U.S. patent application number 13/198082 was filed with the patent office on 2011-11-24 for personal care composition containing leghemoglobin.
Invention is credited to James V. Gruber.
Application Number | 20110286992 13/198082 |
Document ID | / |
Family ID | 27760467 |
Filed Date | 2011-11-24 |
United States Patent
Application |
20110286992 |
Kind Code |
A1 |
Gruber; James V. |
November 24, 2011 |
PERSONAL CARE COMPOSITION CONTAINING LEGHEMOGLOBIN
Abstract
A personal care composition comprising leghemoglobin and at
least one preservative selected from the group consisting of
alcohols, glycols, parabens, hydantoins, quaternary
nitrogen-containing compounds, isothiazolinones, aldehyde-releasing
agents, and halogenated compounds. Preferably, the leghemoglobin is
a nitrogen fixation root nodule extract providing a leghemoglobin
concentration in the composition of between 0.0001% and about 10%
based upon the total weight of the composition. Also disclosed is a
method for preparing the personal care composition.
Inventors: |
Gruber; James V.;
(Somerville, NJ) |
Family ID: |
27760467 |
Appl. No.: |
13/198082 |
Filed: |
August 4, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10366231 |
Feb 13, 2003 |
8021695 |
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13198082 |
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60357544 |
Feb 15, 2002 |
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Current U.S.
Class: |
424/94.4 ;
424/401; 424/94.1; 514/18.8 |
Current CPC
Class: |
A61P 43/00 20180101;
A61Q 19/00 20130101; A61K 8/9728 20170801; A61P 7/00 20180101; A61K
8/645 20130101; A61P 7/02 20180101; A61K 8/068 20130101; A61Q 17/00
20130101; A61K 8/9767 20170801; A61Q 19/04 20130101; A61K 8/9789
20170801 |
Class at
Publication: |
424/94.4 ;
424/401; 424/94.1; 514/18.8 |
International
Class: |
A61K 8/64 20060101
A61K008/64; A61K 8/66 20060101 A61K008/66; A61Q 19/00 20060101
A61Q019/00; A61K 8/02 20060101 A61K008/02 |
Claims
1. A method of preparing a personal care composition containing
leghemoglobin, comprising the steps of: extracting the
leghemoglobin from root nodules infected with bacteria selected
from the group consisting of Cyanobacteria, Anaerobic bacteria,
Purple sulfur bacteria, Purple non-sulfur bacteria, Green sulfur
bacteria, legumes bacteria, and combinations thereof, in order to
provide a leghemoglobin extract, and combining the leghemoglobin
extract with a preservative to provide the personal care
composition.
2. The method of claim 1 wherein the leghemoglobin is obtained from
the roots of plants infected with bacteria from the Genus Rhizobium
or Brachyorhizobium.
3. The method of claim 1 in which the leghemoglobin is taken from a
plant root selected from those found in perennial legumes, annual
legumes, legumeous woody plants, and combinations thereof.
4. The method of claim 1 in which the leghemoglobin is taken from
the roots of annual legumes.
5. The method of claim 1 wherein the preservative is selected from
the group consisting of alcohols, glycols, parabens, quaternary
nitrogen-containing compounds, isothiazolinones, aldehyde-releasing
agents, antioxidants, halogenated compounds and combinations
thereof.
6. The method of claim 1 wherein said combining step further
comprises combining a surfactant with the extract and the
preservative.
7. The method of claim 1 wherein the extract provided in said
extracting step has a leghemoglobin concentration in the
composition of between 0.0001% and about 10% based upon the total
weight of the composition.
8. The method of claim 1 wherein the extract provided in said
extracting step comprises a growth media for other microorganisms
or confluent skin cultures.
9. The method of claim 8 in which the microorganisms are yeast.
10. The method of claim 1, further comprising the step of
encapsulating the leghemoglobin within another component in order
to provide a time-release characteristic to said leghemoglobin.
11. The method of claim 10 wherein the leghemoglobin is
encapsulated within a liposome or a maltodextrin encapsulating
oligosaccharide.
12. The method of claim 1, further comprising the step of providing
at least one additional root nodule extract component in the
composition, the additional root nodule extract component selected
from the group consisting of plant growth regulators, amino acids,
vitamins, proteins, enzymes, minerals carbohydrates,
exopolysaccharides, and combinations thereof
13. The method of claim 12 where the additional root nodule extract
component comprises an enzyme selected from the group consisting of
nitrogenases, superoxide dismutases, catalases, nitric oxide
synthases, sucrose synthases, peroxidases, glutathiones and
combinations thereof
14. The method of claim 12 wherein the additional root nodule
extract component comprises a plant growth regulator selected from
the group consisting of kinetin, zeatin, other furfuryl-type
molecules, complimentary plant growth regulators such as auxins,
and combinations thereof
15. The method of claim 12 wherein the additional root nodule
extract component comprises exopolysaccharides, alone or in
combination with lipopolysaccharides or capsular polysaccharides,
or a combination thereof.
16. A method for binding free radicals on the skin that comprises
contacting the skin with leghemoglobin, thereby causing said
leghemoglobin to act as a scavenger for the free radicals.
17. The method of claim 16 wherein the leghemoglobin acts as a
scavenger for free radicals selected from the group consisting of
reactive oxygen, nitric oxide, and combinations thereof
18. A method for removing free radicals from the skin that
comprises contacting the skin with leghemoglobin in order to cause
free radicals on the skin to bind with the leghemoglobin, thereby
removing said free radicals from the skin.
19. The method of claim 18 wherein said leghemoglobin is provided
by means of personal care composition comprising the leghemoglobin
and at least one preservative selected from the group consisting of
alcohols, glycols, parabens, hydantoins, quaternary
nitrogen-containing compounds, isothiazolinones, aldehyde-releasing
agents, and halogenated compounds.
20. The method of claim 19 wherein said leghemoglobin is in the
form of a nitrogen fixation root nodule extract providing a
leghemoglobin concentration in the composition of between 0.0001%
and about 10% based upon the total weight of the composition.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional application of U.S. application Ser.
No. 10/366,231, filed Feb. 13, 2003, which claims the benefit of
U.S. Provisional Application No. 60/357,544, filed Feb. 15, 2002,
the disclosures of which are incorporated by reference herein in
their entirety.
FIELD OF THE INVENTION
[0002] This invention relates generally to personal care
compositions, and more specifically to such compositions containing
leghemoglobin. The leghemoglobin is suitably extracted from
nitrogen fixation root nodules. The leghemoglobin provides
advantageous properties to the personal care composition during use
by facilitating the control of reactive oxygen and nitric oxide
free radicals on the skin.
BACKGROUND OF THE INVENTION
[0003] Personal care compositions encompass a wide variety of
applications, including soaps, shampoos, skin care medicaments,
cosmetics, as well as therapeutic and homeopathic skin care
formulations. The use of plant-derived raw materials in personal
care compositions is well established. By way of illustration, PCT
patent publication WO 9913855 discloses the use of a combination of
algae extract and exopolysaccharides for cosmetic and
dermapharmaceutical applications.
[0004] Consideration of the use of plant-derived materials in
personal care has become especially important in view of the recent
discovery that certain animal-borne diseases, such as Bovine
Spongiform Encephalopathy (BSE), are spread to humans through
contact with infected meat and meat by-products. The sensitivity of
the personal care industry to these issues is so great that even
the sale of lanolin, a by-product derived from the shearing of
sheep wool, has suffered because it is considered to be an
animal-derived product.
[0005] The use of human-derived ingredients in personal care
compositions is also frowned upon for the same reasons as suggested
above for animal-derived products. Human-derived products can have
even greater concerns than animal-derived constituents because
pathogens borne in the human-derived cosmetic ingredients are even
more likely to have a detrimental effect on human health. It has
been suggested, for example, in U.S. Pat. No. 6,160,021 that human
derived blood constituents, such as hemoglobin, when administered
topically can influence the production of melanin in the skin
providing a method of skin lightening.
[0006] As a consequence of these market pressures, personal care
composition manufacturers are relying more heavily on plant-derived
raw materials, such as plant proteins obtained from corn, soy and
wheat replacing animal proteins. In addition, the cosmetic and
personal care industries look closely to the health food and herbal
supplement industries for trends in product development. Such
trends are summarized, for example, in a recent article in HAPPI 38
(2001) 81. This article summarizes many of the pressures that face
global cosmetic raw material manufactures and the recent trends
towards `natural` based products. Good examples of this are the
recent trends of aromatherapy and natural extracts, particularly
plant extracts, as useful ingredients for providing functionality
and support the labeling claims as to such `natural` based personal
care products.
[0007] Several plants have enjoyed a healthy growth in personal
care compositions because of their purported benefits to humans.
For example, Aloe Vera is a well-known plant that provides an
extract that is known to help aid wound healing. Ginkgo biloba and
grapes are known to contain polyphenols that are purported to
provide anti-oxidative effects to human skin, ameliorating the
harmful effects of UV damage on the skin. Soybean extracts are
extremely popular due to the presence of soy isoflavones that are
suggested to contain plant phytoestrogens, compounds that mimic
human estrogen and help ameliorate the effects of aging,
particularly in post-menopausal women. Products such as extracts
from Asafaetida and Yellow dock are suggested, for example, to
minimize excess melanin formation that is responsible for the
appearance of age spots on the skin. Recently, U.S. Pat. Nos.
5,371,089 and 5,602,139 disclose that plant growth regulators known
as cytokinins appear to extend the longevity of human fibroblasts,
the cells from which human keratinocytes arise. Keratinocytes
become the protective cells that comprise the stratum corneum and
make human life possible on this planet. Certain bacterial and
yeast extracts are known to provide useful cosmetic, therapeutic or
homeopathic effects, in particular, yeast such as Saccharomyces
cerevisiae. In addition, some root extracts, for example, ginseng,
have carried a historical trend for health and energy and are seen
to appear in personal care products purporting such claims.
[0008] A major component of the atmosphere surrounding the earth is
nitrogen gas, N.sub.2. Both plants and animals are bathed in
nitrogen gas from the air and yet, because of the extraordinary
chemical stability of this molecule, nitrogen gas is essentially
chemically inert to both plants and animals. However, molecular
nitrogen is a key component of all of the amino acids, vitamins,
proteins, enzymes, and cells of both Kingdoms. Without a means for
converting nitrogen gas into useful nitrogen components such as
ammonia, life as we know it on this planet would not exist. The
conversion of nitrogen gas into useful nitrogen substrates occurs
through natural and industrial means and comprises what is known as
the nitrogen cycle.
[0009] Fortunately, while plants and animals are incapable of
chemically converting nitrogen into useful nitrogen-containing
components, certain bacteria, especially those of the blue-green
Cyanobacteria Kingdom, such as, for example, Rhizobium, have
evolved a mechanism to convert nitrogen into more useful ammonia.
The process of conversion is known as `Nitrogen Fixation` and
relies on the presence in these organisms of a key enzyme known as
nitrogenase.
[0010] Nitrogen fixation is a complex enzymatic process. The
general enzymatic reaction to convert nitrogen into ammonia can be
summarized as:
N.sub.2+8H++8e.sup.-+16ATP=2NH.sub.3+H.sub.2+16ADP+16Pi
[0011] The process is intimately driven by conversion of adenosine
triphosphate, ATP, into adenosine diphosphate through reduction of
nitrogen to ammonia by nitrogenase enzyme. Because the reaction is
a reduction reaction, it is very sensitive to the presence of
oxygen.
[0012] Some free-living organisms, such as lichen, are capable of
fixing nitrogen but typically on a very small scale. Interestingly,
Rhizobium bacteria, however, will not convert nitrogen into ammonia
when they exist alone. The bacteria are generally unable to keep
the oxygen away that is detrimental to the overall nitrogen
fixation reaction. In order to overcome this problem, the bacteria
have evolved an unusual associative relationship with many green
plants. The bacteria, which can be found readily in the soil, will
infect the roots of various plants, in particular, the roots of
legumes, for example. The bacteria are attracted to the roots by
the excretion of various flavonoids such as luteolin and
hesperetin, such flavonoids being referenced, for example, in
Begum, A. A. et. al., J. Exper. Bot. 2001; 52, 1537-1543 the
contents of which is incorporated in its entirety into the body of
this invention.
[0013] Once within the roots of the plants, the bacteria force
plants to build small, oxygen deficient homes known scientifically
as symbiosomes and more commonly as `root nodules` in which the
bacteria can live and carry out the nitrogen fixation reaction. The
plant, however, does not suffer from the infection, but instead
benefits from the bacterial conversion of nitrogen into ammonia by
using the ammonia for its own life processes. This relationship
between the blue-green bacteria and the green plant is symbiotic.
That is, both organisms benefit from the relationship. The seeds of
many commercial crops that provide safe havens for nitrogen fixing
bacteria are actually sold commercially with the fixing bacteria
mixed with the seeds prior to planting. Such combinations can be
found, for example, at commercial websites such as that provided
for Agrobiologicals at
http://www.agrobiologicals.com/glossary/Cont6.htm.
[0014] The bacteria build the root nodule by infecting the root
hairs of the green plant and switching on the plant growth
regulators that cause a rapid growth of plant cells around the
infected site. The bacteria control the growth of the nodule by
taking over the localized growth mechanisms of the root by using
various plant growth regulators such as, for example, cytokinins as
well as important complimentary plant growth regulators such as
auxins, such plant growth regulators being referenced, for example
in Schultze, M. et. al., Anna. Rev. Genet. 1998; 32, 33-57
incorporated in its entirety into the body of this invention. In
addition, the bacteria begin expanding in size and shedding their
cells walls, becoming bacteroids that begin forming a biofilm-like
lining around themselves made from excreted lipopolysaccharides,
exopolysaccharides and capsular polysaccharides that further
prevents the infiltration of oxygen into the nodule. Such
polysaccharides are referenced, for example in Rodriquez-Carvajal,
M. A. et. al., Biochem. J. 2001; 357, 505-511 incorporated in is
entirety into the body of this invention. Because the process of
nitrogen fixation is so sensitive to the presence of oxygen and
reactive oxygen radicals, the root nodule bacteria also accumulate
and produce a variety of anti-oxidants including but not limited
to, for example, peroxidases, superoxide dismutases, glutathiones,
catalases, oxidases and other protective enzymes and molecules as
might be found described in, for example, Iturbe-Ormaetxe, I. et.
al., Mol. Plant Microbe Interact. 2001; 14, 1189-1196 incorporated
in its entirety into the body of this invention.
[0015] As a further defense mechanism, the bacteria begin to
produce a protein known as `leghemoglobin` that behaves much like
human hemoglobin. The human body has four important globin
molecules (distinguished by the presence of a heme-based porphyrin
ring at the active site of the protein). These include myoglobin,
neuroglobin, hemoglobin and a recently discovered fourth globin
called histoglobin. Globin proteins are distributed throughout the
human body where they capture and control oxygen reactive molecules
such as O.sub.2, CO.sub.2, and NO (nitric oxide). In the
symbiosomes, oxygen will bind to the leghemoglobin and is removed,
through an oxygen transport mechanism, thus further improving the
anaerobic conditions the bacteria require for the nitrogen fixation
reaction to occur.
[0016] The interior of the nitrogen fixation nodule, therefore, is
a complex mixture of plant growth regulators, amino acids,
vitamins, proteins, polysaccharides, minerals and enzymes all which
are either helping the plant grow, protecting or nourishing the
bacteria. The composition of most of these root nodule extracts is
also regionally controlled due to the soil and climates in which
the individual plants are grown. However, regardless of the source
of the plant, the climate or other factors, active root nodules
will always contain leghemoglobin and nitrogenease.
[0017] In addition to binding oxygen, it has recently been
established that globin-type proteins, such as hemoglobin,
myoglobin and leghemoglobin, will bind nitric oxide (see for
example, Hargrove, M et al., J. Mol. Biol. 1997; 266: 1032-1042).
Nitric oxide has been found to be a potent controller of human
blood flow (see, for example, Stix, G. Scientific American 2001,
November). It has also become well established that an enzyme
called nitric oxide synthase controls the presence of nitric oxide
in the skin. Nitric oxide has been found to be part of the
biochemical cascade that occurs in skin when the skin becomes
irritated (see, for example, Bruch-Gerharz, D., et. al., J. Invest.
Dermatol. 1998; 110: 1-7). In particular, the presence of nitric
oxide free radicals in the skin has been demonstrated, along with
the free radicals derived from oxygen such as, for example,
hydrogen peroxide, to have detrimental effects on the health of the
skin (see, for example, Herrling, T. et. al., SOFW J. 2000; 126:
20-26). Accordingly, treatments to reduce nitric oxide levels on
the skin have been disclosed in the literature. Illustratively,
U.S. Pat. No. 6,160,021 discloses, at column 3, lines 28-45
thereof; a skin-treatment method for decreasing the level of nitric
oxide by administering to the skin, either topically or
subcutaneously, an inhibitor of NO synthase or an NO scavenger,
such as a heme compound, such as hemoglobin.
[0018] In addition, it has recently been recognized that nitric
oxide radicals appear to also be responsible for the initiation of
the cascade that causes melanogenesis in human melanocytes. It has
been suggested, for example, that human-derived hemoglobin can
control the production of tyrosinase in fibroblasts as suggest in,
for example, Romero-Graillet, C. et al., J Clin Invest., 1997; 99:
635-642, incorporated in its entirety in the body of this
invention. Therefore, the control and modulation of oxygen and
nitric oxide free radicals in the skin remains an area of
considerable commercial and academic interest and research.
[0019] Plants can be grown and removed from the soil to expose the
root nodules. Interestingly, the contents of the nitrogen fixation
nodules can be extracted using various methods of extraction well
known to those skilled in the art. Such examples may comprise
simple aqueous extractions as, for example, discussed on the Reed
College website under "Nitrogen Fixation. Part II. Physiology and
Anatomy of Nitrogen Fixation" found at
http://webseed.edu/academic/department/biology/nitrogen/Nfix2.html,
or they may be more complex extractions as described, for example,
in Mendonca, EHM. et. al., Phytochemistry 1999; 50: 313-316. In
addition, the extractions may involve the use of super critical
carbon dioxide extraction, methods of which are known to those
skilled in the art. These extractions will comprise a mixture of
the plant growth regulators, amino acids, vitamins, proteins,
polysaccharides, minerals and enzymes that comprise the root
nodules.
SUMMARY OF THE DISCLOSURE
[0020] In one aspect, the present invention relates to a personal
care composition comprising leghemoglobin and at least one
preservative selected from the group consisting of alcohols,
glycols, parabens, quaternary nitrogen-containing compounds,
isothiazolinones, aldehyde-releasing agents, antioxidants and
halogenated compounds, such preservatives being found listed, for
example, in Steinberg, D. C. Cosmet. Toilet. 1997; 112, 57-65, the
contents of which is incorporated in it entirety into the body of
this invention. Preferably, the leghemoglobin is a nitrogen
fixation root nodule extract providing a leghemoglobin
concentration in the composition of between 0.0001% and about 10%
based upon the total weight of the composition.
[0021] In another aspect, the present invention relates to a method
for preparing a personal care composition comprising the steps of:
[0022] (a) extracting the leghemoglobin from root nodules infected
with bacteria selected from the group consisting of Cyanobacteria,
Anaerobic bacteria, Purple sulfur bacteria, Purple non-sulfur
bacteria, Green sulfur bacteria, legumes bacteria, and combinations
thereof, in order to provide a leghemoglobin extract, and [0023]
(b) combining the leghemoglobin extract with a preservative to
provide the personal care composition.
[0024] In yet another aspect, the present invention relates to a
method for binding free radicals on the skin that comprises
contacting the skin with leghemoglobin, thereby causing said
leghemoglobin to act as a scavenger for the free radicals.
[0025] These and other aspects will become apparent upon reading
the following detailed description of the invention.
DETAILED DESCRIPTION
[0026] It has now been now surprisingly found that the extracts of
roots nodules from various plants, for example, perennial legumes
such as peas, beans, and soybeans, annual legumes such as clover
and alfalfa, and woody legumeous plants such as Australian Pine,
contain leghemoglobin, as well as many other useful components,
that provide advantages when incorporated into personal care
compositions. The other useful components of the extracts comprise,
for example, plant growth regulators, amino acids, vitamins,
proteins, polysaccharides, minerals and enzymes. Additionally,
these extracts can function as part of a growth media for other
organisms such as, for example, yeast such as Saccharomyces
cerevisiae, providing key growth factors to these organisms that
can then be further extracted for use in personal care
compositions. Moreover, one or more components of these extracts is
suitably encapsulated within, for example, liposomes, niaosomes,
sub-micron emulsions, polymeric encapsulants, gels, creams or
lotions, to provide a time-release property to the extract
components. Moreover, some of the extract components, notably
leghemoglobin, bind to oxygen and nitric oxide in the skin,
effectively controlling the presence of these molecules, and
minimizing the formation of free radicals.
[0027] Without wishing to be bound by any particular theory, the
present inventor postulates that the presence of leghemoglobin,
topically applied to the skin, will bind to oxygen and nitric oxide
present in the skin, effectively removing these molecules from
their normal radical breakdown cycles and thus, effectively
eliminating the detrimental effects of their free radicals on the
skin. Without being further bound by theory, the inventor suggests
that leghemoglobin can remove nitric oxide from binding with other
critical heme-based enzymes such as, for example, guanylyl cyclase,
the enzyme responsible for upregulating the production of
cyclic-GMP, in a competitive fashion effectively slowing
significant biochemical pathways in human cells dependent on the
upregulation of c-GMP.
[0028] The nitrogen fixation root nodule extracts of the present
invention can be obtained from any number of microorganisms as
summarized, for example, in a table provided on The University of
Edinburgh's website under the heading "The Microbial World. The
Nitrogen Cycle and Nitrogen Fixation" found on their website
located at
[0029] http://helios.bto.ed.ac.uk/bto/microbes/nitrogen.htm, and
incorporated herein by reference in its entirety. Illustrative
origins include, for example, aerobic bacteria such as those from
the Genus' Azotobacter, Beijerinckia, Klebsiella and other
Cyanobacteria, anaerobic bacteria such as, for example, organisms
from the Genus' Clostridium, Desulfovibrio, Purple sulfur bacteria,
Purple non-sulfur bacteria and Green sulfur bacteria, bacteria that
inhabit legumes such as, for example, bacteria from the Genus
Rhizobium and Brachyorhizobium and bacteria that infect other types
of plants such as, for example, bacteria from the Genus Frankia and
Azospirillum. Especially preferred are the nitrogen fixation root
nodule extracts from the Rhizobium and Brachyorhizomium bacteria
that infect annual and perennial legumes, in particular soybean
root nodule extracts.
[0030] Plants from which the root nodules might be taken can
include a variety of species found around the planet. Such plant
species can include, but are not limited to, those listed in the
article by Lindermann, W. C. and Glover, C. R., entitled "Nitrogen
Fixation by Legumes" found at
http://iubio.bio.indiana.edu/R35541-209718-/news/bionet/biology/n2-fixati-
on/9702.newsm and included in its entirety in the body of this
invention. Such plants might include, for example, perennial
legumes such as alfalfa, sweetclover, vetches and true clover or
annual legumes such as, for example, beans, faba beans, peas,
cowpeas, peanuts and soybeans. Certain legumeous woody plants are
known to carry nitrogen fixation bacterial infections such as, but
not limited to, Australian pine. Especially preferred are the root
nodules found on soybeans.
[0031] The personal care composition of the present invention
suitably contains leghemoglobin and at least one preservative
selected from the group consisting of alcohols, glycols, parabens,
quaternary nitrogen-containing compounds, isothiazolinones,
aldehyde-releasing agents, antioxidants and halogenated compounds.
Illustrative alcohols include phenoxyethanol, isopropyl alcohol,
and benzyl alcohol; illustrative glycols include propylene,
butylene, and pentylene glycol; illustrative parabens (also known
as parahydroxybenzoic acids) include methyl, propyl and
butyl-parabens; illustrative quaternary nitrogen-containing
compounds include, benzalkonium chloride, and Quaternium 15;
illustrative isothiazolinones include methylisothiazolinone and
methylchloroisothiazolinone; illustrative aldehyde-releasing agents
include DMDM hydantoin, imidazolidinyl urea and diazolidinyl urea;
illustrative antioxidants include butylated hydroxytoluen and
tocopherol, and illustrative halogenated compounds include
triclosan, and chlorohexidine digluconate.
[0032] Additionally, the personal care composition can optionally
contain other functional ingredients such as, for example, water,
surfactants, emulsifiers, conditioners, emollients, waxes, oils,
polymers, thickeners, fixatives, colorants, humectants,
moisturizers, stabilizers, diluents, solvents, fragrances and the
like, as well as active ingredients such as, for example,
botanicals, neutraceuticals, cosmeceuticals, therapeutics,
pharmaceutics, antifungals, antimicrobials, steroidal hormones,
antidandruff agents, anti-acne components, sunscreens,
preservatives and the like. The concentration of the root extract
component or components in the cosmetic composition is suitably in
the range of 0.0001% to 99% by weight, preferably between 0.1% and
10%, based upon the total weight of the cosmetic composition. The
composition of the present invention can be used in various types
of cosmetic formulations including, but not limited to, lotions,
ointments, creams, sprays, spritzes, aqueous or aqueous alcoholic
mixtures, gels, mousses, patches, pads, masks, moistened cloths and
wipes, solid sticks, clear sticks, lipsticks, aerosol creams,
anhydrous powders, talcs, tonics, oils, emulsions, and bath
salts.
[0033] The root nodule extracts can be obtained from the roots by a
variety of extraction methods known to those skilled in the art.
Such extracts may involve the use of water, water miscible organic
solvents such, for example, acetone, butylenes glcyol or ethanol,
or organic solvents such as, but not limited to, hexane or methyl
ethyl ketone. The extraction may also involve the use of
supercritical fluid extraction, such as, but not limited to, super
critical carbon dioxide extraction. The root nodules may be found
to occur in a moistened state or a dried state, such conditions
influencing the final composition of the extract, but not the
method of extraction. The root nodule extraction may be done alone,
or in the presence of other plant components such as leaves, stems,
flowers, seeds and roots. The presence of these components can
modify the resulting extraction composition. The extraction can be
conducted at normal atmospheric pressure or under greater or lesser
pressures depending on the method of extraction. The extraction can
be conducted at temperatures between -30 .degree. C. and 200
.degree. C., more preferably between 5 .degree. C. and 100 .degree.
C., most preferably between 25 .degree. C. and 45 .degree. C., such
extraction solvents, pressures and temperatures being suitably
selected so as to not denature the leghemoglobin proteins found in
the root nodule extracts.
[0034] The extracts of the present invention may be further
purified by any number of means known to those skilled in the art
including, but not limited to, chromatography, steam distillation,
solvent extraction, centrifugation, decantation, filtration, or
carbon treatment. The extracts of the present invention may be
further concentrated by any means known to those skilled in the art
including, but not limited to, evaporation, spray-drying,
lyophylization, steam distillation or belt or drum drying.
[0035] The following Examples are intended to illustrate the art of
the present invention and are not intended to limit the scope of
the claims below.
EXAMPLE 1
[0036] Two kilograms of freshly pulled soybean (Glycine max) plants
including the roots were washed with a gentle stream of deionized
water to remove residual soil. The nitrogen fixation nodules, which
were readily apparent on well-infected roots, were easily removed
by hand and were collected. To approximately 100 grams of isolated
nodules was added 200 mls of deionized water and the entire mixture
was macerated in a Waring blender at high shear. The resulting dark
heterogeneous lysate was filtered through cheesecloth to remove
large pieces of undissolved material. The resulting hazy, dark
solution was further purified by filtration through fine filter
paper and by carbon and sterile filtration that removed the
organisms responsible for a strong earthy odor in the extract.
[0037] The level of leghemoglobin in this particular extract was
found to be approximately 0.3 mg/ml as determined by the following
analysis method. To a 10 ml sample of the extract was added 1 ml of
0.1M sodium dithionite in 0.1 M Potassium Phosphate Buffer Solution
(PBS) at pH 7.0. This sample was treated with gaseous carbon
monoxide (CO) by gently bubbling a stream of the gas into the
sample for 20 minutes. This procedure permanently binds carbon
monoxide to the heme iron of the leghemoglobin providing a
leghemoglobin-CO complex (referred to as Lba-CO) that has an
extinction coefficient of 200 mM.sup.-1cm.sup.-1 at 416 nm
wavelength in the ultraviolet spectrum. Using 0.4 ml of the Lba-CO
complex solution in a 1-cm pathlength UV cuvette and comparing it
an absorbance blank of PBS at pH 7.0 one determines the
concentration of leghemoglobin using the resulting absorbance and
the known extinction coefficient for the Lba-CO complex.
EXAMPLE 2
[0038] Two kilograms of roots containing active nitrogen fixation
nodules were obtained from Lotus plant (Lotus japonicus) grown
hydroponically in vermiculite inoculated with bacteria from the
Genus Brachyorhizobium. Approximately 100 grams of the nitrogen
fixation nodules were cleaned and removed and the resulting nodules
were extracted by macerating the nodules in a Waring blender with
200 mls of 80% aqueous ethanol. The resulting heterogeneous mixture
was filtered as described above. The ethanol was removed via low
tempertuare vacuum evaporation and replaced with a comparable
amount of deionized water providing a root nodule extract
containing approximately 0.6 mg/ml of leghemoglobin as analyzed
using the method described in Example 1.
EXAMPLE 3
[0039] Samples of root nodules obtained from Alfalfa (Medicago
sativa) were treated in a similar fashion as described above in
Example 1. HPLC analysis of these extracts for the presence of the
cytokinins zeatin (a pure sample of zeatin was obtained from Serva
(Islandia, N.Y.) provided concentrations comparable to those
reported in the literature, in particular, Hashizume, T. et al.,
Agric. Biol. Chem 1985; 49: 3481-3484. The extract was analyzed
using the method described in Example 1 and was found to comprise
approximately 0.5 mg/ml of leghemoglobin.
EXAMPLE 4
[0040] An aqueous extract was prepared from root nodules harvested
from the plant Lupine (Lupinus) using the method described in
Example 1. This extract was found to contain 0.4 mg/ml of
leghemoglobin.
EXAMPLE 5
[0041] Samples of the various aqueous root nodule extracts were
incorporated into a liposome comprising phospholipid and lecithin
obtained from soybean beans. The extract was slurried together with
the phospholipid and lecithin components and the mixture was
homogenized using a high-pressure homogenizer obtained from
Hydraulic Engineering Corporation (Brea, Calif.). The milky white
mixture contained the nitrogen fixation root nodule extract
encapsulated with the liposomal components.
EXAMPLE 6
[0042] A 1:1 mixture of the root nodule extract from Example 1 and
a mixture of maltodextrin encapsulating oligosaccharides available
as a 67% mixture of maltodextrin sugars and water from Roquette
America (Keokuc, Iowa) was spray-dried using a commercial spray
dryer. The resulting encapsulated powder contained approximately
0.15 mg/ml of leghemoglobin as determined by taking a sample and
redissolving it in water and analyzing the mixture for
leghemoglobin using the method described in Example 1.
EXAMPLE 7
[0043] A sample of the aqueous soybean root nodule extract from
Example 1 was placed into an aqueous mixture of Baker's Yeast
growth media obtained from Red Star Yeast (Milwaukee, Wis.). The
media was inoculated with an active yeast culture also obtained
from Red Star and the mixture was allowed to ferment under
controlled aerobic conditions to provide a Live Yeast Cell
Derivative (LYCD) obtained using stress conditions as described in
U.S. Pat. No. 2,239,345. The resulting yeast lysate continued to
test positive for the presence of leghemoglobin as demonstrated by
using the method referenced in Example 1.
[0044] Examples 8-11 illustrate skin care compositions according to
the present invention that can be prepared using various root
nodule extracts as disclosed in examples 1-4.
EXAMPLE 8
[0045] This example illustrates a high internal phase water-in-oil
emulsion incorporating the root nodule extract prepared as
disclosed in Example 1.
TABLE-US-00001 Ingredient wt % 1,3-dimethyl-2-imidazolidinone 0.2
Brij 92.sup.1 5.0 Bentone 38 0.5 MgSO.sub.4.cndot.7H.sub.2O 0.3
DMDM Hydantoin 0.01 Root Nodule Extract 10.0 Water to 100
.sup.1Brij 92 is polyoxyethylene (2) oleyl ether
EXAMPLE 9
[0046] This example illustrates an oil-in-water cream incorporating
the root nodule extract prepared as disclosed in Example 4.
TABLE-US-00002 Ingredient wt % Mineral Oil 4
1,3-dimethyl-2-imidazolidinone 1 Brij 56.sup.1 4 Alfol 16RD.sup.2 4
Triethanolamine 0.75 Butane-1,3-diol 3 Xanthan gum 0.3 Methyl,
Propyl and Butyl Paraben 0.01 Root Nodule Extract 10.0 Water to 100
.sup.1Brij 56 is cetyl alcohol POE (10) .sup.2Alfol 16RD is cetyl
alcohol
[0047] EXAMPLE 10
[0048] This example illustrates an alcoholic lotion incorporating a
root nodule extract prepared as disclosed in Example 2.
TABLE-US-00003 Ingredient wt % 1,3-dimethyl-2-imidazolidinone 0.3
Ethanol 40 Root Nodule Extract 10.0 Water to 100
EXAMPLE 11
[0049] This example illustrates a sub-micron emulsion concentrate
that contains a root nodule extract prepared as described in
Example 3.
TABLE-US-00004 Ingredient wt % Trimethylolpropane
Tricaprylate/Tricaprate 18.0 Glycerin 8.0 Cetearyl alcohol 2.0
Ceteareth 20 2.0 Glyceryl stearate 2.0 BHT 0.01 Root Nodule Extract
10.0 Water to 100
EXAMPLE 12
[0050] To test the ability of a root nodule extract containing
leghemoglobin to control nitric oxide levels in human fibroblasts,
the following assay was preformed.
[0051] Preparation of Fibroblasts
[0052] Fibroblasts were seeded into the individual wells of a 6
well plate with an initial cell density of 3,500 cells/cm.sup.2 in
a total of 2 ml of fibroblast growth medium (FGM). The seeded cells
were incubated overnight in an incubator at 37.+-.2.degree. C. and
5.+-.1% CO.sub.2. On the following day the FGM was removed via
aspiration to eliminate any non-adherent cells and replaced with 2
ml of fresh FGM. The media was changed again every 48 hours of
incubation until the cells were fully confluent.
[0053] Application of Test Material
[0054] The media from the confluent fibroblasts was replaced with
the extract described in Example 1, diluted in FGM to yield a final
concentration of 1%. Gamma-interferon was used as a positive
control (approx 300 U/ml) while untreated cells were used as a
negative control. Each condition was tested with 6 replicates. The
cells were incubated for 48 hours at 37.+-.2.degree. C. and 5.+-.1%
CO.sub.2. After the 48-hour incubation the cell culture medium was
collected and stored frozen for subsequent assay of nitrite.
[0055] Enzymatic Conversion of Nitrate to Nitrite
[0056] A 300 .mu.l aliquot of each cell culture medium sample was
mixed with 10 .mu.l of a nitrate reductase solution (0.1 U/ml
nitrate reductase enzyme, 5 .mu.M FAD, 30 .mu.M NADPH). This
mixture was incubated in a water bath (37.+-.2.degree. C.) for at
least 15 minutes. To remove any residual NADPH that was not
consumed in this initial reaction (the NADPH will interfere with
the subsequent Griess Reaction), 10 .mu.l of a lactate
dehydrogenase solution (100 U/ml lactate dehydrogenase and 0.3 mM
sodium pyruvate) was added and this mixture was allowed to incubate
in a water bath (37.+-.2.degree. C.) for at least 5 minutes.
[0057] Nitrite Assay: Griess Reaction
[0058] A 100 .mu.l aliquot of each cell culture medium sample was
added to a well in a 96-well plate. To each aliquot, 50 .mu.of
sulfanilamide solution (1% sulfanilamide in 5% phosphoric acid) was
added and the plate was incubated at room temperature for 5 to 10
minutes (protected from light). After this first incubation, 50
.mu.l of NED solution (0.1% N-1-napthylethylenediamine
dihydrochloride in DIH.sub.2O) was added to each well and the plate
was allowed to incubate at room temperature for 5 to 10 minutes
(protected from light). After incubating, the absorbance values for
each well of the 96-well plate were read at 540 nm.
[0059] Nitrite Standard Curve
[0060] A nitrite standard curve was generated by performing a
series of dilutions with the nitrite standard to generate the
following series of concentrations: 100 .mu.M, 50 .mu.M, 25.mu.M,
12.5 .mu.M, 6.25 .mu.M, 3.13 .mu.M, 1.56 .mu.M, and 0. These
samples were diluted in cell culture medium.
[0061] Nitrite Assay
[0062] To derive the standard curve for the nitrite assay, the
absorbance versus the nitrite concentration in .mu.M for the
standards was plotted. A linear regression was performed to
establish the line that best fits these data points. Mean
absorbance values for the test materials and untreated samples were
used to estimate the amount of nitrite present in each sample.
Using the analysis method it was determined that the extract from
the soybean root nodules was able to reduce nitric oxide levels in
human fibroblasts by 60% compared to untreated controls.
EXAMPLE 13
[0063] To test the ability of a root nodule extract to improve
human skin fibroblast viability, a sample of the root nodule
extract from Example 4 was tested using the following analytical
procedure.
[0064] Human fibroblasts were grown to confluence using the method
described in Example 12. The fibroblasts were treated with test
material following the procedure described in Example 12. After 48
hours of exposure to the test material, the following test was
run.
[0065] MTT Assay
[0066] The cell culture medium was removed and the fibroblasts were
washed twice with PBS to remove any remaining test material. After
the fmal wash, FGM supplemented with 2 mg/ml MTT was added to each
well and the cells were incubated for approximately 2 hours at
approx. 37.+-.2.degree. C. and 5.+-.1% CO.sub.2. After the 2-hour
incubation, the FGM/MTT solution was removed and the cells were
washed again once with PBS and then 2 ml of isopropyl alcohol was
added to the well to extract the purple formazin crystals. Two
hundred microliters of the isopropyl extracts was transferred to a
96-well plate and the plate was read at 540 nm using isopropyl
alcohol as a blank.
[0067] The Mean MTT absorbance values were calculated for the
negative control wells. Since these cells were not treated this
mean value was used to represent 100% viability (UNTREATED). For
the absorbance values in the wells treated with the test material
(TREATED samples), the percentage of viable cells was calculated
using this equation: [(individual TREATED absorbance)/(mean
UNTREATED absorbance)].times.100. Using this analytical method, it
was determined that the cells treated with the extract from Example
4 had a 30% greater viability than untreated cells showing the
extract improved the cell viability of the human skin
fibroblasts.
EXAMPLE 14
[0068] To test the possibility that a root nodule extract might
promote the growth of soluble human elastin, the following study
was conducted. A soy root nodule extract was prepared as described
in Example 1. The extract and an isotonic saline placebo were
buffered to neutral pH with PBS.
[0069] Neonatal human fibroblast cells were placed in fibroblast
growth medium (FGM) as described above in Example 12 and seeded
into culture flasks. The flasks were incubated at 37.degree. C. and
5% CO.sub.2 until the cells were fully confluent. The media was
changed 24 hours after the initial seeding of the flask to remove
any cells that did not survive and then every 48 hours after that.
Upon reaching confluency the FGM was removed and the cells were
washed once with 9 mis of HEPES Buffered Saline Solution
(HEPES-BSS) to remove any residual proteins from the FGM that can
neutralize trypsin. Following removal of the HEPES-BSS, 6 ml of
Trypsin/EDTA was used to cover the fibroblast monolayer. The
trypsin digests the cellular adhesion proteins that hold the
fibroblasts to the culture flask while the EDTA chelates any
residual calcium to further inactivate the adhesion proteins.
Trypsinization did not exceed 3 minutes. After gathering of the
cells, 12 ml of trypsin neutralizing solution was added and the
released cells were transferred to 15-ml centrifugation tubes. The
flask was rinsed with 6 mls of HEPES-BSS to collect residual cells
that were added to the previous wash. The centrifuge tubes were
then spun at 220.times.g for 5 minutes to pellet the cells. The
supernatant was removed via aspiration and the cells was
resuspended in 4 ml of FGM and seeded into 6-well plates. A small
aliquot was taken to count the number of cells. The cells were then
diluted with FGM to reach a cell density of 17,500 cells/ml. The
seeded cells were incubated overnight as described above. On the
following day, the FGM was removed via aspiration to eliminate any
non-adherent cells and replaced with 2 mls of fresh FGM. The media
was changed again every 48 hours of incubation until the cells are
fully confluent.
[0070] The media of the confluent fibroblasts was replaced with
either fresh media or media containing the root nodule extract from
Example 1. The cells are then incubated for 48 hours at the same
conditions described above.
[0071] After the 48-hour incubation period the cell culture medium
was collected and the insoluble elastin was extracted from the
cells by the addition of 1 ml of 0.25 M oxalic acid to the wells
that house the fibroblasts. The cells were freed from the plastic
wells using a cell scraper and the suspended cells/oxalic acid
mixture was added to centrifuge tubes. The tubes were incubated at
95.degree. C. for 60 minutes to solubilize the cells. The tubes
were then quickly cooled to RT and centrifuged at 3000 rpm for 10
minutes. The supernatants were combined so that both soluble and
insoluble elastins were tested. The supernatant was placed
overnight in dialysis tubing with a low molecular weight cut-off
(.about.15,000 MW) to both concentrate the elastin into a smaller
volume and to remove the oxalic acid.
[0072] A 300 ml aliquot of the sample above was mixed with 1.0 ml
of cold Fastin Precipitating Reagent (available from Biocolor,
Belfast, Northern Ireland) in a 1.5 ml microcentrifuge tube. The
mixture was allowed to incubate for 24 hours at 4.degree. C. to
precipitate elastin. The cooled microcentrifuge tubes were spun at
8,000.times.g for 10 minutes to pack the precipitated elastin. The
supernatant was removed by inverting the tube to drain it and then
by gently tapping it on an absorbent paper towel.
[0073] One (1) ml of Fastin Dye Reagent along with 200 .mu.l of 90%
saturated ammonium sulphate was added to each tube. The
microcentrifuge tubes were then mixed with a vortex mixer and
allowed to incubate for 60 minutes at RT with gentle mechanical
agitation. The Fastin Dye Reagent interacts with the amino acids of
elastin specifically to form a colored elastin/dye complex that
precipitates in the presence of the ammonium sulphate. The
microcentrifuge tubes were spun at 8,000.times.g for 10 minutes to
separate the precipitated elastin/dye complex from the unbound dye.
The supernatant was carefully removed and the tubes were inverted
to drain any residual supernatant.
[0074] One (1) ml of Fastin Destain reagent was added to each
microcentrifuge tube. The tubes were capped and vortexed as
described previously. A 100 ml aliquot from each sample tube was
transferred to a 96-well plate and read at 513 nm with a microplate
reader. The amount of soluble and insoluble elastin was determined
by comparison against a standardization curve prepared from elastin
samples supplied by the manufacturer of the Fastin Dye Kit. Using
the described methodology, it was found that the cells grown in the
presence of the root nodule extract contained statistically greater
quantities of elastin than the cells grown in the untreated
controls. Fibroblast cell viability was determined as described in
Example 13 and the cells were shown to be viable under the
conditions of the experiment.
[0075] EXAMPLE 15
[0076] To test the possibility that an extract from a root nodule
containing leghemoglobin can control the production of cyclic-GMP
in human skin fibroblasts, the following test was run using the
extract produced in Example 2.
[0077] cGMP Assay: Fibroblast Cell Culture Preparation and
Treatment
[0078] Human dermal fibroblasts were seeded at 7.5.times.10.sup.4
cells/ml in 1 ml of cell culture medium into the wells of a 12-well
tissue culture plate (three wells were prepared for each
treatment). After seeding, the cells were incubated overnight at
37.+-.2.degree. C. and 5.+-.1% CO.sub.2. On the next day, the
culture medium was removed via aspiration and the cells were
incubated for 10 minutes in 400 .mu.l of prestimulation buffer
(Stock 800 mM IBMX: 100 mg IBMX dissolved in 563 .mu.l DMSO;
prestimulation buffer: 9.4 .mu.l 800 mM IBMX added to 10 ml
phosphate buffered saline). After this pretreatment, a 200 .mu.l
volume of phosphate buffered saline (negative control), test
material+spermine NONOate (300 mM), or spermine NONOate (300 mM,
positive control) were added to each well. It should be noted that
there is a dilution at this point so materials to be added were
prepared at 3.times. their intended fmal concentration (fmal
concentration of spermine NONOate was 100 mM). The cells were then
incubated for 1.5 hours at 37.+-.2.degree. C. and 5.+-.1% CO.sub.2.
At the end of this treatment period 200 .mu.l of lysis buffer was
added to each well and 12-well plate was placed on a rocking
platform for 10 minutes at room temperature. The cell lysate was
then collected and assayed immediately for cGMP. In addition to the
fibroblasts, blank wells were run (wells without cells in them)
containing known amounts of cGMP. These samples were treated and
collected as described above and used to generate a standard curve
for the cGMP assay.
[0079] Catchpoint cGMP Assay
[0080] The catchpoint cGMP assay is a competitive fluorescence
based ELISA assay. In this type of assay, as the amount of cGMP in
a sample increases, the fluorescent signal will decrease. The assay
procedure is as follows:
[0081] 1. Fifty (50) .mu.l of each sample to be assayed were added
to respective wells in a clear bottom black 96-well plate (these
samples include the cGMP samples to be used for a standard curve).
The wells in the well plate were previously coated with an
anti-rabbit IgG.
[0082] 2. Fifty (50) .mu.l of a rabbit anti-cGMP antibody were then
added to the wells used above.
[0083] 3. Fifty (50) .mu.l of a cGMP-horse radish peroxidase (HRP)
conjugate were then added to each well used above and the well
plate was incubated for 2 hours at room temperature on a rocking
platform.
[0084] 4. After the incubation period, the well plate was washed
four times with 300 .mu.l of wash buffer (supplied with the kit:
0.02 M Tris [pH 7.4], 150 mM CaCl, 0.05% Tween 20, and 0.05%
Proclin).
[0085] 5. After removing the final wash via aspiration, 100 .mu.l
of Stoplight Red fluorescent substrate solution (with 1 mM H.sub.2
O.sub.2) was added to each well used in the well plate and the
plate was incubated for at least 1 hour (10 minutes is the minimum
incubation time, but the signal is stable for up to 24 hours).
During this incubation period the 96-well plate was protected from
light.
[0086] 6. After the incubation period, the 96-well plate was read
using a Fluoroskan Ascent F/L plate reader at these settings:
[0087] a. Excitation filter: 530 nm [0088] b. Emission filter: 590
nm [0089] c. Integration time: 50 msec
[0090] CGMP Assay
[0091] To quantify the amount of cGMP present, a standard curve was
generated using known concentrations cGMP. A regression analysis
was then performed to establish the line that best fits these data
points. Cyclic CMP content in the samples treated via the various
conditions was then estimated via this standard curve. Using this
analysis method, it was found that fibroblasts treated with 100 mM
of Spermine NONOate showed an increased production of c-GMP of
almost 80%. Treatment of the same fibroblasts with 100 mM of
Sperimine NONOate containing 10% of the root nodule extract from
Example 2 reduced the levels of c-GMP back down to levels noted for
untreated cells showing the extract has an ability to control c-GMP
levels in human fibroblasts.
EXAMPLE 14
[0092] To test the possibility that an extract from a root nodule
containing leghemoglobin could influence melanogenesis (tanning) in
human skin, the following in vitro test was performed using the
extract taken from Example 2.
[0093] Tissue Preparation
[0094] Upon arrival, the MatTek MelanoDerm tissues were stored at
4.degree. C. until used. Prior to use, the tissues to be used were
removed from the agarose-shipping tray and placed into a 6-well
plate containing 0.9 ml of assay medium (37.+-.2.degree. C.). All
of the agarose was removed from the outside of the tissue culture
insert since any residual agarose may prevent the assay medium from
reaching the tissue. The tissues were allowed to incubate for at
least 1 hour at 37.+-.2.degree. C. and 5.+-.1% CO.sub.2. After this
initial incubation, the assay medium was replaced with 5.0 ml of
fresh medium (37.+-.2.degree. C.) and the tissues inserts were
placed on 2 sterile stainless steel washers to raise the level of
the bottom of the insert such that it touched the surface of the
assay medium.
[0095] Application of Test Material
[0096] Twenty-five (25) .mu.l or mg of test material was applied
directly onto the surface of the tissue. In addition to the test
material, a positive (10.sup.-7M alpha melanocyte stimulating
hormone and 3 ng/ml bFGF in the assay medium) and negative (no
treatment) control was also applied to respective tissues. The
6-well plates were then incubated at 37.+-.2.degree. C. and 5.+-.1%
CO.sub.2 and identical sets of plates were incubated for 10 days
and 17 days. During this incubation period, the tissues were rinsed
and new test material was applied and the assay medium changed
every other day.
[0097] Macroscopic Observation
[0098] At the end of the incubation period, the assay medium in
some of the 6-well plates was replaced with 0.9 ml of PBS and the
stainless steel washers were removed. The 6-well plates containing
the tissue were placed under an inverted microscope and the tissues
were evaluated to determine if the test material had exerted any
cytotoxic effects on the melanocytes. Toxic effects would be
indicated by melanocytes that had a rounded up and/or non-dendritic
morphology. After the macroscopic evaluation, the Melanoderm
tissues were removed from the culture inserts and stored at
-75.+-.5.degree. C. until assayed for melanin content.
[0099] Melanin Extraction From Tissues
[0100] Two frozen tissues for each treatment and time point were
pooled and homogenized in 0.45 ml of 1% SDS containing 0.05 mM EDTA
and 10 mM Tris (pH 6.8). Twenty (20) .mu.l of a 5 mg/ml Proteinase
K solution was then added to each homogenate and the mixture was
allowed to digest overnight in a water bath set at 45.+-.2.degree.
C. After the overnight digestion, an additional 20 .mu.l of 5 mg/ml
Proteinase K was added to each homogenate and the mixture was
allowed to digest for an additional 4 hours (or longer-until no
clumps of tissue remain). After the digestion, 50 .mu.l of 500 mM
sodium carbonate was added to make the digest basic, followed by
the addition of 10 .mu.l of 30% hydrogen peroxide. The samples were
then incubated at 80.+-.2.degree. C. for 30.+-.5 minutes and then
cooled to room temperature. After cooling, 100 .mu.l of a
chloroform/methanol (2:1) mixture was added to each sample. The
sample was then centrifuged for 10 minutes at 10,000.times.g). The
top phase of each sample was collected and its absorbance at 405 nm
was determined via a 96-well plate reader.
[0101] Melanin Standard
[0102] Known concentrations of synthetic melanin were taken through
the extraction process described above. The absorbance values of
these known concentrations were used to generate a standard curve
of melanin concentrations.
[0103] The absorbance values for the melanin standards were plotted
against their respective concentrations to generate a standard
curve. The equation that best fits this curve was then determined
via regression analysis and this equation was used to calculate the
melanin concentrations of the unknown samples. The tissue melanin
concentrations were then compared using a two-factor analysis of
variance (using treatment and time point as the two factors). It
was found that the tissue sample treated with the extract taken
from Example 2 showed a statistically significant reduction in
melanin concentration verses an untreated control.
[0104] While the invention has been described above with reference
to specific embodiments thereof, it is apparent that many changes,
modifications, and variations can be made without departing from
the inventive concept disclosed herein. Accordingly, it is intended
to embrace all such changes, modifications and variations that fall
within the spirit and broad scope of the appended claims. All
patent applications, patents and other publications cited herein
are incorporated by reference in their entirety.
* * * * *
References