U.S. patent application number 10/293131 was filed with the patent office on 2003-04-24 for whitening compositions containing ascomycete derived enzyme.
Invention is credited to Maes, Daniel H., Mammone, Thomas, Marenus, Kenneth D., Schnittger, Steven F..
Application Number | 20030077236 10/293131 |
Document ID | / |
Family ID | 25128574 |
Filed Date | 2003-04-24 |
United States Patent
Application |
20030077236 |
Kind Code |
A1 |
Mammone, Thomas ; et
al. |
April 24, 2003 |
Whitening compositions containing ascomycete derived enzyme
Abstract
The present invention relates to topical whitening compositions
comprising a whitening effective amount of an Ascomycete-derived
melanin-degrading enzyme extract and methods of preparing the
composition. The compositions provide a whitening effect which
occurs by degrading melanin in the skin, yet the compositions
themselves do not turn dark in color. The enzyme extract can be
derived from Saccharomyces cerevisiae, and is added to the
compositions of the present invention. The compositions containing
the enzyme extract are twice as effective as kojic acid in
producing a whitening effect on the skin.
Inventors: |
Mammone, Thomas;
(Farmingdale, NY) ; Schnittger, Steven F.;
(Huntington Station, NY) ; Marenus, Kenneth D.;
(Dix Hills, NY) ; Maes, Daniel H.; (Huntington,
NY) |
Correspondence
Address: |
Estee Lauder Companies
125 Pinelawn Road
Melville
NY
11747
US
|
Family ID: |
25128574 |
Appl. No.: |
10/293131 |
Filed: |
November 14, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10293131 |
Nov 14, 2002 |
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09783229 |
Feb 14, 2001 |
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6514506 |
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Current U.S.
Class: |
424/59 ; 424/62;
424/94.4 |
Current CPC
Class: |
A61Q 19/02 20130101;
A61K 8/9728 20170801; A61K 38/43 20130101; A61K 8/66 20130101; A61P
17/00 20180101; A61P 17/16 20180101 |
Class at
Publication: |
424/59 ; 424/62;
424/94.4 |
International
Class: |
A61K 007/42; A61K
007/135; A61K 038/44 |
Claims
What we claim is:
1. A topical cosmetic or pharmaceutical composition for application
to the skin comprising a whitening effective amount of an
Ascomycete-derived melanin-degrading enzyme extract and a cosmetic
or pharmaceutical carrier.
2. The composition of claim 1 wherein the enzyme extract is derived
from the genus of Saccharomyces.
3. The composition of claim 2 wherein said Ascomycete-derived
enzyme extract is the species Saccharomyces cerevisiae.
4. The composition of claim 2 wherein said enzyme is a purified
extract solution.
5. The composition of claim 2 wherein said enzyme extract is
present in an amount of about 0.05 to 5.00 percent by weight.
6. A topical cosmetic or pharmaceutical composition for application
to the skin comprising a whitening effective amount of a
Saccharomyces cerevisiae enzyme extract.
7. The composition of claim 6 further comprising a sunscreen.
8. The composition of claim 6 wherein said enzyme is present in an
amount of from about 0.05 to 5.00 percent by weight.
9. A method of whitening the skin comprising topically applying a
composition comprising an Ascomycete derived melanin-degrading
enzyme extract to the skin.
10. The method of claim 9 wherein the Ascomycete is derived from
the order of the group Saccharomyces.
11. The method of claim 9 wherein the Ascomycete is the species
Saccharomyces cerevisiae.
12. The method of claim 9 wherein the enzyme extract is present in
an amount of about 0.05 to about 5.00 percent by weight of the
composition.
13. A method of degrading melanin in the skin which comprises
applying to the skin the composition of claim 6.
14. The method of claim 13 wherein the composition is present in an
amount of about 0.05 to about 5.00 percent by weight of the
composition.
15. The method of claim 14 wherein the Ascomycete-based enzyme
extract is Saccharomyces cerevisiae.
16. A method of inhibiting a UV-B induced tan comprising topically
applying a composition comprising a whitening effective amount of
an Ascomycete melanin-degrading enzyme extract derived from the
genus Saccharomyces.
17. The method of claim 16 wherein the whitening effective amount
is about 0.05 to about 5.00 percent by weight of the composition
and the enzyme extract is derived from the species Saccharomyces
cerevisiae.
Description
[0001] This application is a continuation of application Ser. No.
09/783,229, filed on Feb. 14, 2001.
FIELD OF THE INVENTION
[0002] The invention relates to whitening compositions. More
specifically, the invention relates to a whitening composition
comprising an Ascomycete based enzymatic extract which degrades
epidermal melanin when topically applied to the skin.
BACKGROUND OF THE INVENTION
[0003] Beauty is in the eye of the beholder. The desire to whiten
the skin can be just as appealing to some as achieving a tan is to
others. Thus, while a mole, beauty mark or freckled skin might be
attractive to some, others may consider these dark spots on the
skin to be unattractive. Dark spots are visible on the skin in
areas where the production of melanin is increased. Melanin is
responsible for the "color" of skin and functions to protect the
skin from the harmful effects of UV light. In the skin, the
production of melanin, in response to the stimulus of UV light,
produces the well known tanning effect of the skin, and the natural
increased production of melanin in the skin of certain ethnic
groups of people produces a darker skin tone. For those who dislike
the presence of dark spots on the skin or simply desire a lighter
skin tone, whitening compositions are useful.
[0004] To achieve a whitening effect on the skin, various types of
whitening agents are known. Hydroquinone, 4-isopropylcatechol, and
hydroquinone monobenzyl ether are examples of bleaching agents.
However, bleaching agents require repeat applications to the skin
as the top dead cell layer of the skin sheds. When the new dead
cell layer surfaces, spots darken again or reappear. In addition,
bleaching agents can be irritating because of their strength, and
in some instances, may cause skin conditions such as leucoderma
(vitiligo), and rashing. Another method of whitening the skin is
the use of whitening agents such as ascorbic acid, salicylic acid
and lactic acid, which cause the top layer of dead skin cells to
shed or peel off, and along with it the spots caused by increased
melanin production which have migrated up to the skin surface, are
also shed. This method, however, requires a long period of time,
about 2 to 4 weeks, to produce a whitening effect, and also
requires frequent applications.
[0005] Other known whitening agents are tyrosinase inhibitors such
as, for example, kojic acid, which interfere with the production of
melanin in the skin. Melanin is synthesized in melanocytes, cells
that are present in the epidermal basal layer of the skin. A number
of precursors lead to the production of melanin, such as tyrosine,
dopa, dopa-quinone via the action of tyrosinase, and the precursor
indole-5,6-dihydroquinone which is polymerized into melanin.
Inhibition of any one of the precursors involved in the production
of melanin in the skin thereby prevents melanin from being
produced, and can achieve a depigmenting or "whitening" effect on
the skin.
[0006] While the production of melanin can be inhibited, another
method of whitening the skin involves the decomposition of melanin.
This is described in, for example, U.S. Pat. No. 5,578,296, by
using a cultured product or processed product of a species of
Basidiomycetes fungus or any wood-rotting fungi having melanin
decomposing potency. This method, because it breaks down melanin,
allows melanin to be produced in the skin as protection against
harmful UV light. However, decomposing melanin does not prevent new
spots from being developed in the skin, and therefore, this method
still requires repeat application like other whitening methods.
Further, the Basidiomycete fungus is pre-cultured in nitrogen
limited defined media. The selection of Basidiomycete fungus came
after unsuccessful intensive screenings of microorganisms. In
addition JP 8119843 describes a suppressant for melanin containing
an extract of Armeniacae Semen or Pseudocydonia sinensis Schneid.
as an active ingredient, and JP 6269278 describes a microbial
strain, MEL-1 (FERM P-12991), for decoloring melanin developed on
the skin.
[0007] It is also described by authors Luther and Lipke, in
"Degradation of Melanin by Aspergillus fumigatus", Applied and
Environmental Microbiology, vol. 40, no. 1, pp. 145-155 (July
1980), that a strain of Aspergillus fumigatus, NRRL 6463, isolated
from a compost heap in Watertown, Mass., utilized synthetic
tyrosine and dopa, and deproteinized hair melanins as sole sources
of carbon. The Aspergillus fumigatus described in the study
develops a black color over the course of melanin degradation.
Thus, the use of Aspergillus fumigatus on the skin as a whitening
agent is not described. Further, the structural diversity of
melanin may have an effect on the identity of microorganisms which
are capable of contributing to stages of melanin degradation. The
composition of melanin, although it occurs widely in both marine
and terrestrial animals, varies in the ratio of inolic and phenolic
substituents. The chemical composition and physical properties of a
certain class of melanin are highly dependent on the milieu from
which it is formed. Epidermal melanin is known to have two classes
of melanins, eumelanins and phaeomelanins. Eumelanin in particular
includes a peptide coating. Conclusions regarding a specific
melanin polymer are not a priori applicable to an entire class of
melanin. "Melanin: Its Role in Human Photoprotection", Zeise, L.,
et al. Eds., p. 11 to 12 (Valdenmar Publishing Company 1995). The
study described by Luther and Lipke used deproteinized hair melanin
and synthetic precursors, and the ability of Aspergillus fumigatus
enzyme extract for degrading melanin when topically applied to the
skin has not been described. It is also not known in the prior art
that a fungus derived enzyme extract can surprisingly be twice as
effective in whitening the skin as a commonly used tyrosinase
inhibitor.
[0008] In addition, the effect of divalent metal cations on the
hydrolytic activity on melanin was found using an isolation of a
melanolytic fungus of the Acrostaphylus species, a filamentous
fungus belonging to the Fungi Imperfecti and reclassified as
Nodulisporium. The particular strain NDMC-101 was used as disclosed
by Liu, Yu-tien, in "Isolation of a Melanolytic Fungus and its
Hydrolytic Activity on Melanin", Mycologia, vol. 87, no. 5, pp. 651
to 654 (1995). The Acrostaphylus species is saprophytic on wood and
decaying plant materials, such as logs and stumps, dead twigs and
branches of trees, and dead leaves and stems of herbacious plants.
The fungal strain can utilize melanin as a nitrogen source for
supporting its growth in medium containing an appropriate amount of
divalent metallic ion, and therefore can degrade melanin in
polluted water.
[0009] It has now been surprisingly discovered that an
Ascomycete-derived enzyme extract in a cosmetic or pharmaceutical
composition can be useful as a whitening agent for the skin.
Accordingly, a topical whitening composition is provided by the
present invention that can produce a whitening effect upon
application to the skin without negative side effects and without
the necessity of being combined with other types of whitening
treatment.
SUMMARY OF THE INVENTION
[0010] The present invention relates to a topical cosmetic or
pharmaceutical composition for application to the skin that
comprises a whitening effective amount of a melanase enzyme extract
derived from the Ascomycete phylum. The Ascomycete can be derived
from the group of Aspergillus or Saccharomyces. In particular, the
extract contains a melanin-degrading enzyme derived from a species
of fungus, Aspergillus fumigatus, or yeast, Saccharomyces
cerevisiae, effective in whitening the skin. Preparation of the
compositions of the present invention also includes purifying the
crude enzyme extract before it is added to the cosmetic or
pharmaceutical composition.
[0011] In addition, the present invention includes the method of
whitening the skin by topically applying the compositions
containing these Ascomycete derived enzymes. The method includes
extracting enzymes from Aspergillus fumigatus or Saccharomyces
cerevisiae. The melanase enzyme extract is added to a cosmetic or
pharmaceutical composition, and topically applied to the skin. The
methods of degrading melanin production in the skin and inhibiting
a UV-B induced tan by topical application of the compositions of
the present invention are also included in the present
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a graph illustrating the skin whitening effect on
UV-B induced tan by 2% kojic acid and 1% Aspergillus fumigatus
enzyme extract of the present invention as measured at day 7, day
9, day 12, and day 14 of a treatment regime as indicated by the
change in reflectance values.
[0013] FIG. 2 is a chart illustrating the percent reduction in skin
color between day 9 and day 14 on skin irradiated and treated with
2% kojic acid or 1% Aspergillus fumigatus enzyme extract of the
present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0014] In nature, fungi are regarded as a critical part of the
continuous cycle of life and death of organisms. Particularly,
fungi recycle usually dead organic matter into nutrients that it
needs to feed itself. Fungi release enzymes from hyphae, as part of
their digestive system, into the surrounding environment to break
down organic matter into a form of nutrients that it can absorb. It
has now been surprisingly discovered that topical application of a
cosmetic or pharmaceutical composition containing a melanase enzyme
derived from fungi of the large phylum, Ascomycete is effective in
whitening the skin.
[0015] One particular species of the genus, Aspergillus, also known
as sac fungi, is a saprotroph and can consume almost any
carbonaceous substrate. The preferred Aspergillus is Aspergillus
fumigatus. As shown in FIG. 1, the whitening effect produced with
about 1% of Aspergillus fumigatus melanase enzyme extract is
greater than the whitening effect produced using 2% of kojic acid,
twice the amount of Aspergillus fumigatus extract. The present
invention also includes the surprising discovery that an effective
amount of Aspergillus fumigatus enzyme extract is capable of
degrading epidermal melanin upon topical application to the
skin.
[0016] Aspergillus conidia are ubiquitously spread in the
environment. Phylogenetic analysis of 18S rDNA has supported the
traditional separation between fungi with asci, the Ascomycetes,
and those with basidia, the Basidiomycetes. Also, unlike the spores
of the Basidiomycetes, which are produced external to the basidia
and are discharged only a short distance into the space between the
gills from which they randomly fall out of a cap, as for example,
the cap of a mushroom, the spores of the Ascomycetes can be
explosively discharged into the air as a fine white cloud. One of
the most salient features of the Ascomycetes is the formation of
ascospores and the release of these spores from the asci. Ascopores
are usually aimed and forcibly ejected from the ascocarp. The
concentration of spores in the sac of the Ascomycetes may account
for the rapid growth experienced with this phylum of fungi.
[0017] The genus Aspergillus includes about 132 species and 18
variants. The Aspergillus fumigatus species can be found naturally
in plant materials, compost, soil, and food. Various strains of
Aspergillus fumigatus propagate in a variety of agars at
temperatures ranging from about 24.degree. C. to about 30.degree.
C. Colonies of Aspergillus fumigatus grow rapidly at about
45.degree. C. on Czapek-Dox solution agar. The fungus is velvety to
cottony, and white in color turning to green or gray on top. Its
underside is uncolored, yellow, green or brown. Aspergillus
fumigatus is normally silver-yellow and devoid of dark pigment
unless grown on melanins or melanogens. Luther, et al. p. 146.
Growth of melanase can depend on melanin. Color mutants also can
occur in conidia. The conidia of Aspergillus fumigatus may range
between rough-walled and nearly smooth, and between globose to
ellipsoidal or subglobose. The conidia are also echinulate,
1-celled and about 2.0 to 3.5 .mu.m. Conidiophores, which bear
conidia, are thin-walled, smooth, green and end in a hemispherical
vesicle. Aspergillus fumigatus produces flask-shaped vesicles
bearing phialides in a uniseriate arrangement, a single, compact
column. The phialides cover the upper half of the vesicle. Its
conidial heads also form a single, compact column. The hyphal wall
appears two-layered. One layer is a thick translucent inner layer,
and the other layer is a thin, opaque outer layer.
[0018] The melanin-degrading enzyme extract of the present
invention can be prepared by first growing colonies of Aspergillus
fumigatus, propagated and available commercially from, for example,
American Type Culture Collection (ATCC), Manassas, Va. A preferred
strain is NRRL 6463 available from ATCC. The fungus is available
either in a point of process test tube or freeze-dried. Aspergillus
fumigatus can be grown in malt extract agar, potato dextrose agar,
YpSs agar, PYG medium or Czapek agar. Preferably, the fungus is
grown in potato dextrose agar in a one-liter spinner flask, and is
grown to a very high density of about 10.sup.6 to 10.sup.7 colony
forming units per ml (cfu/ml). These amounts are provided as
guidance and can be adjusted as necessary based on general
knowledge of one skilled in the art of microbiology to achieve the
desired growth rate.
[0019] The enzyme is extracted by centrifuging about 500 ml of the
culture and resuspending it in about 150 mM NaCl, about 50 mM Tris
buffer, and about 1 percent NP40 detergent. The resuspension is
sonicated for about 30 minutes, and can then be added directly to a
cosmetic or pharmaceutical formulation as a crude isolated enzyme.
Preferably, the enzyme is purified by filtering it through a 0.22
.mu.m filter. The amount of enzyme in the purified enzyme extract
solution can be from about 1 to about 95 percent; preferably the
enzyme is about 80 to about 95 percent of the purified
solution.
[0020] The enzyme extract thus prepared is incorporated into a
cosmetic or pharmaceutical formulation in a whitening effective
amount. The term "whitening effective amount" as used herein means
an amount of enzyme that reduces the color of the skin, as measured
using a Minolta Chromameter, by about at least 25 percent,
preferably by at least about 30 percent. Preferably, the whitening
effective amount is from about 0.05 to about 5.00 percent by weight
of the enzyme in the composition. More preferably, the enzyme is
present in an amount of about 0.1 to about 2.0 percent by weight of
the composition.
[0021] It has also been surprisingly discovered that a
yeast-derived enzyme extract can be useful in a cosmetic or
pharmaceutical formulation as a whitening agent for the skin. The
particular single-celled yeast is Saccharomyces cerevisiae (also
known as true yeast or baker's yeast, for example, strain ATCC
60219). Yeasts are characterized by solitary budding cells that
undergo meiotic reproduction by ascospores. Their nucleus, like
that of other eukaryotic organisms, contains a nucleolus and
several chromosomes that are bound by a nuclear membrane. In
yeasts, the ascospores multiply by budding or conidium formation
(fission). The single-cell yeast simply becomes the ascus and
usually has about 4 spores.
[0022] It is known that yeast or baker's yeast is reported to
stimulate respiration of the skin. In the prior art, synergistic
yeast extract is combined with peroxidase enzymes to prevent
interference with peroxidase activity. However, the yeast extract
in European Patent Application EP 1 004 289 is not reported as
having any degradative effect on melanin in the skin. The yeast
extracted for the present invention can be grown in 10% colloidal
minerals (Rockland Corp., Tulsa, Okla.) in YM broth (available from
Difco Labs) for several days. The yeast is sonicated, filtered and
the yeast-derived enzyme is added to a cosmetic or pharmaceutical
composition similar to the fungus described above.
[0023] The present invention also includes the method of whitening
the skin by adding to the cosmetic or pharmaceutical composition
the whitening effective amount of enzyme, either the fungus or the
yeast, and topically applying the composition to the skin. The
compositions of the present invention achieve a whitening effect on
the skin without becoming dark colored. The compositions can be
topically applied to any area of the skin intended for whitening
such as, for example, the face, the legs and arms, and the torso.
The whitening compositions are applied by rubbing them onto an area
on the surface of the skin of about 2 mg/cm.sup.2 and reapplied as
necessary, as for example, on a daily basis. The whitening effect
is produced in about 5 to 10 days. Its superiority as a whitening
agent is observed between about 10 and 15 days. The compositions of
the present invention can be chronically applied. Therefore, the
whitening compositions can be prepared in any form convenient for
topical application to the skin. Such forms include, but are not
limited to gels, creams, colloidal dispersions, emulsions
(water-in-oil or oil-in-water), suspensions, solutions, lotions,
foams, mousses, sprays and the like.
[0024] The whitening compositions are formulated with a variety of
cosmetically and/or pharmaceutically acceptable carriers. The term
"pharmaceutically or cosmetically acceptable carrier" refers to a
vehicle, for either pharmaceutical or cosmetic use, which vehicle
delivers the active components to the intended target and which
will not cause harm to humans or other recipient organisms. As used
herein, "pharmaceutical" or "cosmetic" will be understood to
encompass both human and animal pharmaceuticals or cosmetics.
Useful carriers include, for example, water, acetone, ethanol,
ethylene glycol, propylene glycol, butane-1,3-diol, isopropyl
myristate, isopropyl palmitate, or mineral oil. It will be apparent
to the skilled artisan that the selected carrier must be compatible
and relatively inert with respect to the whitening compositions.
Methodology and components for formulation of compositions are well
known, and can be found, for example, in Remington's Pharmaceutical
Sciences, Eighteenth Edition, A. R. Gennaro, Ed., Mack Publishing
Co., Easton Pa., 1990.
[0025] The whitening compositions of the present invention may be
combined with one or more sunscreens. The term "sunscreen" as used
herein refers to any material which is capable of protecting skin
from ultraviolet radiation having a wavelength of from about 280 to
about 400 nm, by effectively absorbing such radiation, and/or
reflecting or scattering such radiation away from the surface of
skin. Examples of sunscreens with which the compositions of the
present invention can be combined in this context are titanium
dioxide, zinc oxide, benzophenones, p-amino benzoic acid (PABA),
octyl dimethyl PABA, amyldimethyl PABA, octyl methoxycinnamate,
2-ethoxy p-methoxycinnamate, oxybenzone, homosalate, phenyl
salicylate, glyceryl p-aminobenzoate, ethyl-p-glycosylimido
benzoate and the like. In a formulation, the sunscreen agent is
used in the amounts normally used for that agent, and the enzyme is
used in the amounts stated above.
[0026] Various other optional ingredients may be included with the
whitening compositions of the present invention, these include but
are not limited to fragrances, perfumes, flavorings, preservatives,
emollients, antiseptics, pigments, dyes, colorants, humectants,
propellants, waterproofing agents, film formers, vitamins as well
as other classes of materials whose presence may be cosmetically,
pharmaceutically, medicinally or otherwise desirable. Common
examples can be found in the CTFA International Cosmetic Ingredient
Dictionary 4th Edition, The Cosmetic, Toiletry, and Fragrance
Association, Inc., Washington, D.C., 1991, the contents of which
are incorporated herein. The whitening compositions may also be
useful in makeup products.
[0027] The compositions of the present invention may also comprise
additional useful active ingredients which include, but are not
limited to other known whitening agents, such as for example,
tyrosinase inhibitors such as, for example, kojic acid,
antioxidants, antimicrobials, analgesics, anesthetics, anti-acne
agents, antidermatitis agents, antipruritic agents,
anti-inflammatory agents, antihyperkeratolytic agents, anti-dry
skin agents, antiperspirants, antipsoriatic agents, antiseborrheic
agents, antiaging agents, antiwrinkle agents, self-tanning agents,
wound-healing agents, corticosteroids, or hormones. The
incorporation of the active in the formulation is determined by its
solubility and/or stability in combination with the Ascomycete
derived melanin-degrading enzyme of the present invention. The
selection of the mode of delivery for additional active
ingredients, however, is limited to the mode of delivery chosen for
the whitening compositions.
[0028] The invention is further illustrated by the following
non-limiting example.
EXAMPLES
[0029] I. Preparation of a Whitening Composition
1 Material Weight % Phase I Stearic Acid 20 Glycerol Stearate 20
Mineral Oil 120 Petrolatum 4.0 Parabens 0.4 Phase II Water 35.0
Propylene Glycol 5.0 Trisodium EDTA 0.2 Parabens 0.4 Phase III
Water 38.0 Aspergillus 1.0 Furrigatus enzyme extract
[0030] Phase I ingredients and Phase II ingredients are combined in
separate vessels and each combination is heated with stirring to
70.degree. C. The combined Phase I ingredients are then added with
stirring to the combined Phase II ingredients. The mixture is
allowed to cool to 30.degree. C. while stirring. The Phase III
ingredients are combined and added to Phase I and II ingredients to
form a final emulsion. The Aspergillus fumigatus enzyme extract is
NRRL 6463 obtained from ATCC.
[0031] II. Whitening Action of Aspergillus fumigatus Enzyme
Extract
[0032] A comparative study is done to determine the efficacy of an
Aspergillus fumigatus enzyme extract in comparison with the
efficacy of a known tyrosinase inhibitor, kojic acid. Three samples
are prepared. The first sample contains the base formula for a
lotion without any whitening agent added to the formula. The second
sample is the same base formula with 2% kojic acid added to the
formula. Finally, the third sample contains the whitening agent of
the present invention of 1% Aspergillus fumigatus enzyme extract in
the base formula, as similarly described in Example I above.
[0033] Ten volunteer panelists participate in this study. To
qualify for the study, the panelist is required to be in normal
health with no evidence of acute or chronic disease including
dermatologic problems. The panelists are female, ranging in age
from 18 to 45, and having skin type I-II. In addition, the
panelists do not exhibit sunburn effects, rashes, scratches, or
burn marks as these conditions might interfere with the analysis of
the test results. Pregnant or lactating females are also excluded.
Upon examination at the site of testing, the participating
panelists are examined to determine that they are devoid of
excessive warts, nevi, moles, sunburn, suntan, scars or active
dermal lesions. Finally, the panelists do not use systemic or
topical retinoids, antihistamines or similar agents during the
course of the study and two weeks prior to the commencement of the
study.
[0034] Four distinct areas of about 4 cm.sup.2 are marked on the
backs of each of the panelists. Three of the four areas correspond
to the lotion without a whitening agent, lotion with 2% kojic acid,
and the lotion with 1% Aspergillus fumigatus enzyme extract of the
present invention. The fourth area serves as the untreated
irradiated control. Each panelist receives twice the MED of UV-B on
each site area. The sites are radiated with a Xenon Arc Solar
Simulator (150 Watt) with filters (mm UG-5) to expose the skin to
UV-B and UV-A radiation. Tanning is observed for 7 days after
irradiation at which point baseline color measurements are made
using a Minolta Chromameter which measures the difference in
reflectance of the skin, L. The change in the value of the
difference in reflectance is, .DELTA.L*. The delta values are
measured against a baseline skin color value measured on day 7. The
test materials are applied to the respective sites at a rate of 2
mg/cm.sup.2 after the chromameter measurement on day 7, and are
allowed to dry for 10 minutes. Product treatments are continued
once a day for 7 days (i.e., day 8 to day 14 of the test).
Chromameter readings are obtained on day 9, day 12, and day 14
after irradiation. An increase in skin tanning is observed with the
chromameter for about 9 days after irradiation.
[0035] Results are shown in FIGS. 1 and 2. In FIG. 1, the skin
whitening effect of 1% Aspergillus fumigatus enzyme extract
("Aspergillus extract") shows that it is greater than 2% kojic
acid, twice the amount of the enzyme extract. The comparison is
measured in terms of reflectance, (.DELTA.L*), and it indicates
that the enzyme extract containing compositions of the present
invention have a lower reflectance and therefore, greater whitening
effect. Since skin tanning continued to increase until about day 9,
data for days 12 and 14 are normalized against the average data for
day 9. In FIG. 2, comparison of the three formulas in terms of the
percent skin color reduction, between day 9 and day 14 is provided.
The data in FIG. 2 shows that the composition of the present
invention, containing half the amount of enzyme extract (1%) than
the amount of kojic acid (2%), produces a greater reduction in
color of the skin.
[0036] III. Whitening Action of Saccharomyces cerevisiae Enzyme
Extract
[0037] Yeast is grown in 10% colloidal mineral in YM broth for 3
days. The grown yeast is sonicated in a water bath sonicator for
about 30 minutes and filtered. Melanin is prepared by incubating 1
mg/ml, cold dl 3,4-dihydroxy phenylalanine (DOPA) with 250 .mu.Ci
of C.sup.14 DOPA in PBS. The polymerization is started with 10
units/ml of mushroom tyrosinase and is allowed to proceed until it
is dark and insoluble melanin pigment is observed. The melanin thus
prepared is stored at 4.degree. C.
[0038] Insoluble radiolabeled C.sup.14--melanin is prepared from
synthetic melanin by repeated centrifugation or about 1.0 ml at
about 10,000 rpm for about 15 minutes. Centrifugation is repeated
three times. Each time, about 1.0 ml of melanin is added and the
supernatant is discarded. Pooled insoluble melanin is resuspended
in about 100 .mu.ls of PBS and used as substrate. The reaction
mixture consists of 10 .mu.ls of radiolabeled melanin, 70 .mu.ls of
PBS, and intervals of about 0, 25, 50, and 100 .mu.ls of cell
homogenate of fraction (yeast extract). The reaction mixture is
incubated for about one week at 37.degree. C. while it is shaken.
The reactions are stopped by the addition of scintillation fluid
and the melanin count is taken. The treatments are done in
triplicate. A decrease in total melanin count after one week of
incubation is observed. It is found that the yeast extract
decreases the amount of radiolabeled 24 percent, 29 percent and 54
percent for the intervals 25, 50 and 100 .mu.ls of crude yeast
extract, respectively.
[0039] IV. Whitening Action of Saccharomyces cerevisiae Enzyme
Extract
2 Material Weight % Phase I Stearic Acid 20 Glyceryl Stearate 20
Mineral Oil 10.0 Petrolatum 2.0 Parabens 0.4 Phase II Water 40.0
Triethandimine 1.0 Trisodium EDTA 0.2 Propylene Glycol 5.0 Parabens
0.4 Phase III Water 36.0 Saccharomyces 1.0 cerevisiae yeast
extract
[0040] A study is done to determine the efficacy of a Saccharomyces
cerevisiae enzyme extract in comparison with the efficacy of a
known tyrosinase inhibitor, kojic acid. A samples of the yeast
enzyme extract and 2% kojic acid are prepared separately in a
triethanolamine (TEA) stearate base. The first sample contains the
base formula for a lotion 1% Saccharomyces cerevisiae enzyme
extract added to the formula. The second sample is the same base
with 2% kojic acid added to the formula.
[0041] Seven volunteer panelists participate in this study. To
qualify for the study, the panelist is required to be in normal
health with no evidence of acute or chronic disease including
dermatologic problems. The panelists are female, ranging in age
from 18 to 45, and having skin type I-II. In addition, the
panelists do not exhibit sunburn effects, rashes, scratches, or
burn marks as these conditions might interfere with the analysis of
the test results. Pregnant or lactating females are also excluded.
The panelists appear to be in normal health and do not have signs
of acute or chronic disease, including dermatologic problems. Upon
examination at the site of testing, the participating panelists are
examined to determine that they are devoid of excessive warts,
nevi, moles, sunburn, suntan, scars or active dermal lesions.
Finally, the panelists do not use systemic or topical retinoids,
antihistamines or similar agents during the course of the study and
two weeks prior to the commencement of the study.
[0042] Distinct areas of about 4 cm.sup.2 are marked on the backs
of each of the panelists each for applying the two samples, i.e.,
lotion with 2% kojic acid, and the lotion with 1% Saccharomyces
cerevisiae enzyme extract of the present invention. An additional
area is marked as the untreated irradiated control. Each panelist
receives twice the MED of UV-B on each site area. The sites are
radiated with a Xenon Arc Solar Simulator (150 Watt) with filters
(mm UG-5) to expose the skin to UV-B and UV-A radiation. Tanning is
observed for 5 days after irradiation at which point baseline color
measurements are made using a Minolta Chromameter which measures
the difference in reflectance of the skin, L. The change in the
value of the difference in reflectance is, .DELTA.L*. The delta
values are measured against a baseline skin color value measured on
day 7. The test materials are applied to the respective sites at a
rate of 2 mg/cm.sup.2 after the chromameter measurement on day 7,
and are allowed to dry for 10 minutes. Product treatments are
continued once a day for 7 days (i.e., day 8 to day 14 of the
test). Chromameter readings are obtained on day 9, day 12, and day
14 after irradiation. An increase in skin tanning is observed with
the chromameter for about 9 days after irradiation. Results
indicate that the samples containing the yeast enzyme extract (1%)
produces a reduction in color of the skin.
* * * * *