# Patent

## Title
Compositions comprising Lilium candidum extracts and uses thereof

## Abstract

Provided are compositions comprising certain extracts of Lilium Candidum and a carrier. Also provided are methods of lightening the skin comprising the step of applying to skin in need of skin lightening treatment one or more certain extracts of Lilium Candidum.

## Background
FIELD OF INVENTION
The present invention relates to compositions comprising plant extracts for use on skin. More specifically, it relates to compositions comprising extracts of Lilium candidum for lightening the skin.
DESCRIPTION OF RELATED ART
Lilium candidum (White lily) is a member of the family Liliaceae with genus Lilium, which genus contains about 110 plants widespread all over the world. Many of such lily plants are known for their white or colored flowers and are used commonly for ornamental purposes.
Several types of compounds have been isolated from white lily and identified, e.g. organic acids, flavonoids, glycosides, nitrogen containing compounds, saponins, and steroidal components (Eisenreichová, E. Haladová, M. Mu{hacek over (c)}aji, P. Gran{hacek over (c)}ai, D. “THE STUDY OF CONSTITUENTS OF LILIUM CANDIDUM L.”; Acta Facult. Pharm. Univ. Comenianae (2004), 51, pp. 27-37. Additionally, white lily flowers have been identified as being relatively rich in carotenoids, anthocyanins, phenyl propanoids, and volatile aroma components (Norbaek, R., Kondo, T. “Anthocyanins from flowers of Lilium (Liliaceae)”; Phytochemistry (1999), 50: pp. 1181-1184; Grieve. A Modern Herbal. Penguin 1984 ISBN 0-14-046-440-9.; Usher. G. A Dictionary of Plants Used by Man. Constable 1974 ISBN 0094579202.)
Various extracts of Lilium candidum have been shown to exhibit properties such as anti-oxidant, anti-fungal, anti-yeast, and anti-tumor (see, for example: Mucaji, P, Haladova, M, Eisenreichova, E, Sersen, F, Ubik, K & Grancai, D; “Constituents of Lilium candidum L. and their antioxidant activity”, Ceska a Slovenska Farmacie (2007), 56(1), pp. 27-29; Mucaji, P, Hudecova, D, Haladova, M, Eisenreichova, E, “Anti-yeast activity of the ethanolic extracts of Lilium candidum L.”, Ceska a Slovenska Farmacie (2002), 51(6), pp. 297-300; and Vachalkova, A, Eisenreichova, E, Haladova, M, Mucaji, P, Jozova, B & Novotny, L, “Potential carcinogenic and inhibitory activity of compounds isolated from Lilium candidum”, Neoplasma (2000), 47(5), pp. 313-318. Extracts of various parts of Lilium candidum have been described for use in skin lightening applications, and in particular, the bulb of Lilium candidum has been demonstrated to show some tyrosinase inhibition (see, for example, the English translation of Japanese Patent application laid-open disclosure number: Tokkai HEI6-65045 to Kose Corporation, application date Aug. 17, 1992, “External Preparations for skin,” paragraphs [0013], [0020]-[0027], and tables 1-4). Nevertheless, applicants have recognized that certain extracts of the Lilium candidum bulb and flower tend to be less effective, relatively cytotoxic, and/or less suitable for use on human skin for skin lightening.
The present invention is directed to the discovery that certain extracts of Lilium candidum exhibit unexpected combinations of melanogenesis inhibition properties, low cytotoxicity and/or other consumer-desirable properties for use on skin.
SUMMARY OF THE INVENTION
Applicants have discovered unexpectedly that certain extracts of the whole plant, flowers, bulbs, stems, and/or leaves of Lilium candidum may be used in compositions, preferably skin care compositions, and methods for skin lightening.
In particular, applicants have tested various extracts of the present invention as compared to other extracts of Lilium candidum flower and/or bulb, and have discovered that the present extracts tend to exhibit one or more superior properties including more effective UVB-induced melanogenesis inhibition activity, skin lightening, and/or relatively low cytotoxicity. More specifically, as detailed in the Examples herein, applicants have measured the UVB-induced melanogenesis inhibition activity associated with extracts of the present invention, as well as, the skin lightening properties and cytotoxicity of separate extracts of Lilium candidum on 3D skin epidermal equivalent materials. As shown in the Examples, the present extracts provided significant benefits in lightening the skin with relatively low cytotoxicity as compared to other extracts of Lilium candidum. 
Accordingly, in one aspect, the present invention is directed to compositions comprising an extract selected from the group consisting of extracts of Lilium candidum whole plant, Lilium candidum bulb, Lilium candidum flower, Lilium candidum stem, Lilium candidum leaves, and combinations of two or more thereof, and a carrier, wherein said composition comprises about 0.1 wt % or more of at least one polyunsaturated fatty acid having a structure of Formula I:

R—COOH  (I)

wherein R is —(CH2)z—(CH═CH—CH2)n—(CH2)m—CH3, n is from 1 to 6, m is from zero to 6, and z is from 2 to 7.

In another aspect, the present invention is directed to methods of lightening the skin comprising applying to skin in need of skin lightening treatment a composition comprising an extract of Lilium candidum whole plant, Lilium candidum bulb, Lilium candidum flower, Lilium candidum stem, Lilium candidum leaves, or mixtures of two or more thereof, wherein said composition comprises a safe and effective amount of at least one polyunsaturated fatty acid having a structure of Formula I:

R—COOH  (I)

wherein R is —(CH2)z—(CH═CH—CH2)n—(CH2)m—CH3, n is from 1 to 6, m is from zero to 6, and z is from 2 to 7.



DESCRIPTION OF THE INVENTION
As used herein, the term “lightening the skin” refers generally to lightening, brightening, whitening, and/or evening of the skin tone, skin color, and/or shade of skin, and/or to the reduction in shallowness, and/or to the lightening and/or fading of hyperpigmented marks and/or lesions including, but not limited to, pigmented spots, melanin spots, age spots, sun spots, senile lentigos, freckles, lentigos simplex, pigmented solar keratosis, seborrhoeic keratosis, melasma, acne marks, post-inflammatory hyperpigmentation, lentigines, ephelides, combinations of two or more thereof and the like. In certain embodiments, “lightening the skin” also refers to increased skin radiance, glow, translucency and/or luminescence and/or obtaining a more radiant, glowing, translucent or luminous skin tone appearance or a less yellow or sallow skin tone. In certain preferred embodiments, “lightening the skin” refers to lightening and evening the skin tone, increasing skin radiance and/or lightening age spots.
As used herein, the term “skin in need of skin lightening treatment” refers generally to skin that exhibits one or more property selected from the group consisting of: skin having a measured Individual Typology Angle (ITA) value below 41 as determined per the COLIPA GUIDELINE: GUIDELINE FOR THE COLORIMETRIC DETERMINATION OF SKIN COLOUR TYPING AND PREDICTION OF THE MINIMAL ERYTHEMAL DOSE (MED) WITHOUT UV EXPOSURE published in 2007, which is incorporated herein by reference and further described below, darkened and/or sallow skin, including skin darkened by UV, skin with uneven skin tone, or skin with one or more hyperpigmented marks and/or lesions including, but not limited to, pigmented spots, melanin spots, age spots, sun spots, senile lentigos, freckles, lentigos simplex, pigmented solar keratosis, seborrhoeic keratosis, melasma, acne marks, post-inflammatory hyperpigmentation, lentigines, ephelides, combinations of two or more thereof and the like. In the COLIPA guidelines, skin color is defined function of the ITA value as: very light skin >55; Light skin 41-55, Intermediate 28-41, and Tan skin <28. In certain preferred embodiments, “skin in need of skin lightening” refers to individuals with a skin having an ITA value of less than 41, such as about 40 or less, about 35 or less, about 30 or less, or more preferably about 28 or less. In certain other preferred embodiments, the present invention is directed to compositions and methods for use on skin in need of skin lightening treatment selected from sallow and/or darkened skin. In certain other preferred embodiments, the present invention is directed to compositions and methods for use on skin in need of skin lightening treatment selected from the group consisting of age spots, freckles, marks left after acne, and combinations of two or more thereof.
As used herein, a composition that is “essentially free” of an ingredient means the composition that has about 2% or less of that ingredient by weight based on the total weight of the composition. Preferably, a composition that is essentially free of an ingredient has about 1% or less, more preferably about 0.5% or less, more preferably about 0.1% or less, more preferably about 0.05 or less, more preferably about 0.01% or less by weight based on the total weight of composition of the ingredient. In certain more preferred embodiments, a composition that is essentially free of an ingredient is free of the ingredient, i.e. has none of that ingredient in the composition.
As used herein, “cosmetically/dermatologically acceptable” means that the ingredients which the term describes are suitable for use in contact with tissues (e.g., the skin or hair) without undue toxicity, incompatibility, instability, irritation, allergic response, and the like.
As used herein, the term “safe and effective amount” means an amount of the extract or of the composition sufficient to induce the desired effect, but low enough to avoid serious side effects. The safe and effective amount of the compound, extract, or composition will vary with e.g. the age, health and environmental exposure of the end user, the duration and nature of the treatment, the specific extract, ingredient, or composition employed, the particular pharmaceutically-acceptable carrier utilized, and like factors.
Any suitable extracts of Lilium Candidum whole plant, flower, stem, leaves, and/or bulb may be used in accord with the present invention. Suitable extracts of Lilium candidum may be derived from live or dried plant, small cuttings or other portions thereof, and the like.
Suitable extracts of Lilium Candidum whole plant, flower, stem, leaves, and/or bulb may be obtained using conventional methods including, but not limited to, direct extraction of material from the biomass by grinding, macerating, pressing, squeezing, mashing, centrifuging, and/or processes such as cold percolation, agitation/distillation, microwave assisted extraction, supercritical/subcritical CO2 compressed gas extraction with or without polar modifiers, pressurized solvent extraction, accelerated solvent extraction, pressurized or normal hot water extraction, surfactant assisted pressurized hot water extraction, oil extraction, membrane extraction, Soxhlet extraction, the gold finger distillation/extraction and/or processes disclosed, for example, in U.S. Pat. Nos. 7,442,391, 7,473,435, and 7537791 to Integrated Botanical Technologies, LLC, incorporated herein by reference, and the like, or by other methods such as solvent extraction, and the like. Any of a variety of solvents including polar solvents, non-polar solvents, or combinations of two or more thereof may be used in methods of comprising solvent extraction. Suitable polar solvents include polar inorganic solvents such as water and the like, polar organic solvents such as alcohols and corresponding organic acids, for example C1-C8 alcohols including methanol, ethanol, propanol, butanol, and the like and organic acids, including acetic acid, formic acid, propanoic acid, and the like, polyols and glycols, including C1-C8 polyols/glycols and the like, and combinations of two or more thereof. Suitable non-polar solvents include non-polar organic solvents such as alkanes, including C1-C8 alkanes, cycloalkanes, including C1-C8 alkanes, alkyl ethers, including C1-C8 alkyl ethers, Petroleum ethers, ketones, including C1-C8 ketones, methylene chloride, ethyl acetate, xylene, toluene, chloroform, vegetable oil, mineral oil and the like. In another embodiment extraction may be obtained by non-polar solvents described above or supercritical fluid extraction with or without a polar modifier such as C1-C8 alcohols, water, C1-C8 polyols/glycols or C1-C8 organic acids.
In certain preferred embodiments, the extract comprises a non-polar extract prepared by extracting from fresh freeze dried flowers using a non-polar solvent comprising one or more C1-C8 alkanes, C1-C8 cycloalkanes, C1-C8 alkyl ethers, and/or chloroform, more preferably one or more C1-C8 alkanes and/or chloroform. In certain more preferred embodiments, the non-polar extract is extracted from Lilium candidum flower using hexanes, chloroform or a mixture thereof. In even more preferred embodiments, the non-polar extract is extracted from Lilium candidum flower using hexanes. In even more preferred embodiments, the non-polar extract is extracted from Lilium candidum flower using chloroform.
In certain preferred embodiments, the extract of the invention comprises a polar extract prepared by extracting from fresh freeze dried flowers using a polar solvent comprising water, C1-C8 alcohols, C1-C8 polyols, C1-C8 glycols, and combinations of two or more thereof. In certain more preferred embodiments, the polar extract is an aqueous extract extracted from Lilium candidum flower using water.
In certain other preferred embodiments, the extract of the invention comprises a combination of polar and non-polar extracts of from Lilium candidum flower.
In certain preferred embodiments, the extract comprises a non-polar extract prepared by extracting from Lilium candidum bulb using a non-polar solvent comprising one or more C1-C8 alkanes, C1-C8 cycloalkanes, C1-C8 alkyl ethers, and/or chloroform, more preferably one or more C1-C8 alkanes and/or chloroform. In certain more preferred embodiments, the non-polar extract is extracted from Lilium candidum bulb using hexanes, chloroform or a mixture thereof. In even more preferred embodiments, the non-polar extract is extracted from Lilium candidum bulb using hexanes. In even more preferred embodiments, the non-polar extract is extracted from Lilium candidum bulb using chloroform.
In certain preferred embodiments, the extract of the invention comprises a polar extract prepared by extracting from Lilium candidum bulb using a polar solvent comprising water, C1-C8 alcohols, C1-C8 polyols, C1-C8 glycols, and combinations of two or more thereof. In certain more preferred embodiments, the polar extract is an aqueous extract extracted from Lilium candidum bulb using water.
In certain other preferred embodiments, the extract of the invention comprises a combination of polar and non-polar extracts of from Lilium candidum bulb.
In certain preferred embodiments, the extract comprises a non-polar extract prepared by extracting from Lilium candidum stem using a non-polar solvent comprising one or more C1-C8 alkanes, C1-C8 cycloalkanes, C1-C8 alkyl ethers, and/or chloroform, more preferably one or more C1-C8 alkanes and/or chloroform. In certain more preferred embodiments, the non-polar extract is extracted from Lilium candidum stem using hexanes, chloroform or a mixture thereof. In even more preferred embodiments, the non-polar extract is extracted from Lilium candidum stem using hexanes. In even more preferred embodiments, the non-polar extract is extracted from Lilium candidum stem using chloroform.
In certain preferred embodiments, the extract of the invention comprises a polar extract prepared by extracting from Lilium candidum stem using a polar solvent comprising water, C1-C8 alcohols, C1-C8 polyols, C1-C8 glycols, and combinations of two or more thereof. In certain more preferred embodiments, the polar extract is an aqueous extract extracted from Lilium candidum stem using water.
In certain other preferred embodiments, the extract of the invention comprises a combination of polar and non-polar extracts of from Lilium candidum stem.
In certain preferred embodiments, the extract comprises a non-polar extract prepared by extracting from Lilium candidum leaves using a non-polar solvent comprising one or more C1-C8 alkanes, C1-C8 cycloalkanes, C1-C8 alkyl ethers, and/or chloroform, more preferably one or more C1-C8 alkanes and/or chloroform. In certain more preferred embodiments, the non-polar extract is extracted from Lilium candidum leaves using hexanes, chloroform or a mixture thereof. In even more preferred embodiments, the non-polar extract is extracted from Lilium candidum leaves using hexanes. In even more preferred embodiments, the non-polar extract is extracted from Lilium candidum leaves using chloroform.
In certain preferred embodiments, the extract of the invention comprises a polar extract prepared by extracting from Lilium candidum leaves using a polar solvent comprising water, C1-C8 alcohols, C1-C8 polyols, C1-C8 glycols, and combinations of two or more thereof. In certain more preferred embodiments, the polar extract is an aqueous extract extracted from Lilium candidum leaves using water.
In certain other preferred embodiments, the extract of the invention comprises a combination of polar and non-polar extracts of from Lilium candidum leaves.
Applicants have recognized for certain embodiments that preferred extracts of Lilium Candidum whole plant, flower, stem, leaves, and/or bulb comprise one or more polyunsaturated fatty acids having a structure of formula I:

R—COOH  (I)

wherein R is —(CH2)z—(CH═CH—CH2)n—(CH2)m—CH3, where n is from 1 to 6, m is from zero to 6, and z is from 2 to 7. In certain preferred embodiments, R is selected from the group consisting of: —(CH2)7—CH═CH—CH2—(CH2)6—CH3, —(CH2)7—(CH═CH—CH2)2—(CH2)3—CH3, —(CH2)7—(CH═CH—CH2)3—CH3, and combinations of two or more thereof. In certain more preferred embodiments, the polyunsaturated fatty acids are omega-3, omega-6 or omega-9 fatty acids or combinations of two or more thereof. Examples of omega-3 fatty acids include, but are not limited to, α-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, all-cis-6,9,12,15-octadecatetraenoic acid, Eicosatrienoic acid, Eicosatetraenoic acid, Clupanodonic acid, Nisinic acid, and the like. Examples of omega-6 fatty acids include, but are not limited to, linoleic acid, γ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid, Docosapentaenoic acid, Eicosadienoic acid, Docosadienoic acid, Adrenic acid, Calendic acid, and the like. Examples of omega-9 fatty acids include, but are not limited to, oleic acid, Erucic acid, Eicosenoic acid, Eicosatrienoic acid, and the like.

According to certain preferred embodiments, the extracts of Lilium candidum whole plant, flower, stem, leaves, and/or bulb comprise at least about 6 weight % (wt. %) of one or more polyunsaturated fatty acids having a structure of formula I above. In certain embodiments, the extracts comprise from about 6 to about 100 wt. % of polyunsaturated fatty acids having a structure of formula I, more preferably from about 10 to about 95 wt. % of polyunsaturated fatty acids having a structure of formula I, more preferably from about 40 to about 95 wt. %, and more preferably from about 40 to about 80 wt. % of polyunsaturated fatty acids having a structure of formula I. As described herein and claimed, the weight % of polyunsaturated fatty acids in an extract of Lilium candidum is calculated as weight of total solids content of all polyunstatured fatty acid(s) of Formula I in the extract divided by the weight of total solids content of the extract times 100 to get a percent.
According to certain preferred embodiments, the extracts of Lilium candidum whole plant, flower, stem, leaves, and/or bulb comprise one or more hydrophilic materials selected from the group consisting of polysaccharides, oligosaccharides, disaccharides, and combinations of two or more thereof. Examples of polysaccharides include, but are not limited to, Amylose, Amylopectin, Beta-glucans, Glycans, Xylan, Arabinoxylans, glucomannans, combinations of two or more thereof, and the like. Examples of oligosaccharides include, but are not limited to, trisaccharides such as raffinose, melezitose, maltotriose; tetrasaccharides such as acarbose, stachyose; pentasaccharides, combinations of two or more thereof, and the like. Examples of disaccharides include, but are not limited to, maltose, sucrose, lactose, trehalose, turanose, cellobiose, combinations of two or more thereof, and the like.
According to certain preferred embodiments, the extracts of Lilium candidum whole plant, flower, stem, leaves, and/or bulb comprise at least about 0.005 wt. % of one or more polysaccharides, oligosaccharides, disaccharides, and combinations of two or more thereof. In certain embodiments, the extracts comprise from about 0.01 wt. % to about 80 wt. % of polysaccharides, oligosaccharides, disaccharides, and combinations of two or more thereof, more preferably from about 1 to about wt. %, and even more preferably from about 10 to about 20 wt. % of polysaccharides, oligosaccharides, disaccharides, and combinations of two or more thereof.
In certain embodiments, the Lilium candidum whole plant, flower, stem, leaves, and/or bulb extract comprises one or more hydrophilic materials selected from the group consisting of amino acids, pyrroline derivatives, butanedioic acids and their esters, and combinations of two or more thereof. Examples of amino acids include, but are not limited to, threonine, tyrosine, cysteine, methionine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, lysine, histidine, serine, glycine, valine, leucine, phenylalanine, tryptophan, proline, hydroxyproline, γ-aminobutyric acid, lanthionine, isoleucine, β-alanine, glycine, ornithine, hydroxylysine, and combinations of two or more thereof. In certain preferred embodiments, the Lilium candidum extract comprises the amino acid tyrosine. Examples of butanedioic acids and their esters include, but are not limited to malic acid, itatartaric acid, succinic acid, itaconic acid, hydroxyparaconic acids, their alkyl esters, and combinations of two or more thereof. Examples of pyrroline derivatives include, but are not limited to Ethyljatropham, Jatropham and its glucosides, Citraconimide, Pyrroline-2-one and its derivatives including glucosides, Lilaline, 3-methyl-1-(2-oxopyrrolidin-5-yl)-2,5-dihydropyrrol-2-one and its analogs.
According to certain preferred embodiments, the extracts of Lilium candidum whole plant, flower, stem, leaves, and/or bulb comprise at least about 0.001 wt. % of one or more amino acids, pyrroline derivatives, butanedioic acids and their esters, and combinations of two or more thereof. In certain embodiments, the extracts comprise from about 0.0011 to about 60 wt. % of amino acids, pyrroline derivatives, butanedioic acids and their esters, and combinations of two or more thereof, more preferably from about 0.01 wt. % to about 40 wt. %, and even more preferably from about 1 to about 20 wt. % of amino acids, pyrroline derivatives, butanedioic acids and their esters, and combinations of two or more thereof.
According to certain embodiments of the present invention, the Lilium candidum extract preferably comprises a solids weight ratio of lipophilic materials to hydrophilic materials of about 100:0 to about 10:90. As used herein, a “lipophilic material” generally refers to a material that has a dielectric constant of about 1 to about 15, preferably from about 2 to 15, at 22° C. (examples of lipohilic materials include, but are not limited to, (poly)saturated and unsaturated fatty alcohols/acids/esters and the like) and a “hydrophilic material” generally refers to a material that has a dielectric constant of greater than 15 to about 90, preferably greater than 15 to about 80, and in certain more preferred embodiments, from about 35 to about 80, at 22° C. (examples of hydrophilic materials include, but are not limited to, polysaccharides, oligosaccharides, disaccharides, amino acids, pyrroline derivatives, butanedioic acids and their esters, and the like). In certain more preferred embodiments, the extract of the present invention comprises a solids weight ratio of lipophilic materials to hydrophilic materials of about 90:10 to about 20:80 more preferably about 80:20 to about 40:60. In certain particularly preferred embodiments, the extract comprises a solids weight ratio of lipophilic materials to hydrophilic materials of about 80:20.
In certain embodiments, the Lilium candidum extract and/or the composition of the present invention may be prepared to have a relatively low amount of saturated fatty acids therein. In certain preferred embodiments, the extract is essentially free, more preferably free, of one or more saturated fatty acids. In addition, in certain preferred embodiments, the overall composition is essentially free, more preferably free, of one or more saturated fatty acids.
In certain preferred embodiments, the extract has a weight ratio of total polyunsaturated fatty acids of Formula I to total saturated fatty acids (total solids wt. polyunsaturated fatty acids:total solids wt. saturated fatty acids) of about 3:1 or greater. More preferably the weight ratio of total polyunsaturated fatty acids of Formula I to total saturated fatty acids in the extract is from about 4:1 to about 9:1 or greater. In certain more preferred embodiments, the weight ratio of total polyunsaturated fatty acids of Formula I to total saturated fatty acids is about 99:1 or greater.
In certain embodiments, the extract and/or compositions of the present invention may be essentially free of certain other materials. In one embodiment, the extract is essentially free of one or more flavanoids, saponins, and/or glucosides of flavanoids or saponins. In certain embodiments, the extract and the resulting composition is essentially free of flavanoids, saponins and their glucosides. For example, in certain embodiments of the present invention a polar or non-polar extract may be further extracted with, for example, methanol to remove essentially all of the flavanoids, saponins, and/or glucosides of flavanoids or saponins, and/or may be subjected to chromatographical or other methods to remove such materials. Examples of flavanoids, saponins, and/or their glucosides include, but are not limited to: Luteolin, Apigenin, Sapogenin, rutinosides, Tangeritin, Quercetin, Kaempferol, 8-(3-Methylsuccinyl) Kaempferol, Myricetin, Fisetin, Isorhamnetin, Pachypodol, Rhamnazin, Hesperetin, Naringenin, Eriodictyol, Etioline, Homoeriodictyol, Taxifolin (or Dihydroquercetin), Dihydrokaempferol, Genistein, Daidzein, Glycitein, epicatechin, 2-phenylethyl palmitate, Lilaline, Proanthocyanidins, 3,6′-diferuloylsucrose, Helonioside A, Isorhamnetin-3-rutinoside, Kaempferol-3-O-[b-D-xylopyranosyl-(1→2)-b-D-glucopyranoside], Kaempferol-3-O-[b-D-glucopyranosyl-(1→2)-b-D-galactopyranoside], and the like.
Any suitable amounts of Lilium candidum extract may be used in the compositions of the present invention. Preferably, the compositions comprise a safe and effective amount of Lilium candidum extract. In certain preferred embodiments, the compositions comprise from about 0.1 to about 20% Lilium candidum extract. In certain other preferred embodiments, the compositions comprise from about 0.1 to about 10%, from about 0.1 to about 5%, or from about 0.2 to about 2% Lilium candidum extract. In certain other preferred embodiments, the compositions comprise from about 1 to about 5%, preferably from about 2 to about 5% Lilium candidum extract. As used herein, unless otherwise specified, all percentages of ingredients in compositions are weight percent of active/solids ingredient based on the total weight of composition.
In certain preferred embodiments, the compositions of the present invention comprise a total weight percent of about 0.1 or more (based on total weight of polyunsaturated fatty acids in the total weight of the overall composition) of polyunsaturated fatty acids having a structure of formula I (above). The polyunsaturated fatty acids of Formula I may be introduced to the composition as part of the Lilium candidum extract and/or may be introduced to the composition independent of the Lilium candidum extract. In certain embodiments, the composition comprises polyunsaturated fatty acids of Formula I that are not part of the extract. In preferred embodiments, the Lilium candidum extract in the composition comprises at least a portion of the polyunsaturated fatty acids of Formula I in the composition. In more preferred embodiments, the compositions of the present invention comprise a total weight percent of 0.1 to 10 wt. %, more preferably 0.1 to 20 wt. %, more preferably 0.1 to 5 wt. %, and in certain preferred embodiments 0.1 to 3 wt. % or 0.5 to 5 wt. % of polyunsaturated fatty acids of Formula I.
Any suitable carrier may be used in the compositions of the present invention. Preferably, for a skin care composition, the carrier is a cosmetically-acceptable carrier. As will be recognized by those of skill in the art, cosmetically-acceptable carriers comprise carriers that are suitable for use in contact with the body, in particular the skin for skin whitening applications, without undue toxicity, incompatibility, instability, irritation, allergic response, and the like. A safe and effective amount of carrier is from about 50% to about 99.999%, preferably from about 80% to about 99.9%, more preferably from about 99.9% to about 95%, most preferably from about 99.8% to about 98% of the composition. The carrier can be in a wide variety of forms. For example, emulsion carriers, including, but not limited to, oil-in-water, water-in-oil, water-in-oil-in-water, and oil-in-water-in-silicone emulsions, are useful herein. These emulsions can cover a broad range of viscosities, e.g, from about 100 cps to about 200,000 cps. Examples of suitable cosmetically-acceptable carriers include cosmetically-acceptable solvents and materials for cosmetic solutions, suspensions, lotions, creams, serums, essences, gels, toners, sticks, sprays, ointments, liquid washes and soap bars, shampoos, hair conditioners, pastes, foams, mousses, powders, shaving creams, wipes, patches, strips, powered patches, microneedle patches, bandages, hydrogels, film-forming products, facial and skin masks, make-up, liquid drops, and the like. These product types may contain several types of cosmetically-acceptable carriers including, but not limited to solutions, suspensions, emulsions such as microemulsions and nanoemulsions, gels, solids, liposomes, other encapsulation technologies and the like. The following are non-limitative examples of such carriers. Other carriers can be formulated by those of ordinary skill in the art.
In one embodiment, the carrier contains water. In a further embodiment, the carrier may also contain one or more aqueous or organic solvents. Examples of organic solvents include, but are not limited to: dimethyl isosorbide; isopropylmyristate; surfactants of cationic, anionic and nonionic nature; vegetable oils; mineral oils; waxes; gums; synthetic and natural gelling agents; alkanols; glycols; and polyols. Examples of glycols include, but are not limited to, glycerin, propylene glycol, butylene glycol, pentalene glycol, hexylene glycol, polyethylene glycol, polypropylene glycol, diethylene glycol, triethylene glycol, caproyl glycol, glycerol, butanediol and hexanetriol, and copolymers or mixtures thereof. Examples of alkanols include, but are not limited to, those having from about 2 carbon atoms to about 12 carbon atoms (e.g., from about 2 carbon atoms to about 4 carbon atoms), such as isopropanol and ethanol. Examples of polyols include, but are not limited to, those having from about 2 carbon atoms to about 15 carbon atoms (e.g., from about 2 carbon atoms to about 10 carbon atoms) such as propylene glycol. The organic solvents may be present in the carrier in an amount, based upon the total weight of the carrier, of from about 1 percent to about 99.99 percent (e.g., from about 20 percent to about 50 percent). Water may be present in the carrier (prior to use) in an amount, based upon the total weight of the carrier, of from about 5 percent to about 95 percent (e.g., from about 50 percent to about 90 percent). Solutions may contain any suitable amounts of solvent, including from about 40 to about 99.99%. Certain preferred solutions contain from about 50 to about 99.9%, from about 60 to about 99%, from about 70 to about 99%, from about 80 to about 99%, or from about 90 to 99%.
A lotion can be made from such a solution. Lotions typically contain at least one emollient in addition to a solvent. Lotions may comprise from about 1% to about 20% (e.g., from about 5% to about 10%) of an emollient(s) and from about 50% to about 90% (e.g., from about 60% to about 80%) of water.
Another type of product that may be formulated from a solution is a cream. A cream typically contains from about 5% to about 50% (e.g., from about 10% to about 20%) of an emollient(s) and from about 45% to about 85% (e.g., from about 50% to about 75%) of water.
Yet another type of product that may be formulated from a solution is an ointment. An ointment may contain a simple base of animal, vegetable, or synthetic oils or semi-solid hydrocarbons. An ointment may contain from about 2% to about 10% of an emollient(s) plus from about 0.1% to about 2% of a thickening agent(s).
The compositions useful in the present invention can also be formulated as emulsions. If the carrier is an emulsion, from about 1% to about 10% (e.g., from about 2% to about 5%) of the carrier contains an emulsifier(s). Emulsifiers may be nonionic, anionic or cationic.
Lotions and creams can be formulated as emulsions. Typically such lotions contain from 0.5% to about 5% of an emulsifier(s), while such creams would typically contain from about 1% to about 20% (e.g., from about 5% to about 10%) of an emollient(s); from about 20% to about 80% (e.g., from 30% to about 70%) of water; and from about 1% to about 10% (e.g., from about 2% to about 5%) of an emulsifier(s).
Single emulsion skin care preparations, such as lotions and creams, of the oil-in-water type and water-in-oil type are well-known in the art and are useful in the subject invention. Multiphase emulsion compositions, such as the water-in-oil-in-water type or the oil-in-water-in-oil type, are also useful in the subject invention. In general, such single or multiphase emulsions contain water, emollients, and emulsifiers as essential ingredients.
The compositions of this invention can also be formulated as a gel (e.g., an aqueous, alcohol, alcohol/water, or oil gel using a suitable gelling agent(s)). Suitable gelling agents for aqueous and/or alcoholic gels include, but are not limited to, natural gums, acrylic acid and acrylate polymers and copolymers, and cellulose derivatives (e.g., hydroxymethyl cellulose and hydroxypropyl cellulose). Suitable gelling agents for oils (such as mineral oil) include, but are not limited to, hydrogenated butylene/ethylene/styrene copolymer and hydrogenated ethylene/propylene/styrene copolymer. Such gels typically contains between about 0.1% and 5%, by weight, of such gelling agents.
The compositions of the present invention can also be formulated into a solid formulation (e.g., a wax-based stick, soap bar composition, powder, or wipe). The composition of the present invention can also be combined with a solid, semi-solid or dissolvable substrate (eg., a wipe, mask, pad, glove or strip).
The compositions of the present invention may further comprise any of a variety of additional cosmetically active agents. Examples of suitable additional active agents include: additional skin lightening agents, darkening agents, anti-acne agents, shine control agents, anti-microbial agents such as anti-yeast agents, anti-fungal, and anti-bacterial agents, anti-inflammatory agents, anti-parasite agents, external analgesics, sunscreens, photoprotectors, antioxidants, keratolytic agents, detergents/surfactants, moisturizers, nutrients, vitamins, energy enhancers, anti-perspiration agents, astringents, deodorants, hair removers, hair growth enhancing agents, hair growth delaying agents, firming agents, hydration boosters, efficacy boosters, anti-callous agents, agents for skin conditioning, anti-cellulite agents, fluorides, teeth whitening agents, anti-plaque agents, and plaque-dissolving agents, odor-control agents such as odor masking or pH-changing agents, and the like. Examples of various suitable additional cosmetically acceptable actives include hydroxy acids, benzoyl peroxide, D-panthenol, UV filters such as but not limited to avobenzone (Parsol 1789), bisdisulizole disodium (Neo Heliopan AP), diethylamino hydroxybenzoyl hexyl benzoate (Uvinul A Plus), ecamsule (Mexoryl SX), methyl anthranilate, 4-aminobenzoic acid (PABA), cinoxate, ethylhexyl triazone (Uvinul T 150), homosalate, 4-methylbenzylidene camphor (Parsol 5000), octyl methoxycinnamate (Octinoxate), octyl salicylate (Octisalate), padimate O (Escalol 507), phenylbenzimidazole sulfonic acid (Ensulizole), polysilicone-15 (Parsol SLX), trolamine salicylate, Bemotrizinol (Tinosorb S), benzophenones 1-12, dioxybenzone, drometrizole trisiloxane (Mexoryl XL), iscotrizinol (Uvasorb HEB), octocrylene, oxybenzone (Eusolex 4360), sulisobenzone, bisoctrizole (Tinosorb M), titanium dioxide, zinc oxide, carotenoids, free radical scavengers, spin traps, retinoids and retinoid precursors such as retinol, retinoic acid and retinol palmitate, ceramides, polyunsaturated fatty acids, essential fatty acids, enzymes, enzyme inhibitors, minerals, hormones such as estrogens, steroids such as hydrocortisone, 2-dimethylaminoethanol, copper salts such as copper chloride, peptides containing copper such as Cu:Gly-His-Lys, coenzyme Q10, amino acids such a proline, vitamins, lactobionic acid, acetyl-coenzyme A, niacin, riboflavin, thiamin, ribose, electron transporters such as NADH and FADH2, and other botanical extracts such as oat, aloe vera, Feverfew, Soy, Shiitake mushroom extracts, and derivatives and mixtures thereof.
In certain preferred embodiments, the compositions of the present invention are skin care compositions that comprise a Lilium candidum whole plant, flower, stem, leaves, and/or bulb extract and at least one additional skin lightening active agent. Examples of suitable additional skin lightening active agents include, but are not limited to, tyrosinase inhibitors, melanin-inhibiting agents, melanosome transfer inhibiting agents including PAR-2 antagonists, exfoliants, sunscreens, retinoids, antioxidants, Tranexamic acid, skin bleaching agents, allantoin, opacifiers, talcs and silicas, zinc salts, and the like, and other agents as described in Solano et al. Pigment Cell Res. 19 (550-571).
Examples of suitable tyrosinase inhibitors include but, are not limited to, Vitamin C and its derivatives, Vitamin E and its derivatives, Kojic Acid, Arbutin, resorcinols, hydroquinone, Flavones e.g. Licorice flavanoids, Licorice root extract, Mulberry root extract, Dioscorea Coposita root extract, Saxifraga extract and the like, Ellagic acid, Salicylates and derivatives, Glucosamine and derivatives, Fullerene, Hinokitiol, Dioic acid, Acetyl glucosamine, Magnolignane, combinations of two or more thereof, and the like. Examples of vitamin C derivatives include, but are not limited to, ascorbic acid and salts, Ascorbic Acid-2-Glucoside, sodium ascorbyl phosphate, magnesium ascorbyl phosphate, and natural extract enriched in vitamin C. Examples of vitamin E derivatives include, but are not limited to, alpha-tocopherol, beta, tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol, delta-tocotrienol and mixtures thereof, tocopherol acetate, tocopherol phosphate and natural extracts enriched in vitamin E derivatives. Examples of resorcinol derivatives include, but are not limited to, resorcinol, 4-substituted resorcinols like 4-alkylresorcinols such as 4-butyresorcinol (rucinol), 4-hexylresorcinol, phenylethyl resorcinol, 1-(2,4-dihydroxyphenyl)-3-(2,4-dimethoxy-3-methylphenyl)-Propane and the like and natural extracts enriched in resorcinols. Examples of salicylates include, but are not limited to, salicylic acid, acetylsalicylic acid, 4-methoxysalicylic acid and their salts. In certain preferred embodiments, the tyrosinase inhibitors include a 4-substituted resorcinol, a vitamin C derivative, or a vitamin E derivative. In more preferred embodiments, the tyrosinase inhibitor comprises Phenylethyl resorcinol, 4-hexyl resorcinol, or ascorbyl-2-glucoside.
Examples of suitable melanin-degradation agents include, but are not limited to, peroxides and enzymes such as peroxidases and ligninases. In certain preferred embodiments, the melanin-inhibiting agents include a peroxide or a ligninase.
Examples of suitable melanosome transfer inhibiting agents including PAR-2 antagonists such as soy trypsin inhibitor or Bowman-Birk Inhibitor, Vitamin B3 and derivatives such as Niacinamide, Essential soy, Whole Soy, Soy extract. In certain preferred embodiments, the melanosome transfer inhibiting agents includes a soy extract or niacinamide.
Examples of exfoliants include, but are not limited to, alpha-hydroxy acids such as lactic acid, glycolic acid, malic acid, tartaric acid, citric acid, or any combination of any of the foregoing, beta-hydroxy acids such as salicylic acid, polyhydroxy acids such as lactobionic acid and gluconic acid, and mechanical exfoliation such as microdermabrasion. In certain preferred embodiments, the exfolliant include glycolic acid or salicylic acid.
Examples of sunscreens include, but are not limited to, avobenzone (Parsol 1789), bisdisulizole disodium (Neo Heliopan AP), diethylamino hydroxybenzoyl hexyl benzoate (Uvinul A Plus), ecamsule (Mexoryl SX), methyl anthranilate, 4-aminobenzoic acid (PABA), cinoxate, ethylhexyl triazone (Uvinul T 150), homosalate, 4-methylbenzylidene camphor (Parsol 5000), octyl methoxycinnamate (Octinoxate), octyl salicylate (Octisalate), padimate 0 (Escalol 507), phenylbenzimidazole sulfonic acid (Ensulizole), polysilicone-15 (Parsol SLX), trolamine salicylate, Bemotrizinol (Tinosorb S), benzophenones 1-12, dioxybenzone, drometrizole trisiloxane (Mexoryl XL), iscotrizinol (Uvasorb HEB), octocrylene, oxybenzone (Eusolex 4360), sulisobenzone, bisoctrizole (Tinosorb M), titanium dioxide, zinc oxide, and the like.
Examples of retinoids include, but are not limited to, retinol, retinaldehyde, retinoic acid, retinol palmitate, isotretinoin, tazarotene, bexarotene and Adapalene. In certain preferred embodiments, the retinoid is retinol.
Examples of antioxidants include, but are not limited to, water-soluble antioxidants such as sulfhydryl compounds and their derivatives (e.g., sodium metabisulfite and N-acetyl-cysteine, glutathione), lipoic acid and dihydrolipoic acid, stilbenoids such as resveratrol and derivatives, lactoferrin, and ascorbic acid and ascorbic acid derivatives (e.g., ascobyl-2-glucoside, ascorbyl palmitate and ascorbyl polypeptide). Oil-soluble antioxidants suitable for use in the compositions of this invention include, but are not limited to, butylated hydroxytoluene, retinoids (e.g., retinol and retinyl palmitate), tocopherols (e.g., tocopherol acetate), tocotrienols, and ubiquinone. Natural extracts containing antioxidants suitable for use in the compositions of this invention, include, but not limited to, extracts containing flavonoids and isoflavonoids and their derivatives (e.g., genistein and diadzein), extracts containing resveratrol and the like. Examples of such natural extracts include grape seed, green tea, pine bark, feverfew, parthenolide-free feverfew, oat extracts, pomelo extract, wheat germ extract, Hysperedin, Grape extract, Portulaca extract, Licochalcone, chalcone, 2,2′-dihydroxy chalcone, Primula extract, porpolis, and the like.
The additional cosmetically active agent may be present in a composition in any suitable amount, for example, in an amount of from about 0.0001% to about 20% by weight of the composition, e.g., about 0.001% to about 10% such as about 0.01% to about 5%. In certain preferred embodiments, in an amount of 0.1% to 5% and in other preferred embodiments from 1% to 2%.
A variety of other materials may also be present in the compositions of the present invention. These include, for example, chelating agents, humectants, opacifiers, conditioners, preservatives, fragrances and the like. The compositions may include surfactants, for example, those selected from the group consisting of anionic, non-ionics, amphoteric, cationic, or a combination of two or more thereof.
In certain preferred embodiments, the present invention is in the form of a substrate comprising a composition of the present invention. Any suitable substrate may be used in the present invention. Examples of suitable substrates and substrate materials are disclosed, for example, in U.S. Published Application Nos. 2005/0226834 and 2009/0241242 which are incorporated herein by reference in their entirety.
In certain preferred embodiments, the substrate is a wipe or a facial mask. Preferably, such embodiments comprise a water-insoluble substrate as such is defined in the cited references above. For certain embodiments, the water-insoluble substrate may have a size and shape such that it covers the face of a human user to facilitate placing the water-insoluble substrate about the face of the user as a mask substrate. For example, the water-insoluble mask substrate may have openings for a mouth, nose, and/or eyes of the user. Alternatively, the water-insoluble substrate may have no such openings. Such a configuration without openings may be useful for embodiments of the invention in which the water-insoluble substrate is intended to be draped over a non-facial expanse of skin or if the water-insoluble substrate is intended to be used as wipe. The water-insoluble substrate may have various shapes, such as an angular shape (e.g., rectangular) or an arcuate shape such as circular or oval.
In one embodiment of the invention, the product includes a plurality of water-insoluble substrates of different shapes. In one embodiment of the invention, the product includes a first water-insoluble substrate and a second water-insoluble substrate. The first water-insoluble substrate is shaped for application onto the forehead and the second water-insoluble substrate is shaped for application proximate to the mouth, such as areas above and/or below the lips, the chin, and/or the cheeks. In one embodiment of the invention, the first water-insoluble substrate is also applied to the nose region of the face. The first water-insoluble substrate may have a surface area of from about 100 cm2 to about 200 cm2, such as from about 120 cm2 to about 160 cm2 and the second water-insoluble substrate has a surface area of from about 100 cm2 to about 300 cm2, such as from about 150 cm2 to about 250 cm2. In one embodiment of the invention, the water-insoluble substrate has a low stiffness such that it may, for example, readily drape over or conform to the face or other body parts of the user.
The present invention further comprises methods of lightening the skin by applying to skin in need of skin lightening treatment an extract of Lilium candidum whole plant, flower, stem, leaves, and/or bulb, as such extracts and embodiments thereof are described above. In certain embodiments, the method comprises applying a composition of the present invention comprising an extract of Lilium candidum whole plant, flower, stem, leaves, and/or bulb, as such compositions are described above in various embodiments, to skin in need of skin lightening treatment.
The present invention may comprise application to any skin in need of treatment on the body. For example, application may be made to any one or more of the skin of the face, neck, chest, back, arms, axilla, hands and/or legs.
Preferably, the methods of the present invention comprise applying a safe and skin lightening effective amount of Lilium candidum extract to the skin. In certain preferred embodiments, the methods comprise applying from greater than zero to about 20% Lilium candidum extract to the skin in need. In certain other preferred embodiments, the methods comprise applying from about 0.0001 to about 20%, from about 0.001 to about 10%, from about 0.01 to about 5%, from about 0.1 to about 5%, or from about 0.2 to about 2% Lilium candidum extract to the skin in need. In certain other preferred embodiments, the methods comprise from greater than zero to about 1%, from about 0.0001 to about 1%, from about 0.001 to about 1%, or from about 0.01 to about 1% Lilium candidum extract to the skin. In certain other preferred embodiments, the methods comprise applying from about 1 to about 5%, preferably from about 2 to about 5% Lilium candidum extract to the skin.
Any suitable method of applying the extract to the skin in need may be used in accord with the present invention. For example, the extract may be applied directly from a package to the skin in need, by hand to the skin in need, or may be transferred from a substrate such as a wipe or mask, or a combination of two or more thereof. In other embodiments, the extract may be applied via a dropper, tube, roller, spray, patch or added to a bath or otherwise to water to be applied to the skin, and the like.
In certain embodiments, the methods of the present invention further comprise the step of leaving the Lilium candidum extract in contact with the skin for period of time. For example, in certain preferred embodiments after application, the extract is left in contact with the skin for a period of about 15 minutes or greater. In certain more preferred embodiments, the extract is left in contact with the skin for about 20 minutes or greater, more preferably about 1 hour or greater.
In certain embodiments, the method of the present invention comprises a regimen comprising applying the Lilium candidum extract to skin multiple times over a selected period of time. For example, in certain embodiments, the present invention provides a method of skin lightening comprising applying to skin in need of skin lightening a composition comprising a Lilium candidum extract once or twice daily for at least 12 weeks, preferably at least 8 weeks and more preferably for at least 2 weeks.
In certain preferred embodiments, the methods of the present invention comprise applying at least two different compositions or products comprising Lilium candidum extract to the skin. For example, the methods may comprise applying a first composition comprising Lilium candidum extract to skin in need of skin lightening followed by applying a second composition comprising Lilium candidum extract, but that is otherwise different from the first composition, to the skin in need of skin lightening. In certain preferred embodiments, the first and second composition may be independently selected from the group consisting of lotions, cleansers, masks, wipes, creams, serums, gels, and the like. In certain preferred embodiments, at least one of the first and second compositions is a cleanser, lotion, cream, essence, or serum and the other is a facial mask or wipe. In certain other preferred embodiments, at least one of the first and second compositions is a cleanser and the other is a lotion or cream.
In certain other preferred embodiments, the method comprises applying at least three products comprising Lilium candidum extract to skin in need of skin lightening. Preferably such three products are selected from the group consisting of cleansers, lotions, creams, essences, and facial masks.
EXAMPLES
The following test methods were used in the Examples:
Melanin Synthesis Inhibition Test
Control samples of B16(F10) murine melanoma cells were prepared and harvested as indicated below, but without addition of any test sample and without exposure to UVB (untreated control). Other control samples were prepared and harvested as indicated below without addition of test sample and exposed to UVB as described below (treated control). One or more samples of B16(F10) cells were prepared and each pre-treated with a test sample (e.g. E1) followed by UVB exposure as described below. Upon treatment, UVB stimulated melanogenesis in the cells and test compounds were evaluated based on their ability to inhibit or slow down the rate of melanogenesis. The cells were lysed for protein measurement at 595 nm and melanin content at 470 nm. The potency of the test compounds were determined by comparing the % inhibition achieved by the test compounds against the treated control.
Testing Procedure:
On a first day, murine melanoma B16(F10) cells were seeded in 60 mm plates with a density of  ̃1 million cells per plate and incubated for 48 hrs at 37° C., 5% CO2. On day 2, the cells with a confluency rate of 90-100% were treated with test compound at a predetermined concentration (e.g. 25 μg/mL) for two hours (for test compound samples only) followed by exposure to UVB 20 mJ/cm2 (for test samples and treated control). The cells were harvested on day 3 (24 h post UVB irradiation for test samples and treated control) and lysed in protein lysis buffer (50 mM Tris, pH 8, 2 mM EDTA, 150 mM NaCl, and 1% Triton X 100—a nonionic surfactant purchased from BioRad Cat. #: 161-0407), and centrifuged. The resulting supernatant was mixed well with a protein dye assay (Bio-rad protein assay reagent) and a spectrophotometer (Molecular Devices VERSAmax) was used to determine the optical density (protein assay OD) of the sample at 595 nm. The cell pellet remaining after removal of the supernatant was dissolved in alkaline DMSO buffer, and the resulting solution used for melanin absorbance assay at 470 nm to determine melanin assay OD.
Three samples each of the untreated control, treated control, and each test sample were made and the Melanin and Protein OD measured for each. The normalized melanin for each untreated control (3 samples), treated control (3 samples) and test sample (3 samples for each test compound) was calculated via the following equation:

Normalized Melanin=melanin assay OD/protein assay OD. 

The average normalized Melanin of the untreated controls was calculated (sum of the three calculated values/3), and the average normalized Melanin of the treated controls similarly calculated.
The Induction value of the Control was calculated via the equation:

Induction value of Control=average normalized Melanin of treated control−average normalized Melanin of untreated control.

The Induction value with each test sample is then calculated via the equation:

Induction value with Test Sample=normalized Melanin of the test sample−average normalized Melanin of untreated control.

The Inhibition % for each test sample is then calculated via the equation:

100×[(Induction value of Control−Induction value with Test Sample)/Induction value of Control]. The average Inhibition % is calculated as the sum of the three resulting Inhibition % values for each test sample divided by three.

The calculation sequence for % inhibition are explained by a theoretical example, see the following table.













Average normalized melanin
0.98


Untreated control


Average normalized melanin
2.56


UVB treated control


Induction value of control
2.56 − 0.98 = 1.58


Average normalized melanin
1.04


Test sample


Induction value with Test
1.04 − 0.98 = 0.06


sample


Inhibition % for Test sample
[(1.58 − 0.06)/1.58] × 100 = 96.20%









Skin Epidermal Equivalents Model as a skin Lightening Test (ΔL)

Skin epidermal equivalent tissues are available commercially from MatTek's MelanoDermTM System and were used for the following tests. MatTek's MelanoDermTM System consists of normal, human-derived epidermal keratinocytes (NHEK) and melanocytes (NHM) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. Specifically, MEL-300-B tissues, each 9 mm in diameter were used in the following tests.
The test materials prepared in an appropriate vehicle and tested concentrations were applied topically to the skin model daily and the experiment lasted for 8 days. Measurement was taken on day 9.
The macroscopic and microscopic visual tissue darkening end points were measured by taking pictures with a digital camera. The Degree of Lightness for each tissue (L-Value) was measured using a spectrophotometer (Konica Minolta CM-2600d). The ΔL (degree of lightness as compared to control) for each test sample is calculated as per following formula:

ΔL=L-value of treated sample−L-value of control sample.

According to certain preferred embodiments, the compositions of the present invention are effective to achieve a ΔL in accord with this test of greater than zero. More preferably, the compositions of the present invention are effective to achieve a ΔL of about 0.5 or greater, more preferably about 1 or greater, more preferably about 1.5 or greater, and more preferably about 2 or greater.
Cell Viability Test
Cell Viability of the tissue during experiment was evaluated using the MTT assay described as follows. The MTT Tissue Viability Assay is a colorimetric assay system that measures the reduction of a yellow Methylthiazolyldiphenyl-tetrazolium bromide (MTT) into an insoluble purple product by the mitochondria of viable cells.
The skin epidermal tissues used previously to determine degree of lightness for each test material and of untreated tissues were used to determine percent viable cells remaining at the end of the experiment. The skin epidermal tissues after degree of lightness test were incubated with MTT reagent for 3 h. After incubation extraction buffer is added to lyse the cells and allowed to continue overnight. Samples are read using a plate reader at a wavelength of 570 nm and compared against untreated control and expressed in % Cell Viability as of control. A reduction of ≧30% cell viability as of control consider as a significant indication of cell cytotoxicity caused by the test materials. The amount of purple color produced is directly proportional to the number of viable cells.
Example 1
Preparation of Non-Polar Lilium candidum Flower Extract (E1)
Extract (E1): 50.9 gm of fresh freeze dried Lilium candidum flowers (obtained from Prisna, Netherlands) were extracted with 500 ml hexane at ratio of 1:10 (raw material to solvent) and stirred at room temperature for 24 hours at a speed of 1000 rpm. The suspension was filtered and filtrate was evaporated to dryness. The extract was further dried at high vacuum and 2.17 gm hexane extract of Lilium candidum flower (E1) was obtained at yield of 4.26%.
The extract (E1), thus obtained, was analyzed using standard HPLC techniques. HPLC analysis revealed that the extract was composed of lipids, specifically saturated fatty acids and unsaturated omega fatty acids and their phenethyl esters, e.g. Linoleic acid, Linolenic acids, triglycerides, and palmitic acid. Approximately 60-80 wt. % of total extract consist of materials represented by Formula I.
Example 2
Preparation of Polar Lilium candidum Flower Extract (E2)
Extract (E2): 26.6 gm of fresh freeze dried Lilium candidum flowers (obtained from Prisna, Netherlands) were suspended in 400 ml distilled water at a ratio of 1:15 (raw material to solvent) and stirred at room temperature for 24 hours at a speed of 1000 rpm. The suspension was centrifuged at 5000 rpm for 5 minutes and supernatant was filtered using 0.22 μm filter system (Corning Incorporated, NY, USA). The filtrate was then concentrated and freeze dried (MODULYOD, Freeze dryer, Thermo Electron Corporation). 7.87 gm of extract (E2) was obtained at a yield of 29.6%.
The polar extract E2, thus obtained, was analyzed using standard HPLC techniques and found to be essentially free of components represented by Formula I.
Examples 3-4
Preparation of Methanol Lilium candidum Flower Extract (E3) and Mid-Polar (E4)
Methanol extract (E3): 25.5 gm of Lilium candidum flower powder (obtained from residue of example 1) was further extracted with 200 mL of methanol by stirring at a speed of 1000 rpm for 24 h at room temperature. The suspension was filtered using filter paper (#3, Whatman) and filtrate of was evaporated to dryness. The methanol extract was further dried at high vacuum and 6.788 g extract (E3) was obtained at yield of 26.6%.
The methanol extract was analyzed using standard HPLC techniques. The analysis shows that the methanol fraction of Lilium candidum flower is predominantly composed of hydrophilic components.
Mid-polar extract (E4): 245 mg of methanol extract (E3) was loaded on to 5 gm of RP C18 silica gel and successively eluted with water and with water/methanol mixtures. Elution fractions from MeOH/water mixture were combined to obtain mid-polar extract which was essentially free from polar components (Extract E4 with a yield of 12.8% from extract E3 which corresponds to 3.4% from Lilium candidum flower raw materials). HPLC analysis shows mostly flavanoids were present and most of the major components were identified and are listed in Table 1.







TABLE 1







Chemical identities from mid-polar composition


of Lilium candidum flower extract








Serial



Number
Chemical Names











1
Kaempferol-3-O-[b-D-xylopyranosyl-(1→2)-b-D-



glucopyranoside]


2
Kaempferol-3-O-[b-D-glucopyranosyl-(1→2)-b-D-



galactopyranoside]


3
Helonioside A


4
3,6′-diferuloylsucrose


5
Flavonoid glucoside with Mol. wt. 772.2


6
Flavonoid glucoside with Mol. wt. 626.2


7
Phenyl propanoid glucoside with Mol. wt. 416.2


8
Saponin with mol formula C49H76O20


9
Saponin with mol formula C50H78O20


10
3 Saponins identified as a class with Mol wt in the range of 1000









Example 5
Preparation of Combined Non-Polar/Polar Lilium candidum Flower Extract (E5)
A non-polar extract made in accord with E1 and a polar extract made in accord with E2 were combined together to give a Lilium candidum flower extract (E5) that is essentially free of the mid-polar components listed in Table 1. Table 2 lists components identified to be present in Lilium candidum flower extract E5. The hydrophilic and lipophilic part of extract represent natural ratios as were in the Lilium candidum flower raw material.







TABLE 2







Chemical identities from Extract E5









Serial
Polarity



Number
Indication
Extract components












1
Hydrophilic
Polysaccharides


2
Hydrophilic
Pentasaccharides, e.g. Retention Times = 3.86 &




4.5 min; Mol Formula: C30H50O25


3
Hydrophilic
Disaccharides


4
Hydrophilic
Aminoacids, e.g. tyrosine


5
Hydrophilic
Itatartaric acid: CASRN 2957-09-7


6
Hydrophilic
β-Hydroxyparaconic acid: CASRN 19014-10-9


7
Hydrophilic
Alkaloids, e.g. Jatropham: CASRN 50656-76-3


8
Lipophilic
Linoleic acid


9
Lipophilic
Linolenic acid


10
Lipophilic
Omega fatty acid ester: CASRN: 117210-64-7


11
Lipophilic
Omega fatty acid ester: CASRN: 117210-65-8


12
Lipophilic
Omega fatty acid ester: CASRN: 3943-52-0


13
Lipophilic
Palmitic acid









Example 6
Preparation of Combined Non-Polar/Polar/Midpolar Lilium candidum Flower Extract (E6)
Extracts made in accord with E1, E2 and E4 were combined to obtain a total Lilium candidum flower extract (E6) representing natural composition.
Example 7
One Step Preparation of Lilium candidum Extracts (E7 & E8) with Active Composition
In two separate containers 10 g each of Lilium candidum flower and bulb powdered raw material (Prisna, Netherlands) were suspended in 100 mL of Chloroform and stirred at room temperature for 12 h. The suspension was filtered and filtrate dried under low pressure and low heat to obtain dry material (extract). Dry material from flower was 548 mg (E7) and from bulb was 59 mg (E8) with respective yields of 5.4% and 0.66%. HPLC analysis was performed for both extracts and found free of mid-polar components. Extract E8 comprised Lipophilic vs. hydrophilic components in a ratio of approximately 80:20 and the ratio for Extract E7 was closer to 95:5 resp. The ratio of unsaturated Omega fatty acids vs. saturated fatty acids was favored towards Omega fatty acids for E8 (3.5:1). Extract E7 has almost same amounts of unsaturated and saturated fatty acids.
Example 8
UVB Induced Melanogenesis Inhibition Activity in B16F10 Cells and Cytotoxicity
Extracts in accord with E1-E3 were tested for Melanogenesis Inhibition and cytotoxicity in accord with the Melanin Synthesis Inhibition and Cell Viability Tests, respectively, as described above. The results are shown in Table 3 below.







TABLE 3







Melanogenesis Inhbition of Lilium candidum flower Extracts












Extract
E1
E2
E3







IC50 values (%)
0.09
0.1
0.1*







*Inactive at <0.1% and cytotoxic at >0.1%






Example 9
UVB Induced Melanogenesis Inhibition Activity in B16F10 Cells and Cytotoxicity
Extracts in accord with E4-E6 were tested for Melanogenesis Inhibition in accord with the Melanin Synthesis Inhibition and Cell Viability Tests, respectively, as described above. The results are shown in Table 4 below.







TABLE 4







Melanogenesis Inhbition of Extracts












Extract
E4
E5
E6







IC50 values (%)
*
0.065
0.17







* Inactive at <0.1% and cytotoxic at >0.1%






Example 10
Skin Lightening and Cytotoxicity
The following example illustrates the skin lightening properties of extracts E1-E8. All extracts were tested at the concentrations indicated in Table 5 via the Skin Epidermal Equivalents Model as a skin Lightening Test (ΔL) as described above. Cytotoxicity potential was determined by MTT assay for all extracts and calculated as % reduction of cell viability as compared to control, wherein >30% reduction of cell viability constitute significant cytotoxicity issues. The results are shown in Table 5.







TABLE 5







Skin Lightening (ΔL) of Extracts









Extract (concentration (%))
ΔL
Standard deviation (p-value)





E1 (0.01%)
1.7 
0.11


E1 (5%)
1.53
0.04


E2 (0.1%)
2.28
0.06


E2 (5%)
2.96
0.06


E3 (0.01%)
*
—


E3 (0.05%)
*
—


E4 (0.05%)
*
—


E5 (0.1%)
2.14
0.07


E6 (0.1%)
*
—


E7 (5%)
0.75
0.185


E8 (2%)
4.93
0.003





* significant cytotoxicity observed






Example 11
(Comparative): Chemical and Bio-Activity Analysis of Four Commercial White Lily Extracts (C1-C4)
Three commercially available white lily flower extracts and one white lily bulb extract were obtained (C1-C4) as described in Table 6 below. The extracts were analyzed under HPLC via the same method as for extracts E1-E8 above and no compounds of Formula I were detected. The compounds were also tested for skin lightening via the Skin Epidermal Equivalents Model as a skin Lightening Test (ΔL). Results are shown in Table 7 below.







TABLE 6







White Lily Commercial Extracts (C1-C4)














Part of the



Extract
Trade Name
Plant name
plant used
Supplier





C1
Phytofleur
Lilium
Blossoms
Crodarom



lily NP
candidum


C2
White Lily
Lilium
Flowers
Alban Muller



HS
candidum


C3
Lys EG162
Lilium
Flowers
Greentech



Phytelene
candidum


C4
White Lily
Lilium
Bulbs
Maruzen



Extract BG
candidum

Pharmaceuticals
















TABLE 7







Chemical and Skin Lightening results for C1-C4













materials with

ΔL for up




little or no
Bioactives
to 20% of




retention on
as per
the extract



Extract
RP C18 (%)
Formula I (%)
tested







C1
99
Not detected
0



C2
99
Not detected
0



C3
99
Not detected
0



C4
99
Not detected
0










Example 12
(Comparative): Preparation of Lilium candidum Bulb Extract with Polar Solvent System (C5)
In an appropriate glass container 10 g of Lilium candidum bulb dried powder (obtained from Prisna, Netherlands) was suspended in 100 mL of a solvent system comprising Methanol and Dichloromethane (1:1). The suspension was stirred at room temperature for 12 h and filtered. The filtrate dried to obtain a residual material (C5 extract). HPLC analysis shows C5 is mainly composed of polar and mid-polar chemical components, with up to 3% of the extract comprising compositions of Formula I. A composition comprising 2% of the extract (i.e. up to 0.06% of compounds of Formula I in the overall composition) was tested for skin lightening via the Skin Epidermal Equivalents Model as a skin Lightening Test (ΔL). The composition exhibited a ΔL of 0.26±0.26.
Example 13
Preparation of Composition
A product formula in cream with an extract in accord with the present invention is given in the following Table-12:







TABLE 12







Product Formulation










Item
Ingredient/

%


#
Function
Trade/INCI Name
Weight













1
Purified Water
Water
Balance


2
EDTA BD
Disodium EDTA
0.10


3
Emulsifiers
Pemulen TR-1, Brij72, Brij 721,
4.90




Lanette 22, Amphisol K, Simulgel




EG


4
Thickeners
Carbopol Ultrez 20, Xanthan gum
0.20




180


5
Humectants
Butylene Glycol, Glycerin
9.00


6
Skin
Prodew 300, Cetiol SB-45, Edenor
15.75



Conditioning
ST 1 MY, Miglyol 812N, Finsolve



Agent
TN, DC 200 50 cps, DC 345 Fluid,




DC 1403, SP-500


7
Hydrolite-5
Pentylene Glycol
1.00


8
Chlorohexidine
Chlorohexidine Digluconate
0.25



Digluconate



20%


9
Lilium candidum
Lilium candidum
5.00



flower Extract
flower extract


10
Preservatives
Methyl Paraben, Ethyl paraben,
0.60




Propyl Paraben


11
Neutralizing Base
Sodium hydroxide
As per





need









The ingredients are mixed as per standard procedures. A brief general procedure is described here for guidance.

Premix A: Dissolve Lilium candidum flower extract in butylene glycol and water

Premix B: Mix Glycerin and Xanthan 180 until a uniform mixture is achieved

Premix C: Disperse SP 500 in Butylene glycol

Water Phase:

    
    
        Add water into the vessel, begin agitation, add EDTA BD and mix until uniform
        Sprinkle in Pemulen TR-1 and Carbopol Ultrez 20 and mix until a translucent mixture is obtained
        Add Prodew 300, Butylene glycol, and Xantural Premix B until uniform
        Start heating to 80-83° C.
        At 70-75° C., add methyl paraben and mix until uniform
        At 80° C., add sodium hydroxide to neutralize the water phase, Hold Temperature until phasing

Oil Phase:

    
    


Mix Miglyol 815, Finsolve Tenn., Lanette 22, Edenor ST1 MY, Brij 721, Cetiol SB45, Ethyl paraben, Propyl paraben, and heat to 80° C., check that a clear melt is achieved before mix for 20 minutes. At 80° C., add Amphisol K and mix until uniformly dispersed. Hold the temperature at 80-83° C. until phasing.
Phasing:


    
    
        Add Oil phase to water phase under homogenization
        Add Simulgel EG and mix until uniform. Do not proceed until thickening effect is observed.
        Start Cooling to 60-65° C.
        At 60-65° C., slowly add Premix A.
        At 55-60° C., add DC 200 50 cst, DC 345 and DC 1403 and mix until uniform
        At 45° C., add Premix C
        At below 35° C., add Hydrolite 5, Chlorohexidine digluconate, and mix until uniform and homogenize the batch for 5 minutes.

## Claims

1. A method of lightening the skin comprising applying to skin in need of skin lightening treatment a composition comprising a carrier and an effective amount of an extract comprising Lilium candidum bulb extract, Lilium candidum flower extract, or mixtures thereof, wherein said extract comprises at least one polyunsaturated fatty acid having a structure of Formula I:


R—COOH  (I)

wherein R is —(CH2)z—(CH═CH—CH2)n—(CH2)m—CH3, n is from 1 to 6, m is from zero to 6, and z is from 2 to 7, and at least one compound selected from the group consisting of polysaccharides, oligosaccharides, disaccharides, and combinations of two or more thereof, and wherein said composition is effective to achieve a ΔL of greater than zero.

2. The method of claim 1 wherein said composition further comprises polyunsaturated fatty acids of Formula I that are not part of the extract.

3. The method of claim 1 wherein said at least one polyunsaturated fatty acid has a structure of formula I wherein R is selected from the group consisting of: —(CH2)7—CH═CH—CH2—(CH2)6—CH3, —(CH2)7—(CH═CH—CH2)2—(CH2)3—CH3, —(CH2)7—(CH═CH—CH2)3—CH3, and combinations of two or more thereof.

4. The method of claim 1 wherein said at least one polyunsaturated fatty acid of formula I is selected from the group consisting of omega-3, omega-6, omega-9 fatty acids and combinations of two or more thereof.

5. The method of claim 1 wherein said at least one polyunsaturated fatty acid of formula I is selected from the group consisting of α-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, linoleic acid, γ-linolenic acid, di-homo-γ-linolenic acid, arachidonic acid, oleic acid, and combinations of two or more thereof.

6. The method of claim 1 wherein said extract comprises at least one polyunsaturated fatty acid of formula I selected from the group consisting of linoleic acid, linolenic acids, and combinations thereof.

7. The method of claim 1 wherein said extract comprises from about 40 to about 95% of polyunsaturated fatty acids of Formula I.

8. The method of claim 1 wherein said extract is essentially free of flavanoids, saponins, and glucosides of flavanoids or saponins.

9. The method of claim 1 wherein said composition further comprises a material selected from the group consisting of surfactants, chelating agents, emollients, humectants, conditioners, preservatives, opacifiers, fragrances, and combinations of two or more thereof, wherein said composition is a skin care composition in a form selected from the group consisting of lotions, creams, serums, gels, sticks, sprays, ointments, liquid washes, soap bars, shampoos, hair conditioners, pastes, foams, powders, mousses, shaving creams, hydrogels, film-forming products, fluid on wipes, fluid on facial masks, and combinations of two or more thereof.

10. The method of claim 9 wherein said extract comprises from about 40 to about 95% of polyunsaturated fatty acids of Formula I.

11. The method of claim 10 wherein said composition further comprises at least one additional skin lightening active agent.

12. The method of claim 9 wherein said composition is effective to achieve a ΔL of about 0.5 or greater.

13. The method of claim 1 wherein said extract comprises Lilium candidum flower extract.

14. The method of claim 1 wherein said skin in need of treatment is selected from the group consisting of darkened skin, sallow skin, skin with uneven skin tone, skin with one or more hyperpigmented marks, or combinations of two or more thereof.