U.S. patent application number 10/188206 was filed with the patent office on 2003-02-06 for stable, homogeneous natural product extracts containing polar and apolar fractions.
This patent application is currently assigned to Collaborative Technologies, Inc.. Invention is credited to Aust, Duncan T., Wilmott, James M..
Application Number | 20030026856 10/188206 |
Document ID | / |
Family ID | 26826094 |
Filed Date | 2003-02-06 |
United States Patent
Application |
20030026856 |
Kind Code |
A1 |
Aust, Duncan T. ; et
al. |
February 6, 2003 |
Stable, homogeneous natural product extracts containing polar and
apolar fractions
Abstract
Disclosed are stable, homogeneous dispersions, comprising (a)
from about 20 to 90% by weight of a first composition comprising
(i) about 60-95% by weight of a first polar solvent, (ii) about
0-40% by weight of one or more second polar solvents, and (iii)
water soluble components of a first natural product; and (b) from
about 10 to 60% by weight of a second composition comprising: (i)
one or more apolar solvents, and (ii) oil soluble organic
components of a second natural product; and (c) from about 0.01 to
8% by weight of a non-surface active lipid phosphate or a surface
active agent. Also disclosed are methods of forming the stable,
homogeneous dispersions of the invention, comprising forming a
first composition comprising water soluble components of a first
natural product; forming a second composition comprising oil
soluble organic components of a second natural product; mixing the
compositions and subjecting the mixture to high pressure high shear
processing to form a stable, homogeneous dispersion.
Inventors: |
Aust, Duncan T.; (Ridge,
NY) ; Wilmott, James M.; (Shoreham, NY) |
Correspondence
Address: |
DARBY & DARBY P.C.
Post Office Box 5257
New York
NY
10150-5257
US
|
Assignee: |
Collaborative Technologies,
Inc.
|
Family ID: |
26826094 |
Appl. No.: |
10/188206 |
Filed: |
July 1, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10188206 |
Jul 1, 2002 |
|
|
|
09544649 |
Apr 6, 2000 |
|
|
|
60127930 |
Apr 6, 1999 |
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Current U.S.
Class: |
424/725 ;
424/757; 424/766 |
Current CPC
Class: |
A61Q 19/08 20130101;
A61K 2800/75 20130101; A61K 36/28 20130101; A61K 9/1075 20130101;
A61K 36/87 20130101; A61K 36/605 20130101; A61Q 19/02 20130101;
A61K 2800/522 20130101; A61K 36/484 20130101; Y10S 514/937
20130101; A61K 36/53 20130101; A61K 8/9789 20170801 |
Class at
Publication: |
424/725 ;
424/757; 424/766 |
International
Class: |
A61K 035/78 |
Claims
What is claimed
1. A stable, homogeneous dispersion comprising a water soluble
natural product extract and an oil soluble natural product extract,
wherein said composition does not comprise a surface active
agent.
2. The composition of claim 1 wherein said composition comprises a
non-surface active lipid phosphate.
3. A method of forming a stable, homogeneous dispersion comprising
water soluble components of a natural product extract and oil
soluble components of a natural product extract, said composition
formed by the steps of: obtaining a first natural product extract
comprising a polar solvent and water based components of a natural
product; obtaining a second natural product extract comprising an
apolar solvent and oil soluble components of a natural product;
combining said first extract and said second extract to form a
first composition; adding about 0.01 to 8% by weight of a
non-surface active lipid phosphate or a surface active agent; and
subjecting said mixture to high pressure high shear processing to
form a stable, homogeneous dispersion.
4. The method of claim 3 wherein said step of subjecting said
composition to high pressure high shear processing occurs at a
pressure of about 11,000 to about 27,000 psi and a shear rate which
is sufficient to form a dispersion having an average particle size
of from about 200 to about 1,000 nm.
5. The dispersion of claim 3, wherein said polar solvent is
selected from the group consisting of water; a mono, di, tri or
polyhydroxy alkyl derivative; a mono, di, tri or polyhalogenated
alkyl derivative; a mono, di, tri or poly alkyl ether derivative;
or a mono, di, tri or poly carboxy alkyl derivative, or mixtures
thereof.
6. The method of claim 3, wherein said apolar solvent is selected
from the group consisting of a mono, di, tri or polyalkyl ester or
ether of a mono, di, tri or polyhydroxy compound; a saturated,
unsaturated, linear, branched, or cyclic hydrocarbon; a saturated,
unsaturated, linear or branched C.sub.8 to C.sub.30 fatty acid; a
branched, linear, or cyclical silicone or silicone derivative; or a
homopolymer or heteropolymer fluid formed by the polymerization of
alkylene oxide monomers.
7. The method of claim 6, wherein said apolar solvent is selected
from the group consisting of vegetable oil, soybean oil, babasu
oil, castor oil, cottonseed oil, grapeseed oil, rice bran oil,
canola oil, palm oil, palm kernel oil, olive oil, linseed oil,
coconut oil, sunflower oil, safflower oil, peanut oil, corn oil,
mineral oil, petrolatum, hydrogenated polyisobutene, permethyl
fluids, polyisobutene, polybutene, cyclomethicone, dimethicone
polysiloxane, dimethicinol, polysiloxanes, polyalkyl siloxanes,
polyarylsiloxanes, polyalkylaryl siloxanes, polysiloxane
copolymers, polypropylene oxide, polybutylene oxide, isopropyl
palmitate, diisopropyl adipate or mixtures thereof.
8. The method of claim 3, wherein said first composition further
comprises a non-surface active lipid phosphate.
9. The method of claim 3, wherein said polar solvent is a mixture
of water and propylene glycol.
10. The method of claim 3, wherein said first and second natural
products are the same product.
11. The method of claim 3, wherein said first and second natural
products are extracted from different natural products.
12. The method of claim 3, wherein said first and second natural
products are extracted from a mixture of different natural
products.
13. The method of claim 3, wherein said first and second natural
products are extracted from a mixture of different natural
products, respectively.
14. The method of claim 3, wherein said natural product is selected
from the group consisting of mulberry, lavender, licorice root,
arnica, eyebright and grape root.
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 09/544,649, filed Apr. 6, 2000, which claims priority
under 35 U.S.C. .sctn. 119(e) of provisional application serial No.
60/127,930, filed Apr. 6, 1999.
FIELD OF THE INVENTION
[0002] This invention is directed to the field of compositions
comprising natural product extracts for use in personal care
products or pharmaceuticals.
BACKGROUND OF THE INVENTION
[0003] Natural products and natural product extracts are often used
in cosmetic and pharmaceutical applications. Natural products,
particularly botanically sourced natural products, have
demonstrable beneficial properties on the skin and hair. Extracts
of these natural products have demonstrated antimicrobial,
antiseptic, anti-inflammatory, antioxidant, enzyme stimulation or
inhibition, pigmentation enhancement or control, photoprotective
and many other physiological benefits.
[0004] Typically, the solvent systems used to produce these
extracts are polar in nature, and most typically are water or
glycols or a combination thereof. These polar solvents enable the
extraction of only a similarly polar material from the biomass of
the natural product material. Similarly, apolar solvents have been
used to remove the apolar fractions from the biomass of the natural
product materials. There is currently no single universal solvent
capable of only extracting both the desired polar and apolar
fractions. As a result, typical natural product extracts only
provide a portion of the physiologically or aesthetically
beneficial components.
[0005] It has not been possible to date to combine an aqueous
extract with a lipid or other apolar phase into a single system
without the use of surface active agents and special processing
conditions to form emulsions or dispersions. Surface active agents
permit the mixing of a hydrophilic phase and a hydrophobic phase by
lowering the surface tension between the two phases, thereby
creating micellar structures which, when mixed with a suitable
processing procedure, produce stable systems. However, these
surface active agents may be irritating to users, and the processes
used to prepare the emulsions or dispersion make them difficult to
reproduce.
[0006] Since many bioreactive or aesthetic components of the
natural product are located within the structure of the cell wall
or other organelles within the cell, a suitable process is required
to extract the desired components from the cell. Simple solvent
extraction is usually insufficient to remove the protected material
from the cell. The cell wall barrier must be perturbed or ruptured
sufficiently to allow diffusion to occur into the extracting
solvent. A method is therefore needed to rupture cell walls and
membranes to maximize the removal of the physiologically active or
aesthetically pleasing materials. Examples of such process
conditions include the use of heat, high shear mixing, ultrasonic
waves, microwaves, high pressure and prolonged polar or apolar
dialysis.
[0007] It is preferred to combine the process described above with
the use of separate polar and apolar solvents to remove the maximum
amount of all of the materials of interest. It is further preferred
to combine both the polar and apolar extracts into a single,
homogeneous preparation without the use of surface active agents
which can cause irritation.
OBJECTS OF THE INVENTION
[0008] It is therefore an object of the present invention to
provide an improved method of obtaining a composition comprising
both the hydrophobic and hydrophilic components of a natural
product or blend of natural products.
[0009] It is another object of the present invention to provide a
single composition having both oil soluble and water soluble
components of a natural product extract, in order to take advantage
of synergy which is often associated with the use of multiple
components of a natural product.
[0010] It is another object of the present invention to provide a
greater functional activity due to the presence of a full
complement of polar and apolar materials with physiologically
beneficial or aesthetically pleasing properties.
[0011] It is another object of the present invention to provide a
composition having greater potency and stability of the desired
components, in comparison to prior art compositions.
[0012] It is another object of the present invention to provide
better delivery of the physiologically beneficial active agents to
sites within the skin or hair, where the active agents are needed
for activity.
[0013] It is another object of the present invention to provide
ease of manufacture of a finished system containing the full
complement of extracts, yielding cost savings and greater
reproducibility with consistent quality standards.
[0014] It is another object of the present invention to provide a
greater versatility to compositions of natural product extracts
that can be achieved by preparing prior art homogeneous or
heterogeneous complexes.
[0015] It is another object of the present invention to provide a
composition useful for personal care, pharmaceutical or cosmetic
applications, having low irritation due to the absence of
surface-active agents that lower the surface tension between
immiscible phases.
[0016] Applicants have discovered methods of forming stable,
homogeneous compositions comprising both polar and apolar fractions
of natural product extracts. The applicants have now discovered
that aqueous or aqueous/glycolic extracts can be successfully mixed
with apolar extracts (e.g. silicone extracts) using high pressure,
high shear processing to produce a single, stable homogeneous
system that contains the entire complement of polar and apolar
fractions from a selected natural product.
SUMMARY OF THE INVENTION
[0017] The invention is directed to stable, homogeneous dispersions
comprising a water-soluble natural product extract and an oil
soluble natural product extract. In certain embodiments, the
dispersion does not comprise a surface active agent.
[0018] In one embodiment, the invention is directed to stable,
homogeneous dispersions comprising a first composition which
contains one or more polar solvents and water (or polar solvent)
soluble components of a first natural product; and a second
composition comprising one or more apolar solvents and oil (or
apolar solvent) soluble organic components of a second natural
product. Optionally, the dispersion may also comprise from about
0.01 to 8% by weight of a non-surface active lipid phosphate or a
surface active agent.
[0019] In preferred embodiments, the invention is directed to
stable, homogeneous dispersions comprising
[0020] (a) from about 20 to 90% by weight of a first composition
comprising:
[0021] (i) about 60-95% by weight of a first polar solvent;
[0022] (ii) about 0-40% by weight of one or more second polar
solvents; and
[0023] (iii) water soluble components of a first natural product;
and
[0024] (b) from about 10 to 60% by weight of a second composition
comprising:
[0025] (i) one or more apolar solvents; and
[0026] (ii) oil soluble organic components of a second natural
product; and
[0027] (c) from about 0.01 to 8% by weight of a non-surface active
lipid phosphate or a surface active agent.
[0028] In particular embodiments, the first polar solvent may be
water and the second polar solvent is selected from the group
consisting of water; a mono, di, tri or polyhydroxy alkyl
derivative; a mono, di, tri or polyhalogenated alkyl derivative; a
mono, di, tri or poly alkyl ether derivative; and a mono, di, tri
or poly carboxyl alkyl derivative. The apolar solvent may be one or
more of an oil (such as a vegetable oil); a mono, di, tri or
polyalkyl ester or ether of a mono, di, tri or polyhydroxy
compound; a saturated, unsaturated, linear, branched, or cyclic
hydrocarbon; a saturated, unsaturated, linear or branched C.sub.8
to C.sub.30 fatty acid; a branched, linear, or cyclical silicone or
silicone derivative; or a homopolymer or heteropolymer fluid formed
by the polymerization of alkylene oxide monomers.
[0029] The dispersion may comprise components from any of the
natural products which are known to contain physiological
properties. Examplary natural products are mulberry, lavender,
licorice root, arnica, eyebright and grape root. The invention
contemplates the use of water and oil soluble components of natural
products which may be obtained by any of the methods known to those
skilled in the art.
[0030] The invention is also directed to methods of forming the
dispersions of the invention. In one embodiment, the process
includes the steps of obtaining a first natural product extract
comprising a polar solvent and water based components of a natural
product; obtaining a second natural product extract comprising an
apolar solvent and oil soluble components of a natural product;
combining the first and second extracts to form a composition;
optionally adding about 0.01 to 8% by weight of a non-surface
active lipid phosphate or a surface active agent; and subjecting
said mixture to high pressure high shear processing to form a
stable, homogeneous dispersion.
[0031] Preferred rates of high pressure, high shear processing are
at a pressure of about 11,000 to about 27,000 psi, and at a shear
rate which is sufficient to form a dispersion having an average
particle size of from about 200 to about 1,000 nm.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 is a chromatogram of a composition containing the
polar solvent soluble components of mulberry;
[0033] FIG. 2 is a chromatogram of a composition containing the
apolar solvent soluble components of mulberry;
[0034] FIG. 3 depicts the tyrosinase inhibitory activity of Example
15, comprising both the polar and apolar solvent soluble organic
components of mulberry;
[0035] FIG. 4 is a chromatogram of a composition containing the
apolar solvent soluble components of licorice;
[0036] FIG. 5 is a chromatogram of a composition containing the
polar solvent soluble components of licorice;
[0037] FIG. 6 depicts the tyrosinase inhibitory activity of Example
17, comprising both the polar and apolar solvent soluble organic
components of licorice;
[0038] FIG. 7 depicts the melanocyte pigmentation reduction of
Example 17, comprising both the polar and apolar solvent soluble
organic components of licorice;
[0039] FIG. 8 is a chromatogram of a composition containing the
polar solvent soluble components of lavender;
[0040] FIG. 9 is a chromatogram of a composition containing the
apolar solvent soluble components of lavender;
[0041] FIG. 10 depicts the results of a neutrophil elastase
inhibition assay of Example 16, containing both the polar and
apolar solvent soluble organic components of lavender, and a
comparison with the assay results for conventional
prostaglandin;
[0042] FIG. 11 depicts the results of inhibition of UV-induced
PGE.sub.2 release of Example 18, containing both the polar and
apolar solvent soluble organic components of arnica;
[0043] FIG. 12 is a chromatogram a composition containing the polar
solvent soluble components of eyebright;
[0044] FIG. 13 is a chromatogram of a composition containing the
apolar solvent soluble components of eyebright;
[0045] FIG. 14 depicts the results of a cytochrome C reduction
assay of Example 19, containing both the polar and apolar solvent
soluble organic components of eyebright; and
[0046] FIG. 15 depicts the results of a cytochrome C reduction
assay of Example 20, containing both the polar and apolar solvent
soluble organic components of grape root.
DETAILED DESCRIPTION OF THE INVENTION
[0047] All patents, applications, test methods and publications
referenced in this specification are hereby incorporated by
reference in their entirety. In case of conflict, the present
description, including definitions, will prevail.
[0048] As used herein, the terms "water soluble" and "polar solvent
soluble" are used interchangeably, and refer to the property of
being soluble at approximate room temperature and one atmosphere
pressure in water or another polar solvent commonly used in
preparing cosmetic formulations, such as a glycol.
[0049] As used herein, the term "polar solvent soluble components"
refers to the components of a natural product which may be
extracted from the natural product by contacting the natural
product with a polar solvent, for example water or glycol or
mixtures thereof.
[0050] As used herein, the terms "oil soluble" and "apolar solvent
soluble" are used interchangeably, and refer to the property of
being soluble at approximate room temperature and one atmosphere
pressure in an oil or other apolar solvent commonly used in
preparing cosmetic formulations, such as silicone based solvents or
oils or mixtures thereof.
[0051] As used herein, the term "organic oil soluble components"
refers to the components of a natural product which may be
extracted from the natural product by contacting the natural
product with an apolar solvent, such as silicone or oils or
mixtures thereof.
[0052] As used herein, the term "extracts" or "natural product
extracts" are used interchangeably, and refer to the organic
components of a natural product which can be obtained by contacting
the natural product with a polar or apolar solvent.
[0053] As used herein the term "surface-active" or "surface-active
agent" refers to a substance capable of reducing the surface
tension of a liquid in which it is dissolved. A "non-surface active
agent" is a substance which does not effectively reduce the surface
tension of a liquid in which it is dissolved.
[0054] As used herein, the term "surfactant" refers to a
surface-active substance.
[0055] As used herein, the term "surfactant-free dispersion" refers
to a stable or dispersion that is produced without the use of
surface-active ingredients or surfactants.
[0056] The present invention is directed in part to compositions
containing natural product extracts which can be used in personal
care and pharmaceutical formulations and products. In some
embodiments, the invention is directed to a natural product
extracts or combinations of natural product extracts containing
hydrophilic and hydrophobic materials which are processed to
produce a composition with physiologically beneficial and
aesthetically pleasing properties. These natural products can be
either animal or vegetable derived. In preferred embodiments, the
invention is directed to compositions comprising natural product
extracts from land-based or marine-derived botanicals.
[0057] In one embodiment, the compositions of this invention are
dispersions which may comprise both polar solvent soluble
components of a natural product and apolar solvent soluble
components of a natural product. The polar solvent soluble
components may be present in a composition also comprising one or
more polar solvents, and the polar solvent soluble components may
be present in a second composition, also comprising one or more
apolar solvents.
[0058] The first composition used to form the dispersion may
contain the organic polar solvent soluble components of a natural
product. In preferred embodiments, the dispersion may comprise a
first composition, which contains polar solvent soluble components
of a natural product. The first composition may contain first and
second polar solvents. The first polar solvent may be present in
the amount of about 60 to 95% by weight of the first composition.
The first composition may also comprise a second polar solvent,
which may be present in the amount of about 0 to 40% by weight.
[0059] In preferred embodiments, the first polar solvent is water
and the second polar solvent is a glycol, such as propylene glycol
or ethylene glycol.
[0060] The first composition may be present in the amount of about
20 to 90% by weight of the dispersion, and is preferably present in
the amount of 40 to 80 wgt %, more preferably 50 to 70 wgt %, most
preferably 60 to 65 wgt %.
[0061] The dispersion also comprises the organic apolar solvent
soluble components of a natural product, for example in the form of
a second composition which contains apolar solvent soluble organic
components of a natural product and an apolar solvent.
[0062] The second composition maybe present in the amount of about
10 to 60% by weight of the dispersion, and is preferably present in
the amount of 20 to 50 wgt %, more preferably 30 to 40 wgt %, and
most preferably about 30 wgt %.
[0063] In certain embodiments, the natural product extracted in the
first and second composition are the same. However, the invention
also contemplates dispersions in which the natural product extracts
present in the first and second compositions are obtained (or
extracted) from different natural products. In addition, the
invention contemplates dispersions in which either (or both) of the
first and second compositions comprises extracts or components of
more than one natural product.
[0064] In preferred embodiments, the dispersion is a surfactant
free dispersion, and does not contain a surface active agent.
[0065] Phospholipids are one agent which may be used to achieve
surfactant-free dispersions of the invention. Phospholipids in
general are not soluble in water. In contrast, surface active
materials reduce the surface tension of the liquid in which it is
dissolved.
[0066] For example, when lecithin is refined and the concentrations
of phosphatidyl choline increase, the surface tension of
dispersions of the phospholipid in water increase. At
concentrations of 50% phosphatidyl choline and higher, the surface
tension exceeds the surface tension of water alone by a very
considerable level. The conditions involved for the surface tension
measurement are for a condensed, solid monolayer of phosphatidyl
choline. The ability to achieve the monolayer is dependent on first
processing the phosphatidyl choline above its phase transition
temperature of 41.degree. C., and then allowing the condensed
monolayer to cool. This notion of phase transition associated with
increased energy is important in consideration of phospholipids,
and particularly phosphatidyl choline, as a means of achieving
surfactant-free emulsions.
[0067] In particular embodiments, the dispersion may also comprise
from 0.01% to 8% by weight (preferably 0.01 to 5% by weight) of one
or more lipids. Examplary lipids include Phospholipon 80, 80H
(American Lecithin) and Basis LP2OH (Ikeda Corp., Japan).
[0068] The components of the natural products may be obtained by
various methods known to those of ordinary skill in the art. Polar
solvent soluble components of a natural product may be obtained by
contacting the natural product with a polar solvent to form a
solution containing the polar solvent soluble components,
optionally mixing the solution and optionally thereafter diluting
the solution with another polar solvent. For example, a natural
product can be contacted with water to form a solution containing
water soluble components of the natural product, the solution may
be mixed, and thereafter may be diluted with glycol. Alternatively,
the natural product can be contacted with glycol to form a solution
containing organic water soluble components of the natural product,
the solution may be mixed, and thereafter diluting the solution
with water. In still another method, the natural product can be
contacted with both water and glycol to form a solution, and
optionally mixed.
[0069] Polar solvent soluble (or water soluble) components of a
natural product are obtained by contacting the natural product with
a polar solvent, for example water, glycol, or mixtures thereof, to
form a solution containing the polar solvent soluble components. If
necessary, the solution may thereafter be mixed or stirred until
the solution is clear.
[0070] Suitable polar solvents include water; glycols; mono, di,
tri or polyhydroxy alkyl derivatives; mono, di, tri or
polyhalogenated alkyl derivatives; mono, di, tri or polyalkyl ether
derivatives; and mono, di, tri or polycarboxyl acid derivatives and
mixtures thereof. Exemplary glycols include ethylene glycol,
propylene glycol, 1,3-butylene glycol and glycerin.
[0071] Organic apolar solvent soluble (or oil soluble) components
of a natural product are obtained by contacting the natural product
with an apolar solvent, for example cyclomethicone, hydrogenated
polyisobutene, or combinations thereof, to form a solution
containing the organic polar solvent soluble components. If
necessary, the solution may thereafter be mixed or stirred until
the solution is clear.
[0072] Suitbable apolar solvents include mono, di, tri or poly
alkyl ester or ether of a di, tri, or polyhydroxy compound, such as
ethylene glycol, propylene glycol, glycerin, sorbitol or other
polyol compound. Examples of such esters and ethers include, but
are not limited to, saturated and unsaturated, linear and branched
vegetable oils, such as soybean oil, babassu oil, castor oil,
cottonseed oil, chinese tallow oil, crambe oil, perilla oil, danish
rapeseed oil, rice bran oil, palm oil, palm kernel oil, olive oil,
linseed oil, coconut oil, sunflower oil, safflower oil, peanut oil
and corn oil. Preferred saturated and unsaturated vegetable oils
are those having fatty acid components with 6 to 24 carbon atoms. A
more preferred vegetable oil is soybean oil.
[0073] Additional exemplary apolar solvents include compounds
having the formula C.sub.nH.sub.(2n+2-m) where n is an integer
greater than or equal to 6 and m is 0 or an even integer no greater
than n. Such compounds include, but are not limited to, saturated
and unsaturated, linear, branched, and cyclic hydrocarbon chains.
Preferred examples of such compounds include, but are not limited,
mineral oil, petrolatum, permethyl fluids, polybutylenes, and
polyisobutylenes.
[0074] Further apolar solvents contemplated for use in the
invention have the formula 1
[0075] or the formula 2
[0076] where R.sub.1 is a saturated or unsaturated, linear,
branched or cyclic C.sub.1-C.sub.24 alkyl; R.sub.2 is hydrogen or a
saturated or unsaturated, liner, branched or cyclic
C.sub.1-C.sub.24 alkyl; and n is an integer from 0 to 20. Examples
of such aesthetic modifying agents include, but are not limited to,
isopropyl palmitate and diisopropyl adipate.
[0077] Yet another group of apolar solvents is silicone and
silicone derivatives. Silicone may provide lubrication and/or shine
to the composition. Preferably, the silicone is insoluble in water.
Suitable water-insoluble silicone materials include, but are not
limited to, polyalkylsiloxanes, polyarylsiloxanes,
polyalkylarylsiloxanes, polysiloxane gums and polyethersiloxane
copolymers. Examples of suitable silicone materials are disclosed
in U.S. Pat. Nos. 4,788,006; 4,341,799; 4,152,416; 3,964,500;
3,208,911; 4,364,837 and 4,465,619, all of which are incorporated
herein by reference.
[0078] Another suitable hydrophobic material which can be suspended
in the composition has the formula 3
[0079] where R.sub.1 is a saturated or unsaturated, linear,
branched or cyclic alkyl having 2 to 24 carbon atoms; M.sup.(+) is
N.sup.+R.sub.2R.sub.3R.sub.4R.sub.5; R.sub.2, R.sub.3 and R.sub.4
are hydrogen or a saturated or unsaturated, linear or branched
alkyl or hydroxyalkyl having from 1 to 10 carbon atoms; and R.sub.4
is a saturated or unsaturated, linear, branched or cyclic alkyl or
substituted alkyl having 2 to 24 carbon atoms. An example of such a
material is lauramine oleate.
[0080] Another apolar solvent is a polymer formed by polymerization
of alkylene oxide monomers of the formula 4
[0081] where n is from about 2 to about 24. The polymer may be
either a homogenous polymer or a copolymer. Examples of such
homogenous polymers include, but are not limited to, polypropylene
oxide and polybutylene oxide. Generally, the molecular weight of
these polymers ranges from about 100 to about 10,000 daltons.
Additionally, these polymers may be reacted with mono or
polyhydroxyalkyl alcohol, such as UCON fluids from the Union
Carbide Chemical Company, or with a saturated or unsaturated,
linear, branched or cyclic C.sub.1-C.sub.24 alkyl.
[0082] Suitable apolar solvents include oils (both natural and
synthetic), including hydrogenated oils. Exemplary oils include
vegetable oil, soybean oil, babasu oil, castor oil, cottonseed oil,
grapeseed oil, rice bran oil, canola oil, palm oil, palm kernel
oil, olive oil, linseed oil, coconut oil, sunflower oil, safflower
oil, peanut oil, corn oil, mineral oil and petrolatum. Other apolar
solvents contemplated for use in the invention include hydrogenated
polyisobutene, permethyl fluids, polyisobutene, polybutene,
polypropylene oxide, polybutylene oxide, isopropyl palmitate,
disopropyl adipate or mixtures thereof.
[0083] Exemplary silicone and silicone derivatives include branched
or linear cyclical silicone or silicone derivatives,
cyclomethicone, dimethicone polysiloxane, dimethicinol,
polysiloxanes, polysiloxane copolymers, polyalkyl aryl silanes,
polyaryl siloxanes, polyalkyl siloxanes, polyalkyl aryl silanes,
polysiloxane copolymers, low viscosity dimethicone, phenyl
trimethicone (Dow Corning), silicone fluid DC 345 (Dow Corning),
polysynlane (NOF Corp.).
[0084] Still more suitable apolar solvents include mono, di, tri or
polyalkyl esters or ethers or a mono, di, tri or polyhydroxy
compound; saturated or unsaturated, linear or branched
C.sub.8-C.sub.30 fatty acids; and homopolymer or heteropolymer
fluids formed by the polymerization of alkylene oxide monomers.
[0085] The dispersions of the invention may be produced by mixing a
water soluble natural product extract and an oil soluble natural
product extract, using high pressure/high shear conditions to
produce a homogeneous, fluid dispersion which is stable for a
commercially relevant period of time, e.g. between about 180 to 720
days when stored at approximate room temperature, in a commercial
package. The preferred pressure for preparation of this dispersion
is between about 11,000 to about 27,000 psi, more preferably 11,000
to about 21,000 psi, most preferably between about 11,000 and
16,000 psi. The composition may be produced with a shear that
creates average particle size of between about 100 to about 1,000
nm, more preferably between about 100-500 nm.
[0086] The invention is also directed in part to processes for
efficiently rupturing the cell wall or lipid bilayer membrane of
individual cells or subcellular organelles of the natural product
to maximize the removal of the cellular components.
[0087] In certain embodiments, composition of the invention may
comprise non-surface active lipid phosphate phospholipids,
preservatives such as Germazide.TM. MPB, and nonionic detergents
such as polyoxethylene ethers. The dispersion may also comprise
polyethylene glycol and butylene glycol to improve the freeze thaw
stability of the preparations.
[0088] Exemplary non-ionic detergents include polyoxylated ethers
such as Brij detergents, available from Sigma Aldrich Chemical
Co.
[0089] Suitable natural products contemplated for use in the
invention include any of the natural products which are know in the
art to contain components having physiological attributes.
Exemplary natural products include mulberry (morus alba), lavender
(lavandula angustifolia), licorice root (glycyrrhiza glabra),
arnica (arnica montana), eyebright (euphrasia officinalisa), grape
root (mahonia aquifolum), green tea leaves (camelia sinesis),
rosemary powder, echinacea herb powder, evening primrose flowers,
sea parsley powder (palmaria palmata), calendula leaves and tea
tree leaves.
[0090] Recent research has shown that mulberry (morus alba) and
licorice (glycyrrhiza glabra) root extracts act as tyrosinase
inhibitors. Chemical analysis has shown that the plant contains a
number of interesting biochemicals, in particular oxyresveratrol,
umbelliferone, kowano-A, kowano-B, kowano-C, kowano-F, kowano-G,
kowano-H, chalcomoracin, cyclomorusin, cyclomulberrin,
cyclomulberrochromene moracenin-D, morusin, mulberranol,
mulberrochromene, mulberrofuran-B, oxydihydromorusin, sitosterol
and sitosterol-alpha-glucoside. The chemical or chemical(s)
responsible for enzyme inhibition has not been unequivocally
identified however researchers have shown that oxyresveratrol is a
very potent tyrosinase inhibitor (Shin et al., Oxyresveratrol as
the Potent inhibitor on Dopa Oxidase Activity of a Mushroom
Tyrosinase, BBRC, 1998, vol. 243, pp 801-803).
[0091] Licorice root extract is used traditionally in balms for its
powerful anti-inflammatory, anti-allergic and anti toxin
properties. Recently, researchers have discovered that licorice
root extract inhibits enzyme activities, especially tyrosinase and
11-beta-hydroxysteroid dehydrogenase and as a result it causes skin
whitening and potentates the action of hydrocortisone. It is
therefore of great interest to the pharmaceutical and cosmetic
industries.
[0092] The major component of licorice root, glycyrrhizic acid, is
important to the food industry. It is 50 times sweeter than sucrose
(A. Chevallier, The Encyclopedia of Medical Plants p 99, 1996)
making it a very useful food ingredient. Other active components of
Licorice include the triterpine glycosides, glycyrrhizin,
flavonoids, isoflanonoids, kumatakenin; licoricone, glabrol,
glabrone, glyzarin, licoisoflavones A and B, glycyrol,
formononetin, liquiritigenin, liquiritin, neoliguiritin,
rhamnoliquiritin, glyzaglabin, 7-hydroxy-2-methylisoflavone,
4,7-dihydroxyflavone, glabranine, chalcones, coumarins,
triterpenoids, sterols (including betasitosterol, stigmasterol),
amino acids, gums, wax and volatile oil.
[0093] Lavender is associated with youthfulness. It is carminative,
antiseptic and soothing. It is known amongst herbalists as a
holistic relaxant or as "the balancing" herb. Lavender extracts
have been analyzed and the major components, linalool and linalyl
acetate, are just two among of the hundreds of different active
compounds identified. Other significant components include
triterpenes (e.g. ursolic), flavonoids (e.g. luteolin) and
coumarins (A. Y. Leung et al., Encyclopedia of Common Ingredients
Used in Foods and Cosmetics, 1996, pp 339-342). The ursolic acid
and other components are known to be anti-inflammatory. The
cosmeceutical benefits claimed for ursolic acid include restoration
of overall health and functionality of photoaged skin, in addition
to facilitation of tissue repair. Ursolic acid is a potent elastase
inhibitor (Q. Ying et al., "Inhibition of Human Leukocyte Elastase
by Ursolic Acid; Evidence for a Hydrophobic Binding Site for
Pentacyclic Triterpenes, 1991, Biochem. J. 277, 521-526). Skin is a
very dynamic tissue with degradation taking place along side repair
and renewal. Compositions containing lavender extract improve skin
integrity by slowing elastin degradation with respect to natural
ongoing synthesis.
[0094] The mechanism by which lavender extracts `relax` and reduce
muscle tension has been studied in depth. M. Lis-Balchin et al.,
"Studies on the Mode of Action of the Essential Oil of Lavender
(Lavender Angustifolia)", Phytother. Res., 1999, 13(6), 540-542),
showed that muscle tension is reduced through a postsynaptic action
and not via an atropine-like mechanism and that the action of
linalool reflected that of the whole lavender oil. (H.. M. Kim et
al., "Lavender Oil Inhibits Immediate Type Allergic Reaction in
Mice and Rats," J Pharm Pharmacol, 1999, 51(2), 221-226, were more
interested in lavender's `soothing` properties. They studied the
effects of lavender oil on mast cell-mediated allergic reactions in
mice and rats and showed that the components of lavender oil
inhibit immediate-type allergic reactions by inhibition of mast
cell degranulation in vivo and in vitro. Other researchers have
shown that when lavender is applied topically it stimulates the
local circulation (Chevallier, 1996).
[0095] Evening Primrose (oenothera biennis) is a valuable source of
.gamma.-Linolenic Acid (GLA) and other special fatty acids that are
essential for hormone function, energy flow, cell division, immune
responses and many other aspects of metabolism. These critical
fatty acids are used to make powerful tissue-specific compounds
called eicosanoids.
[0096] GLA is effective at very low concentrations (M. S. Manku et
al., Fatty acids in plasma and red cell membranes in normal humans,
Lipids, 1983, 18(2);906). Doses of 1.4 g/d have resulted in
clinically important reduction of the symptoms of rheumatoid
arthritis (L. J. Leventhal et al., "Treatment of Rheumatoid
Arthritis with a Gammalinolenic Acid," Ann Int Med, 1993, 119(9);
867-73). 1-3 g/d is normally recommended for dietary supplements.
When tiny amounts of GLA are applied to dry skin the skin soon
shows signs of improvement. This is not surprising since one of the
early signs of an individual being deficient in GLA is dry skin.
Topical GLA application has been shown to promote the healthy
growth of skin, hair, and nails and GLA has been used to
successfully treat skin conditions such as a topic eczema. As well
as being good for skin disorders, GLA is reported to be good for
arthritis and autoimmune problems. (Leung et al., 1996, L. Galland,
Increased Requirements for Essential Fatty Acids in Atopic
Individuals; Review with Clinical Descriptions, 1999).
[0097] Individuals with normal metabolism can synthesize GLA from
the essential fatty acid linoleic acid. The synthesis is controlled
by the enzyme .delta.-6-desaturase. Interestingly, GLA corrects
most of the biological effects of zinc deficiency (Y. S. Huang et
al., "Moot Biological Effects of Line Deficiency Corrected by
g-Linoleic Acid (18:3 omega-6) but not Atherosclerosis, by Linoleic
Acid (18:2 omega-6), 1982, 41:193-207) indicating that
.delta.-6-desaturase enzyme has a requirement for zinc that is a
first-order essential function of zinc. GLA therefore, although not
technically a vitamin, is an essential requirement for a
significant number of individuals. Especially those whose
.delta.-6-desaturase enzyme is blocked or its activity reduced.
Interestingly, the ability to synthesize GLA is affected by factors
such as the menstrual cycle and diet. GLA synthesis is reduced in
diabetics or in individuals who fast or consume excessive amounts
of carbohydrate (Leung et al., 1996). GLA levels also decrease with
aging.
[0098] GLA can therefore be thought of in similar terms as a
vitamin or an essential fatty acid. Moreover, GLA is being
extensively studied and has been shown to be effective in killing
cancer cells.
[0099] Eyebright is a plant that has been associated with eye care
for generations. Eyebright infusions are mildly astringent but
gentle enough to use on eyes. They are stimulating and have
antioxidant and anti-inflammatory properties. The plant is
recommended both historically and in modern literature for treating
eye inflammations, particularly for conjunctivitis. Eyebright
infusions bring rapid relief of redness, swelling and are very good
at healing recent eye injuries. They are often recommended where
there is a risk of developing serpiginous corneal ulcers (Leung et
al., 1996).
[0100] Eyebright proves to be most effective when the whole plant
is used. The plant is chopped up and applied as a compress
(Chevallier, 1996). The active biochemicals present in Eyebright
plant include the following: Iridoid glycosides, aucubin, catapol
and erostoside, eukovoside, geniposide and luproside, gallotanins,
caffeic acid and ferulic acids. The volatile oil contains trace
amounts of the essential oils as well as beta-sitosterol, oleic
acid, palmitic acid and stearic acid. Eyebright also contains
miscellaneous unidentified alkaloid, amino acids, flavonoids and
tannins.
[0101] In addition to the water soluble and oil soluble components
of natural product extracts, in some embodiments the compositions
of the invention may include various bioactive ingredients or
cosmeceuticals, including antioxidants, skin whitening agents,
elastase inhibitors, vitamins and active agents having
anti-inflammatory, antiseptic, or soothing properties. The
composition of the invention may be used in connection with the
treatment of skin disorders, including eczema, psoriasis, acne,
photoaging, dermatitis, would healing and dry skin.
[0102] In order to further illustrate the present invention, the
experiments described in the following examples were conducted. It
should be understood that the invention is not limited to the
specific examples or the details described therein. The results
obtained from the experiments described in the examples are shown
in the accompanying figures and tables.
EXEMPLARY EMBODIMENTS OF THE INVENTION
[0103] Natural product extracts (both oil soluble and water soluble
extracts) and natural product extract compositions containing both
oil soluble and water soluble components were formed according to
Examples 1 to 26, as shown in Tables 1 to 3 below.
1TABLE 1 Weight % of Oil Soluble Extracts Silcone Extracts 1 2 3 4
5 6 Mulberry, (Morus alba) dust-powder 2.5 grade (China Products)
Lavender, (Lavandula angustifolia) fine 2.5 chopped plant grade
(Aphrodisia) Licorice root, (glycyrrhiza glabra) powder 2.5 grade
(Lotus) Arnica (Arnica montana), herb powder 2.5 (San Francisco
Herb) Eyebright, (euphrasia officinalisa) fine 2.5 chopped plant
grade (Aphrodisia) Grape root, (mahonia aquifolium) coarse 2.5
chopped grade (Aphrodisia) Silicone fluid DC 345 (Dow Corning) 97.5
97.5 97.5 97.5 97.5 97.5 Silicone Extracts 1a 2a 3a 4a 5a 6a Green
tea (Camelia Sinesis) 2.5 2.5 (China Products) Rosemary powder 2.5
(Aphrodisia) Calendula leaves 2.5 Echinacea herb powder (San
Francisco Herb) 2.5 Mulberry, (Morus alba) dust-powder 2.5 grade
(China Products) 0.65 cst silicone fluid DC200 (Dow 97.5 Corning)
Polysynlane (NOF Corp.) 97.5 Phenyl Trimethicone (Dow Corning) 97.5
Silicone fluid DC 345 (Dow Corning) 97.5 97.5 97.5 Method: Mix for
16 hrs and filter through Whatman #1 paper
[0104]
2TABLE 2 Extracts Composition - (Parts Per) Glycol Extracts 7 8 9
10 11 12 13 14 Mulberry, (Morus alba) dust-powder 1 grade (China
Products) Lavender, (Lavandula angustifolia) fine 1 chopped plant
grade (Aphrodisia) Licorice root, (glycyrrhiza glabra) 1 powder
grade (Lotus) Arnica (Arnica montana), herb powder 1 (San Francisco
Herb) Eyebright, (euphrasia officinalisa) fine 1 chopped plant
grade (Aphrodisia) Grape root, (mahonia aquifolium) 1 coarse
chopped grade (Aphrodisia) Tea Tree Leaves 1 Evening Primrose
Flowers 1 Water 125 125 125 125 125 125 125 125 Propylene glycol
(Kramer) 10 10 10 10 10 10 4.5 4.5 Method: Mix botanical and
propylene glycol for 5 hrs at room temperature and filter through
Whatman #1 paper, then add the water at the above ratio and mix to
produce the final extract as defined herein and preserve with an
appropriate concentration of Germazide .TM.. Glycol Extracts 7a 8a
9a 10a Sea Parsley (Palmaria Palmata) powder 1 Gree Tea (Camelia
sinesis) Leaves 1 Rosemary Powder 1 Calendula Leaves 1 Glycerin 3.5
3.5 3.5 Water 5.5 5.5 5.5 1-3,Butylene Glycol (Kramer) 9 Method:
Mix botanical with polar solvent for 3 hrs at 40.degree. C. and
filter through Whatman #1 paper. An appropriate concentration of
Germazide .TM. was then added.
[0105]
3TABLE 3 Compositions Comprising Both Oil Soluble and Water Soluble
Extracts 15 16 17 (Mulberry) Wt. % (Lavender) Wt. % (Licorice) Wt.
% Example 1 30 Example 2 30 Example 3 30 Example 7 65 Example 8 65
Example 9 65 Additive qs Additive qs Additive qs Lipid & Lipid
& Lipid & Preservative Preservative Preservative 18 19 20
(Arnica) Wt. % (Eyebright) Wt. % (Grape Root) Wt. % Example 4 30
Example 5 30 Example 6 30 Example 10 65 Example 11 65 Example 12 65
Additive qs Additive qs Additive qs Lipid & Lipid & Lipid
& Preservative Preservative Preservative 21 22 23 (Arnica) Wt.
% (Eyebright) Wt. % (Grape Root) Wt. % Example 4 30 Example 5 30
Example 6 30 Example 10 65 Example 11 65 Example 12 65 Additive qs
Additive qs Additive qs Lipid & Lipid & Lipid &
Preservative Preservative Preservative 24 25 26 Wt. % Wt. % Wt. %
Tea Tree Oil 30 Evening 30 Evening 30 Primrose Oil Primrose Oil
Example 13 50 Example 14 30 Example 14 66.5 Additive qs Additive qs
Additive qs Lipid & Lipid & Lipid & Preservative
Preservative Preservative 27 28 29 (Green Tea) Wt. % (Ros. Pwdr)
Wt. % (Calendula) Wt. % Example 1a 30 Example 2a 30 Example 3a 30
Example 8a 67.5 Example 9a 67.5 Example 10a 67.5 Additive qs
Additive qs Additive qs Lipid & Lipid & Lipid &
Preservative Preservative Preservative
[0106] The term "qs" means a quantity to sufficient to constitute
the remaining weight percent of the composition.
[0107] The additive lipid and preservative used in each of Examples
15-29 contains 2.35 wt. % phospholipid, 1.6 wt. % Germazide.TM.
MPB, and qs with water.
[0108] Examples 15-29 were formed by mixing the contents of the
composition, making the contents homogenous by using a Silverson
high shear mixer. The composition is then processed through a M110
microfluidizer, manufactured by Microfluidics, Inc. of
Massachusetts, at approximately 17,000 psi.
Mulberry Extract
[0109] In Example 1 (Table 1), a hydrophobic solution comprising
the organic oil soluble materials in mulberry root was prepared by
contacting mulberry with a silicone oil and mixing. The resulting
composition was mixed and filtered to clarity. The Mulberry
silicone extract was examined by HPLC (see FIG. 1) to ensure
complete extraction.
[0110] In Example 7, the polar solvent soluble materials were
derived from Mulberry. The Mulberry powder extract was obtained by
contacting with propylene glycol and constant stirring for 5 hours
at room temperature. The resulting composition was diluted with
water and Germazide.TM. MPB was added.
[0111] The HPLC traces for both the polar solvent soluble and
apolar solvent soluble mulberry extracts are shown in FIGS. 1 and
2. It is clear that their overall composition is very different.
The chromatogram of the apolar soluble material extracted from
mulberry root (FIG. 2) is very different from the materials found
in the chromatogram of the polar solvent soluble components (FIG.
1).
[0112] The two phases were mixed together along with phospholipid
in a high shear mixer and then combined using high pressure, high
shear processing to produce the dispersion of Example 15,
containing both the polar and apolar solvent soluble organic
components of mulberry.
[0113] As shown in FIG. 3, the resulting Mulberry extract
composition (Example 15) is enriched with powerful tyrosinase
inhibitors making it an ideal ingredient in skin whitening
products. HPLC chromatograms show, as predicted by the literature,
that the dispersion of Example 15 contains a vast array of
different polar solvent soluble and apolar solvent soluble
compounds. The apolar solvent soluble compounds are being
effectively extracted into the silicone oil and are therefore
present in the resulting Mulberry extract composition.
Licorice (glycyrrhiza glabra) Root Extract
[0114] The Licorice extract composition of the invention was
prepared from two phases; a hydrophobic phase containing the apolar
solvent soluble materials in Licorice root and a hydrophilic phase
containing the polar solvent soluble materials.
[0115] In Example 3, a silicone based Licorice extract was
prepared. Licorice root powder was contacted with silicone oil and
subject to stirring. The resulting composition was filtered to
clarity.
[0116] In Example 9, polar solvent soluble materials were obtained
derived from Licorice. Licorice powder was extracted by contacting
licorice with propylene glycol and constant stirring for 5 hours at
room temperature. The resulting composition was diluted with water
and Germazide.TM. MPB was added.
[0117] The HPLC trace for silicone and aqueous extracts of licorice
is shown in FIGS. 4 and 5. Glycyrrhizic acid has been identified
and is labeled. FIGS. 4 and 5 show that the Licorice extract
composition of the invention contains a vast array of different
compounds. The chromatograms were run using different wavelengths
solvent systems so it is misleading to compare them directly. The
size of the peak heights for Gylcyrrhizic acid are different
because of the different wavelengths. However, the peaks can be
clearly seen in both chromatograms.
[0118] The two phases were mixed together along with phospholipid
in a high shear mixer and then combined them using high pressure,
high shear processing to produce the dispersion of Example 17,
containing both the polar and apolar solvent soluble organic
components of licorice.
[0119] FIGS. 6 and 7 depict the tyrosinase inhibiting activity and
melanocyte pigmentation reduction activity, respectively, of the
licorice extract composition of Example 17. As shown in FIG. 6,
licorice extract contains powerful tyrosinase inhibitors. The
effect of these inhibitors can be demonstrated by either direct
tyrosinase inhibition or by measuring the reduction of melanocyte
pigmentation. The licorice extract composition of the present
invention is also an antioxidant, making it an ideal ingredient for
skin whitening products.
[0120] In addition, a melanocyte pigmentation assay was conducted
for the composition of Example 17. Results of the assay are
depicted below.
4TABLE 4 Melanocyte Pigmentation Reduction of Example 17 Culture
pigmentation (OD.sub.405 nm) % ID conc. mean SE SD CV % inhibition
p* Example 17 1 0.327 0.004 0.007 2 68 0.00003 Licorice (%) 0.3
0.610 0.015 0.026 4 41 0.0003 0.1 0.621 0.024 0.042 7 40 0.0005
0.03 0.729 0.017 0.029 4 29 0.001 0.01 0.842 0.009 0.016 2 18 0.005
untreated -- 1.032 0.032 0.055 5 0 hydro- 30 0.747 0.013 0.023 3 28
0.001 quinone (.mu.M) *Probability values determined by t test
comparison of pigmentation levels of treated cells with those of
the untreated cells.
[0121] The ID "untreated" refers to a composition which does not
contain any of the organic components of the licorice extract, and
thus is a control.
[0122] FIG. 7 depicts the results of the melanocyte pigmentation
reduction of Example 17. Example 17 has an EC.sub.50 of 0.53% in
this assay (see Table 4 and FIG. 7). It is therefore a potent agent
for reducing pigmentation and skin whitening.
Lavender (lavandula angustifolia) Extract
[0123] Lavender extract compositions of the invention were
obtained.
[0124] In Example 2, a silicone based lavender extract was prepared
by contacting lavender with silicone oil and mixing. The resulting
composition was filtered to clarity. In Example 8, the polar
solvent soluble components of lavender were obtained by contacting
lavender with propylene glycol with constant stirring for 5 hours
at room temperature. The resulting composition was diluted with
water, and Germazide.TM. MPB was added.
[0125] FIGS. 8 and 9 depict the chromatograms of a water soluble
extract and oil soluble extract, respectively, of a lavender
extract composition prepared according to the invention.
[0126] The two phases were mixed together with phospholipid in a
high shear mixer and then combined using high pressure, high shear
processing to produce the dispersion of Example 16, containing both
the polar and apolar solvent soluble organic components of
lavender. The dispersion of Example 16 combines the oil-soluble
actives, mainly linalool and linalyl acetate, with the polar
solvent soluble flavonoids to produce a powerful combination that
will relax, balance and soothe.
[0127] Elastin is the structural protein that is predominately
responsible for the skin's natural elasticity. This elasticity is
gradually lost as skin ages or if skin is damaged because elastin
is slowly removed through the action of the enzyme elastase. If
this enzyme is too active (such as in inflamed skin) the rate of
degradation exceeds the rate of synthesis and the skin quickly
looses its suppleness. Cosmetics that inhibit elastase will
therefore help improve skin, reduce some of the damaging effects of
inflammation and keep skin looking younger for longer.
[0128] FIG. 10 shows elastase inhibition by lavender extract and
conventional polyethylene glycol ("PG") extract. Both these
extracts inhibit elastase, however the lavender extract composition
of the invention is the better inhibitor. Elastase inhibitors in
lavender are water-soluble and so it was expected that both the
conventional PG and the composition of the invention would show
similar inhibition.
[0129] The Example 16 dispersion has the activities necessary to
maintain normal skin and balance combination skin. It is a strong
elastase inhibitor, which helps to maintain skin's elasticity and
youthfulness. By inhibiting elastase, the Example 16 dispersion
also reduces some of the negative effects of inflammation. The
anti-inflammatory properties of Lavender are well supported by
published articles. The data described herein demonstrates that
Lavender may have weak anti-inflammatory properties that calm and
sooth skin. In addition, cell renewal benefits of lavender are well
supported by published literature. The linalool and linalyl acetate
seen in the lavender extract composition of the invention are known
to be antiseptic. These properties of Lavender extract make the
Example 16 dispersion ideal for normal and combination skin.
[0130] Table 5 contains the results of the Extra Cellular Matrix
Degradation assay. The Example 16 dispersion showed some slight
anti-inflammatory activity at the highest concentration tested
(3%). These results indicate that the level of a stronger
anti-inflammatory activity is associated with concentrations of
lavender extracts of greater than 3%.
5TABLE 5 Results of the Extra Cellular Matrix Degradation Assay of
Example 164 ECM degradation by activated % anti- Conc. neutrophils
inflammatory ID (% v/v) mean SE activity Example 16 (Lavender) 3 40
1 30 1 58 1 -3 0.3 56 2 2 0.1 54 1 5 0 57 0.5 0 Conventional PG 3
54 0 5 Lavender 1 54 1 6 0.3 55 1 3 0.1 54 1 5 0 57 0.5 0 Assay
Controls No neutrophils (spontaneous leeching of 9 0.4 --
radiolabeled material from ECM) Neutrophils (degradation of ECM by
27 1 -- unactivated neutrophils) Activated Neutrophils (degradation
of 57 0.5 -- ECM by neutrophils that have been activated with
phorbol ester) Activated Neutrophils + 3% ExCyte .TM. 13 1 77
Heather (degradation of ECM by activated neutrophils in presence of
ExCyte .TM. Heather (positive control for inhibition of matrix
degradation)) ExCyte .TM. Heather is a skin care formulation
containing water, glycerin and calluna vulgaris extract, available
from Collaborative Laboratories of Stony Brook, New York.
Arnica Extract
[0131] Arnica extract compositions of the invention were prepared.
In Example 4, silicone based arnica extract was prepared by
contacting arnica with silicone oil and mixing. The resulting
composition was filtered to clarity. In Example 10, a polar solvent
soluble materials of arnica were obtained by contacting with
propylene glycol with constant stirring for 5 hours at room
temperature. The resulting composition was diluted with water, and
Germazide.TM. MPB was added. The two phases were mixed together
with phospholipid in a high shear mixer and then combined using
high pressure, high shear processing to produce the dispersion of
Example 18, containing both the polar and apolar solvent soluble
organic components of arnica.
[0132] The dispersion of Example 18 was tested for
anti-inflammatory activity. The results of the testing are depicted
in FIG. 11.
Eyebright (euphrasia officinalis)
[0133] An Eyebright extract composition of the invention contains
both the oil-soluble and water-soluble components of the plant, and
thus is very similar to the traditional compresses. The only
components missing that are present in the plant itself are
insoluble fibrous materials. The Eyebright extract composition of
the invention is therefore more effective than conventional
Eyebright extracts that normally consist of either the water
extractable material or the oil extractable material.
[0134] An eyebright extract dispersion was prepared. In Example 5,
a silicone eyebright extract was prepared by contacting with
silicone oil and mixing. The resulting composition was filtered to
clarity. In Example 11, a polar solvent soluble material derived
from eyebright was prepared contacting with propylene glycol with
constant stirring for 5 hours at room temperature. The resulting
composition was diluted with water, and Germazide.TM. MPB was
added.
[0135] FIGS. 12 and 13 depict the chromatograms of polar solvent
soluble (Example 11) and apolar solvent soluble extract (Example 5)
compositions.
[0136] The two phases were mixed together with phospholipid in a
high shear mixer and then combined using high pressure, high shear
processing to produce the dispersion of Example 19, containing both
the polar and apolar solvent soluble organic components of
eyebright.
[0137] FIG. 14 depicts the results of a cytochrome c reduction
assay of the eyebright extact composition of the invention, in
comparison with Sansurf.TM. oil extracted material, a
surfactant-free dispersion of lipophilic materials in water,
manufactured by Collaborative Laboraties of Stony Brook, N.Y.;
conventional PG eyebright; and control compositions of the
composition of the invention and Sansurf.TM.. The Eyebright extract
composition of the invention showed significant activity at the
0.1% concentration. In contrast, the blanks at 0.1% did not have
antioxidant activity. The Eyebright extract composition of the
invention performs better than the other materials tested at all
concentrations up to 0.5%. The apparent activities seen at higher
concentrations are most likely as a result of artifacts such as
light scatter. The Example 19 EC.sub.50 Eyebright extract
composition of the invention in the Cytochrome c Reduction assay
was 0.07% showing that at this concentration the composition of the
invention is a strong antioxidant.
[0138] Lipid Peroxidation Assay was carried out to alleviate the
concerns raised about the antioxidant activity measured in the
Cytochrome c reduction assay. It has been suggested that this assay
may be influenced by factors other than oxidation. The results for
the Example 19 composition in the Lipid Peroxidation assay are
shown in Table 6.
6TABLE 6 Results of the Lipid Peroxidation Assay of Eyebright
Compositions Rate of % OD.sub.532 at indicated Lipid antioxidant
Sample time (min) Peroxidation effect ID conc. % 0 60 120 240
(MOD.sub.532/min) Example 19 1 0.018 0.020 0.021 0.029 46 84
Eyebright 0.2 0.013 0.018 0.028 0.073 257 12 0.04 0.014 0.026 0.056
0.088 320 -9 SanSurf .TM. Oil 1 0.014 0.035 0.060 0.100 360 -23
Extracted 0.2 0.013 0.030 0.062 0.105 396 -35 Material 0.04 0.014
0.030 0.059 0.107 395 -35 Conventional 1 0.014 0.028 0.058 0.109
410 -40 PG 0.2 0.013 0.031 0.061 0.105 390 -33 Eyebright 0.04 0.013
0.034 0.066 0.109 407 -39 Example l9 1 0.020 0.048 0.074 0.111 375
-28 Blank 0.2 0.014 0.034 0.062 0.108 397 -35 0.04 0.015 0.030
0.059 0.099 359 -23 SanSurf .TM. Oil 1 0.020 0.025 0.057 0.105 377
-29 Extract 0.2 0.017 0.024 0.056 0.104 381 -30 Material 0.04 0.017
0.026 0.055 0.111 408 -39 Blank Vitamin C 0.3 0.011 0.012 0.011
0.013 8 97 0.3 0.011 0.011 0.011 0.012 3 99 0.03 0.012 0.013 0.016
0.025 54 82 0.03 0.014 0.0125 0.014 0.0205 30 90 untreated -- 0.013
0.013 0.015 0.023 293 --
[0139] The blanks listed above contain all the elements of the
composition without the organic components of the natural
products.
[0140] The EC.sub.50 for the Eyebright extract dispersion of
Example 19 for lipid peroxidation was found to be 0.6%. The Example
19 dispersion is therefore a strong antioxidant while the
SanSurf.TM. Oil Extracted Material, the Conventional PG Eyebright
and the blank composition of the invention did not significantly
prevent the production of malondialdehyde at any of the
concentrations tested and therefore have no antioxidant
properties.
Grape Root (mahonia aquifolium)
[0141] A Grape Root extract composition of the invention was
prepared.
[0142] In Example 6, a silicone based Grape Root extract is
prepared. Grape Root was extracted by contacting with silicone oil
and mixing. The resulting composition was filtered to clarity. In
Example 12, a polar solvent soluble material derived from Grape
Root is prepared. Grape Root was extracted by contacting with
propylene glycol with constant stirring for 5 hours at room
temperature. The resulting composition was diluted with water, and
Germazide.TM. MPB was added. The two phases were mixed together
with phospholipid in a high shear mixer and then combined using
high pressure, high shear processing to produce the dispersion of
Example 20, containing both the polar and apolar solvent soluble
organic components of grape root.
[0143] FIG. 15 depicts the results of a cytochrome C reduction
assay of Example 20. These results demonstrate that the Grape Root
extract dispersion of the invention (Example 20) is an effective
antioxidant at concentrations of 0.08%.
[0144] The following assays were used for testing the properties of
the natural product extracts of the invention.
Antioxidant Assays
Cytochrome C Reduction Assay Materials
[0145] Citrate-phosphate-dextrose solution ("CPD"; C-7165), sodium
chloride (NaCl; S-5886); phosphate-buffered saline (PBS; 1000-3),
cytochrome c (C-7752), phorbol 12-myristate 13-acetate ("PMA";
P8139), superoxide dismutase ("SOD"; S-5395) and staurosporine
("STA"; S-4400) were obtained from Sigma. Hank's balanced salt
solution ("HBSS"; 14025-035) and Lymphoprep.TM. 1.077 were obtained
from Gibco BRL. Dextran T-500 (17-0320-01) was obtained from
Pharmacia. Wright's stain (LeukoStat.TM. stain kit) was obtained
from Fisher (CS-430). Optical densities were determined using a
Dynatech MR5000 spectrophotometer.
Methods
[0146] Polymorphonuclear leukocytes (neutrophils or "PMN") were
isolated using standard procedures. Blood was drawn from healthy
donors using citrate-phosphate-dextrose as an anticoagulant. An
equal volume of 3% Dextran in 0.9% NaCl was added to settle the
majority of erythrocytes. After 20 minutes incubation, the cleared
top layer was harvested and centrifuged at 250 Xg for 10 minutes.
The cell pellet was resuspended in 0.9% NaCl, layered onto
Lymphoprep.TM. and centrifuged at 400 Xg for 40 minutes. The
resulting pellet was then subjected to several cycles of hypotonic
lysis (typically 3 cycles) to remove residual erythrocytes. Each
lysis cycle involved resuspension of the cell pellet in ice-cold
0.2% NaCl for 30 seconds, restoration of isotonicity by the
addition of an equal volume of 1.6% NaCl, followed by
centrifugation at 250 g for 10 minutes at 4.degree. C. When the
erythrocyte removal was complete, the PMN were resuspended in PBS
and stored at 4.degree. C. until required. Purity of the
preparation was checked by staining a cell smear with Wright's
stain.
Lipid Peroxidation Assay Materials
[0147] Thiobarbituric acid ("TBA"; T5500), sodium dodecyl sulfate
(L4509), butylated hydroxytoluene (B1378), glycine (G7126) and
phosphate-buffered saline ("PBS"; 1000-3) were obtained from Sigma.
Lecithin (429415) was obtained from Calbiochem. Ethanol (122898)
was obtained from Aaper Alcohol and Chemical Co. Vitamin C (95209)
was obtained from Fluka. Ferric chloride (AR5029) was obtained from
Mallinckrodt. Optical densities were determined using a SpectraMax
250 spectrophotometer (Molecular Devices). Ultraviolet irradiation
was performed using a model VWR M-20E Chromato-Vue transilluminator
(VWR Scientific).
Methods
[0148] The Lipid Peroxidation assay measures inhibition of
UV-induced lipid oxidation. The reaction was performed at room
temperature by irradiating liposome solutions, containing varying
concentrations of test samples, with ultraviolet C light.
[0149] The extent of lipid peroxidation induced by the UV light was
determined by measuring the consequent production of
malondialdehyde that results from breakdown of oxidized lipid.
Irradiated liposome solutions contained 0.8% lecithin and 18%
ethanol in PBS, along with test sample or control. Vitamin C, a
peroxyl radical scavenger, was included in each assay as a control
for inhibition of lipid oxidation. Duplicate aliquots of the
irradiation mixtures were harvested at several time points and
their malondialdehyde contents were measured using the
"thiobarbituric acid reaction". TBA reaction mixtures contained 300
.mu.M butylated hydroxytoluene, 300 .mu.M FeCl.sub.3, 16 mM TBA,
0.14% sodium dodecyl sulfate, 90 .mu.M glycine (pH 3.6) and 3%(v/v)
irradiation mixture aliquot. The mixtures were heated at
100.degree. C. for 15 minutes, cooled to room temperature and their
absorbencies were read at 532 and 650 nm. The readings at 650 nm
were subtracted from those at 532 nm to correct for turbidity.
These corrected absorbencies were plotted as a function of
irradiation time and the rate of lipid peroxidation was determined
by linear regression curve fit. An EC.sub.50 (the concentration of
test sample that inhibits 50% of the rate of lipid peroxidation)
was calculated for each sample.
Anti-Inflammation Assay
Extra Cellular Matrix Degradation (ECM) Assay Materials
[0150] Ammonium hydroxide (A-6899), streptomycin sulfate (S-0890)
citrate-phosphate-dextrose solution ("CPD"; C-7165), sodium
chloride (NaCl; S-5886); phosphate-buffered saline (PBS; 1000-3),
phorbol 12-myristate 13-acetate ("PMA"; P8139), and sodium azide
(S-8032) were obtained from Sigma. Minimal Essential Medium ("MEM";
11095-072), tryptose phosphate (18050-013) heat-inactivated fetal
bovine serum ("FBS"; 10082-147) Hank's balanced salt solution
("HBSS"; 14025-035) and Lymphoprep.TM. 1.077 were obtained from
Gibco BRL. R-22 cells were obtained from Dr. S. Simon (Dept. of
Pathology, SUNY Stony Brook). Dextran T-500 (17-0320-01) was
obtained from Pharmacia. Wright's stain (LeukoStat.TM. stain kit)
was obtained from Fisher (CS-430). ExCyte.TM. Heather was obtained
from Collaborative Laboratories.
[0151] For the production of radiolabeled ECM plates, R-22 smooth
muscle cells were seeded into 24-well plates and grown in
maintenance medium (MEM containing 10% FBS, 2% tryptose phosphate,
100.mu.g/ml streptomycin). Upon reaching confluence, the cells were
switched into labeling medium (maintenance medium supplemented with
50 mg/ml ascorbic acid and 0.5 .mu.Ci/ml
L-[2,3,4,5-.sup.3H]-proline) and maintained in this medium for 2
weeks. The plates were then harvested by aspirating the labeling
medium and lysing the cells by 5 minute incubation in 25 mM
ammonium hydroxide followed by 3 cycles of 5 minute incubation in
sterile deionized water. Prior to use, the plates were stored at
4.degree. C. with 50 .mu.l/well of 0.02% sodium azide.
[0152] Polymorphonuclear leukocytes (neutrophils or "PMN") were
isolated using standard procedures. Blood was drawn from healthy
donors using citrate-phosphate-dextrose as anticoagulant. An equal
volume of 3% Dextran in 0.9% NaCl was added to settle the majority
of erythrocytes. After 20 minutes incubation, the cleared top layer
was harvested and centrifuged at 250 Xg for 10 minutes. The cell
pellet was resuspended in 0.9% NaCl, layered onto Lymphoprep.TM.
and centrifuged at 400 Xg for 40 minutes. The resulting pellet was
then subjected to several cycles of hypotonic lysis (typically 3
cycles) to remove residual erythrocytes. Each lysis cycle involved
resuspending the cell pellet in ice-cold 0.2% NaCl for 30 seconds,
restoration of isotonicity by addition of an equal volume of 1.6%
NaCl, followed by centrifugation at 250 g for 10 minutes at
4.degree. C. When erythrocyte removal was complete, the PMN were
resuspended in PBS and stored at 4.degree. C. until use. Purity of
the preparation was checked by staining a cell smear with Wright's
stain.
Methods
[0153] For the ECM degradation assay, radiolabeled ECM plates were
washed 3 times with HBSS to remove azide. To start the degradation
reaction, 1 ml of HBSS containing 5 nM PMA, 1.times.10.sup.6 PMN,
and the indicated test sample concentration was added to 3 wells of
an ECM plate. In each assay, there were also 4 control conditions
(triplicate wells for each). These were HBSS alone (to correct for
leeching of unincorporated radiolabel), cells in HBSS (to monitor
degradation by "unstimulated" PMN), cells in HBSS with 5 nM PMA
(maximal stimulated matrix degradation), and cells in HBSS with 5
nM PMA and 3% ExCyte.TM. Heather (positive control for inhibition
of matrix degradation), available from Collaborative Laboratories.
Upon addition of reaction mixtures, the plates were incubated at
37.degree. C. for 4 hours. ECM degradation in each well was scored
by scintillation counting to measure radioactivity released into
the supernatant as well as that remaining in the residual matrix.
Using these 2 measures, the % ECM degradation was calculated for
each well. An EC.sub.50, the concentration of test sample that
decreased ECM degradation by 50% relative to the maximal stimulated
matrix degradation condition, was calculated where possible.
Elastase Inhibition Assay Materials
[0154] Tris[hydroxymethyl]aminomethane ("Tris"; T-1410), sodium
chloride (NaCl; S-5886), dimethyl sulfoxide ("DMSO"; D-8779),
sodium acetate (S-8625) and hydrochloric acid ("HCl"; H-7020) were
obtained from Sigma. Human neutrophil elastase ("HNE";
16-14-051200) was obtained from Athens Research.
MethoxysuccinylAAPVpNA ("peptide"; L-1335) was obtained from
Bachem. Optical densities were determined using a Dynatech MR5000
spectrophotometer.
Methods
[0155] The assay was performed in a 96-well plate with triplicate
wells for each reaction condition. Reaction mixtures contained 63
mM Tris-HCl (pH 8.0), 195 mM NaCl, 5 mM sodium acetate, 1.5% DMSO,
300 .mu.g/.mu.l peptide, 1.5 .mu.g/.mu.l HNE and test sample as
indicated. The reaction was started by addition of HNE and followed
by measuring A.sub.405 at minute intervals for 10 minutes. The
reaction rate was determined from the slope of a straight line
fitted to the data plot.
Inhibition of UV-Induced PGE.sub.2 Production
[0156] This assay measures production of PGE.sub.2 by keratinocytes
exposed to UVB radiation.
Materials
[0157] HaCaT cells, a spontaneously immortalized human keratinocyte
line (Boukamp et al, J Cell Biology 106 (1988) 761-771), were
obtained from Dr. Norbert Fusenig (German Cancer Research Center).
Tris[hydroxymethyl]aminomethane (T-1410), sodium chloride (S-5886),
ethylenediaminetetraacetic acid (E-4884), and phosphate-buffered
saline ("PBS"; 1000-3), Neutral Red (N-6634) and glucose (G-5400)
were obtained from Sigma. Ethanol (A405P-4) was obtained from
Fisher Scientific. Acetic acid (AC110) was obtained from Spectrum
Chemical Corp. Dulbecco's Modified Eagle's Medium ("DMEM";
11885-076), heat-inactivated fetal bovine serum ("FBS"; 10082-147),
and 5000 units/ml penicillin/5000 .mu.g/.mu.l streptomycin
(15070-063) were obtained from Gibco BRL. PGE.sub.2 EIA kits
(DE0100) were obtained from R&D Systems. UBL model
FSX24T12/UVB-HO bulbs (National Biological Corp.) were used for
cell irradiation. Bulb output was measured with an International
Light Model IL1700 radiometer. Absorbances were measured using a
Dynatech MR5000 spectrophotometer.
Methods
[0158] Cells were seeded into 12-well plates in DMEM containing 10%
FBS, 50 units/ml penicillin and 50 .mu.g/.mu.l streptomycin
("medium"). When the cells were approximately 65% confluent they
were switched into medium containing varying concentrations of test
samples or indomethacin (positive control for inhibition of
PGE.sub.2 production). After a 12-hour preincubation, the media
were removed and the cells were washed with, and transferred into
PBS-glucose (PBS containing 5.5 mM glucose). They were immediately
irradiated with 11 mJ/cm.sup.2 of UVB (approx. 45 second exposure)
and transferred back into media containing the same concentrations
of test samples or controls as for the preincubation period. After
a further 8 hour incubation, the media were collected and stored at
-70.degree. C. After removal of the media, the cells were fed with
medium containing Neutral Red dye and incubated at 37.degree. C.
for 3 hours. The cells were then washed with buffer and
internalized dye was extracted with an ethanolacetic acid solution.
Extracted Neutral Red was determined by measuring its absorbance at
550 nm. The levels of PGE.sub.2 in the supernatants were measured
using a commercially available EIA kit.
Skin Whitening Assays
Tyrosinase Inhibition Activity Materials
[0159] The following materials were used in the tyrosinase
assay:--Tyrosine T8909, Tyrosinase (mushroom) T7755, Hydroquinone
H9003, and Sodium Phosphate S7907 and S8282 were obtained from
Sigma. Optical density readings were determined using a Dynatech
MR5000 spectrophotometer.
Methods
[0160] Tyrosinase activity was determined by measuring the rate of
change of optical density at 490 nm as tyrosine was converted into
dopachrome. (Tyrosinase catalyzes the conversion of tyrosine into
DOPA quinone, which spontaneously converts into dopachrome.) The
rate of tyrosinase activity in the presence of various
concentrations of test sample was measured at room temperature in a
reaction mixture containing 50 mM sodium phosphate (pH 6.75), 275
.mu.M tyrosine and 25 U/ml mushroom tyrosinase. Readings were made
at one minute intervals for ten minutes and the reaction rate was
calculated by linear regression. Each concentration was run in
triplicate. Inhibition of tyrosinase activity was expressed as a
percentage of the activity measured for the untreated control
mixture (no test sample). An EC.sub.50 (the concentration of test
sample that inhibits 50% of tyrosinase activity) was calculated.
Hydroquinone was tested in each assay as a positive control for
inhibition of tyrosinase activity.
Melanocyte Pigmentation Assay Materials
[0161] The following materials were used in the melanocyte
pigmentation assay. Cloudman S91 cells (36-1-38C8-16) were obtained
from American Type Culture Collection. Dulbecco's Modified Eagle's
Medium ("DMEM"; 11885-076), heat-inactivated horse serum
(26050-088), heat-inactivated fetal bovine serum (10082-147) and
5000 units/ml penicillin/50 .mu.g/ml streptomycin (15070-063) were
obtained from Gibco BRL. Phosphate-buffered saline (PBS; 1000-3),
dimethyl sulfoxide (D2650), hydroquinone (H9003),
.alpha.-melanocyte stimulating hormone (M4135), trichloroacetic
acid (T9159) and sodium hydroxide (S8045) were obtained from Sigma.
Optical densities were determined using a Dynatech MR5000
spectrophotometer.
Methods
[0162] The melanocyte pigmentation assay measures a test sample's
ability to inhibit pigmentation. Cloudman S91 melanocytes were
seeded into multiwell plates in medium (Dulbecco's Modified Eagle's
Medium containing 15% heat-inactivated horse serum, 2.5%
heat-inactivated fetal bovine serum, 50 units/ml penicillin and 50
.mu.g/ml streptomycin) containing 10 nM .varies.-melanocyte
stimulating hormone and varying concentrations of the test sample
or 30 .mu.M hydroquinone (the positive control for inhibition of
pigmentation). After a 5-day treatment period with each condition
tested in triplicate wells, the cells were harvested by washing
with PBS and adding 5% trichloroacetic acid to each well. After a
15 minute incubation the acid was aspirated and replaced with 200
.mu.l/well of 10% dimethyl sulfoxide/1N sodium hydroxide. The
plates were sealed and incubated at 65.degree. C. for 30 minutes.
200 .mu.l of each digestion mixture was then transferred to a well
of a 96-well microtiter plate and optical densities were read at
405 nm.
[0163] Inhibition of pigmentation is expressed as the percentage
decrease in optical density compared to that for untreated cells
(no test sample or hydroquinone). An EC.sub.50 (the concentration
of test sample that inhibits 50% of pigmentation) was calculated
for each sample.
* * * * *