U.S. patent application number 17/734548 was filed with the patent office on 2022-08-11 for cosmetic compositions and methods for their use in firming skin.
The applicant listed for this patent is MARY KAY INC.. Invention is credited to Tiffany CARLE, David GAN, Michelle HINES, Geetha KALAHASTI.
Application Number | 20220249359 17/734548 |
Document ID | / |
Family ID | |
Filed Date | 2022-08-11 |
United States Patent
Application |
20220249359 |
Kind Code |
A1 |
KALAHASTI; Geetha ; et
al. |
August 11, 2022 |
COSMETIC COMPOSITIONS AND METHODS FOR THEIR USE IN FIRMING SKIN
Abstract
A method of reducing the appearance of loose, sagging, or
flaccid skin is disclosed. The method can include topically
applying to skin in need thereof a composition that includes 0.0001
wt. % to 10 wt. % Argania spinosa kernel extract, 0.0001 wt. % to
10 wt. % dill extract, 0.0001 wt. % to 10 wt. % Myrciaria dubia
fruit extract, and 0.0001 wt. % to 10 wt. % Croton lechleri extract
or Morus alba fruit extract. Topical application of the composition
can increase elastin expression, increase elastin secretion, or
increase elastin fiber formation in the skin in need thereof, and
the appearance of loose, sagging, or flaccid skin can be
reduced.
Inventors: |
KALAHASTI; Geetha; (Plano,
TX) ; HINES; Michelle; (Hickory Creek, TX) ;
GAN; David; (Southlake, TX) ; CARLE; Tiffany;
(Dallas, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MARY KAY INC. |
Addison |
TX |
US |
|
|
Appl. No.: |
17/734548 |
Filed: |
May 2, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16007143 |
Jun 13, 2018 |
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17734548 |
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62518847 |
Jun 13, 2017 |
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International
Class: |
A61K 8/9789 20060101
A61K008/9789; A61Q 19/08 20060101 A61Q019/08 |
Claims
1. A method of reducing the appearance of loose, sagging, or
flaccid skin, the method comprising topically applying to skin in
need thereof a composition comprising 0.0001 wt. % to 10 wt. %
Argania spinosa kernel extract, 0.0001 wt. % to 10 wt. % dill
extract, 0.0001 wt. % to 10 wt. % Myrciaria dubia fruit extract,
and 0.0001 wt. % to 10 wt. % Croton lechleri extract or Morus alba
fruit extract, wherein topical application of the composition
increases elastin expression, increases elastin secretion, or
increases elastin fiber formation in the skin in need thereof, and
wherein the appearance of loose, sagging, or flaccid skin is
reduced.
2. The method of claim 1, wherein topical application of the
composition increases skin elasticity in the skin in need
thereof.
3. The method of claim 1, wherein topical application of the
composition increases elastin expression in the skin in need
thereof.
4. The method of claim 1, wherein topical application of the
composition increases elastin expression by at least 75% in the
skin in need thereof as compared to skin which has not been treated
with the composition.
5. The method of claim 1, wherein topical application of the
composition increases collagen expression in the skin in need
thereof.
6. The method of claim 1, wherein topical application of the
composition increases laminin or fibronectin production in the skin
in need thereof.
7. The method of claim 1, wherein topical application of the
composition increases fibulin-5 production in the skin in need
thereof.
8. The method of claim 1, wherein the skin in need thereof is skin
of a jowl.
9. The method of claim 1, wherein the skin in need thereof is neck
skin.
10. The method of claim 1, wherein the skin in need thereof is
facial skin.
11. The method of claim 1, wherein the composition is an
emulsion.
12. The method of claim 11, wherein the emulsion is an oil-in-water
emulsion.
13. The method of claim 1, wherein the composition is a gel.
14. The method of claim 1, wherein the composition is an aqueous
solution.
15. The method of claim 1, wherein the composition is a
hydro-alcoholic solution.
16. The method of claim 1, wherein each extract is an aqueous
extract, an alcoholic extract, or an aqueous-alcoholic extract.
17. The method of claim 1, wherein the composition is applied
daily.
18. The method of claim 1, wherein the composition comprises 0.0001
wt. % to 10 wt. % Croton lechleri extract.
19. The method of claim 1, wherein the composition comprises 0.0001
wt. % to 10 wt. % Morus alba fruit extract.
20. The method of claim 1, wherein the composition comprises 0.1
wt. % to 5 wt. % Argania spinosa kernel extract, 0.1 wt. % to 5 wt.
% dill extract, 0.1 wt. % to 5 wt. % Myrciaria dubia fruit extract,
and 0.1 wt. % to 5 wt. % Croton lechleri extract or Morus alba
fruit extract.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 16/007,143 filed Jun. 13, 2018, which claims priority to U.S.
Provisional Application No. 62/518,847 filed Jun. 13, 2017. The
contents of the referenced applications are incorporated into the
present application by reference.
BACKGROUND OF THE INVENTION
A. Field of the Invention
[0002] The present invention relates generally to cosmetic
compositions that can be used to improve the skin's visual
appearance by firming or tightening the skin. The composition can
include a combination of Argania spinosa kernel extract, dill
extract, Myrciaria dubia fruit extract, and Croton lechleri extract
and/or Morus alba fruit extract to achieve these effects. The
combination is chemically compatible and can be incorporated into a
wide-range of product formulations (e.g., masks, serums, creams,
cleansers, toners, gels, emulsions, gel emulsions, gel serums,
etc.).
B. Description of Related Art
[0003] Ageing, chronic exposure to adverse environmental factors,
malnutrition, fatigue, etc., can change the visual appearance,
physical properties, or physiological functions of skin in ways
that are considered visually undesirable. Some of the more notable
and obvious changes include increased sagging, loss of firmness,
and loss of elasticity. This can result in an "aged" appearance
where the person looks older than their age. Further, loose skin
can also result in the development of fine lines and wrinkles as
well as coarse surface texture. Less obvious but measurable changes
which occur as skin ages or endures chronic environmental insult
include a general reduction in cellular and tissue vitality,
reduction in cell replication rates, reduced cutaneous blood flow,
reduced moisture content, accumulated errors in structure and
function, alterations in the normal regulation of common
biochemical pathways, and a reduction in the skin's ability to
remodel and repair itself. Many of the alterations in appearance
and function of the skin are caused by changes in the outer
epidermal layer of the skin, while others are caused by changes in
the lower dermis.
[0004] One proposed solution to treating loose or sagging skin is
cosmetic/plastic surgery. This can involve making incisions in
facial or neck skin, pulling the skin tight, and then suturing the
incisions. Some drawbacks with such surgery are risk of infection
and the expenses associated with the surgery. Further, the skin can
have an unnatural waxy or plastic visual appearance that may not be
desirable.
[0005] Another solution involves the use of lasers, which can be
less invasive when compared to the aforementioned cosmetic/plastic
surgery option. This is oftentimes referred to as laser skin
tightening, which uses an infrared light source to tighten skin by
heating the collagen under the skin's surface. As the collagen is
heated, it can cause the skin to contract or tighten. A benefit of
this process is that you can see an immediate result. A downside is
that the skin tightening effect dissipates over time, which can
require multiple treatments per month. Further, laser treatments
are usually performed by a professional, which can require office
visits.
[0006] There have also been numerous attempts to tighten loose skin
through the use of topical skin compositions. Such compositions
typically include active ingredients that claim to help reduce the
appearance of sagging or loose skin. By way of example, hyaluronic
acid or salts thereof (e.g., sodium hyaluronate) are ingredients
that are designed to attract and retain water at the skin. In
particular, water can be used to fill up spaces between the
connective fibers collagen and elastin in the dermis layer of skin,
thereby making the skin appear to have a more tightened or
less-loose appearance. One of the downsides of hyaluronic acid is
that it may not have a lasting effect, thereby requiring large
amounts and multiple applications, which can result in increased
expenses and complications associated with its use. There have also
been attempts to use plant extracts and chemical compounds to
tighten skin. By way of example, US Publication 2008/0292651 claims
that a combination of Polygonum fagopyrum seed extract, Chlorella
vulgaris extract, palmitoyl wheat protein hydrolysate, algae
extract, and a tripeptide (e.g., glutathione
(2-amino-5-{[2-[(carboxymethyl)amino]-1-(mercaptomethyl)-2-oxoethyl]amino-
}-5-oxopentanoic acid or .gamma.-glutamylcysteinylglycine) can be
used to improve skin firmness, lift the skin, and prevent skin
sagging. The use of compounds such as chemically modified peptides
can be expensive and can result in skin irritation.
[0007] While there have been countless efforts to treat loose or
sagging skin, these efforts oftentimes require surgery, special
tools, or use ingredients that can be costly, ineffective over
longer periods of time, and/or cause skin irritation. The current
slate of options are failing to meet the needs of people having
loose or sagging skin.
SUMMARY OF THE INVENTION
[0008] The inventors have identified a solution to the problems
associated with loose/sagging/faccid skin. The solution is premised
on a combination of plant-based ingredients that includes Argania
spinosa kernel extract, dill extract, Myrciaria dubia fruit
extract, and Croton lechleri extract and/or Morus alba fruit
extract. This combination can be used to create topical skin
compositions that reduce the appearance of loose, sagging, and
flaccid skin, such as under the neck, and improve facial
contouring. Without wishing to be bound by theory, the efficacy of
this combination can be linked to its ability to modify specific
biochemical pathways in skin that can result in improved skin
firmness, skin elasticity, and/or facial contouring. In particular,
and as illustrated in a non-limiting manner in the Examples
section, this combination of plant-based ingredients can be used to
increase the expression of elastin, collagen, laminin, and/or
fibronectin in skin cells, which are proteins associated with the
structure of skin. By increasing the amount of these proteins in
the skin, it is believed that the skin will have a more firm,
taught, supple, and/or elastic appearance. Even further, it has
been discovered that the particular combination of Morus alba fruit
extract and Croton lechleri extract can increase the production of
fibulin-5 in skin. Fibulin-5 is an elastin-binding protein that can
have the ability to provide mechanical elasticity to skin tissue,
which further helps with improving skin tightness and/or
elasticity. Further, the combination of plant-based ingredients are
compatible with one another and can therefore exist in a single
topical skin care composition, which can be beneficial in that only
one composition needs to be applied to skin rather than multiple
compositions that oftentimes come in the form of a kit or regimen.
While kits and regimens are contemplated in the context of the
present invention, they are not required to obtain the beneficial
effects offered by the compositions of the present invention.
[0009] In some aspects, there is disclosed a topical composition.
In some aspects, the topical composition includes any one of, any
combination of, or all of Argania spinosa kernel extract, dill
extract, Myrciaria dubia fruit extract, Croton lechleri extract,
and Morus alba fruit extract.
[0010] In some instances, the topical composition includes an
effective amount of Argania spinosa kernel extract, dill extract,
Myrciaria dubia fruit extract, Croton lechleri extract, and/or
Morus alba fruit extract to reduce the appearance of loose,
sagging, and flaccid skin, and improve facial contouring. In some
instances, the topical composition includes an effective amount of
Argania spinosa kernel extract, dill extract, Myrciaria dubia fruit
extract, and Croton lechleri extract to increase elasticity of
skin. In some instances, the topical composition increases elastin
synthesis. In some instances, the topical composition increases
collagen expression. In some instances, the topical composition
increases production of laminin. In some instances, the topical
composition increases production of fibronectin. In some instances,
the topical composition increases expression of proteins that are
involved in elastin organization in fibroblasts (e.g., fibulin-5).
In some instances, the Argania spinosa kernel extract and/or dill
extract is a water extract. In some instances, the Croton lechleri
extract and/or Morus alba fruit extract is a water and glycerol
extract. In some instances, the Myrciaria dubia fruit extract is a
dry fruit powder.
[0011] The topical compositions disclosed herein may further
comprise one or more ingredients described herein. For example, the
composition may comprise one or more additional ingredients
selected from one or more conditioning agents, moisturizing agents,
pH adjusters, structuring agents, inorganic salts, and
preservatives. In some instances, the topical composition further
includes water. The amounts of the ingredients within the
composition can vary (e.g., amounts can be as low as 0.000001% to
as high as 98% w/w or any range therein). In some instances, the
topical composition is an emulsion, serum, gel, gel emulsion, or
gel serum. In some instances, the topical composition can further
contain one or more, or all of, water, disodium EDTA,
HDI/trimethylol hexyllactone crosspolymer, nylon-12,
acrylates/C10-30 alkyl crosspolymer, glyceryl stearate, PEG-100
stearate, ethylhexyl palmitate, pentylene glycol, dimethyl
isosorbide, triethanolamine, iodopropynyl butylcarbamate,
dimethicone, dimethicone crosspolymer, phenoxyethanol, decylene
glycol, 1,2-hexanediol, glycerin, and sodium polyacrylate. In some
instances, the topical composition contains 40 to 80% w/w of water,
0.001 to 1% w/w of disodium EDTA, 0.1 to 5% w/w of HDI/trimethylol
hexyllactone crosspolymer, 0.01 to 3% w/w of nylon-12, 0.001 to 1%
w/w of acrylates/C10-30 alkyl crosspolymer, 1 to 10% w/w of a
combination of glyceryl stearate and PEG-100 stearate, 1 to 15% w/w
of ethylhexyl palmitate, 1 to 5% w/w of pentylene glycol, 0.01 to
3% w/w of dimethyl isosorbide, 0.01 to 3% w/w of triethanolamine,
0.001 to 1% w/w of iodopropynyl butylcarbamate, 5 to 20% w/w of a
combination of dimethicone and dimethicone crosspolymer, 0.01 to 3%
w/w of a combination of phenoxyethanol, decylene glycol, and
1,2-hexanediol, and 5 to 20% w/w of a combination of glycerin,
water, and sodium polyacrylate.
[0012] Methods of use for the compositions disclosed herein are
also disclosed. In some aspects, a method is disclosed of improving
a condition or appearance of skin, comprising applying any one of
the compositions disclosed herein to skin in need thereof. In one
aspect, any one of the compositions disclosed herein are applied to
skin and the composition is left on the skin, or alternatively
removed from the skin after a period of time. In another aspect,
the compositions disclosed herein are used to treat and/or reduce
the appearance of loose, sagging, and flaccid skin. In another
aspect, the compositions disclosed here are used to improve facial
contouring. In another aspect, a method for increasing skin
elasticity is disclosed herein. In another aspect, a method for
increasing elastin synthesis is disclosed herein. In another
aspect, a method for increasing expression of proteins that are
involved in elastin organization is disclosed herein. In another
aspect, a method for increasing the production of fibulin-5 is
disclosed herein. In another aspect, a method for increasing the
production of fibronectin is disclosed herein. In another aspect, a
method for increasing the production of laminin is disclosed
herein. In another aspect, a method for increasing the expression
of collagen is disclosed herein. In some aspects, the methods
include applying any one of the topical compositions described
herein to skin. In some aspects, the methods include applying the
composition to skin of the neck. In some aspects, the methods
include applying the composition to skin of the face. In some
aspects, the methods include applying the composition to skin of a
jowl.
[0013] In particular aspects, the compositions of the present
invention are formulated as a topical skin composition. The
composition can have a dermatologically acceptable vehicle or
carrier for the compounds and extracts. The composition can further
include a moisturizing agent or a humectant, a surfactant, a
silicone containing compounds, a UV agent, an oil, and/or other
ingredients identified in this specification or those known in the
art. The composition can be a mask, lotion, cream, gel, serum,
emulsion (e.g., oil-in-water, water-in-oil, silicone-in-water,
water-in-silicone, water-in-oil-in-water, oil-in-water-in-oil,
oil-in-water-in-silicone, etc.), solutions (e.g., aqueous or
hydro-alcoholic solutions), anhydrous bases (e.g., lipstick or a
powder), ointments, milk, paste, aerosol, solid forms, eye jellies,
gel serums, gel emulsions, etc. The composition can be formulated
for topical skin application at least 1, 2, 3, 4, 5, 6, 7, or more
times a day during use. In other aspects of the present invention,
compositions can be storage stable or color stable, or both. It is
also contemplated that the viscosity of the composition can be
selected to achieve a desired result, e.g., depending on the type
of composition desired, the viscosity of such composition can be
from about 1 cps to well over 1 million cps or any range or integer
derivable therein (e.g., 2 cps, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,
40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,
900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000,
20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000,
200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000,
1000000, 2000000, 3000000, 4000000, 5000000, 10000000, cps, etc.,
as measured on a Brookfield Viscometer using a TC spindle at 2.5
rpm at 25.degree. C.).
[0014] The compositions in non-limiting aspects can have a pH of
about 6 to about 9. In other aspects, the pH can be 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, or 14. The compositions can include a
triglyceride. Non-limiting examples include small, medium, and
large chain triglycerides. In certain aspects, the triglyceride is
a medium chain triglyceride (e.g., caprylic capric triglyceride).
The compositions can also include preservatives. Non-limiting
examples of preservatives include methylparaben, propylparaben, or
a mixture of methylparaben and propylparaben. In some embodiments,
the composition is paraben-free.
[0015] Compositions of the present invention can have UVA and UVB
absorption properties. The compositions can have an sun protection
factor (SPF) of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20,
25, 30, 35, 40, 45, 50, 55, 60, or more, or any integer or
derivative therein. The compositions can be sunscreen lotions,
sprays, or creams.
[0016] The compositions of the present invention can also include
any one of, any combination of, or all of the following additional
ingredients: water, a chelating agent, a moisturizing agent, a
preservative, a thickening agent, a silicone containing compound,
an essential oil, a structuring agent, a vitamin, a pharmaceutical
ingredient, or an antioxidant, or any combination of such
ingredients or mixtures of such ingredients. In certain aspects,
the composition can include at least two, three, four, five, six,
seven, eight, nine, ten, or all of these additional ingredients
identified in the previous sentence. Non-limiting examples of these
additional ingredients are identified throughout this specification
and are incorporated into this section by reference. The amounts of
such ingredients can range from 0.0001% to 99.9% by weight or
volume of the composition, or any integer or range in between as
disclosed in other sections of this specification, which are
incorporated into this paragraph by reference.
[0017] Kits that include the compositions of the present invention
are also contemplated. In certain embodiments, the composition is
comprised in a container. The container can be a bottle, dispenser,
or package. The container can dispense a pre-determined amount of
the composition. In certain aspects, the compositions is dispensed
in a spray, mist, dollop, or liquid. The container can include
indicia on its surface. The indicia can be a word, an abbreviation,
a picture, or a symbol.
[0018] It is also contemplated that the compositions disclosed
throughout this specification can be used as a leave-on or
rinse-off composition. By way of example, a leave-on composition
can be one that is topically applied to skin and remains on the
skin for a period of time (e.g., at least 5, 6, 7, 8, 9, 10, 20, or
30 minutes, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours, or overnight or
throughout the day). Alternatively, a rinse-off composition can be
a product that is intended to be applied to the skin and then
removed or rinsed from the skin (e.g., with water) within a period
of time such as less than 5, 4, 3, 2, or 1 minute. An example of a
rinse off composition can be a skin cleanser, shampoo, conditioner,
or soap. An example of a leave-on composition can be a skin
moisturizer, sunscreen, mask, overnight cream, or a day cream.
[0019] It is contemplated that any embodiment discussed in this
specification can be implemented with respect to any method or
composition of the invention, and vice versa. Furthermore,
compositions of the invention can be used to achieve methods of the
invention.
[0020] In one embodiment, compositions of the present invention can
be pharmaceutically or cosmetically elegant or can have pleasant
tactile properties. "Pharmaceutically elegant," "cosmetically
elegant," and/or "pleasant tactile properties" describes a
composition that has particular tactile properties which feel
pleasant on the skin (e.g., compositions that are not too watery or
greasy, compositions that have a silky texture, compositions that
are non-tacky or sticky, etc.). Pharmaceutically or cosmetically
elegant can also relate to the creaminess or lubricity properties
of the composition or to the moisture retaining properties of the
composition.
[0021] Also contemplated is a product comprising a composition of
the present invention. In non-limiting aspects, the product can be
a cosmetic product. The cosmetic product can be those described in
other sections of this specification or those known to a person of
skill in the art. Non-limiting examples of products include a
moisturizer, a cream, a lotion, a skin softener, a gel, a wash, a
foundation, a night cream, a lipstick, a cleanser, a toner, a
sunscreen, a mask, an anti-aging product, a deodorant, an
antiperspirant, a perfume, a cologne, etc.
[0022] Also disclosed are the following Embodiments 1 to 24 of the
present invention. Embodiment 1 is a method of treating skin, the
method comprising topically applying to the skin an effective
amount of a topical composition comprising Argania spinosa kernel
extract, dill extract, Myrciaria dubia fruit extract, and Croton
lechleri extract and/or Morus alba fruit extract, wherein the skin
is treated. Embodiment 2 is the method of Embodiment 1, wherein the
skin is treated to improve the appearance of loose, sagging, and/or
flaccid skin and/or to improve facial contouring, and wherein the
appearance of loose, sagging, and/or flaccid skin is improved
and/or facial contouring is improved. Embodiment 3 is the method of
any of Embodiments 1 and 2, wherein the skin is treated to increase
elasticity and wherein skin elasticity is increased. Embodiment 4
is the method of any of Embodiments 1 to 3, wherein the skin is
treated to increase the expression of elastin, and wherein
expression of elastin is increased. Embodiment 5 is the method of
any of Embodiments 1 to 4, wherein the skin is treated to increase
the expression of collagen, and wherein the expression of collagen
is increased. Embodiment 6 is the method of any of Embodiments 1 to
5, wherein the skin is treated to increase the production of
laminin and/or fibronectin, and wherein the production of laminin
and/or fibronectin is increased. Embodiment 7 is the method of any
of Embodiments 1 to 6, wherein the skin is treated to increase the
production of fibulin-5, and wherein the production of fibulin-5 is
increased. Embodiment 8 is the method of any of Embodiments 1 to 7,
wherein the topical composition further comprises water. Embodiment
9 is the method of any of Embodiments 1 to 8, wherein the Argania
spinosa kernel extract is a water extract, dill extract is a water
extract, Croton lechleri extract is a water and glycerol extract,
the Morus alba fruit extract is a water and glycerol extract,
and/or the Myrciaria dubia fruit extract is a dried fruit powder.
Embodiment 10 is the method of any of Embodiments 1 to 9, wherein
the topical composition is an emulsion, serum, gel, gel emulsion,
or gel serum. Embodiment 11 is the method of any of Embodiments 1
to 10, wherein the topical composition is an oil in water emulsion
or a water in oil emulsion. Embodiment 12 is the method of any of
Embodiments 1 to 11, wherein the composition is applied to skin of
a jowl. Embodiment 13 is a topical composition comprising Argania
spinosa kernel extract, dill extract, Myrciaria dubia fruit
extract, and Croton lechleri extract and/or Morus alba fruit
extract. Embodiment 14 is the topical composition of Embodiment 13,
wherein the topical composition comprises an effective amount of
Argania spinosa kernel extract, dill extract, Myrciaria dubia fruit
extract, and Croton lechleri extract and/or Morus alba fruit
extract to improve the appearance of loose, sagging, and/or flaccid
skin and/or to improve facial contouring. Embodiment 15 is the
topical composition of any of Embodiments 13 and 14, wherein the
topical composition comprises an effective amount of Argania
spinosa kernel extract, dill extract, Myrciaria dubia fruit
extract, and Croton lechleri extract and/or Morus alba fruit
extract to increase skin elasticity. Embodiment 16 is the topical
composition of any of Embodiments 13 to 15, wherein the topical
composition comprises an effective amount of Argania spinosa kernel
extract, dill extract, Myrciaria dubia fruit extract, and Croton
lechleri extract and/or Morus alba fruit extract to increase
elastin synthesis, increase collagen expression, increase laminin
production, increase fibronectin production, and/or increase
fibulin-5 production. Embodiment 17 is the topical composition of
any of Embodiments 13 to 16, wherein the Argania spinosa kernel
extract is a water extract, dill extract is a water extract, Croton
lechleri extract is a water and glycerol extract, the Morus alba
fruit extract is a water and glycerol extract, and/or the Myrciaria
dubia fruit extract is a dried fruit powder. Embodiment 18 is the
topical composition of any of Embodiments 13 to 17, further
comprising water. Embodiment 19 is the topical composition of any
of Embodiments 13 to 18, wherein the topical composition is an
emulsion, serum, gel, gel emulsion, or gel serum. Embodiment 20 is
the topical composition of any of Embodiments 13 to 19, wherein the
topical composition is an oil in water emulsion or water in oil
emulsion. Embodiment 21 is the topical composition of any of
Embodiments 13 to 20, wherein the topical composition comprises
water, disodium EDTA, HDI/trimethylol hexyllactone crosspolymer,
nylon-12, acrylates/C10-30 alkyl crosspolymer, glyceryl stearate,
PEG-100 stearate, ethylhexyl palmitate, pentylene glycol, dimethyl
isosorbide, triethanolamine, iodopropynyl butylcarbamate,
dimethicone, dimethicone crosspolymer, phenoxyethanol, decylene
glycol, 1,2-hexanediol, glycerin, and sodium polyacrylate.
Embodiment 22 is the topical composition of Embodiment 21,
comprising 40 to 80% w/w of water, 0.001 to 1% w/w of disodium
EDTA, 0.1 to 5% w/w of HDI/trimethylol hexyllactone crosspolymer,
0.01 to 3% w/w of nylon-12, 0.001 to 1% w/w of acrylates/C10-30
alkyl crosspolymer, 1 to 10% w/w of a combination of glyceryl
stearate and PEG-100 stearate, 1 to 15% w/w of ethylhexyl
palmitate, 1 to 5% w/w of pentylene glycol, 0.01 to 3% w/w of
dimethyl isosorbide, 0.01 to 3% w/w of triethanolamine, 0.001 to 1%
w/w of iodopropynyl butylcarbamate, 5 to 20% w/w of a combination
of dimethicone and dimethicone crosspolymer, 0.01 to 3% w/w of a
combination of phenoxyethanol, decylene glycol, and 1,2-hexanediol,
and 5 to 20% w/w of a combination of glycerin, water, and sodium
polyacrylate. Embodiment 23, is the method of any of Embodiments 1
to 12, wherein the topical composition comprises water, disodium
EDTA, HDI/trimethylol hexyllactone crosspolymer, nylon-12,
acrylates/C10-30 alkyl crosspolymer, glyceryl stearate, PEG-100
stearate, ethylhexyl palmitate, pentylene glycol, dimethyl
isosorbide, triethanolamine, iodopropynyl butylcarbamate,
dimethicone, dimethicone crosspolymer, phenoxyethanol, decylene
glycol, 1,2-hexanediol, glycerin, and sodium polyacrylate.
Embodiment 24 is the method of Embodiment 23, wherein the topical
composition comprises 40 to 80% w/w of water, 0.001 to 1% w/w of
disodium EDTA, 0.1 to 5% w/w of HDI/trimethylol hexyllactone
crosspolymer, 0.01 to 3% w/w of nylon-12, 0.001 to 1% w/w of
acrylates/C10-30 alkyl crosspolymer, 1 to 10% w/w of a combination
of glyceryl stearate and PEG-100 stearate, 1 to 15% w/w of
ethylhexyl palmitate, 1 to 5% w/w of pentylene glycol, 0.01 to 3%
w/w of dimethyl isosorbide, 0.01 to 3% w/w of triethanolamine,
0.001 to 1% w/w of iodopropynyl butylcarbamate, 5 to 20% w/w of a
combination of dimethicone and dimethicone crosspolymer, 0.01 to 3%
w/w of a combination of phenoxyethanol, decylene glycol, and
1,2-hexanediol, and 5 to 20% w/w of a combination of glycerin,
water, and sodium polyacrylate
[0023] "Topical application" means to apply or spread a composition
onto the surface of lips or keratinous tissue. "Topical skin
composition" includes compositions suitable for topical application
on skin and/or keratinous tissue. Such compositions are typically
dermatologically-acceptable in that they do not have undue
toxicity, incompatibility, instability, allergic response, and the
like, when applied to skin and/or keratinous tissue. Topical skin
care compositions of the present invention can have a selected
viscosity to avoid significant dripping or pooling after
application to skin and/or keratinous tissue.
[0024] "Keratinous tissue" includes keratin-containing layers
disposed as the outermost protective covering of mammals and
includes, but is not limited to, lips, skin, hair, and nails.
[0025] The term "about" or "approximately" are defined as being
close to as understood by one of ordinary skill in the art. In one
non-limiting embodiment the terms are defined to be within 10%,
preferably within 5%, more preferably within 1%, and most
preferably within 0.5%.
[0026] The term "substantially" and its variations are refers to
ranges within 10%, within 5%, within 1%, or within 0.5%.
[0027] The terms "inhibiting" or "reducing" or "preventing" or
"avoiding" or any variation of these terms includes any measurable
decrease or complete inhibition to achieve a desired result. The
terms "promote" or "increase" or any variation of these terms
includes any measurable increase or production of a protein or
molecule (e.g., matrix proteins such as fibronectin, laminin,
collagen, or elastin or molecules such as hyaluronic acid) to
achieve a desired result.
[0028] The term "effective," as that term is used in the
specification and/or claims, means adequate to accomplish a
desired, expected, or intended result.
[0029] The use of the word "a" or "an" when used in conjunction
with the terms "comprising," "including," "having," or
"containing," or any variations of these terms, in the claims
and/or the specification may mean "one," but it is also consistent
with the meaning of "one or more," "at least one," and "one or more
than one."
[0030] The terms "wt. %," "vol. %," or "mol. %" refers to a weight,
volume, or molar percentage of a component, respectively, based on
the total weight, the total volume of material, or total moles,
that includes the component. In a non-limiting example, 10 grams of
component in 100 grams of the material is 10 wt. % of
component.
[0031] As used in this specification and claim(s), the words
"comprising" (and any form of comprising, such as "comprise" and
"comprises"), "having" (and any form of having, such as "have" and
"has"), "including" (and any form of including, such as "includes"
and "include") or "containing" (and any form of containing, such as
"contains" and "contain") are inclusive or open-ended and do not
exclude additional, unrecited elements or method steps.
[0032] The compositions and methods for their use can "comprise,"
"consist essentially of," or "consist of" any of the ingredients or
steps disclosed throughout the specification. With respect to the
phrase "consisting essentially of," a basic and novel property of
the compositions and methods of the present invention is the
ability to reduce the appearance of loose skin under the neck
and/or improve facial contouring.
[0033] Other objects, features, and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the examples, while indicating specific embodiments
of the invention, are given by way of illustration only.
Additionally, it is contemplated that changes and modifications
within the spirit and scope of the invention will become apparent
to those skilled in the art from this
DETAILED DESCRIPTION
Description of Illustrative Embodiments
[0034] As noted above, several of the unique aspects of the present
invention are to combine in a topical cosmetic composition Argania
spinosa kernel extract, dill extract, Myrciaria dubia fruit
extract, and Croton lechleri extract and/or Morus alba fruit
extract. This allows for the benefits of reducing the appearance of
loose, sagging, and flaccid skin, such as under the neck, and
improving facial contouring.
[0035] These and other non-limiting aspects of the present
invention are described in the following sections.
A. Active Ingredients
[0036] The present invention is premised on a determination that a
combination of active ingredients--Argania spinosa kernel extract,
dill extract, Myrciaria dubia fruit extract, and Croton lechleri
extract and/or Morus alba fruit extract--can be used to reduce the
appearance of loose, sagging, and flaccid skin, and improving
facial contouring.
[0037] This combination of ingredients can be used in different
products to treat various skin conditions. By way of non-limiting
examples, the combination of ingredients can be formulated in an
emulsion (e.g. oil in water, water in oil), a gel, a serum, a gel
emulsion, a gel serum, a lotion, a mask, or a body butter.
[0038] Argania spinosa kernel extract is an extraction from the
kernel of the Argan tree, which is native to the Mediterranean
region. In some instances, Argania spinosa kernel extract is
commercially available. In some instances, Argania spinosa kernel
extract can be supplied by BASF under the tradename Argatensyl. In
some instances, the extract can be an aqueous extract or an alcohol
extract or a combination thereof, wherein the extractant is water,
an alcohol (e.g., methanol, ethanol, propanol, butanol, a
polyhydric alcohol such as glycerol, etc.) or a mixture of water
and alcohol. In some instances, the extract is a water extract. The
extract can be in liquid form or can be dried to be in powdered
form by removing the extracting liquid (e.g., water or alcohol or
both).
[0039] Dill extract is an extract from the annual herb, Peucedanum
graveolens. In some instances, dill extract is commercially
available. In some instances, dill extract can be supplied by BASF
under the tradename LYS'LASTINE.TM.. In some instances, the extract
can be an aqueous extract or an alcohol extract or a combination
thereof, wherein the extractant is water, an alcohol (e.g.,
methanol, ethanol, propanol, butanol, a polyhydric alcohol such as
glycerol, etc.) or a mixture of water and alcohol. In some
instances, the extract is a water extract. The extract can be in
liquid form or can be dried to be in powdered form by removing the
extracting liquid (e.g., water or alcohol or both).
[0040] Myrciaria dubia fruit extract is an extract from a small
bushy river side tree commonly known as camu camu. Camu camu is
endogenous to the Amazon Rainforest vegetation in Peru and Brazil
and bears a red/purple cherry-like fruit rich in vitamin C. In some
instances, Myrciaria dubia fruit extract is commercially available.
In some instances, Myrciaria dubia fruit extract can be supplied by
AMAX under the tradename Camu Camu. In some instances, the extract
can be pulp, an aqueous extract, or an alcohol extract or a
combination thereof, wherein the extractant is water, an alcohol
(e.g., methanol, ethanol, propanol, butanol, a polyhydric alcohol
such as glycerol, etc.) or a mixture of water and alcohol. The
extract can be in liquid form or can be dried to be in powdered
form by removing the extracting liquid (e.g., water or alcohol or
both). In one instance, the extract is a dried fruit powder where
the fruit is obtained and dried without the use of any extracting
solvent. In some instances, the Myrciaria dubia fruit extract does
not contain an extract of Myrciaria dubia seed.
[0041] Croton lechleri extract is an extract from a tree native to
the foot of the Peruvian Andes. The Croton lechleri tree is also
known as dragon's blood because of its thick red latex. In some
instances, Croton lechleri extract is commercially available. In
some instances, Croton lechleri extract can be supplied by Naturex
under the tradename Dragon's Blood. In some instances the Croton
lechleri extract is an extract of Croton lechleri resin. In some
instances, the extract can be an aqueous extract or an alcohol
extract or a combination thereof, wherein the extractant is water,
an alcohol (e.g., methanol, ethanol, propanol, butanol, a
polyhydric alcohol such as glycerol, etc.) or a mixture of water
and alcohol. The extract can be in liquid form or can be dried to
be in powdered form by removing the extracting liquid (e.g., water
or alcohol or both). In some instances, the extract is a
water+glycerol extract.
[0042] Morus alba fruit extract is an extract of white mulberry
fruit, a tree native to northern China. In some instances, Morus
alba fruit extract is commercially available. In some instances,
Morus alba fruit extract can be supplied by Rahn AG under the
tradename DERMOFEEL.RTM. Enlight. In some instances, the extract
can be an aqueous extract or an alcohol extract or a combination
thereof, wherein the extractant is water, an alcohol (e.g.,
methanol, ethanol, propanol, butanol, a polyhydric alcohol such as
glycerol, etc.) or a mixture of water and alcohol. The extract can
be in liquid form or can be dried to be in powdered form by
removing the extracting liquid (e.g., water or alcohol or both). In
some instances, the extract is a water+glycerol extract.
[0043] It is also contemplated that any combination of Argania
spinosa kernel extract, dill extract, Myrciaria dubia fruit
extract, Croton lechleri extract and/or Morus alba fruit extract
can be used. For example, at least the following combinations are
contemplated to be useful in the context of the present invention
such as reducing the appearance of loose, sagging, and flaccid
skin, and/or improving facial contouring: (1) Argania spinosa
kernel extract and dill extract; (2) Argania spinosa kernel extract
and Myrciaria dubia fruit extract; (3) Argania spinosa kernel
extract and Croton lechleri extract; (4) Argania spinosa kernel
extract and Morus alba fruit extract; (5) Argania spinosa kernel
extract, dill extract and Myrciaria dubia fruit extract; (6)
Argania spinosa kernel extract, dill extract and Croton lechleri
extract; (7) Argania spinosa kernel extract, dill extract, and
Morus alba fruit extract; (8) Argania spinosa kernel extract, dill
extract, Myrciaria dubia fruit extract and Croton lechleri extract;
(9) Argania spinosa kernel extract, dill extract, Myrciaria dubia
fruit extract, and Morus alba fruit extract; (10) dill extract and
Myrciaria dubia fruit extract; (11) dill extract and Croton
lechleri extract; (12) dill extract and Morus alba fruit extract;
(13) dill extract, Myrciaria dubia fruit extract and Croton
lechleri extract; (14) dill extract, Myrciaria dubia fruit extract
and Morus alba fruit extract; (15) dill extract, Myrciaria dubia
fruit extract, Croton lechleri extract and Morus alba fruit
extract; (16) Myrciaria dubia fruit extract and Croton lechleri
extract; (17) Myrciaria dubia fruit extract and Morus alba fruit
extract; (18) Myrciaria dubia fruit extract, Croton lechleri
extract and Morus alba fruit extract; and (19) Croton lechleri
extract and Morus alba fruit extract.
[0044] The extracts described herein can be extracts made through
extraction methods known in the art and combinations thereof.
Non-limiting examples of extraction methods include the use of
liquid-liquid extraction, solid phase extraction, aqueous
extraction, ethyl acetate, alcohol (e.g., monohydric and polyhydric
alcohols), acetone, oil, supercritical carbon dioxide, heat,
pressure, pressure drop extraction, ultrasonic extraction, etc.
Extracts can be a liquid, solid, dried liquid, re-suspended solid,
etc.
B. Amounts of Ingredients
[0045] It is contemplated that the compositions of the present
invention can include any amount of the ingredients discussed in
this specification. The compositions can also include any number of
combinations of additional ingredients described throughout this
specification (e.g., pigments, or additional cosmetic or
pharmaceutical ingredients). The concentrations of the any
ingredient within the compositions can vary. In non-limiting
embodiments, for example, the compositions can comprise, consisting
essentially of, or consist of, in their final form, for example, at
least about 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%,
0.0007%, 0.0008%, 0.0009%, 0.0010%, 0.0011%, 0.0012%, 0.0013%,
0.0014%, 0.0015%, 0.0016%, 0.0017%, 0.0018%, 0.0019%, 0.0020%,
0.0021%, 0.0022%, 0.0023%, 0.0024%, 0.0025%, 0.0026%, 0.0027%,
0.0028%, 0.0029%, 0.0030%, 0.0031%, 0.0032%, 0.0033%, 0.0034%,
0.0035%, 0.0036%, 0.0037%, 0.0038%, 0.0039%, 0.0040%, 0.0041%,
0.0042%, 0.0043%, 0.0044%, 0.0045%, 0.0046%, 0.0047%, 0.0048%,
0.0049%, 0.0050%, 0.0051%, 0.0052%, 0.0053%, 0.0054%, 0.0055%,
0.0056%, 0.0057%, 0.0058%, 0.0059%, 0.0060%, 0.0061%, 0.0062%,
0.0063%, 0.0064%, 0.0065%, 0.0066%, 0.0067%, 0.0068%, 0.0069%,
0.0070%, 0.0071%, 0.0072%, 0.0073%, 0.0074%, 0.0075%, 0.0076%,
0.0077%, 0.0078%, 0.0079%, 0.0080%, 0.0081%, 0.0082%, 0.0083%,
0.0084%, 0.0085%, 0.0086%, 0.0087%, 0.0088%, 0.0089%, 0.0090%,
0.0091%, 0.0092%, 0.0093%, 0.0094%, 0.0095%, 0.0096%, 0.0097%,
0.0098%, 0.0099%, 0.0100%, 0.0200%, 0.0250%, 0.0275%, 0.0300%,
0.0325%, 0.0350%, 0.0375%, 0.0400%, 0.0425%, 0.0450%, 0.0475%,
0.0500%, 0.0525%, 0.0550%, 0.0575%, 0.0600%, 0.0625%, 0.0650%,
0.0675%, 0.0700%, 0.0725%, 0.0750%, 0.0775%, 0.0800%, 0.0825%,
0.0850%, 0.0875%, 0.0900%, 0.0925%, 0.0950%, 0.0975%, 0.1000%,
0.1250%, 0.1500%, 0.1750%, 0.2000%, 0.2250%, 0.2500%, 0.2750%,
0.3000%, 0.3250%, 0.3500%, 0.3750%, 0.4000%, 0.4250%, 0.4500%,
0.4750%, 0.5000%, 0.5250%, 0.0550%, 0.5750%, 0.6000%, 0.6250%,
0.6500%, 0.6750%, 0.7000%, 0.7250%, 0.7500%, 0.7750%, 0.8000%,
0.8250%, 0.8500%, 0.8750%, 0.9000%, 0.9250%, 0.9500%, 0.9750%,
1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%,
2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%,
3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%,
4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%,
5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.0%, 6.1%, 6.2%, 6.3%, 6.4%,
6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%,
7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%,
8.7%, 8.8%, 8.9%, 9.0%, 9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%, 9.7%,
9.8%, 9.9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,
21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%,
50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or any range
derivable therein, of at least one of the ingredients that are
mentioned throughout the specification and claims. In non-limiting
aspects, the percentage can be calculated by weight or volume of
the total composition. A person of ordinary skill in the art would
understand that the concentrations can vary depending on the
addition, substitution, and/or subtraction of ingredients in a
given composition.
C. Vehicles
[0046] The compositions of the present invention can include or be
incorporated into all types of vehicles and carriers. The vehicle
or carrier can be a pharmaceutically or dermatologically acceptable
vehicle or carrier. Non-limiting examples of vehicles or carriers
include water, glycerin, alcohol, oil, a silicon containing
compound, a silicone compound, and wax. Variations and other
appropriate vehicles will be apparent to the skilled artisan and
are appropriate for use in the present invention. In certain
aspects, the concentrations and combinations of the compounds,
ingredients, and agents can be selected in such a way that the
combinations are chemically compatible and do not form complexes
which precipitate from the finished product.
D. Structure
[0047] The compositions of the present invention can be structured
or formulated into a variety of different forms. Non-limiting
examples include emulsions (e.g., water-in-oil,
water-in-oil-in-water, oil-in-water, silicone-in-water,
water-in-silicone, oil-in-water-in-oil, oil-in-water-in-silicone
emulsions), creams, lotions, solutions (both aqueous and
hydro-alcoholic), anhydrous bases (such as lipsticks and powders),
gels, masks, peels, and ointments. Variations and other structures
will be apparent to the skilled artisan and are appropriate for use
in the present invention.
E. Additional Ingredients
[0048] In addition to the combination of ingredients disclosed by
the inventors, the compositions can also include additional
ingredients such as cosmetic ingredients and pharmaceutical active
ingredients. Non-limiting examples of these additional ingredients
are described in the following subsections.
1. Cosmetic Ingredients
[0049] The CTFA International Cosmetic Ingredient Dictionary and
Handbook (2004 and 2008) describes a wide variety of non-limiting
cosmetic ingredients that can be used in the context of the present
invention. Examples of these ingredient classes include: fragrance
agents (artificial and natural; e.g., gluconic acid,
phenoxyethanol, and triethanolamine), dyes and color ingredients
(e.g., Blue 1, Blue 1 Lake, Red 40, titanium dioxide, D&C blue
no. 4, D&C green no. 5, D&C orange no. 4, D&C red no.
17, D&C red no. 33, D&C violet no. 2, D&C yellow no.
10, and D&C yellow no. 11), flavoring agents/aroma agents
(e.g., Stevia rebaudiana (sweetleaf) extract, and menthol),
adsorbents, lubricants, solvents, moisturizers (including, e.g.,
emollients, humectants, film formers, occlusive agents, and agents
that affect the natural moisturization mechanisms of the skin),
water-repellants, UV absorbers (physical and chemical absorbers
such as para-aminobenzoic acid ("PABA") and corresponding PABA
derivatives, titanium dioxide, zinc oxide, etc.), essential oils,
vitamins (e.g., A, B, C, D, E, and K), trace metals (e.g., zinc,
calcium and selenium), anti-irritants (e.g., steroids and
non-steroidal anti-inflammatories), botanical extracts (e.g., Aloe
vera, chamomile, cucumber extract, Ginkgo biloba, ginseng, and
rosemary), anti-microbial agents, antioxidants (e.g., BHT and
tocopherol), chelating agents (e.g., disodium EDTA and tetrasodium
EDTA), preservatives (e.g., methylparaben and propylparaben), pH
adjusters (e.g., sodium hydroxide and citric acid), absorbents
(e.g., aluminum starch octenylsuccinate, kaolin, corn starch, oat
starch, cyclodextrin, talc, and zeolite), skin bleaching and
lightening agents (e.g., hydroquinone and niacinamide lactate),
humectants (e.g., sorbitol, urea, methyl gluceth-20, saccharide
isomerate, and mannitol), exfoliants, waterproofing agents (e.g.,
magnesium/aluminum hydroxide stearate), skin conditioning agents
(e.g., aloe extracts, allantoin, bisabolol, ceramides, dimethicone,
hyaluronic acid, biosaccharide gum-1, ethylhexylglycerin, pentylene
glycol, hydrogenated polydecene, octyldodecyl oleate, and
dipotassium glycyrrhizate). Non-limiting examples of some of these
ingredients are provided in the following subsections.
a. UV Absorption and/or Reflecting Agents
[0050] UV absorption and/or reflecting agents that can be used in
combination with the compositions of the present invention include
chemical and physical sunblocks. Non-limiting examples of chemical
sunblocks that can be used include para-aminobenzoic acid (PABA),
PABA esters (glyceryl PABA, amyldimethyl PABA and octyldimethyl
PABA), butyl PABA, ethyl PABA, ethyl dihydroxypropyl PABA,
benzophenones (oxybenzone, sulisobenzone, benzophenone, and
benzophenone-1 through 12), cinnamates (octyl methoxycinnamate
(octinoxate), isoamyl p-methoxycinnamate, octylmethoxy cinnamate,
cinoxate, diisopropyl methyl cinnamate, DEA-methoxycinnamate, ethyl
diisopropylcinnamate, glyceryl octanoate dimethoxycinnamate and
ethyl methoxycinnamate), cinnamate esters, salicylates (homomethyl
salicylate, benzyl salicylate, glycol salicylate, isopropylbenzyl
salicylate, etc.), anthranilates, ethyl urocanate, homosalate,
octisalate, dibenzoylmethane derivatives (e.g., avobenzone),
octocrylene, octyl triazone, digalloyl trioleate, glyceryl
aminobenzoate, lawsone with dihydroxyacetone, ethylhexyl triazone,
dioctyl butamido triazone, benzylidene malonate polysiloxane,
terephthalylidene dicamphor sulfonic acid, disodium phenyl
dibenzimidazole tetrasulfonate, diethylamino hydroxybenzoyl hexyl
benzoate, bis diethylamino hydroxybenzoyl benzoate, bis
benzoxazoylphenyl ethylhexylimino triazine, drometrizole
trisiloxane, methylene bis-benzotriazolyl tetramethylbutylphenol,
and bis-ethylhexyloxyphenol methoxyphenyltriazine,
4-methylbenzylidene camphor, and isopentyl 4-methoxycinnamate.
Non-limiting examples of physical sunblocks include, kaolin, talc,
petrolatum and metal oxides (e.g., titanium dioxide and zinc
oxide).
b. Moisturizing Agents
[0051] Non-limiting examples of moisturizing agents that can be
used with the compositions of the present invention include amino
acids, chondroitin sulfate, diglycerin, erythritol, fructose,
glucose, glycerin, glycerol polymers, glycol, 1,2,6-hexanetriol,
honey, hyaluronic acid, hydrogenated honey, hydrogenated starch
hydrolysate, inositol, lactitol, maltitol, maltose, mannitol,
natural moisturizing factor, PEG-15 butanediol, polyglyceryl
sorbitol, salts of pyrrolidone carboxylic acid, potassium PCA,
propylene glycol, saccharide isomerate, sodium glucuronate, sodium
PCA, sorbitol, sucrose, trehalose, urea, and xylitol.
[0052] Other examples include acetylated lanolin, acetylated
lanolin alcohol, alanine, algae extract, Aloe barbadensis, Aloe
barbadensis extract, Aloe barbadensis gel, Althea officinalis
extract, apricot (Prunus armeniaca) kernel oil, arginine, arginine
aspartate, Arnica montana extract, aspartic acid, avocado (Persea
gratissima) oil, barrier sphingolipids, butyl alcohol, beeswax,
behenyl alcohol, beta-sitosterol, birch (Betula alba) bark extract,
borage (Borago officinalis) extract, butcherbroom (Ruscus
aculeatus) extract, butylene glycol, Calendula officinalis extract,
Calendula officinalis oil, candelilla (Euphorbia cerifera) wax,
canola oil, caprylic/capric triglyceride, cardamom (Elettaria
cardamomum) oil, carnauba (Copernicia cerifera) wax, carrot (Daucus
carota sativa) oil, castor (Ricinus communis) oil, ceramides,
ceresin, ceteareth-5, ceteareth-12, ceteareth-20, cetearyl
octanoate, ceteth-20, ceteth-24, cetyl acetate, cetyl octanoate,
cetyl palmitate, chamomile (Anthemis nobilis) oil, cholesterol,
cholesterol esters, cholesteryl hydroxystearate, citric acid, clary
(Salvia sclarea) oil, cocoa (Theobroma cacao) butter,
coco-caprylate/caprate, coconut (Cocos nucifera) oil, collagen,
collagen amino acids, corn (Zea mays) oil, fatty acids, decyl
oleate, dimethicone copolyol, dimethiconol, dioctyl adipate,
dioctyl succinate, dipentaerythrityl hexacaprylate/hexacaprate,
DNA, erythritol, ethoxydiglycol, ethyl linoleate, Eucalyptus
globulus oil, evening primrose (Oenothera biennis) oil, fatty
acids, Geranium maculatum oil, glucosamine, glucose glutamate,
glutamic acid, glycereth-26, glycerin, glycerol, glyceryl
distearate, glyceryl hydroxystearate, glyceryl laurate, glyceryl
linoleate, glyceryl myristate, glyceryl oleate, glyceryl stearate,
glyceryl stearate SE, glycine, glycol stearate, glycol stearate SE,
glycosaminoglycans, grape (Vitis vinifera) seed oil, hazel (Corylus
americana) nut oil, hazel (Corylus avellana) nut oil, hexylene
glycol, hyaluronic acid, hybrid safflower (Carthamus tinctorius)
oil, hydrogenated castor oil, hydrogenated coco-glycerides,
hydrogenated coconut oil, hydrogenated lanolin, hydrogenated
lecithin, hydrogenated palm glyceride, hydrogenated palm kernel
oil, hydrogenated soybean oil, hydrogenated tallow glyceride,
hydrogenated vegetable oil, hydrolyzed collagen, hydrolyzed
elastin, hydrolyzed glycosaminoglycans, hydrolyzed keratin,
hydrolyzed soy protein, hydroxylated lanolin, hydroxyproline,
isocetyl stearate, isocetyl stearoyl stearate, isodecyl oleate,
isopropyl isostearate, isopropyl lanolate, isopropyl myristate,
isopropyl palmitate, isopropyl stearate, isostearamide DEA,
isostearic acid, isostearyl lactate, isostearyl neopentanoate,
jasmine (Jasminum officinale) oil, jojoba (Buxus chinensis) oil,
kelp, kukui (Aleurites moluccana) nut oil, lactamide MEA,
laneth-16, laneth-10 acetate, lanolin, lanolin acid, lanolin
alcohol, lanolin oil, lanolin wax, lavender (Lavandula
angustifolia) oil, lecithin, lemon (Citrus medica limonum) oil,
linoleic acid, linolenic acid, Macadamia ternifolia nut oil,
maltitol, matricaria (Chamomilla recutita) oil, methyl glucose
sesquistearate, methylsilanol PCA, mineral oil, mink oil,
mortierella oil, myristyl lactate, myristyl myristate, myristyl
propionate, neopentyl glycol dicaprylate/dicaprate, octyldodecanol,
octyldodecyl myristate, octyldodecyl stearoyl stearate, octyl
hydroxystearate, octyl palmitate, octyl salicylate, octyl stearate,
oleic acid, olive (Olea europaea) oil, orange (Citrus aurantium
dulcis) oil, palm (Elaeis guineensis) oil, palmitic acid,
pantethine, panthenol, panthenyl ethyl ether, paraffin, PCA, peach
(Prunus persica) kernel oil, peanut (Arachis hypogaea) oil, PEG-8
C12-18 ester, PEG-15 cocamine, PEG-150 distearate, PEG-60 glyceryl
isostearate, PEG-5 glyceryl stearate, PEG-30 glyceryl stearate,
PEG-7 hydrogenated castor oil, PEG-40 hydrogenated castor oil,
PEG-60 hydrogenated castor oil, PEG-20 methyl glucose
sesquistearate, PEG-40 sorbitan peroleate, PEG-5 soy sterol, PEG-10
soy sterol, PEG-2 stearate, PEG-8 stearate, PEG-20 stearate, PEG-32
stearate, PEG-40 stearate, PEG-50 stearate, PEG-100 stearate,
PEG-150 stearate, pentadecalactone, peppermint (Mentha piperita)
oil, petrolatum, phospholipids, plankton extract, polyamino sugar
condensate, polyglyceryl-3 diisostearate, polyquaternium-24,
polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80,
polysorbate 85, potassium myristate, potassium palmitate, propylene
glycol, propylene glycol dicaprylate/dicaprate, propylene glycol
dioctanoate, propylene glycol dipelargonate, propylene glycol
laurate, propylene glycol stearate, propylene glycol stearate SE,
PVP, pyridoxine dipalmitate, retinol, retinyl palmitate, rice
(Oryza sativa) bran oil, RNA, rosemary (Rosmarinus officinalis)
oil, rose oil, safflower (Carthamus tinctorius) oil, sage (Salvia
officinalis) oil, sandalwood (Santalum album) oil, serine, serum
protein, sesame (Sesamum indicum) oil, shea butter (Butyrospermum
parkii), silk powder, sodium chondroitin sulfate, sodium
hyaluronate, sodium lactate, sodium palmitate, sodium PCA, sodium
polyglutamate, soluble collagen, sorbitan laurate, sorbitan oleate,
sorbitan palmitate, sorbitan sesquioleate, sorbitan stearate,
sorbitol, soybean (Glycine soja) oil, sphingolipids, squalane,
squalene, stearamide MEA-stearate, stearic acid, stearoxy
dimethicone, stearoxytrimethylsilane, stearyl alcohol, stearyl
glycyrrhetinate, stearyl heptanoate, stearyl stearate, sunflower
(Helianthus annuus) seed oil, sweet almond (Prunus amygdalus
dulcis) oil, synthetic beeswax, tocopherol, tocopheryl acetate,
tocopheryl linoleate, tribehenin, tridecyl neopentanoate, tridecyl
stearate, triethanolamine, tristearin, urea, vegetable oil, water,
waxes, wheat (Triticum vulgare) germ oil, and ylang ylang (Cananga
odorata) oil.
c. Antioxidants
[0053] Non-limiting examples of antioxidants that can be used with
the compositions of the present invention include acetyl cysteine,
ascorbic acid polypeptide, ascorbyl dipalmitate, ascorbyl
methylsilanol pectinate, ascorbyl palmitate, ascorbyl stearate,
BHA, BHT, t-butyl hydroquinone, cysteine, cysteine HCl,
diamylhydroquinone, di-t-butylhydroquinone, dicetyl
thiodipropionate, dioleyl tocopheryl methylsilanol, disodium
ascorbyl sulfate, distearyl thiodipropionate, ditridecyl
thiodipropionate, dodecyl gallate, erythorbic acid, esters of
ascorbic acid, ethyl ferulate, ferulic acid, gallic acid esters,
hydroquinone, isooctyl thioglycolate, kojic acid, magnesium
ascorbate, magnesium ascorbyl phosphate, methylsilanol ascorbate,
natural botanical anti-oxidants such as green tea or grape seed
extracts, nordihydroguaiaretic acid, octyl gallate,
phenylthioglycolic acid, potassium ascorbyl tocopheryl phosphate,
potassium sulfite, propyl gallate, quinones, rosmarinic acid,
sodium ascorbate, sodium bisulfite, sodium erythorbate, sodium
metabisulfite, sodium sulfite, superoxide dismutase, sodium
thioglycolate, sorbityl furfural, thiodiglycol, thiodiglycolamide,
thiodiglycolic acid, thioglycolic acid, thiolactic acid,
thiosalicylic acid, tocophereth-5, tocophereth-10, tocophereth-12,
tocophereth-18, tocophereth-50, tocopherol, tocophersolan,
tocopheryl acetate, tocopheryl linoleate, tocopheryl nicotinate,
tocopheryl succinate, and tris(nonylphenyl)phosphite.
d. Structuring Agents
[0054] In other non-limiting aspects, the compositions of the
present invention can include a structuring agent. Structuring
agent, in certain aspects, assist in providing rheological
characteristics to the composition to contribute to the
composition's stability. In other aspects, structuring agents can
also function as an emulsifier or surfactant. Non-limiting examples
of structuring agents include stearic acid, palmitic acid, stearyl
alcohol, cetyl alcohol, behenyl alcohol, stearic acid, palmitic
acid, the polyethylene glycol ether of stearyl alcohol having an
average of about 1 to about 21 ethylene oxide units, the
polyethylene glycol ether of cetyl alcohol having an average of
about 1 to about 5 ethylene oxide units, and mixtures thereof.
e. Emulsifiers
[0055] In certain aspects of the present invention, the
compositions do not include an emulsifier. In other aspects,
however, the compositions can include one or more emulsifiers.
Emulsifiers can reduce the interfacial tension between phases and
improve the formulation and stability of an emulsion. The
emulsifiers can be nonionic, cationic, anionic, and zwitterionic
emulsifiers (See McCutcheon's (1986); U.S. Pat. Nos. 5,011,681;
4,421,769; 3,755,560). Non-limiting examples include esters of
glycerin, esters of propylene glycol, fatty acid esters of
polyethylene glycol, fatty acid esters of polypropylene glycol,
esters of sorbitol, esters of sorbitan anhydrides, carboxylic acid
copolymers, esters and ethers of glucose, ethoxylated ethers,
ethoxylated alcohols, alkyl phosphates, polyoxyethylene fatty ether
phosphates, fatty acid amides, acyl lactylates, soaps, TEA
stearate, DEA oleth-3 phosphate, polyethylene glycol 20 sorbitan
monolaurate (polysorbate 20), polyethylene glycol 5 soya sterol,
steareth-2, steareth-20, steareth-21, ceteareth-20, cetearyl
glucoside, cetearyl alcohol, C12-13 pareth-3, PPG-2 methyl glucose
ether distearate, PPG-5-ceteth-20, bis-PEG/PPG-20/20 dimethicone,
ceteth-10, polysorbate 80, cetyl phosphate, potassium cetyl
phosphate, diethanolamine cetyl phosphate, polysorbate 60, glyceryl
stearate, PEG-100 stearate, arachidyl alcohol, arachidyl glucoside,
and mixtures thereof.
f. Silicone Containing Compounds
[0056] In non-limiting aspects, silicone containing compounds
include any member of a family of polymeric products whose
molecular backbone is made up of alternating silicon and oxygen
atoms with side groups attached to the silicon atoms. By varying
the --Si--O-- chain lengths, side groups, and crosslinking,
silicones can be synthesized into a wide variety of materials. They
can vary in consistency from liquid to gel to solids.
[0057] The silicone containing compounds that can be used in the
context of the present invention include those described in this
specification or those known to a person of ordinary skill in the
art. Non-limiting examples include silicone oils (e.g., volatile
and non-volatile oils), gels, and solids. In certain aspects, the
silicon containing compounds includes a silicone oils such as a
polyorganosiloxane. Non-limiting examples of polyorganosiloxanes
include dimethicone, cyclomethicone, polysilicone-11, phenyl
trimethicone, trimethylsilylamodimethicone,
stearoxytrimethylsilane, or mixtures of these and other
organosiloxane materials in any given ratio in order to achieve the
desired consistency and application characteristics depending upon
the intended application (e.g., to a particular area such as the
skin, hair, or eyes). A "volatile silicone oil" includes a silicone
oil have a low heat of vaporization, i.e. normally less than about
50 cal per gram of silicone oil. Non-limiting examples of volatile
silicone oils include: cyclomethicones such as Dow Corning 344
Fluid, Dow Corning 345 Fluid, Dow Corning 244 Fluid, and Dow
Corning 245 Fluid, Volatile Silicon 7207 (Union Carbide Corp.,
Danbury, Conn.); low viscosity dimethicones, i.e. dimethicones
having a viscosity of about 50 cst or less (e.g., dimethicones such
as Dow Corning 200-0.5 cst Fluid). The Dow Corning Fluids are
available from Dow Corning Corporation, Midland, Mich.
Cyclomethicone and dimethicone are described in the Third Edition
of the CTFA Cosmetic Ingredient Dictionary (incorporated by
reference) as cyclic dimethyl polysiloxane compounds and a mixture
of fully methylated linear siloxane polymers end-blocked with
trimethylsiloxy units, respectively. Other non-limiting volatile
silicone oils that can be used in the context of the present
invention include those available from General Electric Co.,
Silicone Products Div., Waterford, N.Y. and SWS Silicones Div. of
Stauffer Chemical Co., Adrian, Mich.
g. Exfoliating Agent
[0058] Exfoliating agents include ingredients that remove dead skin
cells on the skin's outer surface. These agents may act through
mechanical, chemical, and/or other means. Non-limiting examples of
mechanical exfoliating agents include abrasives such as pumice,
silica, cloth, paper, shells, beads, solid crystals, solid
polymers, etc. Non-limiting examples of chemical exfoliating agents
include acids and enzyme exfoliants. Acids that can be used as
exfoliating agents include, but are not limited to, glycolic acid,
lactic acid, citric acid, alpha hydroxy acids, beta hydroxy acids,
etc. Other exfoliating agents known to those of skill in the art
are also contemplated as being useful within the context of the
present invention.
h. Essential Oils
[0059] Essential oils include oils derived from herbs, flowers,
trees, and other plants. Such oils are typically present as tiny
droplets between the plant's cells, and can be extracted by several
method known to those of skill in the art (e.g., steam distilled,
enfleurage (i.e., extraction by using fat), maceration, solvent
extraction, or mechanical pressing). When these types of oils are
exposed to air they tend to evaporate (i.e., a volatile oil). As a
result, many essential oils are colorless, but with age they can
oxidize and become darker. Essential oils are insoluble in water
and are soluble in alcohol, ether, fixed oils (vegetal), and other
organic solvents. Typical physical characteristics found in
essential oils include boiling points that vary from about
160.degree. to 240.degree. C. and densities ranging from about
0.759 to about 1.096.
[0060] Essential oils typically are named by the plant from which
the oil is found. For example, rose oil or peppermint oil are
derived from rose or peppermint plants, respectively. Non-limiting
examples of essential oils that can be used in the context of the
present invention include sesame oil, macadamia nut oil, tea tree
oil, evening primrose oil, Spanish sage oil, Spanish rosemary oil,
coriander oil, thyme oil, pimento berries oil, rose oil, anise oil,
balsam oil, bergamot oil, rosewood oil, cedar oil, chamomile oil,
sage oil, clary sage oil, clove oil, cypress oil, eucalyptus oil,
fennel oil, sea fennel oil, frankincense oil, geranium oil, ginger
oil, grapefruit oil, jasmine oil, juniper oil, lavender oil, lemon
oil, lemongrass oil, lime oil, mandarin oil, marjoram oil, myrrh
oil, neroli oil, orange oil, patchouli oil, pepper oil, black
pepper oil, petitgrain oil, pine oil, rose otto oil, rosemary oil,
sandalwood oil, spearmint oil, spikenard oil, vetiver oil,
wintergreen oil, or ylang ylang. Other essential oils known to
those of skill in the art are also contemplated as being useful
within the context of the present invention.
i. Thickening Agents
[0061] Thickening agents, including thickener or gelling agents,
include substances which that can increase the viscosity of a
composition. Thickeners includes those that can increase the
viscosity of a composition without substantially modifying the
efficacy of the active ingredient within the composition.
Thickeners can also increase the stability of the compositions of
the present invention. In certain aspects of the present invention,
thickeners include hydrogenated polyisobutene, trihydroxystearin,
ammonium acryloyldimethyltaurate/vp copolymer, or a mixture of
them.
[0062] Non-limiting examples of additional thickening agents that
can be used in the context of the present invention include
carboxylic acid polymers, crosslinked polyacrylate polymers,
polyacrylamide polymers, polysaccharides, and gums. Examples of
carboxylic acid polymers include crosslinked compounds containing
one or more monomers derived from acrylic acid, substituted acrylic
acids, and salts and esters of these acrylic acids and the
substituted acrylic acids, wherein the crosslinking agent contains
two or more carbon-carbon double bonds and is derived from a
polyhydric alcohol (see U.S. Pat. Nos. 5,087,445; 4,509,949;
2,798,053; CTFA International Cosmetic Ingredient Dictionary,
Fourth edition, 1991, pp. 12 and 80). Examples of commercially
available carboxylic acid polymers include carbomers, which are
homopolymers of acrylic acid crosslinked with allyl ethers of
sucrose or pentaerytritol (e.g., Carbopol.TM. 900 series from B. F.
Goodrich).
[0063] Non-limiting examples of crosslinked polyacrylate polymers
include cationic and nonionic polymers. Examples are described in
U.S. Pat. Nos. 5,100,660; 4,849,484; 4,835,206; 4,628,078;
4,599,379).
[0064] Non-limiting examples of polyacrylamide polymers (including
nonionic polyacrylamide polymers including substituted branched or
unbranched polymers) include polyacrylamide, isoparaffin and
laureth-7, multi-block copolymers of acrylamides and substituted
acrylamides with acrylic acids and substituted acrylic acids.
[0065] Non-limiting examples of polysaccharides include cellulose,
carboxymethyl hydroxyethylcellulose, cellulose acetate propionate
carboxylate, hydroxyethylcellulose, hydroxyethyl ethylcellulose,
hydroxypropylcellulose, hydroxypropyl methylcellulose, methyl
hydroxyethylcellulose, microcrystalline cellulose, sodium cellulose
sulfate, and mixtures thereof. Another example is an alkyl
substituted cellulose where the hydroxy groups of the cellulose
polymer is hydroxyalkylated (preferably hydroxy ethylated or
hydroxypropylated) to form a hydroxyalkylated cellulose which is
then further modified with a C10-C30 straight chain or branched
chain alkyl group through an ether linkage. Typically these
polymers are ethers of C10-C30 straight or branched chain alcohols
with hydroxyalkylcelluloses. Other useful polysaccharides include
scleroglucans comprising a linear chain of (1-3) linked glucose
units with a (1-6) linked glucose every three unit.
[0066] Non-limiting examples of gums that can be used with the
present invention include acacia, agar, algin, alginic acid,
ammonium alginate, amylopectin, calcium alginate, calcium
carrageenan, carnitine, carrageenan, dextrin, gelatin, gellan gum,
guar gum, guar hydroxypropyltrimonium chloride, hectorite,
hyaluronic acid, hydrated silica, hydroxypropyl chitosan,
hydroxypropyl guar, karaya gum, kelp, locust bean gum, natto gum,
potassium alginate, potassium carrageenan, propylene glycol
alginate, sclerotium gum, sodium carboxymethyl dextran, sodium
carrageenan, tragacanth gum, xanthan gum, and mixtures thereof.
j. Preservatives
[0067] Non-limiting examples of preservatives that can be used in
the context of the present invention include quaternary ammonium
preservatives such as polyquaternium-1 and benzalkonium halides
(e.g., benzalkonium chloride ("BAC") and benzalkonium bromide),
parabens (e.g., methylparabens and propylparabens), phenoxyethanol,
benzyl alcohol, chlorobutanol, phenol, sorbic acid, thimerosal or
combinations thereof.
2. Pharmaceutical Ingredients
[0068] Pharmaceutical active agents are also contemplated as being
useful with the compositions of the present invention. Non-limiting
examples of pharmaceutical active agents include anti-acne agents,
agents used to treat rosacea, analgesics, anesthetics, anorectals,
antihistamines, anti-inflammatory agents including non-steroidal
anti-inflammatory drugs, antibiotics, antifungals, antivirals,
antimicrobials, anti-cancer actives, scabicides, pediculicides,
antineoplastics, antiperspirants, antipruritics, antipsoriatic
agents, antiseborrheic agents, biologically active proteins and
peptides, burn treatment agents, cauterizing agents, depigmenting
agents, depilatories, diaper rash treatment agents, enzymes, hair
growth stimulants, hair growth retardants including DFMO and its
salts and analogs, hemostatics, kerotolytics, canker sore treatment
agents, cold sore treatment agents, dental and periodontal
treatment agents, photosensitizing actives, skin protectant/barrier
agents, steroids including hormones and corticosteroids, sunburn
treatment agents, sunscreens, transdermal actives, nasal actives,
vaginal actives, wart treatment agents, wound treatment agents,
wound healing agents, etc.
F. Kits
[0069] Kits are also contemplated as being used in certain aspects
of the present invention. For instance, compositions of the present
invention can be included in a kit. A kit can include a container.
Containers can include a bottle, a metal tube, a laminate tube, a
plastic tube, a dispenser, a pressurized container, a barrier
container, a package, a compartment, a lipstick container, a
compact container, cosmetic pans that can hold cosmetic
compositions, or other types of containers such as injection or
blow-molded plastic containers into which the dispersions or
compositions or desired bottles, dispensers, or packages are
retained. The kit and/or container can include indicia on its
surface. The indicia, for example, can be a word, a phrase, an
abbreviation, a picture, or a symbol.
[0070] The containers can dispense a pre-determined amount of the
composition. In other embodiments, the container can be squeezed
(e.g., metal, laminate, or plastic tube) to dispense a desired
amount of the composition. The composition can be dispensed as a
spray, an aerosol, a liquid, a fluid, or a semi-solid. The
containers can have spray, pump, or squeeze mechanisms. A kit can
also include instructions for employing the kit components as well
the use of any other compositions included in the container.
Instructions can include an explanation of how to apply, use, and
maintain the compositions.
EXAMPLES
[0071] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
[0072] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit, and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
A. Example 1
[0073] Combinations of active ingredients disclosed herein can be
included in a wide-range of topical product formulations for skin
and/or hair. Non-limiting examples of combinations of active
ingredients include those found in Table 1 and Table 2.
TABLE-US-00001 TABLE 1 Ingredient Croton Lechleri extract (Dragon's
Blood by Naturex) Argania Spinosa Kernel Extract (Argatensyl by
BASF) Dill (Peucedanum graveolens) Extract (LYS'LASTINE .TM. by
BASF) Myrciaria dubia fruit extract (Camu Camu by AMAX)
TABLE-US-00002 TABLE 2 Ingredient Morus Alba Fruit Extract Argania
Spinosa Kernel Extract (Argatensyl by BASF) Dill (Peucedanum
graveolens) Extract (LYS'LASTINE .TM. by BASF) Myrciaria dubia
fruit extract (Camu Camu by AMAX)
B. Example 2
[0074] Tables 3 and 4 describe generic formulations or skin testing
formulations in which an active ingredient can be incorporated
into. These formulations can also be used to determine the types of
skin benefits that can be attributed to the active ingredient.
These formulations are prepared in such a manner that any resulting
skin benefit from topical application of the formula to skin can be
directly attributed to the active ingredient being tested. In the
context of aspects of the present invention, the active ingredient
that can be tested can be Argania spinosa kernel extract, dill
extract, Myrciaria dubia fruit extract, Croton lechleri extract,
Morus alba fruit extract, or any combination thereof, or all of
such active ingredients, or at least 1, 2, 3, 4, and/or 5 of such
active ingredients. It should be recognized that other standard
testing vehicles can also be used to determine the skin benefit
properties of active ingredient and that the following formulations
are non-limiting testing vehicles.
TABLE-US-00003 TABLE 3* Ingredient % Concentration (by weight)
Phase A Water 84.80 Xanthan gum 0.1 M-paraben 0.15 P-paraben 0.1
Citric acid 0.1 Phase B Cetyl alcohol 4.0 Glyceryl stearate + PEG
100 4.0 Octyl palmitate 4.0 Dimethicone 1.0 Tocopheryl acetate 0.2
Phase C Active Ingredient** 2.0 TOTAL 100 *Procedure for making
composition: Sprinkle Xanthan gum in water and mix for 10 min.
Subsequently, add all ingredients in phase A and heat to
70-75.degree. C. Add all items in phase B to separate beaker and
heat to 70-75.degree. C. Mix phases A and B at 70-75.degree. C.
Continue mixing and allow composition to cool to 30.degree. C.
Subsequently, add phase C ingredient while mixing. **The active
ingredients identified throughout this specification can be
incorporated into composition as the active ingredient. The active
ingredients can be individually used or combined in this
composition. The concentration ranges of the active ingredients (or
combination of active ingredients) can be modified as desired or
needed by increasing or decreasing the amount of water.
TABLE-US-00004 TABLE 4* Ingredient % Concentration (by weight)
Phase A Water 78.6 M-paraben 0.2 P-paraben 0.1 Na.sub.2 EDTA 0.1
Shea butter 4.5 Petrolatum 4.5 Glycerin 4.0 Propylene Glycol 2.0
Finsolve TN 2.0 Phase B Sepigel 305 2.0 Phase C Active Ingredient
** 2.0 TOTAL 100 *Add ingredients in phase A to beaker and heat to
70-75.degree. C. while mixing. Subsequently, add the phase B
ingredient with phase A and cool to 30.degree. C. with mixing.
Subsequently, add phase C ingredient while mixing. ** The active
ingredients identified throughout this specification can be
incorporated into composition as the active ingredient. The active
ingredients can be individually used or combined in this
composition. The concentration ranges of the active ingredients (or
combination of active ingredients) can be modified as desired or
needed by increasing or decreasing the amount of water.
C. Example 3
[0075] The formulations represented in Table 5-11 are non-limiting
examples of the types of formulations that can be prepared in the
context of the present invention. Any standard method can be used
to prepare such formulations. For instance, simple mixing of the
ingredients in a beaker can be used. One should mix such
ingredients and add heat as necessary to obtain a homogenous
composition. The active ingredients that can be used in the
formulations can include Argania spinosa kernel extract, dill
extract, Myrciaria dubia fruit extract, Croton lechleri extract,
Morus alba fruit extract, or any combination thereof, or all of
such active ingredients, or at least 1, 2, 3, 4, and/or 5 of such
active ingredients.
[0076] Table 5 includes a non-limiting example of a composition of
the present invention. The composition can be formulated into an
emulsion (e.g., oil in water (o/w), water in oil (w/o), oil in
water in oil (o/w/o), water in oil in water (w/o/w), etc.) and the
additional ingredients identified throughout the specification can
be included into the Table 5 composition (e.g., by adjusting the
water content of composition). Further, the concentration ranges of
the ingredients identified in Table 5 can vary depending on a
desired formulation (e.g., cream, lotion, moisturizer cleanser,
etc.).
TABLE-US-00005 TABLE 5 Ingredient % Concentration (by weight) Water
q.s. Active Ingredient * 0.1% to 10% Glycerin 3 to 40% Butylene
glycol 0.0001 to 10% Propylene glycol 0.0001 to 10% Phenoxyethanol
0.0001 to 10% Disodium EDTA 0.0001 to 10% Steareth-20 0.0001 to 10%
Chlorhexidine Diglunonate 0.0001 to 10% Potassium Sorbate 0.0001 to
10% Preservative** 0.0001 to 2% TOTAL 100 * The active ingredients
identified throughout this specification can be incorporated into
composition as the active ingredient. The active ingredients can be
individually used or combined in this composition. The
concentration ranges of the active ingredients (or combination of
active ingredients) can be modified as desired or needed by
increasing or decreasing the amount of water. **Any preservative
can be used identified in the specification or those known in the
art.
[0077] Table 6 includes a non-limiting example of a composition of
the present invention. The composition can be formulated into an
emulsion (e.g., o/w, w/o, o/w/o, w/o/w, etc.) and the additional
ingredients identified throughout the specification can be included
into the Table 6 composition (e.g., by adjusting the water content
of composition). Further, the concentration ranges of the
ingredients identified in Table 6 can vary depending on a desired
formulation (e.g., cream, lotion, moisturizer cleanser, etc.).
TABLE-US-00006 TABLE 6 Ingredient % Concentration (by weight) Water
q.s. Active Ingredient* 0.1% to 10% Dimethicone 0.0001 to 10%
Triethanolamine 0.0001 to 10% Phenonip 0.0001 to 10% Betaine 0.0001
to 10% Disodium EDTA 0.0001 to 10% Tocopheryl acetate 0.0001 to 10%
Prodew 400 0.0001 to 10% Preservative** 0.0001 to 2% TOTAL 100 *
The active ingredients identified throughout this specification can
be incorporated into composition as the active ingredient. The
active ingredients can be individually used or combined in this
composition. The concentration ranges of the active ingredients (or
combination of active ingredients) can be modified as desired or
needed by increasing or decreasing the amount of water. **Any
preservative can be used identified in the specification or those
known in the art.
[0078] Table 7 includes a non-limiting example of a composition of
the present invention. The composition can be formulated into an
emulsion (e.g., o/w, w/o, o/w/o, w/o/w, etc.) and the additional
ingredients identified throughout the specification can be included
into the Table 7 composition (e.g., by adjusting the water content
of composition). Further, the concentration ranges of the
ingredients identified in Table 7 can vary depending on a desired
formulation (e.g., cream, lotion, moisturizer cleanser, etc.). In
particular embodiments, the Table 7 composition can be a
moisturizer.
TABLE-US-00007 TABLE 7 Ingredient % Concentration (by weight) Water
q.s. Active Ingredient * 0.1% to 10% Glycerin 0.0001 to 10%
Pentylene Glycol 0.0001 to 10% Capryl Glycol 0.0001 to 10% Disodium
EDTA 0.0001 to 10% Capric/Caprylic Triglyceride 0.0001 to 10% Lipex
205 (Shea Butter) 0.0001 to 10% Squalane 0.0001 to 10% Cetyl
Alcohol 0.0001 to 10% Dimethicone 0.0001 to 10% Ceramide II 0.0001
to 10% Stearic Acid 0.0001 to 10% Super Sterol Ester 0.0001 to 10%
Arlacel 165 0.0001 to 10% Simulgel 600 0.0001 to 10% TOTAL 100 *The
active ingredients identified throughout this specification can be
incorporated into composition as the active ingredient. The active
ingredients can be individually used or combined in this
composition. The concentration ranges of the active ingredients (or
combination of active ingredients) can be modified as desired or
needed by increasing or decreasing the amount of water.
[0079] Table 8 includes a non-limiting example of a composition of
the present invention. The composition can be formulated into an
emulsion (e.g., o/w, w/o, o/w/o, w/o/w, etc.) and the additional
ingredients identified throughout the specification can be included
into the Table 8 composition (e.g., by adjusting the water content
of composition). Further, the concentration ranges of the
ingredients identified in Table 8 can vary depending on a desired
formulation (e.g., cream, lotion, moisturizer cleanser, etc.). In
particular embodiments, the Table 8 composition can be a
moisturizer.
TABLE-US-00008 TABLE 8 Ingredient % Concentration (by weight) Water
q.s. Active Ingredient * 0.1% to 10% Glycerin 0.0001 to 10%
Pentylene Glycol 0.0001 to 10% Capryl Glycol 0.0001 to 10% Disodium
EDTA 0.0001 to 10% Petrolatum 0.0001 to 10% Squalane 0.0001 to 10%
Cetyl Alcohol 0.0001 to 10% Arlacel 165 0.0001 to 10% Dimethicone
0.0001 to 10% Simulgel 600 0.0001 to 10% TOTAL 100 * The active
ingredients identified throughout this specification can be
incorporated into composition as the active ingredient. The active
ingredients can be individually used or combined in this
composition. The concentration ranges of the active ingredients (or
combination of active ingredients) can be modified as desired or
needed by increasing or decreasing the amount of water.
[0080] Table 9 includes a non-limiting example of a composition of
the present invention. The composition can be formulated into an
emulsion (e.g., o/w, w/o, o/w/o, w/o/w, etc.) and the additional
ingredients identified throughout the specification can be included
into the Table 9 composition (e.g., by adjusting the water content
of composition). Further, the concentration ranges of the
ingredients identified in Table 9 can vary depending on a desired
formulation (e.g., cream, lotion, moisturizer cleanser, etc.). In
particular embodiments, the Table 9 composition can be a sunscreen
lotion.
TABLE-US-00009 TABLE 9 Ingredient % Concentration (by weight) Water
q.s. Active Ingredient * 0.1% to 10% Xanthan Gum 0.0001 to 10%
Disodium EDTA 0.0001 to 10% Pentylene Glycol 0.0001 to 10% Capryl
Glycol 0.0001 to 10% Pemulen TR-1 0.0001 to 10% Triethanolamine
0.0001 to 10% PVP/Hexadecene Copolymer 0.0001 to 10% Finsolv TN 10
to 30% Sorbitan Isostearate 0.0001 to 10% Sunscreen Ingredient** 2
to 25% TOTAL 100 * The active ingredients identified throughout
this specification can be incorporated into composition as the
active ingredient. The active ingredients can be individually used
or combined in this composition. The concentration ranges of the
active ingredients (or combination of active ingredients) can be
modified as desired or needed by increasing or decreasing the
amount of water. **Sunscreen ingredient can be any sunscreen
ingredient, or combination of such ingredients, identified in the
specification (e.g. UV absorbing and/or reflecting agents) or known
to those of ordinary skill in the art. In one embodiment, the
sunscreen ingredient is a combination of zinc oxide and titanium
dioxide.
[0081] Table 10 includes a non-limiting example of a composition of
the present invention. The additional ingredients identified
throughout the specification can be included into the Table 10
composition (e.g., by adjusting the water content of composition).
Further, the concentration ranges of the ingredients identified in
Table 10 can vary depending on a desired formulation (e.g., cream,
lotion, moisturizer cleanser, etc.). In particular embodiments, the
Table 10 composition can be a cleanser.
TABLE-US-00010 TABLE 10 Ingredient % Concentration (by weight)
Water q.s. Active Ingredient* 0.1% to 10% Disodium EDTA 0.0001 to
10% Citric Acid 0.0001 to 10% Pentylene Glycol 0.0001 to 10% Capryl
Glycol 0.0001 to 10% sodium methyl cocoyl taurate 10 to 30% sodium
cocoamphodiacetate 1 to 10% TOTAL 100 *The active ingredients
identified throughout this specification can be incorporated into
composition as the active ingredient. The active ingredients can be
individually used or combined in this composition. The
concentration ranges of the active ingredients (or combination of
active ingredients) can be modified as desired or needed by
increasing or decreasing the amount of water.
[0082] Table 11 includes a non-limiting example of a composition of
the present invention. The composition can be formulated into an
emulsion (e.g., o/w, w/o, o/w/o, w/o/w, etc.) and the additional
ingredients identified throughout the specification can be included
into the Table 11 composition (e.g., by adjusting the water content
of composition). Further, the concentration ranges of the
ingredients identified in Table 11 can vary depending on a desired
formulation (e.g., cream, lotion, moisturizer cleanser, etc.). In
particular embodiments, the Table 11 composition can be a
cream.
TABLE-US-00011 TABLE 11.sup..LAMBDA. Ingredient % Concentration (by
weight) Water 51 Disodium EDTA 0.1 HDI/Trimethylol Hexyllactone 2.0
Crosspolymer Nylon-12 0.5 Acrylates/C10-30 Alkyl Crosspolymer 0.2
Dow Corning 9041** 10 Glyceryl Stearate & PEG-100 Stearate 4.5
Ethylhexyl Palmitate 8.0 Pentylene Glycol 3.0 Dimethyl Isosorbide
1.0 Argatensyl LS 9735** 2.0 Peucedanum graveolens (dill) extract
1.0 Myrciaria dubia fruit extract 1.0 Croton lechleri resin extract
2.0 Triethanolamine 0.3 SYMOCIDE .RTM. PS** 1.1 Iodopropynyl
butylcarbamate 0.2 Inagel V.V.OP** 13 Excipients q.s. TOTAL 100
.sup..LAMBDA.Formulation can be prepared by mixing the ingredients
in a beaker under heat 70-75.degree. C. until homogenous.
Subsequently, the formulation can be cooled to standing room
temperature (20-25.degree. C.). Further, and if desired, additional
ingredients can be added, for example, to modify the rheological
properties of the composition or ingredients that provide benefits
to skin. *Excipients can be added, for example, to modify the
rheological properties of the composition. Alternatively, the
amount of water can be varied so long as the amount of water in the
composition is at least 30% w/w, and preferably between 50 to 80%
w/w. **Dow Corning 9041 comprises dimethicone and dimethicone
crosspolymer; Argatensyl LS 9735 comprises Argania spinosa kernel
extract; SYMOCIDE .RTM. PS comprises phenoxyethanol, decylene
glycol, and 1,2-hexanediol; Inagel V.V.OP comprises glycerin,
water, and sodium polyacrylate.
D. Example 4 (Clinical Results)
[0083] In a twelve week, controlled clinical study, it has been
determined that the combination of Argania spinosa kernel extract,
dill extract, Myrciaria dubia fruit extract, and Croton lechleri
extract can increase elasticity of skin. See Table 12.
[0084] Briefly, twenty-one women subjects with mild to moderate
neck sagging were instructed to apply a cream containing Argania
spinosa kernel extract, dill extract, Myrciaria dubia fruit
extract, and Croton lechleri extract twice daily to the jawline,
morning and evening after cleansing. The subjects used the product
for twelve consecutive weeks. Elasticity measurements were taken
before the first application of the composition (baseline) and then
at three weeks, six weeks, and twelve weeks of consecutive use.
[0085] The elasticity measurements were taken using a DERMALAB.RTM.
Suction Cup along the jawline between the chin and neck.
Appropriate statistical analysis were performed comparing the
baseline to the measurements at three, six, and twelve weeks. For
all analyses, p<0.05 was taken as the level of significance.
Reduction in reaction time indicates an increase in elasticity of
the skin.
TABLE-US-00012 TABLE 12 Descriptive Reaction Time (ms) Statistics
Baseline Week 3 Week 6 Week 12 Mean 261.5 246.1 213.9 215.8 Percent
-5.91% -18.19% -17.49% Change P-Value P > 0.05 P < 0.01 P
< 0.01
E. Example 5 (In Vitro Assays)
[0086] It has been determined that Argania spinosa kernel extract,
dill extract, Myrciaria dubia fruit extract, Croton lechleri
extract, and/or Morus alba fruit extract can increase elastin
synthesis, increase collagen expression, increase production of
laminin, increase production of fibronectin, and/or increase the
expression of proteins that are involved in elastin organization in
fibroblasts (e.g., Fibulin-5). A summary of the results are found
in Table 13 and the methods used to determine the properties of the
ingredients are provided below. Taken together, a combination of
these ingredients is expected to also have the same activities as
the individual ingredients therein. These activities can reduce the
appearance of loose, sagging, and flaccid skin, improve facial
contouring (e.g., change the appearance of the shape of the face by
tightening the skin), and increase elasticity of skin.
TABLE-US-00013 TABLE 13 Assay Ingredient Activity Elastin Argania
spinosa kernel extract +200% Expression Croton lechleri extract
+81% Collagen Myrciaria dubia fruit extract +250% Expression Croton
lechleri extract +281% Morus alba fruit extract (0.1% extract) +27%
Laminin Myrciaria dubia fruit extract +240% Production Argania
spinosa kernel extract +78% Morus alba fruit extract (0.1% extract)
+14.9% Fibronectin Argania spinosa kernel extract +33% Production
Morus alba fruit extract (0.1% extract) +12.9% Fibulin-5 Morus alba
fruit extract Significant Production Increase Croton lechleri
extract Significant Increase
[0087] Elastin Stimulation Assay: Elastin is a connective tissue
protein that helps skin resume shape after stretching or
contracting. Elastin is also an important load-bearing protein used
in places where mechanical energy is required to be stored. Elastin
is made by linking many soluble tropoelastin protein molecules, in
a reaction catalyzed by lysyl oxidase. Elastin secretion and
elastin fibers were monitored in cultured human fibroblasts by
staining of cultured human fibroblasts using immunofluorescent
antibodies directed against elastin by a direct ELISA sandwich
method. A Meso Scale Discovery system SECTOR 2400 Imaging system
was used to analyze the results. Changes in elastin secretion and
elastin fibers caused by Argania spinosa kernel extract and Croton
lechleri extract were determined by incubating cultured human
fibroblasts with the active ingredient for a period of time before
probing the cells or a lysate thereof with antibodies directed
against elastin. It was shown that Argania spinosa kernel extract
and Croton lechleri extract increased elastin synthesis by 200% and
81%, respectively.
[0088] Collagen Stimulation Assay: Collagen is an extracellular
matrix protein critical for skin structure. Increased synthesis of
collagen helps improve skin firmness and elasticity. This bioassay
was used to examine the effect of Myrciaria dubia fruit extract,
Croton lechleri extract, and Morus alba fruit extract (Morus alba
fruit extract at 0.1% extract concentration) on the production of
type I procollagen peptide (a precursor to collagen) by human
epidermal fibroblasts. The endpoint of this assay was a
spectrophotometric measurement that reflects the presence of
procollagen peptide and cellular viability. The assay employed the
quantitative sandwich enzyme immunoassay technique whereby a
monoclonal antibody specific for procollagen peptide has been
pre-coated onto a microplate. Standards and samples are pipetted
into the wells and any procollagen peptide present was bound by the
immobilized antibody. After washing away any unbound substances, an
enzyme-linked polyclonal antibody specific for the procollagen
peptide was added to the wells. Following a wash to remove any
unbound antibody-enzyme reagent, a substrate solution was added to
the wells and color was developed in proportion to the amount of
procollagen peptide bound in the initial step. Color development
was stopped and the intensity of the color at 450 nm was measured
using a microplate reader.
[0089] For generation of samples and controls, subconfluent normal
human adult epidermal fibroblasts (Cascade Biologics) were
cultivated in standard DMEM growth medium with 10% fetal bovine
serum (Mediatech) at 37.degree. C. in 10% CO.sub.2. The cells were
treated with each of the tested ingredients and controls for 3
days. Following incubation, cell culture medium was collected and
the amount of procollagen peptide secretion was quantified using
the sandwich enzyme linked immuno-sorbant assay (ELISA) from Takara
(#MK101) as explained above. It was shown that Myrciaria dubia
fruit extract, Croton lechleri extract, and Morus alba fruit
extract increased collagen production by 250%, 81%, and 27%,
respectively.
[0090] Laminin and Fibronectin Stimulation Assay: Laminin and
fibronectin are major proteins in the dermal-epidermal junction
(DEJ) (also referred to as the basement membrane). The DEJ is
located between the dermis and the epidermis interlocks forming
fingerlike projections called rete ridges. The cells of the
epidermis receive their nutrients from the blood vessels in the
dermis. The rete ridges increase the surface area of the epidermis
that is exposed to these blood vessels and the needed nutrients.
The DEJ provides adhesion of the two tissue compartments and
governs the structural integrity of the skin. Laminin and
fibronectin are two structural glycoproteins located in the DEJ.
Considered the glue that holds the cells together, laminin and
fibronectin are secreted by dermal fibroblasts to help facilitate
intra- and inter-cellular adhesion of the epidermal calls to the
DEJ. Laminin and fibronectin secretion was monitored by quantifying
laminin and fibronectin in cell supernatants of cultured human
fibroblasts treated for 3 days with culture medium with or without
1.0% final concentration of the test ingredient(s), except that
Morus alba fruit extract was tested at 0.1% final concentration.
Following incubation, laminin and fibronectin content was measured
using immunofluorescent antibodies directed against each protein in
an enzyme linked immuno-sorbant assay (ELISA). Measurements were
normalized for cellular metabolic activity, as determined by
bioconversion of
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
-2H-tetrazolium (MTS). For laminin production, Myrciaria dubia
fruit extract, Argania spinosa kernel extract, and Morus alba fruit
extract were shown to increase laminin production by 240%, 78%, and
14.9%, respectively. For fibronectin, Argania spinosa kernel
extract and Morus alba fruit extract were shown to increase
fibronectin production by 33% and 12.9%, respectively.
[0091] Fibulin-5 Expression Assay: Fibulin-5 is a secreted
extracellular-matrix protein that regulates elastic fiber assembly.
Fibulin-5 expression levels were determined by immunostaining of
Fibulin 5 with a monoclonal anti-fibulin 5 antibody, clone 393904
(R&D Systems, Ref.MAB3095) of paraffinized sections of human
ex-vivo skin explants. The antibody was diluted at 1:50 in PBS-BSA
0.3% and incubated with the paraffinized sections overnight at room
temperature. The staining was enhanced with a biotin/streptavidin
enhancement system (VECTASTAIN.RTM. R.T.U. Universal ,VECTOR.RTM.)
and revealed by VIP (VECTOR.RTM., Ref. SK-4600). The immunostaining
was assessed by observation using a microscope. Croton lechleri
extract and Morus alba fruit extract significantly increased the
expression of fibulin-5.
F. Example 6 (Additional Assays)
[0092] Assays that can be used to determine the efficacy of any one
of the ingredients or any combination of ingredients or
compositions having said combination of ingredients disclosed
throughout the specification and claims can be determined by
methods known to those of ordinary skill in the art. The following
are non-limiting assays that can be used in the context of the
present invention. It should be recognized that other testing
procedures can be used, including, for example, objective and
subjective procedures.
[0093] Appearance of Loose, Sagging, and Flaccid Skin: Skin
looseness, sagging, and flaccidity can be evaluated with clinical
grading techniques. Evaluations can be made independently by two
clinicians and averaged. Changes in skin looseness, sagging, and/or
flaccidity caused by any one of, all of, or any combination of the
active ingredients disclosed herein can be determined by measuring
the skin looseness, sagging, and flaccidity at a baseline time,
then applying the active ingredient(s) to skin once, twice, three
times, or more over a period of time, such as 1 minute, 10 minute,
1 hour, 6 hours, 12 hours, 1 day, 2 days, 1 week, 2 weeks, 4 weeks,
6 weeks, 12 weeks, or more, or any time therein, before measuring
the skin looseness, sagging, and flaccidity again. Negative
controls can be used to subtract out any effects of the carrier of
the active ingredient(s).
[0094] Surface Contour of the Skin Assay with a Profilometer/Stylus
Method: The surface contour of the skin can be measured by using
the profilometer/Stylus method. This includes either shining a
light or dragging a stylus across the replica surface. The vertical
displacement of the stylus can be fed into a computer via a
distance transducer, and after scanning a fixed length of replica a
cross-sectional analysis of skin profile can be generated as a
two-dimensional curve. This scan can be repeated any number of
times along a fix axis to generate a simulated 3-D picture of the
skin. Ten random sections of the replicas using the stylus
technique can be obtained and combined to generate average values.
The values of interest include Ra which is the arithmetic mean of
all roughness (height) values computed by integrating the profile
height relative to the mean profile height. Rt which is the maximum
vertical distance between the highest peak and lowest trough, and
Rz which is the mean peak amplitude minus the mean peak height.
Values are given as a calibrated value in mm. Equipment should be
standardized prior to each use by scanning metal standards of know
values. Ra Value can be computed by the following equation:
R.sub.a=Standardize roughness; l.sub.m=the traverse (scan) length;
and y=the absolute value of the location of the profile relative to
the mean profile height (x-axis).
[0095] Changes in facial contouring caused by any one of, all of,
or any combination of the active ingredients disclosed herein can
be determined by measuring the contour at a baseline time, then
applying the active ingredient(s) to skin once, twice, three times,
or more over a period of time, such as 1 minute, 10 minute, 1 hour,
6 hours, 12 hours, 1 day, 2 days, 1 week, 2 weeks, 4 weeks, 6
weeks, 12 weeks, or more, or any time therein, before measuring the
skin contour again. Negative controls can be used to subtract out
any effects of the carrier of the active ingredient(s).
[0096] B16 Pigmentation Assay: Melanogenesis is the process by
which melanocytes produce melanin, a naturally produced pigment
that imparts color to skin, hair, and eyes. Inhibiting
melanogenesis is beneficial to prevent skin darkening and lighten
dark spots associated with aging. This bioassay utilizes B16-F1
melanocytes (ATCC), an immortalized mouse melanoma cell line, to
analyze the effect of compounds on melanogenesis. The endpoint of
this assay is a spectrophotometric measurement of melanin
production and cellular viability. B16-F1 melanocytes, can be
cultivated in standard DMEM growth medium with 10% fetal bovine
serum (Mediatech) at 37.degree. C. in 10% CO.sub.2 and then treated
with any one of the active ingredients, combination of ingredients,
or compositions having said combinations disclosed in the
specification for 6 days. Following incubation, melanin secretion
is measured by absorbance at 405 nm and cellular viability is
quantified.
[0097] Fibrillin-1 Expression Assay: Fibrillin-1 is an
extracellular matrix protein that forms a scaffold for the
deposition of elastin and provides structural support in elastic
and nonelastic connective tissue. Fibrillin-1 is secreted by
fibroblasts. Fibrillin-1 expression levels can be determined by a
quantitative sandwich enzyme immunoassay technique using a
microtiter plate pre-coated with an anti-fibrillin-1 antibody.
Samples containing, cells, lysed cells, and/or culture medium of
cells from cells cultured in the presence of negative controls or
any one of, all of, or any combination of the active ingredients
disclosed herein can be tested for the presence and concentration
of fibrillin-1 under each test condition. Samples can be added to
the microtiter wells to allow the fibrillin-1 to bind. The unbound
contents of the well can then be washed out and the concentration
of the fibrillin-1 bound in the well determined by a substrate
solution that develops color in proportion to the amount of
fibrillin-1 bound in the initial step. Color can be determined
using a microplate reader for detection.
[0098] Tumor Necrosis Factor Alpha (TNF-.alpha.) Assay: The
prototype ligand of the TNF superfamily, TNF-.alpha., is a
pleiotropic cytokine that plays a central role in inflammation.
Increase in its expression is associated with an up regulation in
pro-inflammatory activity. This bioassay can be used to analyze the
effect of any one of the active ingredients, combination of
ingredients, or compositions having said combinations disclosed in
the specification on the production of TNF-.alpha. by human
epidermal keratinocytes. The endpoint of this assay can be a
spectrophotometric measurement that reflects the presence of
TNF-.alpha. and cellular viability. The assay employs the
quantitative sandwich enzyme immunoassay technique whereby a
monoclonal antibody specific for TNF-.alpha. has been pre-coated
onto a microplate. Standards and samples can be pipetted into the
wells and any TNF-.alpha. present is bound by the immobilized
antibody. After washing away any unbound substances, an
enzyme-linked polyclonal antibody specific for TNF-.alpha. can be
added to the wells. Following a wash to remove any unbound
antibody-enzyme reagent, a substrate solution can be added to the
wells and color develops in proportion to the amount of TNF-.alpha.
bound in the initial step using a microplate reader for detection
at 450 nm. The color development can be stopped and the intensity
of the color can be measured. Subconfluent normal human adult
keratinocytes (Cascade Biologics) cultivated in EpiLife standard
growth medium (Cascade Biologics) at 37.degree. C. in 5% CO.sub.2,
can be treated with phorbol 12-myristate 13-acetate (PMA, 10 ng/ml,
Sigma Chemical, #P1585-1MG) and any one of the active ingredients,
combination of ingredients, or compositions having said
combinations disclosed in the specification for 6 hours. PMA has
been shown to cause a dramatic increase in TNF-.alpha. secretion
which peaks at 6 hours after treatment. Following incubation, cell
culture medium can be collected and the amount of TNF-.alpha.
secretion quantified using a sandwich enzyme linked immuno-sorbant
assay (ELISA) from R&D Systems (#DTA00C).
[0099] Antioxidant (AO) Assay: An in vitro bioassay that measures
the total anti-oxidant capacity of any one of the ingredients,
combination of ingredients, or compositions having said
combinations disclosed in the specification. The assay relies on
the ability of antioxidants in the sample to inhibit the oxidation
of ABTS.RTM. (2,2'-azino-di-[3-ethylbenzthiazoline sulphonate]) to
ABTS.RTM.+by metmyoglobin. The antioxidant system of living
organisms includes enzymes such as superoxide dismutase, catalase,
and glutathione peroxidase; macromolecules such as albumin,
ceruloplasmin, and ferritin; and an array of small molecules,
including ascorbic acid, .alpha.-tocopherol, .beta.-carotene,
reduced glutathione, uric acid, and bilirubin. The sum of
endogenous and food-derived antioxidants represents the total
antioxidant activity of the extracellular fluid. Cooperation of all
the different antioxidants provides greater protection against
attack by reactive oxygen or nitrogen radicals, than any single
compound alone. Thus, the overall antioxidant capacity may give
more relevant biological information compared to that obtained by
the measurement of individual components, as it considers the
cumulative effect of all antioxidants present in plasma and body
fluids. The capacity of the antioxidants in the sample to prevent
ABTS oxidation is compared with that of Trolox, a water-soluble
tocopherol analogue, and is quantified as molar Trolox equivalents.
Anti-Oxidant capacity kit #709001 from Cayman Chemical (Ann Arbor,
Mich. USA) can be used as an in vitro bioassay to measure the total
anti-oxidant capacity of each of any one of the active ingredients,
combination of ingredients, or compositions having said
combinations disclosed in the specification. The protocol can be
followed according to manufacturer recommendations.
[0100] ORAC Assay: Oxygen Radical Absorption (or Absorbance)
Capacity (ORAC) of any one of the active ingredients, combination
of ingredients, or compositions having said combinations disclosed
in the specification can also be assayed by measuring the
antioxidant activity of such ingredients or compositions.
Antioxidant activity indicates a capability to reduce oxidizing
agents (oxidants). This assay quantifies the degree and length of
time it takes to inhibit the action of an oxidizing agent, such as
oxygen radicals, that are known to cause damage to cells (e.g.,
skin cells). The ORAC value of any one of the active ingredients,
combination of ingredients, or compositions having said
combinations disclosed in the specification can be determined by
methods known to those of ordinary skill in the art (see U.S.
Publication Nos. 2004/0109905 and 2005/0163880; and commercially
available kits such as Zen-Bio ORAC Anti-oxidant Assay kit
(#AOX-2)). The Zen-Bio ORAC Anti-oxidant Assay kit measures the
loss of fluorescein fluorescence over time due to the
peroxyl-radical formation by the breakdown of AAPH
(2,2'-axobis-2-methyl propanimidamide, dihydrochloride). Trolox, a
water soluble vitamin E analog, serves as positive control
inhibition fluorescein decay in a dose dependent manner.
[0101] Mushroom tyrosinase activity assay: In mammalian cells,
tyrosinase catalyzes two steps in the multi-step biosynthesis of
melanin pigments from tyrosine (and from the polymerization of
dopachrome). Tyrosinase is localized in melanocytes and produces
melanin (aromatic quinone compounds) that imparts color to skin,
hair, and eyes. Purified mushroom tyrosinase (Sigma) can be
incubated with its substrate L-Dopa (Fisher) in the presence or
absence of each of the active ingredients, any one of the
combination of ingredients, or compositions having said
combinations disclosed in the specification. Pigment formation can
be evaluated by colorimetric plate reading at 490 nm. The percent
inhibition of mushroom tyrosinase activity can be calculated
compared to non-treated controls to determine the ability of test
ingredients or combinations thereof to inhibit the activity of
purified enzyme. Test extract inhibition was compared with that of
kojic acid (Sigma).
[0102] Matrix Metalloproteinase 3 and 9 Enzyme Activity (MMP3;
MMP9) Assay: An in vitro matrix metalloprotease (MMP) inhibition
assay. MMPs are extracellular proteases that play a role in many
normal and disease states by virtue of their broad substrate
specificity. MMP3 substrates include collagens, fibronectins, and
laminin; while MMP9 substrates include collagen VII, fibronectins
and laminin. Using Colorimetric Drug Discovery kits from BioMol
International for MMP3 (AK-400) and MMP-9 (AK-410), this assay is
designed to measure protease activity of MMPs using a thiopeptide
as a chromogenic substrate
(Ac-PLG-[2-mercapto-4-methyl-pentanoyl]-LG-OC2H5)5,6. The MMP
cleavage site peptide bond is replaced by a thioester bond in the
thiopeptide. Hydrolysis of this bond by an MMP produces a
sulfhydryl group, which reacts with DTNB
[5,5'-dithiobis(2-nitrobenzoic acid), Ellman's reagent] to form
2-nitro-5-thiobenzoic acid, which can be detected by its absorbance
at 412 nm (.epsilon.=13,600 M-1 cm-1 at pH 6.0 and above 7). The
active ingredients, any one of the combination of ingredients, or
compositions having said combinations disclosed in the
specification can be assayed.
[0103] Matrix Metalloproteinase 1 Enzyme Activity (MMP1) Assay: An
in vitro matrix metalloprotease (MMP) inhibition assay. MMPs are
extracellular proteases that play a role in many normal and disease
states by virtue of their broad substrate specificity. MMP1
substrates include collagen IV. The Molecular Probes Enz/Chek
Gelatinase/ Collagenase Assay kit (#E12055) utilizes a fluorogenic
gelatin substrate to detect MMP1 protease activity. Upon
proteolytic cleavage, bright green fluorescence is revealed and may
be monitored using a fluorescent microplate reader to measure
enzymatic activity.
[0104] The Enz/Chek Gelatinase/Collagenase Assay kit (#E12055) from
Invitrogen is designed as an in vitro assay to measure MMP1
enzymatic activity. The active ingredients, any one of the
combination of ingredients, or compositions having said
combinations disclosed in the specification can be assayed. The
assay relies upon the ability of purified MMP1 enzyme to degrade a
fluorogenic gelatin substrate. Once the substrate is specifically
cleaved by MMP1 bright green fluorescence is revealed and may be
monitored using a fluorescent microplate reader. Test materials are
incubated in the presence or absence of the purified enzyme and
substrate to determine their protease inhibitor capacity.
[0105] Cyclooxygenase (COX) Assay: An in vitro cyclooxygenase-1 and
-2 (COX-1, -2) inhibition assay. COX is a bifunctional enzyme
exhibiting both cyclooxygenase and peroxidase activities. The
cyclooxygenase activity converts arachidonic acid to a hydroperoxy
endoperoxide (Prostaglandin G2; PGG2) and the peroxidase component
reduces the endoperoxide (Prostaglandin H2; PGH2) to the
corresponding alcohol, the precursor of prostaglandins,
thromboxanes, and prostacyclins. This COX Inhibitor screening assay
measures the peroxidase component of cyclooxygenases. The
peroxidase activity is assayed colorimetrically by monitoring the
appearance of oxidized N,N,N',N'-tetramethyl-p-phenylenediamine
(TMPD). This inhibitor screening assay includes both COX-1 and
COX-2 enzymes in order to screen isozyme-specific inhibitors. The
Colormetric COX (ovine) Inhibitor screening assay (#760111, Cayman
Chemical) can be used to analyze the effects of each of the active
ingredients, any one of the combination of ingredients, or
compositions having said combinations disclosed in the
specification on the activity of purified cyclooxygnase enzyme
(COX-1 or COX-2). According to manufacturer instructions, purified
enzyme, heme and test extracts can be mixed in assay buffer and
incubated with shaking for 15 min at room temperature. Following
incubation, arachidonic acid and colorimetric substrate can be
added to initiate the reaction. Color progression can be evaluated
by colorimetric plate reading at 590 nm. The percent inhibition of
COX-1 or COX-2 activity can be calculated compared to non-treated
controls to determine the ability of test extracts to inhibit the
activity of purified enzyme.
[0106] Lipoxygenase (LO) Assay: An in vitro lipoxygenase (LO)
inhibition assay. LOs are non-heme iron-containing dioxygenases
that catalyze the addition of molecular oxygen to fatty acids.
Linoleate and arachidonate are the main substrates for LOs in
plants and animals. Arachadonic acid may then be converted to
hydroxyeicosotrienenoic (HETE) acid derivatives, that are
subsequently converted to leukotrienes, potent inflammatory
mediators. This assay provides an accurate and convenient method
for screening lipoxygenase inhibitors by measuring the
hydroperoxides generated from the incubation of a lipoxygenase (5-,
12-, or 15-LO) with arachidonic acid. The Colorimetric LO Inhibitor
screening kit (#760700, Cayman Chemical) can be used to determine
the ability of each of the active ingredients, any one of the
combination of ingredients, or compositions having said
combinations disclosed in the specification to inhibit enzyme
activity. Purified 15-lipoxygenase and test ingredients can be
mixed in assay buffer and incubated with shaking for 10 min at room
temperature. Following incubation, arachidonic acid can be added to
initiate the reaction and the mixtures can be incubated for an
additional 10 min at room temperature. Colorimetric substrate can
be added to terminate catalysis and color progression can be
evaluated by fluorescence plate reading at 490 nm. The percent
inhibition of lipoxyganse activity can be calculated compared to
non-treated controls to determine the ability of each of the active
ingredients, any one of the combination of ingredients, or
compositions having said combinations disclosed in the
specification to inhibit the activity of purified enzyme.
[0107] Elastase Assay: EnzChek.RTM. Elastase Assay (Kit #E-12056)
from Molecular Probes (Eugene, Oreg. USA) can be used as an in
vitro enzyme inhibition assay for measuring inhibition of elastase
activity for each of the active ingredients, any one of the
combination of ingredients, or compositions having said
combinations disclosed in the specification. The EnzChek kit
contains soluble bovine neck ligament elastin that can be labeled
with dye such that the conjugate's fluorescence can be quenched.
The non-fluorescent substrate can be digested by elastase or other
proteases to yield highly fluorescent fragments. The resulting
increase in fluorescence can be monitored with a fluorescence
microplate reader. Digestion products from the elastin substrate
have absorption maxima at .about.505 nm and fluorescence emission
maxima at .about.515 nm. The peptide,
N-methoxysuccinyl-Ala-Ala-Pro-Val- chloromethyl ketone, can be used
as a selective, collective inhibitor of elastase when utilizing the
EnzChek Elastase Assay Kit for screening for elastase
inhibitors.
[0108] Oil Control Assay: An assay to measure reduction of sebum
secretion from sebaceous glands and/or reduction of sebum
production from sebaceous glands can be assayed by using standard
techniques known to those having ordinary skill in the art. In one
instance, the forehead can be used. Each of the active ingredients,
any one of the combination of ingredients, or compositions having
said combinations disclosed in the specification can be applied to
one portion of the forehead once or twice daily for a set period of
days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more
days), while another portion of the forehead is not treated with
the composition. After the set period of days expires, then sebum
secretion can be assayed by application of fine blotting paper to
the treated and untreated forehead skin. This is done by first
removing any sebum from the treated and untreated areas with moist
and dry cloths. Blotting paper can then be applied to the treated
and untreated areas of the forehead, and an elastic band can be
placed around the forehead to gently press the blotting paper onto
the skin. After 2 hours the blotting papers can be removed, allowed
to dry and then transilluminated. Darker blotting paper correlates
with more sebum secretion (or lighter blotting paper correlates
with reduced sebum secretion.
[0109] Erythema Assay: An assay to measure the reduction of skin
redness can be evaluated using a Minolta Chromometer. Skin erythema
may be induced by applying a 0.2% solution of sodium dodecyl
sulfate on the forearm of a subject. The area is protected by an
occlusive patch for 24 hrs. After 24 hrs, the patch is removed and
the irritation-induced redness can be assessed using the a* values
of the Minolta Chroma Meter. The a* value measures changes in skin
color in the red region. Immediately after reading, the area is
treated with the active ingredients, any one of the combination of
ingredients, or compositions having said combinations disclosed in
the specification. Repeat measurements can be taken at regular
intervals to determine the formula's ability to reduce redness and
irritation.
[0110] Skin Moisture/Hydration Assay: Skin moisture/hydration
benefits can be measured by using impedance measurements with the
Nova Dermal Phase Meter. The impedance meter measures changes in
skin moisture content. The outer layer of the skin has distinct
electrical properties. When skin is dry it conducts electricity
very poorly. As it becomes more hydrated increasing conductivity
results. Consequently, changes in skin impedance (related to
conductivity) can be used to assess changes in skin hydration. The
unit can be calibrated according to instrument instructions for
each testing day. A notation of temperature and relative humidity
can also be made. Subjects can be evaluated as follows: prior to
measurement they can equilibrate in a room with defined humidity
(e.g., 30-50%) and temperature (e.g., 68-72.degree. C.). Three
separate impedance readings can be taken on each side of the face,
recorded, and averaged. The T5 setting can be used on the impedance
meter which averages the impedance values of every five seconds
application to the face. Changes can be reported with statistical
variance and significance. Each of the active ingredients, any one
of the combination of ingredients, or compositions having said
combinations disclosed in the specification can be assayed
according to this process.
[0111] Skin Clarity and Reduction in Freckles and Age Spots Assay:
Skin clarity and the reduction in freckles and age spots can be
evaluated using a Minolta Chromometer. Changes in skin color can be
assessed to determine irritation potential due to product treatment
using the a* values of the Minolta Chroma Meter. The a* value
measures changes in skin color in the red region. This is used to
determine whether each of the active ingredients, any one of the
combination of ingredients, or compositions having said
combinations disclosed in the specification is inducing irritation.
The measurements can be made on each side of the face and averaged,
as left and right facial values. Skin clarity can also be measured
using the Minolta Meter. The measurement is a combination of the
a*, b, and L values of the Minolta Meter and is related to skin
brightness, and correlates well with skin smoothness and hydration.
Skin reading is taken as above. In one non-limiting aspect, skin
clarity can be described as L/C where C is chroma and is defined as
(a.sup.2+b.sup.2).sup.1/2.
[0112] Skin Dryness, Surface Fine Lines, Skin Smoothness, and Skin
Tone Assay: Skin dryness, surface fine lines, skin smoothness, and
skin tone can be evaluated with clinical grading techniques. For
example, clinical grading of skin dryness can be determined by a
five point standard Kligman Scale: (0) skin is soft and moist; (1)
skin appears normal with no visible dryness; (2) skin feels
slightly dry to the touch with no visible flaking; (3) skin feels
dry, tough, and has a whitish appearance with some scaling; and (4)
skin feels very dry, rough, and has a whitish appearance with
scaling. Evaluations can be made independently by two clinicians
and averaged.
[0113] Clinical Grading of Skin Tone Assay: Clinical grading of
skin tone can be performed via a ten point analog numerical scale:
(10) even skin of uniform, pinkish brown color. No dark,
erythremic, or scaly patches upon examination with a hand held
magnifying lens. Microtexture of the skin very uniform upon touch;
(7) even skin tone observed without magnification. No scaly areas,
but slight discolorations either due to pigmentation or erythema.
No discolorations more than 1 cm in diameter; (4) both skin
discoloration and uneven texture easily noticeable. Slight
scaliness. Skin rough to the touch in some areas; and (1) uneven
skin coloration and texture. Numerous areas of scaliness and
discoloration, either hypopigmented, erythremic or dark spots.
Large areas of uneven color more than 1 cm in diameter. Evaluations
were made independently by two clinicians and averaged.
[0114] Clinical Grading of Skin Smoothness Assay: Clinical grading
of skin smoothness can be analyzed via a ten point analog numerical
scale: (10) smooth, skin is moist and glistening, no resistance
upon dragging finger across surface; (7) somewhat smooth, slight
resistance; (4) rough, visibly altered, friction upon rubbing; and
(1) rough, flaky, uneven surface. Evaluations were made
independently by two clinicians and averaged.
[0115] Skin Smoothness and Wrinkle Reduction Assay With Methods
Disclosed in Packman et al. (1978): Skin smoothness and wrinkle
reduction can also be assessed visually by using the methods
disclosed in Packman et al. (1978). For example, at each subject
visit, the depth, shallowness and the total number of superficial
facial lines (SFLs) of each subject can be carefully scored and
recorded. A numerical score was obtained by multiplying a number
factor times a depth/width/length factor. Scores are obtained for
the eye area and mouth area (left and right sides) and added
together as the total wrinkle score.
[0116] Skin Firmness Assay with a Hargens Ballistometer: Skin
firmness can be measured using a Hargens ballistometer, a device
that evaluates the elasticity and firmness of the skin by dropping
a small body onto the skin and recording its first two rebound
peaks. The ballistometry is a small lightweight probe with a
relatively blunt tip (4 square mm-contact area) was used. The probe
penetrates slightly into the skin and results in measurements that
are dependent upon the properties of the outer layers of the skin,
including the stratum corneum and outer epidermis and some of the
dermal layers.
[0117] Skin Softness/Suppleness Assay with a Gas Bearing
Electrodynamometer: Skin softness/suppleness can be evaluated using
the Gas Bearing Electrodynamometer, an instrument that measures the
stress/strain properties of the skin. The viscoelastic properties
of skin correlate with skin moisturization. Measurements can be
obtained on the predetermined site on the cheek area by attaching
the probe to the skin surface with double-stick tape. A force of
approximately 3.5 gm can be applied parallel to the skin surface
and the skin displacement is accurately measured. Skin suppleness
can then be calculated and is expressed as DSR (Dynamic Spring Rate
in gm/mm).
[0118] Appearance of Lines and Wrinkles Assay with Replicas: The
appearance of lines and wrinkles on the skin can be evaluated using
replicas, which is the impression of the skin's surface. Silicone
rubber like material can be used. The replica can be analyzed by
image analysis. Changes in the visibility of lines and wrinkles can
be objectively quantified via the taking of silicon replicas form
the subjects' face and analyzing the replicas image using a
computer image analysis system. Replicas can be taken from the eye
area and the neck area, and photographed with a digital camera
using a low angle incidence lighting. The digital images can be
analyzed with an image processing program and are of the replicas
covered by wrinkles or fine lines was determined.
[0119] MELANODERM.TM. Assay: In other non-limiting aspects, the
efficacy of each of the active ingredients, any one of the
combination of ingredients, or compositions having said
combinations disclosed in the specification can be evaluated by
using a skin analog, such as, for example, MELANODERM.TM..
Melanocytes, one of the cells in the skin analog, stain positively
when exposed to L-dihydroxyphenyl alanine (L-DOPA), a precursor of
melanin. The skin analog, MELANODERM.TM., can be treated with a
variety of bases containing each of the active ingredients, any one
of the combination of ingredients, or compositions having said
combinations disclosed in the specification or with the base alone
as a control. Alternatively, an untreated sample of the skin analog
can be used as a control.
[0120] Production of Filaggrin: Changes in the production of
filaggrin in keratinocytes due to each of the active ingredients,
any one of the combination of ingredients, or compositions having
said combinations disclosed in the specification can be measured.
Filaggrin is the precursor to Natural Moisturizing Factor (NMF) in
the skin. Increased NMF increases the moisture content of the skin.
Filaggrin production in treated and non-treated keratinocytes can
be determined using a bioassay that analyzes filaggrin
concentration in keratinocyte cell lysates. A non-limiting example
of a bioassay that can be used to quantify filaggrin production is
the PROTEINSIMPLE.RTM. Simon.TM. western blotting protocol. For
each sample, normal human epidermal keratinocytes (NHEK) are grown
in EPI-200--Mattek Epilife.RTM. growth media with calcium from Life
Technologies (M-EP-500-CA). NHEK are incubated in growth medium
overnight at 37.degree. C. in 5% CO.sub.2 prior to treatment. NHEK
are then incubated in growth medium with 1% test compound/extract
or no compound/extract (negative control) for 24 to 36 hours. The
NHEK can then be washed, collected, and stored on ice or colder
until lysed on ice using a lysis buffer and sonication. The protein
concentrations of the samples can be determined and used to
normalize the samples. The lysates can be stored at -80.degree. C.
until use in the quantification assay.
[0121] The PROTEINSIMPLE.RTM. Simon.TM. western blotting bioassay
assay employs a quantitative western blotting immunoassay technique
using an antibody specific for filaggrin to quantitatively detect
filaggrin in the test samples. Cell samples are lysed and
normalized for protein concentration. Normalized samples and
molecular weight standards can then be loaded and ran on a
denatured protein separation gel using capillary electrophoresis.
The proteins in the gel are immobilized and immunoprobed using a
primary antibody specific for filaggrin. The immobilized proteins
can then be immunoprobed with an enzyme-linked detection antibody
that binds the primary antibody. A chemiluminescent substrate
solution can then be added to the immobilized proteins to allow
chemiluminescent development in proportion to the amount of
filaggrin bound in the immobilization. The chemiluminescent
development is stopped at a specific time and the intensity of the
chemiluminescent signal can be measured and compared to positive
and negative controls.
[0122] Production of Occludin: Changes in the production of
occludin in keratinocytes due to each of the active ingredients,
any one of the combination of ingredients, or compositions having
said combinations disclosed in the specification can be measured.
Occludin is a protein critical to the formulation of tight
junctions and the skin's moisture barrier function. A non-limiting
example of how occludin production in treated and non-treated
keratinocytes can be determined is by the use of a bioassay that
analyzes occludin concentration in keratinocyte cell lysates. The
bioassay can be performed using PROTEINSIMPLE.RTM. Simon.TM.
western blotting protocol. For the samples, adult human epidermal
keratinocytes (HEKa) from Life Technologies (C-005-5C) can be grown
at 37.degree. C. and 5% CO.sub.2 for 24 hours in Epilife growth
media with calcium from Life Technologies (M-EP-500-CA)
supplemented with Keratinocyte Growth Supplement (HKGS) from Life
Technologies (S-101-5). HEKa are then incubated in growth medium
with test compound/extract, no compound/extract for negative
control, or with 1 mM CaCl.sub.2 for positive control for 24 to 48
hours. The HEKa are then washed, collected, and stored on ice or
colder until lysed on ice using a lysis buffer and sonication. The
protein concentrations of the samples can be determined and used to
normalize the samples. The lysates are stored at -80.degree. C.
until use in the bioassay.
[0123] The PROTEINSIMPLE.RTM. Simon.TM. western blotting bioassay
assay employs a quantitative western blotting immunoassay technique
using an antibody specific for occludin to quantitatively detect
occludin in the test samples. Cell samples are lysed and normalized
for protein concentration. Normalized samples and molecular weight
standards are then loaded and ran on a denatured protein separation
gel using capillary electrophoresis. The proteins in the gel are
then immobilized and immunoprobed using a primary antibody specific
for occludin. The immobilized proteins are immunoprobed with an
enzyme-linked detection antibody that binds the primary antibody. A
chemiluminescent substrate solution is then added to the
immobilized proteins to allow chemiluminescent development in
proportion to the amount of occludin bound in the immobilization.
The chemiluminescent development can be stopped at a specific time
and the intensity of the chemiluminescent signal can be measured
and compared to positive and negative controls.
[0124] Keratinocyte Monolayer Permeability: Changes in the
permeability of a keratinocyte monolayer due to each of the active
ingredients, any one of the combination of ingredients, or
compositions having said combinations disclosed in the
specification can be measured. Keratinocyte monolayer permeability
is a measure of skin barrier integrity. Keratinocyte monolayer
permeability in treated and non-treated keratinocytes can be
determined using, as a non-limiting example, the In Vitro Vascular
Permeability assay by Millipore (ECM642). This assay analyzes
endothelial cell adsorption, transport, and permeability. Briefly,
adult human epidermal keratinocytes from Life Technologies
(C-005-5C) can be seeded onto a porous collagen-coated membrane
within a collection well. The keratinocytes are then incubated for
24 hours at 37.degree. C. and 5% CO.sub.2 in Epilife growth media
with calcium from Life Technologies (M-EP-500-CA) supplemented with
Keratinocyte Growth Supplement (HKGS) from Life Technologies
(S-101-5). This incubation time allows the cells to form a
monolayer and occlude the membrane pores. The media is then
replaced with fresh media with (test sample) or without
(non-treated control) test compounds/extracts and the keratinocytes
are incubated for an additional 48 hours at 37.degree. C. and 5%
CO.sub.2. To determine permeability of the keratinocyte monolayer
after incubation with/without the test compound/extract, the media
is replaced with fresh media containing a high molecular weight
Fluorescein isothiocyanate (FITC)-Dextran and the keratinocytes are
incubated for 4 hours at 37.degree. C. and 5% CO.sub.2. During the
4 hours incubation, FITC can pass through the keratinocytes
monolayer and porous membrane into the collection well at a rate
proportional to the monolayer's permeability. After the 4 hour
incubation, cell viability and the content of FITC in the
collection wells can be determined. For the FITC content, the media
in the collection well is collected and fluorescence of the media
determined at 480 nm (Em) when excited at 520 nm. Percent
permeability and percent change in comparison to the non-treated
controls can be determined by the following equations: Percent
Permeability=((Mean Ex/Em of test sample)/Mean Ex/Em untreated
control)*100; Percent Change=Percent Permeability of test
sample-Percent Permeability of untreated control.
[0125] Production of Hyaluronic Acid: Changes in the production of
hyaluronic acid in human dermal fibroblasts due to each of the
active ingredients, any one of the combination of ingredients, or
compositions having said combinations disclosed in the
specification can be measured. HA is a polysaccharide involved in
stabilization of the structure of the matrix and is involved in
providing turgor pressure to tissue and cells. As one non-limiting
example, HA production in treated and non-treated adult human
dermal fibroblasts (HDFa) cells can be determined using the
Hyaluronan DuoSet ELISA kit from R&D Systems (DY3614). In this
assay, for production of samples, subconfluent HDFa cells from
Cascade Biologics (C-13-5C) are incubated at 37.degree. C. and 10%
CO.sub.2 in starvation medium (0.15% fetal bovine serum and 1%
Penicillin Streptomycin solution in Dulbecco's Modified Eagle
Medium) for 72 hours prior to treatment. The cells are then
incubated with fresh starvation medium with either test compound,
positive control (phorbol 12-myristate 13-acetate from
Sigma-Aldrich (P1585) and platelet derived growth factor from
Sigma-Aldrich (P3201)), or no additive for 24 hours. Media is then
collected and frozen at -80.degree. C. until use in the ELISA
assay.
[0126] Briefly, the ELISA assay employs a quantitative sandwich
enzyme immunoassay technique whereby a capture antibody specific
for HA can be pre-coated onto a microplate. Standards and media
from treated and untreated cells are pipetted into the microplate
wells to enable any HA present to be bound by the immobilized
antibody. After washing away any unbound substances, an
enzyme-linked detection antibody specific for HA is added to the
wells. Following a wash to remove any unbound antibody-enzyme
reagent, a substrate solution is added to the wells to allow color
development in proportion to the amount of HA bound in the initial
step. The color development is stopped at a specific time and the
intensity of the color at 450 nm can be measured using a microplate
reader.
[0127] Inhibition of Hyaluronidase Activity: Changes in the
activity of hyaluronidase due to each of the active ingredients,
any one of the combination of ingredients, or compositions having
said combinations disclosed in the specification can be measured.
Hyaluronidase is an enzyme that degrades HA. HA is a polysaccharide
involved in stabilization of the structure of the matrix and is
involved in providing turgor pressure to tissue and cells. As one
non-limiting example, hyaluronidase activity can be determined
using an in vitro protocol modified from Sigma-Aldrich protocol #EC
3.2.1.35. Briefly, hyaluronidase type 1-S from Sigma-Aldrich
(H3506) is added to microplate reaction wells containing test
compound or controls. Tannic acid can be used as a positive control
inhibitor, no test compound can be added for the control enzyme,
and wells with test compound or positive control but without
hyaluronidase can be used as a background negative control. The
wells are incubated at 37.degree. C. for 10 minutes before addition
of substrate (HA). Substrate is added and the reactions incubated
at 37.degree. C. for 45 minutes. A portion of each reaction
solution is then transferred to and gently mixed in a solution of
sodium acetate and acetic acid pH 3.75 to stop that portion of the
reaction (stopped wells). The stopped wells and the reaction wells
should both contain the same volume of solution after addition of
the portion of the reaction solution to the stopped wells. Both the
reaction wells and the stopped wells are incubated for 10 minutes
at room temperature. Absorbance at 600 nm is then measured for both
the reaction wells and the stopped wells. Inhibition can be
calculated using the following formulas: Inhibitor (or control)
activity=(Inhibitor stopped wells absorbance at 600 nm--inhibitor
reaction wells absorbance at 600 nm); Initial activity=control
enzyme absorbance at 600 nm; Percent Inhibition=[(Initial
activity/Inhibitor Activity)*100]-100.
[0128] Peroxisome Proliferator-Activated Receptor Gamma
(PPAR-.gamma.) Activity: Changes in the activity of PPAR-.gamma.
due to each of the active ingredients, any one of the combination
of ingredients, or compositions having said combinations disclosed
in the specification can be measured. PPAR-.gamma. is a receptor
critical for the production of sebum. As one non-limiting example,
the activity of PPAR-.gamma. can be determined using a bioassay
that analyzes the ability of a test compound or composition to
inhibit binding of a ligand. Briefly, fluorescent small-molecule
pan-PPAR ligand, FLUORMONE.TM. Pan-PPAR Green, available from Life
Technologies (PV4894), can be used to determine if test compounds
or compositions are able to inhibit binding of the ligand to
PPAR-.gamma.. The samples wells include PPAR-.gamma. and
fluorescent ligand and either: test compound or composition (test);
a reference inhibitor, rosiglitazone (positive control); or no test
compound (negative control). The wells are incubated for a set
period of time to allow the ligand opportunity to bind the
PPAR-.gamma.. The fluorescence polarization of each sample well can
then be measured and compared to the negative control well to
determine the percentage of inhibition by the test compound or
composition.
[0129] Cytokine array: Human epidermal keratinocytes are cultured
to 70-80% confluency. The media in the plate is aspirated and
0.025% trypsin/EDTA is added. When the cells became rounded, the
culture dish is gently tapped to release the cells. The
trypsin/EDTA containing cells are removed from the culture dish and
neutralized. Cells are centrifuged for 5 min. at 180.times.g to
form a pellet of cells. The supernatant is aspirated. The resulting
pellet is resuspended in EpiLife media (Cascade Biologics). The
cells are seeded in 6-well plates at approximately 10-20%
confluency. After the cells became approximately 80% confluent, the
media is aspirated and 1.0 ml of EpiLife.TM., along with phorbol
13-Myristate 12-acetate ("PMA") (a known inducer of inflammation)
and the test composition dilutions are added to two replicate wells
(i.e., 1.0% (100 .mu.l of 100X stock) and 0.1% (10 .mu.l of 100X
stock) test compositions are diluted into a final volume of 1 ml
EpiLife Growth Medium). The media is gently swirled to ensure
adequate mixing. In addition, 1.0 ml of EpiLife.TM. is added to the
control wells, with and without additional PMA. The plates are then
incubated at 37.+-.1.degree. C. and 5.0.+-.1% CO.sub.2 for
approximately 5 hours after dosing. Following this 5-hour
incubation, all media is collected in conical tubes and frozen at
-70.degree. C.
[0130] For analysis, a 16-pad hybridization chamber is attached to
16-pad FAST slides arrayed in triplicate with 16 anti-cytokine
antibodies plus experimental controls (Whatman BioSciences), and
the slides are placed into a FASTFrame (4 slides per frame) for
processing. Arrays are blocked for 15 min. at room temp. using 70
ml S&S Protein Array Blocking buffer (Whatman Schleicher and
Scheull). Blocking buffer is removed and 70 ml of each supernatant
sample is added to each array. Arrays are incubated for 3 hours at
room temp. with gentle agitation. Arrays are washed 3 times with
TBS-T. Arrays are treated with 70 ml of an antibody cocktail,
containing one biotinylated antibody corresponding to each of the
arrayed capture antibodies. Arrays are incubated for 1 hour at room
temp. with gentle agitation. Arrays are washed 3 times with TBS-T.
Arrays are incubated with 70 ml of a solution containing
streptavidin-Cy5 conjugate for 1 hour at room temp. with gentle
agitation. Arrays are washed 3 times with TBS-T, quickly rinsed in
de-ionized water, and dried.
[0131] Slides can be imaged in a Perkin-Elmer ScanArray 4000
confocal fluorescent imaging system. Array images can be saved and
analyzed using Imaging Research ArrayVision software. Briefly, spot
intensities are determined by subtracting background signal. Spot
replicates from each sample condition can be averaged and then
compared to the appropriate controls.
[0132] Endothelial Tube Formation: Endothelial tube formation is
involved in angiogenesis and micro-vessel capillary formation.
Capillary formation and angiogenesis may contribute to redness and
rosacea of the skin. The ability for endothelial cells to form
tubes in the presence or absence of test extracts and compounds may
be determined using a capillary tubule disruption assay with
pre-formed primary human umbilical vein endothelial cells (HUVEC)
in a cell culture system.
[0133] Briefly, HUVECs are cultured in vitro on Extracellular
Matrix, which stimulates the attachment and tubular morphogenesis
of endothelial cells to form capillary-like lumen structures. These
in vitro formed capillary tubules are similar to human blood vessel
capillaries in many aspects. The capillary tube assay is based on
this phenomenon and is used for evaluation of potential vasculature
targeting agents.
[0134] HUVEC cultures are grown in a 5% CO.sub.2 37.degree. C. cell
incubator. The full growth medium for HUVECs is Endothelial Cell
Basal Medium (EBM) supplemented with 2% fetal bovine serum (FBS),
12 .mu.g/ml bovine brain extract, 1 .mu.g/ml hydrocortisone, and 1
.mu.g/ml GA-1000 (gentamicin-amphothericin). HUVEC cultures between
passage 3 and 8 may be used for all assay experiments.
[0135] HUVECs are pre-labeled with fluorescent agent Calcein AM and
seeded in Extracellular Matrix coated 96-well culture plate with
their full growth medium. After about four hours of the
morphogenesis process, the endothelial capillary tubes should be
formed. Then, test agent in designed doses in 50 .mu.l volume is
applied into the formed capillary tubule cultures as treatment
conditions. The no-treatment controls can be added with vehicle of
test agents. Sutent, a FDA approved anti-angiogenic drug one
concentration can be included as assay performance control. After
about six hours of treatment, the endothelial tubule morphology in
each well is examined by microscopy, imaged, and the capillary
disrupting activities under treatment conditions can be
quantitatively analyzed. Each test conditions can be conducted in
duplicate wells, including controls.
[0136] All of the compositions and/or methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and/or methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
References
[0137] The following references, to the extent that they provide
exemplary procedural or other details supplementary to those set
forth herein, are specifically incorporated herein by
reference.
[0138] 1. Cosmetic Ingredient Dictionary, Third Edition, CTFA,
1982
[0139] 2. International Cosmetic Ingredient Dictionary, Fourth
edition, CTFA, 1991
[0140] 3. International Cosmetic Ingredient Dictionary and
Handbook, Tenth Edition, CTFA, 2004
[0141] 4. International Cosmetic Ingredient Dictionary and
Handbook, Twelfth Edition, CTFA, 2008
* * * * *