Abstract:
Disclosed are extracts of  Nigrospora sphaerica  as cosmetic skin benefit agents, preferably as skin lightening agents alone or in combination with other skin benefit agents and together with a cosmetic vehicle. The inventive extracts, compositions and methods have effective skin lightening properties, may be easier to deliver to the skin, and are commercially available or cost-effective and available in nature.

Description:
FIELD OF THE INVENTION 
     The invention relates to compositions for topical application to human skin which compositions contain extracts of  Nigrospora sphaerica  and to methods of using the compositions for treatment and conditioning, and particularly for lightening, of skin. 
     BACKGROUND OF THE INVENTION 
     Many people are concerned with the degree of pigmentation of their skin. For example, people with age spots or freckles may wish such pigmented spots to be less pronounced. Others may wish to reduce the skin darkening caused by exposure to sunlight or to lighten their natural skin color. To meet this need, many attempts have been made to develop products that reduce the pigment production in the melanocytes. However, the substances identified thus far tend to have either low efficacy or undesirable side effects, such as, for example, toxicity or skin irritation. Therefore, there is a continuing need for new skin lightening agents, with improved overall effectiveness, as well as agents that lend themselves to ease of processing in their manufacture. 
     The present invention is based at least in part on the discovery that extracts of the fungus  Nigrospora sphaerica  have at least comparable and/or demonstrably better skin lightening activity than known skin lightening agents. The use of  Nigrospora sphaerica  for cosmetic applications has not heretofore been known. 
     SUMMARY OF THE INVENTION 
     The present invention alleviates the deficiencies of the prior art and includes, in part, a novel composition containing a pharmaceutically or cosmetically acceptable carrier and an extract of  Nigrospora spaerica . The inventive compositions contain about 0.000001 to about 50% of extract of  Nigrospora sphaerica , preferably, an organic solvent extract thereof. Preferably, the amount of the extract is about 0.00001% to about 10%, more preferably about 0.001% to about 7%, and even more preferably about 0.01% to about 5%, to attain optimum skin lightening activity at a minimum cost. 
     The present invention is based at least in part on the discovery that extracts of the fungus  Nigrospora sphaerica  have at least comparable skin and/or better skin lightening activity than known skin lightening agents. 
     In the preferred embodiment of the invention, further skin benefit agents may be included in the compositions and inventive method, such as alpha-hydroxy acids, beta-hydroxy acids, polyhydroxy acids, hydroquinone, t-butyl hydroquinone; Vitamin B and/or C derivatives; dioic acids, retinoids, resorcinol derivatives, betulinic acid, vanillic acid, betulinic acid, hydrolactin, and mixtures thereof. Organic and inorganic (e.g. micronized metal oxides) sunscreens may also be included. Organic sunscreens may include Benzophenone-3, Benzophenone-4, Benzophenone-8, Methoxycinnamate, Ethyl dihydroxypropyl-PABA, Glyceryl PABA, Homosalate, Methyl anthranilate, Octocrylene, Octyl dimethyl PABA, Octyl methoxycinnamate (PARSOL MCX), Octyl salicylate, PABA, 2-Phenylbenzimidazole-5-sulphonic acid, TEA salicylate, 3-(4-methylbenzylidene)-camphor, Benzophenone-1, Benzophenone-2, Benzophenone-6, Benzophenone-12, 4-Isopropyl dibenzoyl methane, Butyl methoxy dibenzoyl methane (PARSOL 1789), Etocrylene, and mixtures thereof. 
     The invention also includes methods of treating and conditioning skin by applying topically thereto the inventive compositions containing extracts of  Nigrospora sphaerica . The invention also preferably includes a method of lightening the skin by applying thereto the inventive compositions. Compositions are useful in preventing or repairing such skin conditions as wrinkling, laxity, and photo damage. 
     The inventive compositions and methods have effective skin lightening properties, may be easier to deliver to the skin, and are cost-effective and available from natural sources. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention relates to compositions for topical application to human skin which compositions contain extracts of  Nigrospora sphaerica  and to methods of using the compositions for treatment and conditioning, and particularly for lightening, of skin. 
       Nigrospora  is a filamentous dematiaceous fungus widely distributed in soil, decaying plants, and seeds.  Nigrospora  colonies are rapid growing, compact, woolly, at first white, becoming gray with black areas and a black reverse color.  Nigrospora sphaerica  produce conidiophores which forcibly discharge the conidia. Conidia are black, solitary, unicellular, slightly flattened horizontally, and have a thin equatorial germ slit.  Nigrospora sphaerica , the best known species of the genus  Nigrospora , may be found in the Hawaiian Islands and around the Pacific Ocean, among other places. The extracts of  Nigrospora sphaerica  suitable for use in present compositions are organic solvent extracts, e.g., alcoholic extracts (methanol) or ethyl acetate extracts. 
     According to the present invention, extracts of  Nigrospora sphaerica  must be presented in the composition in an amount of about 0.000001% to about 50% by weight of the composition. Preferably, the amount of the extract is about 0.00001% to about 10%, more preferably about 0.001% to about 7%, and even more preferably about 0.01% to about 5%, to attain optimum skin lightening activity at a minimum cost. 
     As used herein, the term “cosmetic composition” is intended to describe compositions for topical application to human skin. 
     As used herein, the term “comprising” means including, made up of, composed of, consisting and/or consisting essentially of. 
     The term “skin” as used herein includes the skin on the face, neck, chest, back, arms, axilla, hands, legs, and scalp. 
     Except in the examples, or where otherwise explicitly indicated, all numbers in this description indicating amounts of material or conditions of reaction, physical properties of materials and/or use are to be understood as modified by the word “about”. All amounts are by weight of the composition, unless otherwise specified. 
     It should be noted that in specifying any range of concentration, any particular upper concentration can be associated with any particular lower concentration. 
     
       Nigrospora sphaerica  
     
     The extracts according to the present invention are from a material of the Kingdom of fungi; Phylum of Ascomycota; Genus of  Nigrospora ; and Species of  sphaerica. Nigrospora sphaerica , as used in accordance with the present invention, was harvested in the Hawaiian Islands and around the Pacific Ocean. After harvesting, the organisms are grown in a first culture medium, from which the specific strain is isolated and removed to be grown in a second culture medium. The  Nigrospora sphaerica  is then isolated from the second culture medium, followed by extraction. The culture and extraction techniques used in accordance with the present invention are those known or further developed by those skilled in the art of microbiology. The particular culture protocol under which the microorganism was grown and the extraction protocol that was used to prepare the extracts that were screened are set forth hereinbelow. The culture and extraction protocols are further translated below into detailed recipes for media preparation and methods of extraction. 
     Lead Collection Details 
     The following table contains information about the geographic location of collection, the media used to isolate the microorganism, and the morphological characteristics of the microorganism growing on agar isolation plates. 
                                                     TABLE 1               Location/   Collection   Type   Salinity   Recovery   Recovery   Isolation   Spore   Fungal       Description   Taken   Description   ppt   Media   Temp.   Media   Formation   Texture                   Hawaiian   Subtidal   Red   36   PDA   25 deg. C.   YPG (yeast,   FALSE   Dense       Islands:               (potato       glucose, peptone)       tufts       Coral reefs,               starch,       with SPC (soy,       rubble and               dextrose)       potato,       lava tubes                       carbohydrate)                    
Fermentation Media for Fungal Cultures and Protocol
 
     
       
         
               
               
               
             
           
               
                   
                 TABLE 2 
               
               
                   
                   
               
               
                   
                 Media Component 
                 Quantity 
               
               
                   
                   
               
             
             
               
                   
                 Sodium Citrate 
                   8 g 
               
               
                   
                 Cottonseed meal 
                   5 g 
               
               
                   
                 Mannitol 
                   5 g 
               
               
                   
                 Peptone 
                   4 g 
               
               
                   
                 L-arginine 
                 1.5 g 
               
               
                   
                   
               
             
          
         
       
         
         Add Media components to 1 L seawater adjusted to pH=4.0±0.2. 
         Ferment in Stationary liquid for 16 days @25° C.
 
Extraction Protocol
 
       
    
     Add approximately equal volume of HP-20 resin beads (DIAION brand, Mitsubishi, Japan) to the whole broth culture. 
     Shake for 2 hr. 
     Filter, wash twice with equal volume H 2 O 
     Hold vacuum for 45 min. to dry 
     Extract residue with an equal volume of MeOH (methanol). Other suitable organic solvents include but are not limited to ethanol, acetone, ethyl acetate, chloroform, etc. 
     Filter, remove solvent. 
     Identification of Microorganisms 
     The identity of the microorganisms was confirmed to be  Nigrospora sphaerica  using the following procedure. Sample results, including fatty acid profiles and library matches were performed by Microbial ID Inc., Newark, Del. 
     Optional Skin Benefit Agents 
     Preferred cosmetic compositions are those suitable for the application to human skin according to the method of the present invention, which optionally, but preferably, include a skin benefit agent in addition to the inventive coumarin derived compounds. 
     Suitable additional skin benefit agents include anti-aging, wrinkle-reducing, skin whitening, anti-acne, and sebum reduction agents. Examples of these include alpha-hydroxy acids, beta-hydroxy acids, polyhydroxy acids, hydroquinone, t-butyl hydroquinone, Vitamic B and C derivatives, dioic acids, retinoids; betulinic acid; vanillic acid; allantoin, a placenta extract; hydrolactin; and resorcinol derivatives. 
     Cosmetically Acceptable Carrier 
     The cosmetically acceptable vehicle may act as a dilutant, dispersant or carrier for the skin benefit ingredients in the composition, so as to facilitate their distribution when the composition is applied to the skin. 
     The vehicle may be aqueous, anhydrous or an emulsion. Preferably, the compositions are aqueous or an emulsion, especially water-in-oil or oil-in-water emulsion, preferentially oil in water emulsion. Water when present will be in amounts which may range from 5 to 99%, preferably from 20 to 70%, optimally between 40 and 70% by weight. 
     Besides water, relatively volatile solvents may also serve as carriers within compositions of the present invention. Most preferred are monohydric C 1 -C 3  alkanols. These include ethyl alcohol, methyl alcohol and isopropyl alcohol. The amount of monohydric alkanol may range from 1 to 70%, preferably from 10 to 50%, optimally between 15 to 40% by weight. 
     Emollient materials may also serve as cosmetically acceptable carriers. These may be in the form of silicone oils and synthetic esters. Amounts of the emollients may range anywhere from 0.1 to 50%, preferably between 1 and 20% by weight. 
     Silicone oils may be divided into the volatile and non-volatile variety. The term “volatile” as used herein refers to those materials which have a measurable vapor pressure at ambient temperature. Volatile silicone oils are preferably chosen from cyclic or linear polydimethylsiloxanes containing from 3 to 9, preferably from 4 to 5, silicon atoms. Linear volatile silicone materials generally have viscosities less than about 5 centistokes at 25° C. while cyclic materials typically have viscosities of less than about 10 centistokes. Nonvolatile silicone oils useful as an emollient material include polyalkyl siloxanes, polyalkylaryl siloxanes and polyether siloxane copolymers. The essentially non-volatile polyalkyl siloxanes useful herein include, for example, polydimethyl siloxanes with viscosities of from about 5 to about 25 million centistokes at 25° C. Among the preferred non-volatile emollients useful in the present compositions are the polydimethyl siloxanes having viscosities from about 10 to about 400 centistokes at 25° C. 
     Among the ester emollients are:
         (1) Alkenyl or alkyl esters of fatty acids having 10 to 20 carbon atoms. Examples thereof include isoarachidyl neopentanoate, isononyl isonanonoate, oleyl myristate, oleyl stearate, and oleyl oleate.   (2) Ether-esters such as fatty acid esters of ethoxylated fatty alcohols.   (3) Polyhydric alcohol esters. Ethylene glycol mono and di-fatty acid esters, diethylene glycol mono- and di-fatty acid esters, polyethylene glycol (200-6000) mono- and di-fatty acid esters, propylene glycol mono- and di-fatty acid esters, polypropylene glycol 2000 monooleate, polypropylene glycol 2000 monostearate, ethoxylated propylene glycol monostearate, glyceryl mono- and di-fatty acid esters, polyglycerol poly-fatty esters, ethoxylated glyceryl mono-stearate, 1,3-butylene glycol monostearate, 1,3-butylene glycol distearate, polyoxyethylene polyol fatty acid ester, sorbitan fatty acid esters, and polyoxyethylene sorbitan fatty acid esters are satisfactory polyhydric alcohol esters.   (4) Wax esters such as beeswax, spermaceti, myristyl myristate, stearyl stearate and arachidyl behenate.   (5) Sterol esters, of which cholesterol fatty acid esters are examples.       

     Fatty acids having from 10 to 30 carbon atoms may also be included as cosmetically acceptable carriers for compositions of this invention. Illustrative of this category are pelargonic, lauric, myristic, palmitic, stearic, isostearic, hydroxystearic, oleic, linoleic, ricinoleic, arachidic, behenic and erucic acids. 
     Humectants of the polyhydric alcohol-type may also be employed as cosmetically acceptable carriers in compositions of this invention. The humectant aids in increasing the effectiveness of the emollient, reduces scaling, stimulates removal of built-up scale and improves skin feel. Typical polyhydric alcohols include glycerol, polyalkylene glycols and more preferably alkylene polyols and their derivatives, including propylene glycol, dipropylene glycol, polypropylene glycol, polyethylene glycol and derivatives thereof, sorbitol, hydroxypropyl sorbitol, hexylene glycol, 1,3-butylene glycol, 1,2,6-hexanetriol, ethoxylated glycerol, propoxylated glycerol and mixtures thereof. For best results the humectant is preferably propylene glycol or sodium hyaluronate. The amount of humectant may range anywhere from 0.5 to 30%, preferably between 1 and 15% by weight of the composition. 
     Thickeners may also be utilized as part of the cosmetically acceptable carrier of compositions according to the present invention. Typical thickeners include crosslinked acrylates (e.g. Carbopol 982), hydrophobically-modified acrylates (e.g. Carbopol 1382), taurate polymer, cellulosic derivatives and natural gums. Among useful cellulosic derivatives are sodium carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, ethyl cellulose and hydroxymethyl cellulose. Natural gums suitable for the present invention include guar, xanthan, sclerotium, carrageenan, pectin and combinations of these gums. Amounts of the thickener may range from 0.0001 to 5%, usually from 0.001 to 1%, optimally from 0.01 to 0.5% by weight. 
     Collectively the water, solvents, silicones, esters, fatty acids, humectants and/or thickeners will constitute the cosmetically acceptable carrier in amounts from 1 to 99.9%, preferably from 80 to 99% by weight. 
     An oil or oily material may be present, together with an emulsifier to provide either a water-in-oil emulsion or an oil-in-water emulsion, depending largely on the average hydrophilic-lipophilic balance (HLB) of the emulsifier employed. 
     Surfactants may also be present in cosmetic compositions of the present invention. Total concentration of the surfactant will range from 0.1 to 40%, preferably from 1 to 20%, optimally from 1 to 5% by weight of the composition. The surfactant may be selected from the group consisting of anionic, nonionic, cationic and amphoteric actives. Particularly preferred nonionic surfactants are those with a C 10 -C 20  fatty alcohol or acid hydrophobe condensed with from 2 to 100 moles of ethylene oxide or propylene oxide per mole of hydrophobe; C 2 -C 10  alkyl phenols condensed with from 2 to 20 moles of alkylene oxide; mono- and di-fatty acid esters of ethylene glycol; fatty acid monoglyceride; sorbitan, mono- and di- C 8 -C 20  fatty acids; block copolymers (ethylene oxide/propylene oxide); and polyoxyethylene sorbitan as well as combinations thereof. Alkyl polyglycosides and saccharide fatty amides (e.g. methyl gluconamides) are also suitable nonionic surfactants. 
     Preferred anionic surfactants include soap, alkyl ether sulfate and sulfonates, alkyl sulfates and sulfonates, alkylbenzene sulfonates, alkyl and dialkyl sulfosuccinates, C 8 -C 20  acyl isethionates, acyl glutamates, C 8 -C 20  alkyl ether phosphates and combinations thereof. 
     Optional Components 
     In the cosmetic compositions of the invention, there may be optionally added plasticizers; calamine; antioxidants; chelating agents; as well as sunscreens. 
     Other adjunct minor components may also be incorporated into the cosmetic compositions. These ingredients may include coloring agents, pigments, opacifiers, and perfumes. Amounts of these other adjunct minor components may range anywhere from 0.001% up to 20% by weight of the composition. 
     Sunscreens. For use as sunscreen, metal oxides may be used alone or in mixture and/or in combination with organic sunscreens. Examples of organic sunscreens include but are not limited those set forth in the table below. 
     The amount of the organic sunscreens in the cosmetic composition is preferably in the range of about 0.1 wt % to about 10 wt %, more preferably about 1 wt % to 5 wt %. Preferred organic sunscreens are PARSOL MCX and Parsol 1789, due to their effectiveness and commercial availability. 
     
       
         
               
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                 CTFA Name 
                 Trade Name 
                 Supplier 
               
               
                   
               
             
             
               
                 Benzophenone-3 
                 UVINUL M-40 
                 BASF Chemical Co. 
               
               
                 Benzophenone-4 
                 UVINUL MS-40 
                 BASF Chemical Co. 
               
               
                 Benzophenone-8 
                 SPECRA-SORB UV-24 
                 American Cyanamide 
               
               
                 Methoxycinnamate 
                 BERNEL HYDRO 
                 Bernel Chemical 
               
               
                 Ethyl dihydroxypropyl-PABA 
                 AMERSCREEN P 
                 Amerchol Corp. 
               
               
                 Glyceryl PABA 
                 NIPA G.M.P.A. 
                 Nipa Labs. 
               
               
                 Homosalate 
                 KEMESTER HMS 
                 Hunko Chemical 
               
               
                 Methyl anthranilate 
                 SUNAROME UVA 
                 Felton Worldwide 
               
               
                 Octocrylene 
                 UVINUL N-539 
                 BASF Chemical Co. 
               
               
                 Octyl dimethyl PABA 
                 AMERSCOL 
                 Amerchol Corp. 
               
               
                 Octyl methoxycinnamate 
                 PARSOL MCX 
                 Bernel Chemical 
               
               
                 Octyl salicylate 
                 SUNAROME WMO 
                 Felton Worldwide 
               
               
                 PABA 
                 PABA 
                 National Starch 
               
               
                 2-Phenylbenzimidazole-5-sulphonic acid 
                 EUSOLEX 232 
                 EM Industries 
               
               
                 TEA salicylate 
                 SUNAROME W 
                 Felton Worldwide 
               
               
                 3-(4-methylbenzylidene)-camphor 
                 EUSOLEX 6300 
                 EM Industries 
               
               
                 Benzophenone-1 
                 UVINUL 400 
                 BASF Chemical Co. 
               
               
                 Benzophenone-2 
                 UVINUL D-50 
                 BASF Chemical Co. 
               
               
                 Benzophenone-6 
                 UVINUL D-49 
                 BASF Chemical Co. 
               
               
                 Benzophenone-12 
                 UVINUL 408 
                 BASF Chemical Co. 
               
               
                 4-Isopropyl dibenzoyl methane 
                 EUSOLEX 8020 
                 EM Industries 
               
               
                 Butyl methoxy dibenzoyl methane 
                 PARSOL 1789 
                 Givaudan Corp. 
               
               
                 Etocrylene 
                 UVINUL N-35 
                 BASF Chemical Co. 
               
               
                   
               
             
          
         
       
     
     Use of the Composition 
     The fungal extracts, compositions and methods according to the invention are intended primarily as a personal care product for topical application to human skin, as well as to lighten the skin, to reduce the degree of pigmentation in the skin, or to even the skin tone. 
     In use, a small quantity of the composition, for example from 1 to 5 ml, is applied to exposed areas of the skin, from a suitable container or applicator and, if necessary, it is then spread over and/or rubbed into the skin using the hand or fingers or a suitable device. 
     Product Form and Packaging 
     The cosmetic composition of the invention can be formulated as a lotion having a viscosity of from 4,000 to 10,000 mPas, a fluid cream having a viscosity of from 10,000 to 20,000 mPas, or a cream having a viscosity of from 20,000 to 100,000 mPas or above. The composition can be packaged in a suitable container to suit its viscosity and intended use by the consumer. For example, a lotion or fluid cream can be packaged in a bottle or a roll-ball applicator or a propellant-driven aerosol device or a container fitted with a pump suitable for finger operation. When the composition is a cream, it can simply be stored in a non-deformable bottle or squeeze container, such as a tube or a lidded jar. When the composition is a solid or semi-solid stick, it may be packaged in a suitable container for manually or mechanically pushing out or extruding the composition. 
     The invention accordingly also provides a closed container containing a cosmetically acceptable composition as herein defined. 
     The following examples are by way of example, not by way of limitation, of the principles of the present invention, to illustrate the best mode of carrying out the invention. 
     EXAMPLE 1 
     This example illustrates reduction in new melanin synthesis using the inventive extracts of  Nigrospora sphaerica.    
     The extracts of  Nigrospora shpaerica  used throughout the examples that follow were prepared in accordance with the procedures set forth hereinabove. 
     Organ Culture Method 
     Waste skin from 8-month old pigmented pigs was shaved to remove hairs and washed thoroughly to remove debris and reduce microbial content. The cleaned skin was then dermatomed to approximately 1-mm thickness×75-cm 2  and placed in tissue culture flasks containing Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM Cat#1188507) containing penicillin (100-units/ml), streptomycin (100-ug/ml), kanamycin (200-ug/ml), glutamine (2-mM), and fungizone (0.5-ug/ml). All flasks were incubated overnight at 37° C. in the presence of 5% carbon dioxide. This allowed the skin to equilibrate for 18 hours prior to the addition of melanin inhibitors. Areas of the skin that had scratches, erythema, or uneven pigmentation were discarded. 
     The following morning, the skin was punched into 4-mm diameter biopsies and transferred to a 96-well tissue culture plate. To each well containing a biopsy, 180-microliters of fresh DMEM containing penicillin (100-units/ml), streptomycin (100-ug/ml), glutamine (2-mM), hydrocortisone (100-μg/ml) and 0.4-μCi/ml  14 C-thiouracil (Moravek Biochemicals) was added along with 20-microliters of natural extract. (Extracts were initially dissolved in DMSO, i.e., dimethyl sulfoxide, and diluted with phosphate buffered saline.) All plates were returned to the incubator for three days. 
     Following the three day incubation, excess unincorporated radiolabel was removed by washing the biopsies at room temperature two times with 0.1 M carbonate buffer at pH 10 for 30 minutes and 2 hours respectively using an automated plate washer. Biopsies were then bleached and dissolved by addition of 100-microliters of 1 N NaOH/3% H 2 O 2  followed by incubation overnight at 60° C. 
     Once dry, 50-microliters of 2N-HCl was added to each well to re-dissolve/neutralize the samples followed by the addition of 220-microliters of scintillation cocktail (Packard MicroScint-40). Samples were mixed several times to ensure even dispersion, sealed, and assessed for radiolabel incorporation using a Packard TopCount scintillation counter. A reduction in radiolabel incorporation translates to a reduction in new melanin synthesis. 
     
       
         
               
               
               
             
               
               
               
             
           
               
                 TABLE 4 
               
               
                   
               
               
                   
                   
                 % Radiolabel 
               
               
                 Treatment 
                 Concentration 
                 Reduction* 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   Nigrospora sphaerica  extract 
                  80 ug/ml 
                 45 
               
               
                   
                   8 ug/ml 
                 4 
               
               
                 Kojic Acid 
                 1.4 mg/ml 
                 56 
               
               
                   
                 (10 mM) 
               
               
                   
               
               
                 *Statistically significant an p-value of less than 0.05 using students t-test. 
               
             
          
         
       
     
     EXAMPLE 2 
     MelanoDerm Tissue Model Method 
     A MelanoDerm tissue equivalent model (MEL-300 from Mat Tek, Ashland, Mass.) containing melanocytes obtained from dark skin individuals was utilized to confirm the results obtained in the pig organ culture. MelanoDerms were cultured as per the supplier instructions. 
     Medium was replaced every two days over a 14-day period and treatments including the natural extracts were added to the medium phase at a final concentration of 50 and 100-micrograms per ml. Positive control was included, i.e., kojic acid, which is a known skin lightening agent. During each medium change, a portion of the waste was analyzed for levels of lactate dehydrogenase as an indicator of cellular toxicity and stress. At no time was the level of lactate dehydrogenase above the vehicle control solvents (water or ethanol). 
     Determination of melanin content within each MelanoDerm was performed using the assay provided by the manufacturer. Each MelanoDerm was homogenized in 450-microliters of buffer (1% SDS, 0.05-mM EDTA, 10-mM Tris-HCl, pH 6.8) containing 20-microliters of Proteinase K (5 mg/ml) and incubated overnight at 45° C. The following day, 50-microliters of sodium carbonate (500-mM) and 10-microliters of hydrogen peroxide (30% solution) was added to each sample and incubated at 80° C. for 30 minutes and then cooled. The optical density of the top phase was determined (405-nm) following extraction of the sample with 100-microliters of chloroform:methanol (2:1) and centrifugation (10,000 g) for ten minutes. Optical density readings relative to the untreated or vehicle control (water or ethanol) were then calculated. 
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 MelanoDerm Skin Equivalent Model Results 
               
             
          
           
               
                   
                   
                   
                 % Melanin 
               
               
                   
                 Treatment 
                 Concentration 
                 Reduction 
               
               
                   
                   
               
               
                   
                 Kojic Acid 
                   14 micro-g/ml 
                 9.5 
               
               
                   
                   
                 (100 uM) 
               
               
                   
                   Nigrospora sphaerica  Extract 
                   50 ug/ml 
                 60.9* 
               
               
                   
                   
                  100 ug/ml 
                 48.9* 
               
               
                   
                   
               
               
                   
                 *Statistically significant an p-value of less than 0.05 using students t-test. 
               
             
          
         
       
     
     EXAMPLE 3 
     Cosmetic compositions within the scope of the invention were prepared. 
     A base formulation shown in the Table below was made by heating phase A ingredients to 70 to 85° C. with stirring. Phase B ingredients were heated in a separate container to 70 to 85° C. with stirring. Then, phase A was added into phase B while both phases were kept at 70 to 85° C. The mixture was stirred for at least 15 minutes at 70 to 85° C., then cooled. 
     
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                 TABLE 6 
               
               
                   
                   
               
               
                   
                   
                 a 
                 b 
                   
               
               
                   
                 Ingredients 
                 % wt. 
                 % wt. 
                 Phase 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Isostearyl Palmitate 
                 6.00 
                 6.00 
                 A 
               
               
                   
                 C12-C15 Alkyl Octanoate 
                 3.00 
                 3.00 
                 A 
               
               
                   
                 PEG-100 Stearate 
                 2.00 
                 2.00 
                 A 
               
               
                   
                 Glyceryl Hydroxystearate 
                 1.50 
                 1.50 
                 A 
               
               
                   
                 Stearyl Alcohol 
                 1.50 
                 1.50 
                 A 
               
               
                   
                 Stearic acid 
                 3.00 
                 4.00 
                 A 
               
               
                   
                 TEA, 99% 
                 1.20 
                 1.20 
                 B 
               
               
                   
                 Dimethicone 
                 1.00 
                 1.00 
                 A 
               
               
                   
                 Sorbitan Monostearate 
                 1.00 
                 1.00 
                 A 
               
               
                   
                 Magnesium Aluminum Silicate 
                 0.60 
                 0.60 
                 B 
               
               
                   
                 Vitamin E acetate 
                 0.10 
                 0.10 
                 A 
               
               
                   
                 Cholesterol 
                 0.50 
                 0.50 
                 A 
               
               
                   
                 Simethicone 
                 0.01 
                 0.01 
                 B 
               
               
                   
                 Xanthan gum 
                 0.20 
                 0.20 
                 B 
               
               
                   
                 Hydroxyethylcellulose 
                 0.50 
                 0.50 
                 B 
               
               
                   
                 Propylparaben 
                 0.10 
                 0.10 
                 B 
               
               
                   
                 Disodium EDTA 
                 0.05 
                 0.05 
                 B 
               
               
                   
                 Butylated hydroxytoluene 
                 0.05 
                 0.05 
                 B 
               
               
                   
                   Nigrospora sphaerica  extract 
                 0.05 
                 2.00 
                 B 
               
               
                   
                 Niacinamide 
                 1.00 
                 1.00 
                 B 
               
               
                   
                 Metal oxide 
                 2.50 
                 5.00 
                 B 
               
               
                   
                 Methylparaben 
                 0.15 
                 0.15 
                 B 
               
               
                   
                 Water 
                 BAL* 
                 BAL* 
                 B 
               
               
                   
                 Total 
                 100.00 
                 100.00 
                 B 
               
               
                   
                   
               
               
                   
                 *BAL means Balance. 
               
             
          
         
       
     
     EXAMPLE 4 
     An additional cosmetic composition within the scope of the invention was prepared as follows:
         1. Heat Phase A to 80° C.   2. Heat Phase B to 75° C. in a separate container   3. Add B to A and mix with heat off for 30 min.   4. At 50° C. add Phase C and mix for 10 min.       

     
       
         
               
               
               
               
             
           
               
                   
                 TABLE 7 
               
               
                   
                   
               
               
                   
                 Component 
                 Wt % 
                 Phase 
               
               
                   
                   
               
             
             
               
                   
                 water, DI 
                 BALANCE 
                 A 
               
               
                   
                 disodium EDTA 
                 0.05 
                 A 
               
               
                   
                 magnesium aluminum silicate 
                 0.6 
                 A 
               
               
                   
                 methyl paraben 
                 0.15 
                 A 
               
               
                   
                 simethicone 
                 0.01 
                 A 
               
               
                   
                 butylene glycol 1,3 
                 3.0 
                 A 
               
               
                   
                 hydroxyethylcellulose 
                 0.5 
                 A 
               
               
                   
                 glycerine, USP 
                 2.0 
                 A 
               
               
                   
                 xanthan gum 
                 0.2 
                 A 
               
               
                   
                 triethanolamine 
                 1.2 
                 B 
               
               
                   
                 stearic acid 
                 3.0 
                 B 
               
               
                   
                 propyl paraben NF 
                 0.1 
                 B 
               
               
                   
                 glyceryl hydroxystearate 
                 1.5 
                 B 
               
               
                   
                 stearyl alcohol 
                 1.5 
                 B 
               
               
                   
                 isostearyl palmitate 
                 6.0 
                 B 
               
               
                   
                 C12-15 alcohols octanoate 
                 3.0 
                 B 
               
               
                   
                 dimethicone 
                 1.0 
                 B 
               
               
                   
                 cholesterol NF 
                 0.5 
                 B 
               
               
                   
                 sorbitan stearate 
                 1.0 
                 B 
               
               
                   
                 Micronized titanium dioxide 
                 5.0 
                 C 
               
               
                   
                 tocopheryl acetate 
                 0.1 
                 B 
               
               
                   
                 PEG-100 stearate 
                 2.0 
                 B 
               
               
                   
                 sodium stearoyl lactylate 
                 0.5 
                 B 
               
               
                   
                 hydroxycaprylic acid 
                 0.1 
                 C 
               
               
                   
                   Nigrospora sphaerica  extract 
                 10.0 
                 C 
               
               
                   
                 PARSOL MCX 
                 2.4 
                 C 
               
               
                   
                 alpha-bisabolol 
                 0.2 
                 C 
               
               
                   
                   
               
             
          
         
       
     
     EXAMPLES 5-12 
     A set of additional compositions were prepared within the scope of the present invention and are listed in the table below. 
     
       
         
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                   
                 TABLE 8 
               
             
             
               
                   
                   
               
               
                   
                 Examples (wt. %) 
               
             
          
           
               
                   
                   
                 5 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                 acid 
               
               
                   
                   
                 soap 
               
               
                 Ingredients 
                 Phase 
                 base 
                 6 
                 7 
                 8 
                 9 
                 10 
                 11 
                 12 
               
               
                   
               
             
          
           
               
                 Stearic acid 
                 A 
                 17.9 
                 17.9 
                 17.9 
                 17.9 
                 17.9 
                 17.9 
                 17.9 
                 17.9 
               
               
                 Sodium cetearyl 
                 A 
                   
                 2.2 
                   
                 1 
                 1.5 
                 2 
                 3 
                 2 
               
               
                 sulfate* (emulsifier) 
               
               
                 Myrj 59* 
                 A 
                   
                   
                 2 
                 2 
                 2 
                 2 
                 2 
                 1 
               
               
                 (emulsifier) 
               
               
                 Span 60* 
                 A 
                   
                   
                 2 
                 2 
                 2 
                 2 
                 2 
                 1 
               
               
                 (emulsifiers) 
               
               
                 
                   Nigrospora sphaerica 
                 
                 B 
                 0.05 
                 0.05 
                 2.0 
                 2.0 
                 3.5 
                 3.5 
                 5.0 
                 10.0 
               
               
                 extract 
               
               
                 Micronized Zinc 
                 B 
                 2.50 
                 5.00 
                 5.00 
                 2.50 
                 2.50 
                 5.00 
                 2.50 
                 5.00 
               
               
                 Oxide 
               
               
                 KOH, 22% (form in 
                   
                 2.20 
               
               
                 situ soap with 
               
               
                 stearic acid) 
               
               
                 Octyl 
                   
                 2.50 
                   
                   
                 2.50 
                 2.50 
                   
                 2.50 
               
               
                 methoxycinnamate 
               
               
                 Water 
                 B 
                 BAL 
                 BAL 
                 BAL 
                 BAL 
                 BAL 
                 BAL 
                 BAL 
                 BAL 
               
               
                 Glycerin 
                 B 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
               
               
                   
               
             
          
         
       
     
     It should be understood that the specific forms of the invention herein illustrated and described are intended to be representative only. Changes, including but not limited to those suggested in this specification, may be made in the illustrated embodiments without departing from the clear teachings of the disclosure. Accordingly, reference should be made to the following appended claims in determining the full scope of the invention.