictrum plant may be used to produce materials used in the compositions, including, but not limited to, roots, stems, rhizomes, leaves, flowers, fruits, and/or extracts of these parts. Thalictrum foeniculaceum may be used in raw, suspended, dehydrated, concentrated or extracted form. Typically, thalictrum is in dry or liquid form. Extracts of this component 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. Exemplary extraction methods are described below.

In many embodiments, the composition comprises an extract from a thalictrum petersonii species. As used herein, reference to "Thalictrum extract" generally refers to a composition containing an extract from the genus Thalictrum, including species of Thalictrum from the genus Thalictrum, either alone (i.e., "Thalictrum extract") or in combination with one or more other species of the genus Thalictrum. Thalictrum extract is commercially available from a variety of sources. In addition, suitable thalictrum extract may be obtained by using any conventional extraction technique.

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, thalictrum extract can be obtained using organic solvent extraction techniques. In another example, thalictrum extract may be obtained using solvent sequential fractionation. Total water-ethanol extraction techniques can also be used to obtain Thalictrum aquilegifolium extract. 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 phytochemical components. 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 thalictrum extract is obtained by alcohol extraction of plant material of the thalictrum species.

Another example of an extraction technique that may be used to obtain a thalictrum extract is supercritical fluid carbon dioxide supercritical 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.

Thalictrum extract may be added to the composition in any amount. Typically, the thalictrum extract is present in an amount effective to reduce (or inhibit) melanogenesis and/or eliminate melanin in the skin of the subject. In various embodiments, the thalictrum extract is present in an amount of about 10 to about 300 μ g/mL of the composition. In specific embodiments, the thalictrum extract is present in an amount of about 10, about 20, about 25, about 50, about 75, or about 100 μ g/mL of the composition. Also contemplated are various subranges and amounts from about 10 to about 300 μ g/mL of the composition, as well as amounts less than or greater than these amounts.

The amount of thalictrum extract present in the composition may depend on several factors, including the desired level of melanin inhibition, the level of melanin inhibition in a particular extract or composition, and other factors. In certain embodiments, thalictrum extract is present in an amount of about 0.01 to about 20 parts by weight ("pbw") based on 100pbw of the composition. In a further embodiment, the thalictrum extract is present in an amount of 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 used in the compositions 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 comprises a supplemental active ingredient in addition to thalictrum petersonii. The supplementary active ingredients are generally selected from the following groups: hovenia dulcis (Hovenia dulcis), Cortex Lycii radicis or fructus Lycii (Lyci Cortex or Lycium chinense), Galium aparine (Galium aparine) and combinations thereof. In these embodiments, the supplemental active ingredient can be an extract from hovenia dulcis thunb, lycium barbarum, and protoladium species. Such extracts may be obtained commercially, or by one of the extraction techniques described above. Although optional, it is contemplated that any one or combination of these supplemental active ingredients may be used in combination with the thalictrum extract to further aid in skin lightening. If used, the supplemental active ingredient may be used in an amount as described for thalictrum extract, for example, in an amount of about 10 to about 300 μ g/mL of the composition or about 0.01 to about 20pbw of the composition.

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 "traditional Chinese medicines") other than thalictrum. 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 may be formulated to include a cosmetically acceptable carrier (or vehicle) and prepared and/or packaged and labeled to increase skin lightening or whitening, inhibit, reduce or reduce melanogenesis or pigmentation. 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 method of preparation includes the step of extracting plant material of the thalictrum species to obtain the thalictrum extract. The method of preparation further comprises the step of combining the thalictrum petersonii extract 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 may be used for skin lightening in various ways. As an example, a cosmetic method for lightening a subject's skin includes the step of applying Thalictrum petitkii to the subject's skin. Thalictrum petalinum may be applied in various ways, including applying the composition to the skin of a subject. 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 melanin production 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

An exemplary extraction method is described below. The extraction method is used to obtain the thalictrum petersonii extract of the composition of the present disclosure. This extraction process may be referred to as methanol fractionation or methanol extraction. One skilled in the art will appreciate that other extraction methods may be used to obtain the desired extract, including those described above. Other extraction methods and analytical methods (e.g., assays) relevant to determining skin lightening efficacy are described in international publication nos. WO 2011/019468 a2, WO2011/109139 a2, and WO 2013/169634 a2 to Rana et al, the disclosures of which are incorporated herein by reference.

Exemplary extraction method

Any material or extract used should be in powder form. Samples were weighed and recorded (to the nearest 0.1g) into flasks according to the following table:

	feeding of the feedstock	Extract of plant
			Plant/botanical weight (g)	50	10
Flask volume (mL)	500	150
			Volume of methanol (mL)	300	60

A stir bar was added and the amount of methanol indicated in the table was poured into the flask. The flask opening was capped with aluminum foil. The flask was placed on a magnetic stir plate and stirred using a slow/medium stirring rate for at least 12 hours. The sample was kept from direct light. The flask was removed from the stir plate and sonicated for one hour at room temperature with occasional vortexing.

The sample solution was filtered through GF/A filter paper directly into a 500mL round bottom long neck flask. The solvent remaining in the round bottom long neck flask was evaporated using a rotary evaporator. The solvent volume was reduced to less than 10 mL. The concentrated extract (still in liquid form) was transferred to a pre-weighed scintillation vial (weighing without lid) using a glass pipette. Further dilution with methanol was used for transfer purposes as needed.

The tube was placed under a nitrogen evaporator and the volume was reduced to the minimum possible using a slow nitrogen flow. The recommended bath temperature is 40 ℃. The tube was removed from the nitrogen evaporator and placed in the desiccator without a lid until dry (approximately 12 hours). The final dry weight of the fractions in the scintillation vial (no lid) was recorded (calculated by difference). The tube is capped and stored in a refrigerator for further use.

After obtaining the thalictrum extract using the aforementioned extraction methods, various tests were conducted and corresponding data sets were generated to determine the effectiveness of the thalictrum extract in skin lightening. In relevant part, cytotoxicity, tyrosinase inhibition, melanin synthesis inhibition, and melanin elimination are evaluated using potency assays and methods known to those skilled in the art. The tabular data is provided in the following data 1, data 2, data 3, and data 4 sets and the respective corresponding FIGS. 1, 2, 3, and 4.

Data 1

Cytotoxicity of Thalictrum petaloides in B16F 10-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. Base according to CTG illustrated by data 1 and FIG. 1In the evaluation of the safety of the cells, the Thalictrum petasillum has no cytotoxicity risk.

Based on in vitro bioassay data, the herb extract of thalictrum petersonii was found to have skin lightening and depigmenting effects. Inhibition of tyrosinase activity, inhibition of melanin synthesis, and the presence of melanin-eliminating activity were monitored by cell-based assays administered by model cell 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 tyrosinase activity inhibition and melanin synthesis inhibition, while hydroquinone was used as a positive control for the melanin-eliminating activity present. Summarizing the results described below, thalictrum petersonii has outstanding activity as a skin lightening or depigmenting agent in vitro tests without the risk of cytotoxicity.

Data 2

Tyrosinase inhibition in B16F10 by Thalictrum petaloides (arbutin as reference)

Herb of common Meadowrue

Arbutin

The thalictrum petersonii extract exerts excellent tyrosinase inhibitory activity in B16F10 cells, with an inhibition rate of 93.2% at a concentration of 100 μ g/mL relative to the positive control arbutin at the same concentration. Specifically, as illustrated in data 2 and fig. 2, thalictrum petersonii acted comparable or much better than arbutin in tyrosinase inhibitory activity in B16F10 cells. This data set is generally obtained by the assay methods outlined immediately below.

Tyrosinase inhibition assay method:

the purpose is as follows: this procedure describes a standard method for the determination of tyrosinase activity inhibition 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)

24 orifice plate (Costar)

96 well plate (Costar)

·L-Dopa

The procedure is as follows:

day 1: plated cells

1. The plates were trypsinized and cell density was determined.

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

3. 1 ml/well of the cell paste was dispensed onto a 24 well assay plate.

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 an assay plate (final concentration: 1X).

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

Day 5: imaging a plate

1. The inverted plate was shaken on a media waste receiver and blotted dry with a cleaning paper towel.

2. Add 80. mu.l 0.05mM PBS (0.1% Triton-X) and freeze at 80 ℃ for 4 hours.

3. The plates were melted at room temperature. Repeat steps 2&3 twice.

4. The sample was transferred into a 96-well V-bottom plate and spun down at 3000RPM for 10 minutes.

5. A60. mu.l sample of the supernatant was transferred to a UV plate. 140 μ L of fresh 0.1% L-DOPA was added to each well and incubated at 37 ℃ for 1 hour.

6. Read absorbance at 475 nm.

7. A5. mu.l sample of the supernatant was transferred to another UV plate to determine the protein concentration with BCA.

Data processing:

GraphPad was used.

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

The basal signal was obtained from cells without L-DOPA.

The maximum signal is obtained from the effect of L-DOPA.

Data 3

Melanin synthesis inhibition in B16F10 by Thalictrum petiolatus (arbutin as reference)

Herb of common Meadowrue

Arbutin

In the melanin synthesis inhibitory activity in B16F10 cells, the inhibitory rate of thalictrum petersonii was 107.0% at 100 μ g/mL relative to the positive control arbutin. Specifically, as exemplified by data 3 and fig. 3, thalictrum petersonii acted comparable to arbutin in melanin synthesis inhibitory activity in B16F10 cells. This means that Thalictrum petaloides has the ability to strongly interrupt (block) 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

1. The plates were trypsinized and cell density was determined.

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

Day 2: addition of test Compounds

Day 5: 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.

The maximum signal is obtained from the effect of DMSO.

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

Data 4

Melanin elimination rate of Thalictrum petiolatus in B16F10 (hydroquinone as reference)

Herb of common Meadowrue

Hydroquinone

In the melanin elimination assay present, the elimination of 14.0% pigment in B16F10 cultured cells was found to be eliminated after 72 hours of addition of 100. mu.g/mL Thalictrum petaloides, relative to the positive control hydroquinone at 76.0% elimination at 100. mu.g/mL. This means that Thalictrum petaloides 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. The plates were trypsinized and cell density was determined.

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

Day 2: cell processing

1. Add 10. mu.M forskolin and 50. mu.M 8-MOP per 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.

Data processing:

GraphPad was used.

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.

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

1. A composition for skin lightening comprising a thalictrum petitkii extract obtained by alcohol extraction of plant material of the thalictrum petitkii species.

2. A topical composition for skin lightening, the active ingredient of the composition consisting of an extract of Thalictrum petitkii obtained by alcohol extraction of plant material of the Thalictrum petitkii species.

3. The composition of claim 1 or 2, wherein the thalictrum petersonii extract is present in an amount effective to reduce melanin production in a subject and/or eliminate melanin in a subject.

4. The composition of claim 1 or 2, wherein the thalictrum petersonii extract obtained by alcohol extraction of plant material of the species thalictrum petersonii is present in an amount of from 10 to 300 μ g/mL of the composition.

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

6. The composition of claim 5, wherein the cosmetically acceptable carrier is selected from the group consisting of: water, wax, alcohol, vegetable oil, mineral oil, silicone, and combinations thereof.

7. The composition of claim 1, further comprising a supplemental active ingredient selected from the group consisting of: semen Hoveniae, cortex Lycii, and caulis et folium piperis.

8. The composition of claim 7, wherein the supplementary active ingredient comprises extracts from hovenia dulcis thunb seed, lycium bark seed and solanum lyratum seed.

9. The composition of claim 8, wherein the supplemental active ingredient extract is present in an amount of 10 to 300 μ g/mL of the composition.

10. The composition of any one of claims 7 to 9, further comprising a cosmetically acceptable carrier.

11. The composition of claim 10, wherein the cosmetically acceptable carrier is selected from the group consisting of: water, wax, alcohol, vegetable oil, mineral oil, silicone, and combinations thereof.

12. A topical composition for skin lightening, the composition consisting of a Thalictrum petitoides extract obtained by alcohol extraction of plant material of the Thalictrum petitoides species.

13. Non-therapeutic use of a composition of any one of claims 1, 2 or 12 for lightening the skin of a subject.

14. A method of preparing the composition of any one of claims 1, 2 or 12, the method comprising the step of extracting plant material of the thalictrum andraeanum species by an organic solvent extraction technique, a solvent continuous fractionation technique, or a total water-ethanol extraction technique to obtain a thalictrum andraeanum extract.

15. The method as set forth in claim 14 wherein the step of extracting is further defined as methanol extracting the plant material of the thalictrum species to obtain the thalictrum extract.

16. The method of claim 14 further comprising the step of combining said thalictrum petersonii extract with a cosmetically acceptable carrier.

17. A non-therapeutic cosmetic method for lightening a subject's skin, the method comprising the step of applying thalictrum petersonii to the subject's skin.

18. The method of claim 17, wherein the applying step is further defined as applying a composition to the skin of the subject, wherein the composition comprises an extract of thalictrum ramosissimum.

19. The method of claim 18 wherein the thalictrum petersonii extract is obtained by alcohol extraction of plant material of the species thalictrum petersonii.

20. The method of claim 18 or 19, wherein the thalictrum petersonii extract is present in an amount effective to reduce melanin production in the skin of the subject and/or eliminate melanin in the skin of the subject.