Specification
COMPOSITIONS COMPRISING PAULOWNIA
TOMENTOSA WOOD EXTRACTS AND USES THEREOF
CROSS-REFERENCE TO RELATED APPLICATION
This application is a continuation-in-part of U.S. Application No. 13/21 1,626, filed August
17,201 1, which is a continuation-in-part of U.S. ApplicationNo. 121859,323, filed August 19,
2010.
FLELD OF INVENTION
The present invention relates to compositions comprising Paulownin for use on skin. The
present invention also relates to compositions comnprising plant extracts fiom genus Paulownia for
use on skin. More specifically, it relates to conlpositions comprising extracts of Pa~rlou~nia
tottietitosa wood for a variety of uses on the skin.
DESCRIPTION OF RELATED ART
Paulownia is a genus of plants native to Asia which has spread gradually to Europe and
the USA. Species from the Paulownia genus are generally considered ornamental trees with
wide craft and ecological applications for their wood. For example, the wood of such trees is
popular for making soundboards of stringed instruments in Japan, China, and Korea. It is also
popular with timber merchants for use in making furniture. Paulownia trees have ecological uses
and are considered phyto-remediators, that is, they can process industrial contaminauts through
their vascular system to help clean and reclaim land.
In Japan, Paulownia is called kiri which refeis specifically to one species, Pa~rlo~onia
fottietitosa, also called "Princess Tree." Other names which are commonly used are "empress
tree", "Foxglove Tree", "Royal Paulownia", "Pao tong" (in China) and "Odong-Namoo" (in
Korea). The scientific name is "Paulownia tomentosa" with a number of synonyms reported in
various literature, i.e. "Paulownia imperialis", "Paulo~vnia recurva", and "Bignonia tomentosa".
Paulownia tomentosa belongs to the family "Paulowniaceae" or sometimes refer to
"Scrophulariaceae". The United States Department of Agriculture @lants.USDA.gov) Plant
database identifies Princess tree by a unique symbol "PAT02", with Paulownia tomentosa and
Paulownia imperialis as synonym natnes.
The flower oil of Pazrlo~eiiiaio iireiitosa is well studied and found to he richer in aroma as
compared to other species. A number of bioactivities are associated with extracts of various
parts of Paulownia, e.g. anti-cancer components from flower extract, anthelminthic activity from
non-specified extract, antibacterial activities from fruit and flower extracts, antioxidant activity
froin flower extract, and anti-viral properties from stem bark. Leaf extracts ofPaulownia are
described for hair growth and hair promoting properties.
Paulownin, also known as isopaulownin or neopaolownin, is a lignan isolated from aerial
parts of various plant. The chemical structure can be given as follows:
The present invention relates to applicant's discovery that extracts ofParrlo~vniato itrentosa wood
are beneficial for use in compositions on skin. For example, applicants have discovered that
such compositions tend to exhibit significant and unexpected properties for skin including skin
lightening, improving signs of aging, and reducing inflammation. The present invention also
relates to the applicant's discovery that the compound Paulownin exhibits significant and
unexpected skin lightening, anti-aging benefits and other properties.
SUMMARY OF THE INVENTION
In one aspect, the present invention is directed to compositions comprising Paulownin
and a carrier.
In another aspect, the present invention is directed to methods of improving a sign of
aging in skin comprising the step of applying to skin in need of improving the signs of aging a
composition comprising Paulownin and a carrier.
In another aspect, the present invention is directed to compositions comprising an extract
of Pa~rloiviiiat onrentosa wood and a carrier.
In another aspect, the present invention is directed to methods of lightening the skin
comprising the step of applying to skin in need of skin lightening treatment an extract of
Parrloivnia tontetitosa wood.
In another aspect, the present invention is directed to methods of improving a sign of
aging in skin comprising the step of applying to skin in need of improving the signs of aging an
extract of Pauloisnia ton~entosaw ood.
In yet another aspect, the present invention is directed to methods of reducing skin
inflammation comprising the step of applying to a skin in need of reducing skin inflammation an
extract of Parrlo~vniato nienfosa wood.
DESCRIPTION OF THE INVENTION
As noted above, applicants have discovered unexpectedly that Paulownin and/or extracts
of the wood of Pazrlo~sniat ontentosa may be used in compositions, preferably skin care
compositions, and for methods of use including, but not limited to, improving signs of aging,
skin lightening, and reducing inflammation.
As used herein, the term "lightening the skin" refers generally to lightening, brightening,
whitening, and/or evening of the skin tone, skin color, and/or shade of skin, and/or to the
reduction in sallowness, and/or to the lightening and/or fading of hyperpigmented marks and/or
lesions including, but not limited to, pigmented spots, melanin spots, age spots, sun spots, senile
lentigos, freckles, lentigos simplex, pigmented solar keratosis, seborrhoeic keratosis, melasma,
acne marks, post-inflammatory hyperpigmentation, lentigines, ephelides, combinations of two or
more thereof and the like. In certain embodiments, "lightening the skin" also refers to increased
skin radiance, glow, translucency and/or luminescence and/or obtaining a more radiant, glowing,
tratislucent or luminous skin tone appearance or a less yellow or sallow skin tone. In certain
preferred embodiments, "lightening the skin" refers to lightening atid evening the skin tone,
increasing skin radiance and/or lightening age spots.
As used herein, the term "skin in need of skin lightening treatment" refers generally to
skin that exhibits one or more property selected from the group consisting of: skin having a
measured Individual Typology Angle (ITA) value below 41 as determined per the COLIPA
GUIDELINE: GUIDELINE FOR THE COLORIMETRIC DETERMINATION OF SKIN
COLOUR TYPING AND PREDICTION OF THE MINIMAL ERYTHEMAL DOSE (MED)
WITHOUT UV EXPOSURE published in 2007, which is incorporated herein by reference and
further described below, darkened and/or sallow skin, including skin darkened by UV, skin with
uneven skin tone, or skin with one or more hyperpigmented marks andlor lesions including, but
not limited to, pigmented spots, melanin spots, age spots, sun spots, senile lentigos, freckles,
lentigos simplex, pigmented solar keratosis, seborrhoeic keratosis, melasma, acne marks, postinflammatory
hyperpigmentation, lentigines, ephelides, combinations of two or more thereof and
the like. In the COLIPA guidelines, skin color is defined function of the ITA value as: very light
skin >55; Light skin 41-55, Interlnediate 28-41, and Tan skin <28. In certain preferred
embodiments, "skin in need of skin lightening" refers to individuals with a skin having an ITA
value of less than 41, such as about 40 or less, about 35 or less, about 30 or less, or more
preferably about 28 or less. In certain other preferred embodiments, the present invention is
directed to compositions and methods for use on skin in need of skin lightening treatment
selected from sallow and/or darkened skin. In certain other preferred embodiments, the present
invention is directed to compositions and methods for use on skin in need of skin lightening
treatment selected from the group consisting of age spots, freckles, marks left after acne, and
combinations of two or more thereof.
As used herein, "skin in need of improving the signs of aging" means a skin that is, but
not limited to, sagging, loose, lax, rough, wrinkly, thinned, uneven. Improving the signs of aging
means improving the firmness of the skin, improving the texture of the skin, improving the
appearance of wrinkles in skin, improving the skin tone or the treatment of external aggressions
in skin.
As used herein, "improving the firmness of skin" means the enhancing of the firmness or
elasticity of the skin, preventing the loss of firtnuess or elasticity of skin, or preventing or
treating sagging, lax and loose skin. The firmness or elasticity of the skin can be measured by
use of a cutometer. See Handbook ofNon-Invasive Methods and the Skin, eds. J. Serup, G.
Jetnec & G. Grove, Chapter 66.1 (2006). The loss of skin elasticity or firmness may be a result of
a number of factors, including but not limited to aging, environmental damage, or the result of an
application of a cosmetic to the skin.
As used herein, "improving the texture of skin" means the smoothing of the surface of the
skin to remove either bumps or crevasses on the skin surface.
As used herein, "improving the appearance of wrinkles in skin" means preventing,
retarding, arresting, or reversing the process of wrinkle and fine line formation in skin.
As used herein, "treatment of external aggressions in skin" means the reduction or
prevention of the damage from external aggressions in skin. Examples of external aggressions
include, but are not limited to, damage to the skin from the use of cleansers (e.g., topical
cleansers containing surfactants), make-up, shaving as well as environmental damage such as
from UV light (e.g., sun damage from sunlight or damage from non-natural sources such as UV
lamps and solar simulators), ozone, exhaust, pollution, chlorine and chlorine containing
compounds, and cigarette smoke. Effects of external aggressions on the skin include, but are not
limited to, oxidative and/or nitrosative damage to and modifications on lipids, carbohydrates,
peptides, proteins, nucleic acids, and vitamins. Effects of external aggressions on the skin also
include, but are not limited to, loss of cell viability, loss or alteration of cell functions, and
changes in gene and/or protein expression.
As used herein, "improving the skin tone" means the lightening of the appearance of the
skin (e.g., lightening pigmented marks or lesions, reducing skin sallowness, and/or evening the
color of the skin).
As used herein, "skin in need of reducing skin inflammation" means a skin exhibiting
redness or erythema, edema, or being reactive or sensitive to external elements. External
elements include, but are not limited to, sun rays (UV, visible, IR), microorgat~isms,a tmospheric
pollutants such as ozone, exhaust pollutants, chlorine and chlorine generating compounds,
cigarette smoke, cold temperature, heat. Inflammatory disorders and related conditions which
may be treated or prevented by use of the compositions of this invention include, but are not
limited to the following: arthritis, bronchitis, contact dermatitis, atopic dermatitis, psoriasis,
seborrheic dermatitis, eczema, allergic dermatitis, polymorphous light eruptions, inflammatory
dermatoses, folliculitis, alopecia, poison ivy, insect bites, acne inflammation, irritation induced
by extrinsic factors including, but not limited to, chemicals, trauma, pollutants (such as cigarette
smoke) and sun exposure, secondary conditions resulting from inflammation includit~gb ut not
limited to xerosis, hyperkeratosis, pruritus, postinflammatory hyperpigmentation, scarring and
the like. Preferably, the infla~nmatoryd isorders and related conditions which may be treated or
prevented using the methods of the invention are arthritis, inflammatory dermatoses, contact
dermatitis, allergic dermatitis, atopic dermatitis, polymorphous light eruptions, irritation,
including erythema induced by extrinsic factors, acne inflammation, psoriasis, sebosrheic
dermatitis, eczema, poison ivy, insect bites, folliculitus, alopecia, and secondary conditions and
the like.
As used herein, unless otherwise specified, all percentages of ingredients in compositions
are weight percent of active/solids ingredient based on the total weight of composition.
As used herein, a composition that is "essentially fsee" of an ingredient means the
composition that has about 2% or less ofthat ingredient by weight based on the total weight of
the composition. Preferably, a composition that is essentially fsee of an ingredient has about 1%
or less, more preferably about 0.5% or less, more preferably about 0.1% or less, more preferably
aboot 0.05 or less, rnore preferably about 0.01% or less by weight based on the total weight of
composition of the ingredient. In certain more preferred embodiments, a composition that is
essentially free of an ingredient is free of the ingredient, i.e., has none ofthat ingredient in the
composition.
As used herein, "cosmetically/ dermatologically acceptable" means that the ingredients
which the term describes are suitable for use in contact with tissues (e.g., the skin or hair)
without undue toxicity, incompatibility, instability, irritation, allergic response, and the like.
The extracts comprising Paulownin may be, or may be made from, one or more fractions
of materials derived from Puitlo~siiia tonieiitosa wood via any of the extraction methods as
described herein. In certain embodiments, the extracts comprise a fraction, or a combination of
two or more fractions, derived from Pazrlonaiia torrielitosa wood.
Any suitable extracts of Parrlo~sniato n7eiitosa wood may be used in accord with the
present invention. In general, the wood of the Paitlo~ioiiat onleiitosa tree includes wood from the
stem, branches, or a combination of both. Suitable extracts of Pa7rlo11wiat onientosa wood may
be derived from wood chips, wood dusts, and/or small cuttings, and the like.
Suitable extracts of Paitlo~~~tnoimae ntosa wood may be obtained using conventional
methods including, but not limited to, direct extraction of material from the wood by grinding,
macerating, pressing, squeezing, mashing, centrifuging, and/or processes such as cold
percolation, agitation/distillation, microwave assisted extraction, soaication,
supercritical/subcritical C02 compressed gas extraction with or without polar modifiers,
pressurized solvent extraction, accelerated solvent extraction, pressurized or normal hot water
extraction, surfactant assisted pressurized hot water extraction, oil extraction, membrane
extraction, Soxhlet extraction, the gold finger distillation/extraction and/or processes disclosed,
for example, in US Pat. Nos. 7442391,7473435, and 7537791 to Integrated Botanical
Technologies, LLC, incorporated herein by reference, and the like, or by other methods such as
solvent extraction, and the like. Any of a variety of solvents including polar solvents, non-polar
solvents, or combinations of two or more thereof may be used in methods of comprising solvent
extraction. Suitable polar solvents include polar inorganic solvents such as water and the like,
polar organic solve~lts uch as alcohols and corresponding organic acids, for example CI-Cs
alcohols itlcluding methanol, ethanol, propanol, butanol, and the like and organic acids,
including acetic acid, formic acid, propanoic acid, and the like, polyols and glycols, including
CI-Cs polyols/glycols and the like, and combinations of two or more thereof. Suitable non-polar
solvents include non-polar organic solvents such as alkanes, including CI-Cg alkanes,
cycloalkanes, including CI-Cs alkanes, alkyl ethers, including CI-Cg alkyl ethers, Petroleum
ethers, ketones, including CI-Cg ketones, methyiene chloride, ethyl acetate, xylene, toluene,
chloroform, vegetable oil, mineral oil and the like. In another embodiment extraction may be
obtained by non-polar solvents described above or supercritical fluid extraction with or without a
polar modifier such as C1-C8 alcohols, water, CI-Cgpolyols/glycols, or CI-Cg organic acids. In
certain preferred embodiments, the extract of the invention is a polar extract prepared by
pulverizing the wood and extracting using a polar solvent having a dielectric constant value of
between 1 and 100 at 20°C, preferably a dielectric constaut of a value between 4 and 60 at 20°C,
more preferably a dielectric constant of a value between 4 and 50 at 20°C, and even more
preferably a dielectric constant of a value between 4 and 40 at 20°C. Examples of preferred
polar solvents include CI-Cs alcohols, c1-C~po IyoI~/glycoIC~,I -Cgo rganic acids, water and
combinations of two or more thereof having a dielectric constant value of between 1 and 100,
preferably between 4 and 60, and more preferably between 5 and 40 at 20°C, including, but not
limited to, those solvents and combinations of solve~~htasv ing the desired dielectric constant
value as disclosed in "Dielectric Constants of Some Organic Solvent-Water Mixtures at Various
Temperatures," Akerlof, Gosta; JACS, Vol. 54, No. 11 (Nov. 1932), pp. 4125-4139, incorporated
herein by reference. In certain preferred embodiments, the polar extract is extracted using one or
more CI-Cg alcohols, c1-C~po lyols, Cl-Cg glycols, and combinations of two or more thereof. In
certain more preferred embodiments, the extract is extracted using one or more CI-Ca~lc ohols,
C1-C4 polyols, and/or CI-C4 glycols. In certain more preferred embodiments, the extract is
prepared using a solvent comprising methanol, ethanol, or a combination thereof with or without
presence of water. In more preferred embodiment, the extract is prepared using anhydrous
alcohol or reagent grade denatured alcohol and dried Kiri wood dust agitating at room
temperature for 3 days. In certain preferred embodiments, the extract may be further refined by
charcoal (also referred to as active carbon) treatment.
In certain embodime~ltst,h e Partlo~stiiat otiientosa extract may be prepared to be
essentially free of certain materials. In one embodiment, the extract is essentially free of Ursolic
acid, beta-Sitosterol, or both.
In certain embodiments, the composition may additionally include extracts from other
parts of Pazdowtiia tonier7tosa, for example, one or more of the bark, leaves, roots, fruits, seeds,
or flowers. In other embodiments, the composition is essentially free from extracts of other nonwood
parts of Pazrlo~viiiat ortreitfosa.
The present invention further comprises skin care compositions comprising Paulownin
and methods of treating the skin, for example methods of treating one or more signs of aging in
skin, methods of lightening the skin, treating inflammation andlor the like, by applying to skin in
need of improving signs of aging a composition comprising Paulownin, i.e., a compound ofthe
formula:
Paulownin
Paulownin for use herein may be derived via any of a variety of natural sources, such as
extraction from botanicals, or may be synthesized using known synthetic methods (see, for
example, Stereoselective Synthesis of 3-Alkyl-2-aryltetrahydrofi~ran-4-01s: Total Synthesis of
(2)-Paulownin. Angle, Steven R.; Choi, Inchang; Tham, Fook S. Department of Chemistry.
Wright State University, Dayton, OH, USA. Journal of Organic Chemistry (2008), 73(16), 6268-
6278; and Total synthesis of (+)-Paolownin. Okazaki, Momotoshi; Ishibashi, Fumito; Shuto,
Yoshihiro; Taniguchi, Eiji. Faculty Agric., Ehime Univ., Matsuyama, Japan. Bioscience,
Biotechnology, and Biochemistry (1997), 61(4), 743-745). In certain preferred embodiments, the
Paulownin is extracted from botanicals. Examples of suitable botanicals include plants of the
genus Paulownia, such as Paulonviia tonierifosa,P aulo~snifao rttitlei, Pazlloi~~niealo ngata,
Parrlolenia kmsakattrii, as well as other plants such as Aritorioa obloiigifolia, Dolicliandrone
crispa, Firrniatia platanifolia, Grrteliria arborea, Grtieliiiu asiafica, Gitieliria vitieiisis, Isodori
paivifolizrs, Kigeliapirinata, A4arkliartriapla@calyx, Markliariria stiptilrte, Millirigtoriia
horterisis, botanicals of the species Olea, Pliyllartliroii coinorense, Tabebtria incana, Vitex
trijolia, Prasitrni tnajzis, combinations of two or more thereof, and the like. In certain preferred
embodiments the Paulownin is extracted from the wood of Pazrlo~niiato tttetitosrr.
In cei-tain embodiments, Paulownin may be obtained via extraction of cell cultures of
various plants, including cell cultures of plants of the genus Paulownia, such as Pazrlo~eiiia
tott~eiifosaT. he cell cultures which are extracted to obtain extractslPaulownin for use in the
present invention may be of any form iucluding suspension cell cultures and tlie like.
Any suitable amounts of Paulownin or Pazrloic~iiiufo nientosa wood extract may be used
in the compositions of the present invention. Preferably, the compositions comprise a safe and
effectivea mount ofP aulownin or Pairlo~vniato itieritosa wood extract. As used herein, a "safe
and effective atnount" means an amount of the extract or of the composition sufficient to induce
the desired effect, but low enough to avoid serious side effects, including cytotoxicity and tlie
like. For embodiments comprisiug skin lightening uses of the composition, a "skin lightelling
effective amount" means an atnount of extract that is effective to achieve a AL value that is
greater than zero in the Skin Epidermal Equivalents Model as a skin Lightening Test (AL) as
described below. In certain preferred embodiments, tlie skin lightening effective amount is an
amount effective to achieve a AL value of about 1 or greater.
For ernbodi~nelitso f the present invention related to uses oft he cor~~positionfosr
improving a sign of aging in skin, an "effective amount for improviug a sign of aging" means an
amount that provides a percent inhibition of MMP-1 or MMP-9 production, measured in accord
with the Inhibition of UV-Induced MMP induction procedure of Example 1 1 below, that is
greater than zero. In certain preferred embodiments, the effective amount for improving a sign
of aging is an amount that provides a percent inhibitiou of MMP-1 or MMP-9 production,
measured in accord with the Inhibition of UV-Induced MMP induction procedure of Example I1
below, that is about 10% or greater. In certain preferred embodiments, the effective amount for
improving a sign of aging is an amount that provides a percent inhibition of Reactive Oxygeu
Species (ROS), measured in accord with the Inhibitiotl of Reactive Oxygen Species Formation in
reconstituted epidermis and in human epithelial cells methods given below in Examples 8 and 9
respectively, that is about 10% or greatel; aud preferably 20% or greater.
For embodiments of the present invention related to uses of the compositions for
reducing inflammation, an "effective amount for reducing inflammation" means an amount that
provides a percent inhibition of skin inflammation (IL-8), measured in accord with the Anti-
Inflammatory effects on Release of UV-Induced Pro-inflammatory mediators on Reconstituted
Epidermis procedure for IL-8 of Example 7 below, that is greater than zero. In certain preferred
embodiments, the effective amount for improviug a sigu of aging is an amount that provides a
percent inhibition of skin inflamlnatiou (IL-8), measured in accord with the Anti-I~iflammatory
effects on Release of UV-Induced Pro-inflammatory mediators on Reconstituted Epidermis
procedure for IL-8 of Example 7 below, that is about 10% or greater.
In certain preferred embodiments, the compositions comprise from greater than zero to
about 20% Paulownin or Puulon~tiiat ottientosa wood extract. In certain other preferred
embodiments, the compositions comprise from about 0.0001 to about 20%, from about 0.001 to
about lo%, from about 0.01 to about 5%, from about 0.1 to about 5%, or from about 0.2 to about
2% Paulo\vnin or Paz~lo~~~totritileat ltosa wood extract. In certain other preferred embodiments,
the compositions comprise from greater than zero to about 1%, from about 0.0001 to about 1%,
from about 0.001 to about 1%, or from about 0.01 to about 1% Paulownin or Pairloisnia
tontetitosa wood extract. In certain other preferred embodiments, the compositions comprise
from about 1 to about 5%, preferably from about 2 to about 5% Paulownin or Pattlo~snia
totiientosa wood extract.
Any suitable carrier may be used in the compositions of the present invention.
Preferably, for a skin care composition, the carrier is a cosmetically-acceptable carrier. As will
be recognized by those of skill in the art, cosmetically-acceptable carriers comprise carriers that
are suitable for use in contact with the body, in particular the skin for skin whitening
applications, without undue toxicity, incompatibility, instability, irritation, allergic response, and
the like. A safe and effective amount of carrier is from aboot 50% to about 99.999%, preferably
from about 80% to about 99.9%, more preferably from about 99.9% to about 95%, most
preferably from about 99.8% to about 98% of the composition. The carrier can be in a wide
variety of fonns. For example, emulsion carriers, including, but not limited to, oil-in-water,
water-in-oil, water-in-oil-in-water, and oil-in-water-in-silicone emulsions, are useful herein.
These emulsions can cover a broad range of viscosities, e.g., from about 100 cps to about
200,000 cps. Examples of suitable cosmetically-acceptable carriers include cosmeticallyacceptable
solvents and materials for cosmetic solutions, suspensions, lotions, creams, serums,
essences, gels, toners, sticks, sprays, ointments, liquid washes and soap bars, shampoos, hair
conditioners, pastes, foams, mousses, powders, shaving creams, wipes, patches, strips, powered
patches, microneedle patches, bandages, hydrogels, film-forming products, facial and skin
masks, make-up, liquid drops, and the like. These product types may contain several types of
cosmetically- acceptable carriers including, but not limited to solutions, suspensions, emulsions
such as tnicroe~nulsionsa nd nanoemalsions, gels, solids, liposomes, other encapsulation
technologies and the like. The following are non-limitative examples of such carriers. Other
carriers can be formulated by those of ordinary skill in the art.
In one embodiment, the carrier contains water. In a further embodiment, the carrier may
also contain one or more aqueous or organic solvents. Examples of organic solvents include, but
are not limited to: dimethyl isosorbide; isopropylmyristate; surfactants of cationic, anionic and
tlonionic nature; vegetable oils; mineral oils; waxes; gums; synthetic and natural gelling agents;
alkanols; glycols; atld polyols. Examples of glycols include, brit are not limited to, glycerin,
propylene glycol, butylene glycol, pentalene glycol, hexyletle glycol, polyethylene glycol,
polypropylene glycol, diethylene glycol, triethylene glycol, capryl glycol, glycerol, butanediol
and hexanetriol, and copolymers or mixtures thereof. Examples of alkanols include, but are not
limited to, those having from about 2 carbon atoms to about 12 carbon atoms (e.g., from about 2
carbon atoms to about 4 carbon atoms), such as isopropatlol and ethanol. Examples of polyols
include, but are not limited to, those having from about 2 carbon atoms to about 15 carbon atoms
(e.g., from about 2 carbon atoms to about 10 carbon atoms) such as propylene glycol. The
organic solvents may be present in the carrier in an amount, based upon the total weight of the
carrier, of from about 1 percent to about 99.99 percent (e.g., from about 20 percent to about 50
percent). Water may be present in the carrier (prior to use) in an amount, based upon the total
weight of the carrier, of from about 5 percent to about 95 percent (e.g., frotn about 50 percent to
about 90 percent). Solutions may contain any suitable amounts of solvent, including from about
40 to about 99.99%. Certain preferred solutions contain from about 50 to about 99.9%, frotn
about 60 to about 99%, frotn about 70 to about 99%, from about 80 to about 99%, or from about
90 to 99%.
A lotion can be made from such a solution. Lotions typically contain at least one
etnollient in addition to a solvent. Lotions may comprise from about 1% to about 20% (e.g.,
from about 5% to about 10%) of an emollient(s) and from about 50% to about 90% (e.g., from
about 60% to about 80%) ofwater.
Another type of product that may be formulated from a solution is a cream. A cream
typically contains from about 5% to about 50% (e.g., frotn about 10% to about 20%) of an
emollient(s) and frotn about 45% to about 85% (e.g., from about 50% to about 75%) ofwater.
Yet atlother type of product that may be formulated from a solution is an ointment. An
ointment may contain a simple base of animal, vegetable, or synthetic oils or semi-solid
hydrocarbons. An ointment may contain from about 2% to about 10% of an emollient(s) plus
from about 0.1% to about 2% of a thickening agent(s).
The compositions usefbl in the present invention can also be formulated as emulsions. If
the carrier is an emulsion, from about 1% to about 10% (e.g., from about 2% to about 5%) of the
carrier contains an emulsifier(s). Emulsifiers may be nonionic, anionic, or cationic.
Lotions and creams can be formulated as emulsions. Typically such lotions contain from
0.5% to about 5% of an emulsifier(s), while such creams would typically contain from about 1%
to about 20% (e.g., from about 5% to about 10%) of an emollient(s); from about 20% to about
80% (e.g., from 30% to about 70%) of water; and from about 1% to about 10% (e.g., from about
2% to about 5%) of an emulsifier(s).
Single emulsion skin care preparations, such as lotions and creams, of the oil-in-water
type and water-in-oil type are well-known in the art and are useful in the subject invention.
Multiphase emulsion compositions, such as the water-in-oil-in-water type or the oil-in-water-inoil
type, are also useful in the subject invention. In general, such single or ~naltiphasee mulsions
contain water, emollients, and emulsifiers as essential ingredients.
The compositions of this invention can also be formulated as a gel (e.g., an aqueous,
alcohol, alcohol/water, or oil gel using a suitable gelling agent(s)). Suitable gelling agents for
aqueous andlor alcoholic gels include, but are not limited to, natural gums, acrylic acid and
acrylate polymers and copolymers, and cellulose derivatives (e.g., hydroxymethyl cellulose and
hydroxypropyl cellulose). Suitable gelling agents for oils (such as mineral oil) include, but are
not limited to, hydrogenated butylene/ethyletle/styrene copolymer and hydrogenated
ethylenelpropylenelstyrene copolymer. Such gels typically contains between about 0.1% and
5%, by weight, of such gelling agents.
The compositions of the present invention can also be formulated into a solid formulation
(e.g., a wax-based stick, soap bar composition, powder, or wipe). The composition of the present
invention can also be combined with a solid, semi-solid, or dissolvable substrate (e.g., a wipe,
mask, pad, glove or strip).
The compositions of the present invention can also be formulated into formulation used
for the oral cavity, such as toothpaste, gel, rinse, solution, patch, and the like. The compositions
may also be formulated for use in the eye, such as in solutions, emulsions, suspensions used as
drops or washes and the like, or formulated for use in the vaginal mucosa such as via gels,
lotions, lubricants, and the like.
The compositions of the present invention may further comprise any of a variety of
additional cosmetically active agents. Examples of suitable additional active agents include:
additional skin lightening agents, darkening agents, additional anti-aging agents, tropoelastin
promoters, collagen promoters, anti-acne agents, shine control agelits, anti-microbial agents
such as anti-yeast agents, anti-fungal, atid anti-bacterial agents, anti-inflammatory agents, antiparasite
agents, external analgesics, sunscreens, photoprotectors, antioxidants, keratolytic agents,
detergents/surfactants, moisturizers, nutrients, vitamins, energy enhancers, anti-perspiration
agents, astringents, deodorants, hair removers, hair growth enhancing agents, hair growth
delaying agents, firming agents, hydration boosters, efficacy boosters, anti-callous agents, agents
for skin conditioning, anti-cellulite agents, fluorides, teeth whitening agents, anti-plaque agents,
and plaque-dissolving agents, odor-control agents such as odor masking or pH-changing agents,
and the like. Examples of various suitable additional cosmetically acceptable actives include
hydroxy acids, benzoyl peroxide, D-panthenol, , UV filters such as but not limited to avobenzone
(Parsol 1789), bisdisulizole disodium (Neo Heliopao AP), diethylamino hydroxybenzoyl hexyl
benzoate (Uvinul A Plus), ecamsole (Mexoryl SX), methyl anthranilate, 4-amitiobenzoic acid
(PABA), cinoxate, ethylhexyl triazone (Uvinul T 150), homosalate, 4-methylbenzylidene
catnplior (Parsol 5000), octyl ~nethoxycinnamate(O ctinoxate), octyl salicylate (Octisalate),
padimate 0 (Escalol507), plienylbenzimidazoie sulfonic acid (Ensulizole), polysilicone-15
(Parsol SLX), trolamine salicylate, Bemotrizinol (Tinosorb S), benzophenones 1-12,
dioxybe~izoned, rometrizole trisiloxane (Mexoryl XL), iscotrizinol (Uvasorb HEB), octocrylene,
oxybenzone (Eusolex 4360), sulisobenzone, bisoctrizole (Tinosorb M), titanium dioxide, zinc
oxide, carotenoids, free radical scavengers, spin traps, retinoids and retinoid precursors such as
retinol, retinoic acid and retinyl palmitate, ceratnides, polyunsaturated fatty acids, essential fatty
acids, enzymes, enzyme inhibitors, minerals, hormones such as estrogens, steroids such as
liydrocortisone, 2-dimethylaminoethanol, copper salts such as copper chloride, peptides
containing copper such as Cu:Gly-His-Lys, coenzyme Q10, amino acids such a proline, vitamins,
lactobionic acid, acetyl-coenzyme A, niacin, riboflavin, thiamin, ribose, electron transporters
such as NADH atid FADH2, and other botanical extracts such as oat, aloe Vera, Feverfew, Soy,
Shiitake mushroom extracts. and derivatives and mixtures thereof.
In certain preferred embodiments, the compositions of the present invention are skin care
compositions that comprise Paulownin and/or an extract ofP uz~1o~etf~oitutt entosaw ood and at
least one additional skin lightening active agent. Examples of suitable additional skin lightening
active agents include, but are not limited to, tyrosinase inhibitors, melanin-degradation agents,
melanoso~netr ansfer inhibiting agents including PAR-2 antagonists, exfoliants, sunscreens,
retinoids, antioxidants, Tranexamic acid, tranexamic acid cetyl ester hydrochloride, skin
bleaching agents, linoleic acid, adenosine non no phosphate disodium salt, Chamomilla extract,
allantoin, opacifiers, talcs and silicas, zinc salts, and the like, and other agents as described in
Solano et 01. Pigment Cell Res. 19 (550-571) and Ando et al. Int J Mol Sci 11 (2566-2575).
Examples of suitable tyrosinase inhibitors include but, are not limited to, Vitamin C and
its derivatives, Vitamin E and its derivatives, Kojic Acid, Arbutin, resorcinols, hydroquinone,
Flavones e.g. Licorice flavanoids, Licorice root extract, Mulberry root extract, Dioscorea
Coposita root extract, Saxifraga extract and the like, Ellagic acid, Salicylates and derivatives,
Glucosamine and derivatives, Fullerene, Hinokitiol, Dioic acid, Acetyl glucosatnine, 5,s'-
dipropyl-biphenyl-2,2'-diol (Magnolignan), 4-(4-11ydroxyphenyl)-2-butanol (4-HPB),
combinations of two or more thereof, and the like. Examples of vitamin C derivatives include,
but are not limited to, ascorbic acid and salts, Ascorbic Acid-2-Glucoside, sodium ascorbyl
phosphate, magnesium ascorbyl phosphate, and natural extract enriched in vitamin C. Examples
of vitamin E derivatives include, but are not limited to, alpha-tocopherol, beta, tocopherol,
gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol,
delta-tocotrienol and mixtures thereof, tocopherol acetate, tocopherol phosphate and natural
extracts enriched in vitamin E derivatives. Examples of resorcinol derivatives include, but are
not limited to, resorcinol, 4-substituted resorcinols like 4-alkylresorcinols such as 4-
butyresorcinol (rucinol), 4-hexylresorcinol (Synovea HR, Sytheon), phenylethyl resorcinol
(Symwhite, Symrise), 1-(2,4-dihydroxypheny1)-3-(2,4-dimetl1oxy-3-methylp1~enyl)-Propane
(nivitol, Unigen) and the like and natural extracts enriched in resorcinols. Examples of
salicylates include, but are not limited to, 4-methoxy potassium salicylate, salicylic acid,
acetylsalicylic acid, 4-methoxysalicylic acid and their salts. In certain preferred embodiments,
the tyrosinase inhibitors include a 4-substituted resorcinol, a vitamin C derivative, or a vitamin E
derivative. In more preferred embodiments, the tyrosinase inhibitor comprises Phenylethyl
resorcitloi, 4-hexyl resorcinol, or ascorbyl-2-glucoside.
Examples of suitable melanin-degradation agents include, but are not limited to,
peroxides and enzymes such as peroxidases and ligninases. In certain preferred embodiments,
the melanin-inhibiting agents include a peroxide or a ligninase.
Examples of suitable melanosome transfer inhibiting agents including PAR-2 antagonists
such as soy trypsin inhibitor or Bowman-Birk Inhibitor, Vitamin B3 and derivatives such as
Niacinamide, Essential soy, Whole Soy, Soy extract. In certain preferred embodiments, the
melanosotne transfer inhibiting agents includes a soy extract or niacinamide.
Examples of exfolliants include, but are not limited to, alpha-hydroxy acids such as lactic
acid, glycolic acid, malic acid, tartaric acid, citric acid, or atiy combination of any of the
foregoing, beta-hydroxy acids such as salicylic acid, polyhydroxy acids such as lactobionic acid
and gloconic acid, and mechanical exfoliation such as microdermabrasion. In certain preferred
embodiments, the exfolliatit iticlude glycolic acid or salicylic acid.
Exatnples of sunscreens include, but are not limited to, avobenzone (Parsol 1789),
bisdisulizole disodium (Neo Heliopan AP), diethylamino hydroxybenzoyl hexyl benzoate
(Uvinul A Plus), ecamsule (Mexoryl SX), methyl antbranilate, 4-atninobenzoic acid (PABA),
cinoxate, ethylhexyl triazone (Uvinul T 150), homosalate, 4-methylbenzylidene camphor (Parsol
5000), octyl methoxycinnamate (Octinoxate), octyl salicylate (Octisalate), padimate 0 (Escalol
507), phenylbenzimidazole sulfonic acid (Ensulizole), polysilicone-15 (Parsol SLX), trolamine
salicylate, Bemotrizinol (Tinosorb S), benzophenones 1-12, dioxybenzone, drometrizole
trisiloxane (Mexoryl XL), iscotriziuol (Uvasorb HEB), octocrylene, oxybenzone (Eusolex 4360),
sulisobenzone, bisoctrizole (Tinosorb M), titanium dioxide, zinc oxide, and the like.
Exatnples of retinoids include, but are not limited to, retinol (Vitamin A alcohol), retinal
(Vitamin A aldehyde), retinyl acetate, retinyl propionate, retiuyl linoleate, retinoic acid, retinyl
palmitate, isotretinoin, tazarotene, bexarotene, Adapaletie, colnbinations of two or more thereof
atld the like. It1 certain preferred embodiments, the retinoid is selected fsotn the group consisting
of retinol, retinal, retinyl acetate, retinyl propionate, retinyl linoleate, and cotnbinations of two or
more thereof. In certain more preferred embodiments, the retinoid is retinol.
Examples of antioxidants include, but are not limited to, water-soluble antioxidants such
as sulfhydryl compounds and their derivatives (e.g., sodium metabisulfite and N-acetyl-cysteine,
glutathione), lipoic acid atid dihydrolipoic acid, stilbenoids such as resveratrol and derivatives,
lactoferrin, iron and copper chelators and ascorbic acid and ascorbic acid derivatives (e.g.,
ascobyl-2-glucoside, ascorbyl palmitate and ascorbyl polypeptide). Oil-soluble antioxidants
suitable for use in the compositions of this iuvention include, but are not limited to, butylated
hydroxytoluene, retinoids (e.g., retinol and retinyl palmitate), tocopherols (e.g., tocopherol
acetate), tocotrienols, and ubiquinones. Natural extracts containing antioxidants suitable for use
in the compositions of this invention, include, but not limited to, extracts containing flavonoids
and isoflavonoids and their derivatives (e.g., genistein and diadzein), extracts containing
resveratrol and the like. Examples of such natural extracts include grape seed, green tea, black
tea, white tea, pine bark, feverfew, parthenolide-free feverfew, oat extracts, blackberry extract,
cotinus extract, soy extract, pomelo extract, wheat germ extract, Hesperedin, Grape extract,
Portulaca extract, Licochalcone, chalcone, 2,2'-dihydroxy chalcone, Primula extract, propolis,
and the like.
The additional cosmetically active agent may be present in a composition in any suitable
amount, for example, in an amount of from about 0.0001% to about 20% by weight oftbe
composition, e.g., about 0.001% to about 10% such as about 0.01% to about 5%. In certain
preferred embodiments, in an amount of 0.1% to 5% and in other preferred embodiments from
1% to 2%.
In certain preferred embodiments, the compositions of the present invention are skin care
compositions that comprise Paulownin and/or an extract of Purrlo~~~tnoniule ritosu wood and at least
one additional anti-inflammatoiy agent. Suitable additional anti-inflammatory active agents
include, but are not limited to, cornpounds that have an IC50 (concentration at which a compound
achieves 50% inhibition of inflammation) of less than or equal to 100 pdml for Interleukin-2 in the
ANTI-INFLAMMATORY ASSAY set foi-th below. In a preferred embodiment, the IC50 for the
second anti-inflammatory compounds is less than about 70 pglml, more preferably less than about
50 pdml, more preferably less than about 40 pg/ml, more preferably less than about 30 pdml.
The ANTI-INFLAMMATORY ASSAY assesses the ability of an agent to reduce the
production of cytokines by human lymphocytes stimulated with the T-cell receptor (TCR)
activating agent phytohaemagglutinin (PHA), and is conducted in the following manner. Human
leukocytes are collected from a healthy adult male via leukopheresis, aud adjusted to a density of
1 x lo6 cells/mL in serum free lymphocyte growth medium (ExVivo-15, Biowhittaker,
Walkersville, Md.). PBLs are stimulated with 10 pg/mL PHA in the presence or absence of test
samples following published methods (Hamamoto Y., et al. Exp Der1t1ufol2:23 1-235, 1993).
Following a 48 hour incubation at 37' C. with 5% CO2, the supernatant is removed and
evaluated for cytokine content using commercially available multiplex cytokine detection kit.
Examples of suitable anti-inflammato~ya gents include substituted resorcinols, (E)-3-(4-
methylphenylsulfony1)-2-propenenitrile( such as "Bay 11-7082," co~n~nercialalyva ilable from
Sigma-Aldrich of St. Louis, Missouri), tetrabydrocurcuminoids (such as Tetrahydrocurcuminoid
CG, available from Sabinsa Corporation of Piscataway, NJ),ex tracts and materials derived from the
following:
Phellodendron Amurense Cortex Extract (PCE)
Non-Denatured Soy (Glycine max)
Feverfew (Tanacetum parthenium)
Ginger (Zingiber officinale)
Ginko (Ginko Biloba)
Madecassoside (centella asiatica extract ingredient)
Cotinus (Cotinus coggygria)
Butterbur Extract @etasites hybridus)
Goji Berry (Lycium barbarum )
Milk Thistle Extract (Silybum marianum))
Honeysuckle (Lonicera japonica )
Basalm of Peru (Myroxylon pereirae)
Sage (Salvia officinalis)
Cranberry Extract (Vaccinium oxycoccos)
Amaranth Oil (Amaranthus cruentus)
Pomegranate (Punica granatum)
Yerbe Mate (Ilex paraguariensis Leaf Extract)
White Lily Flower Extract (Lilium Candidurn)
Olive Leaf Extract (Olea europaea)
Phloretin (apple extract)
Oat Flour (Aveena Sativa)
Lifenol (Hops: Hulnulus lupulus) Extract
Bugrane P (Ononis spinosa)
Licochalcone (Licorice: Glycyrrhiza inflate extract ingredient)
Symrelief (Bisabolol and Ginger extract)
combinations of two or more thereof, and the like.
Resorcinol is a dihydroxy phenol compound (i.e., 1,3 dihydroxybenzene) having by the
following structure:
As used herein, "substituted resorcinol" means resorcinol comprising at least one substituent in the
2,4,5, or 6 position. Thus, the substituted resorcinol may have as few as one and as many as four
substituents. Positions 1 and 3 of the s~tbstihitedr esorcinol comprise-OH groups, as shown above.
In embodiments wherein substituted resorcinol is used for anti-inflammation, it is highly
preferred that all of the si~bstituentso f the substituted resorcinol are free of phenyl (-C6H5
aromatic) moieties. In ce~taine mbodiments, all of the s~~bstituenatrse free of aromatic moieties
(with or without heteroatorns). In certain such embodiments, it is preferred that all of the
substituents of the substituted resorcinol are free of ketone functionalities (carbonyls bonded to
two other carbon atoms). In certain other such embodiments, all ofthe substituents of the
substituted resorcinol are free of both phenyl functionalities and ketone functionalities. In
certain other such embodiments, the substituted resorcinol comprises at least one substituent
comprising 5 to 11 carbon atoms, preferably 5 to 10 carbon atoms, more preferably 5 to 9 carbon
atoms, most preferably 5 to 8 carbon atoms. In certain other such embodiments, at least one
substituent comprises an alkyl group, such as one having the number of carbon atoms described
above. The alkyl group is preferably unsaturated.
In certain embodime~ltst,h e 4 position of the resorcinol is substituted, and, in certain
embodiments, only the 4 position is substituted. In another embodiment, the 4 position is
substituted with an alkyl group. hl certain preferred embodiments, the substihited resorcinol
comprises a single substihient at the 4 position that comprises an alkyl group. In certain other
preferred embodiments, the substituted resorcinol comprises a single substituent at the 4 position
that consists of an alkyl group directly bonded to the benzene ring.
Pa~ticularlys uitable substituted resorcinols for anti-inflammation agents include 4-hexyl
resorcinol and 4-octylresorcinol, particularly 4-hexyl resorcinol. The st~uctureos f 4-hexylresorcinol
and 4-octylresorcinol are shown below:
4-hexyl resorcinol
4-Hexyl resorcinol is commercially available as "SYNOVEA HR" fiom Sytheon of Lincoln Park,
NJ. 4-Octylresorcinol is commercially available from City Chemical LLC of West Haven,
Connecticut.
In certain embodiments, the substituted resorcinol comprises at least two substituents in the
2, 4, 5, or 6 positions. Such substituents may optionally be linked to form a ring, such as a cyclic
aliphatic hydrocarbon optionally comprising heteroatoms such as sulfur or oxygen. Such a linked
sobstituent may comprise 5 to 10 carbon atoms, e.g., 8 to 10 carbon atoms, and optionally include 1
to 3 heteroatoms. Examples of suitable substituted resorcinols comprising cyclic aliphatic
substituents joining the 2 and 3 positions include Zearalanone and P-Zearalanol:
Zearalanone and P-Zearalanol are commercially available from Sigma Chemicals of St. Louis,
Missouri.
In certain other embodiments, the substituted resorcinol comprises halide-containing and/or
nitroso-containing st~bstituents. Suitable examples contain ~iia
Based on the example above, topical application of the Patrloitoiia totrlentosa extract was able to
significantly reduce the UV-stimulated release of inflammatory mediators. Therefore, Pairlo~vliia
tonlelitosa extracts would be expected to provide an effective anti-inflammatory benefit when
applied to skin.
91.4
0%
Example 8
I~ihibitiono f Reactive Oxygen Species Formation in Reconstituted Epidermis UV-induced
hydrogen peroxide formation was determined using a modification of the method of Martin et
al., A~.cli Dertnatol Res. (2008) 300:69-80, in reconstituted epidermis and the human epithelial
cell litle, KB. Epidermal equivalents (EPI 200 HCF), multilayer and differentiated epidermis
consisting of normal human epidermal keratinocytes, were purchased froin MatTek (Ashland,
MA). Upon receipt, epidermal equivalents were incubated for 24 hours at 37°C in maintenance
medium without hydrocortisone. After 24 hours, the tissues were incubated for 30 minutes with
5 pM of the hydrogen peroxide-sensitive fluorescent probe 5-(and-6)-chloromethyl-2',7'-
dichlorodihydro-fluorescein diacetate, acetyl ester (CM-H2DCFDA) (Invitrogen Corp.,
Carlsbad, CA). After incubation, the plate was rinsed to remove excess probe and equivalents
were topically treated (2ing/cm2) with Pa~rlo~sntioat ilentosa extract (E3) in 70% ethanol/30%
propylene glycol vehicle. The plate was immediately read on a fluorescent plate reader set at
wavelengths 48511m excitation/ 530nm emission to detect basal peroxide formation. The plate
was then exposed to UV (1000W-Oriel solar simulator equipped with a 1 tnm Schott WG 320
161.2
183.3
55.2%
42.7%
filter; UV dose applied 4.2 k.T/mZ as measured at 36Onm). The plate was read 60 minutes post
UV exposure.
Table 5
I Treatment (Dose. as %\v/v) I Mean Fluorescent Units I Percent Inhibition of ROS I
I Propylene Glycol) I I I
Production
Based on the example, topical application of Parrloi~~nfioar rlenfosae xtracts was able to
significantly reduce the UV-stimulated production of ROS in reconstituted epidermis. Therefore
when applied to skin, Pazrloisnia fo~r~eritoesxat racts would be expected to provide protection
against induction of ROS from solar irradiation.
UV + Vehicle (70:30 Ethanol: I 761.5
Example 9
0%
Inhibition of Reactive Oxygen Species Pornlation in human epithelial cells
KB cells obtained from ATCC (ATCC#CCL-17, Manassas, VA) were plated in 96-well tissue
culture treated plates at a density of 5000 cellslwell in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 10% fetal bovine serum (Invitrogen Corp., San Diego, CA). After
48 hours, cells were incubated for 30 minutes with 5 pM of the hydrogen peroxide-sensitive
fluorescent probe 5-(and-6)-chloromethyl-2',7'-dicI~lorodihydro-fluorescedinia cetate, acetyl ester
(CM-H2DCFDA) (hlvitrogen Corp., Carlsbad, CA). After incubation, the plate was rinsed to
remove excess probe and Pazrloisriia tonrentosa extract (E3) was added at the indicated
concentrations. The plate was immediately read on a fluorescent plate reader set at wavelengths
485nm excitation1 530nm emission to detect basal peroxide formation. The plate was then
exposed to UV (1000W-Oriel solar simulator equipped with a 1 mm Schott WG 320 filter; UV
dose applied 4.2 k.J/m2 as measured at 360nm). The plate was read 60 minutes post UV
exposure.
Table 6
Percent Inhibition of ROS
Production (over vehicle)
-
Treatment (Dose, as %w/v)
untreated
I I
Mean Fluorescertt Units
79.7
I I
Based on the examnples, treatment with Paulo~el~tioan tenfosa extract was able to significantly
reduce the UV-stimulated production of ROS in humatl epithelial cells. Therefore when applied
UV treated -
UV + Pazrlo~sniat orr~eittosa
(0.005%)
UV + Paulo~t~ritioan te~itosa
(0.01%)
UV + Pazrlo~snialo nientosa
(0.02%)
to skin, Pazilo~~~itioinai erttosa extracts would be expected to provide protection against induction
of ROS from solar irradiation.
220.5
UV + Vehicle (DMSO) 0%
Example 10
Protection from Elastase Degradation
Human leukocyte elastase (HLE) was purchased from Sigma (St. Louis, Mo.), and reconstituted
at 1 unit/ml in phosphate buffered saline (PBS, Invitrogen life Technologies, Carlsbad, Calif.).
Soluble bovine neck ligament elastin labeled with BODIPY FL dye was purchased from
Molecular Probes, Inc. (Eugene, Or.), such that the fluoresceuce was quenched in the conjugate,
and could be activated upon elastase digestion. Human leukocyte elastase (0.0625 Ulml), elastin
substrate (25 pglml), and increasing concentrations of test material were incubated for two hours
at 37C. Fluorescence was measured at excitation at 490 ntn and emission at 520 nm using a
fluorescent plate reader Gemini from Molecular Devices (Sunnyvale, CA). Background
fluorescence of substriate alone had been subtracted from each measurement. Pazrlo~~~nia
torrientosa extract (E3) was prepared in DMSO at a stock concentration of 10 tnglml and serially
219.3
160.9
146.2
140.2
26.6%
33.3%
36.1%
diluted. Paulo~vriiato nteritosa extract inhibited HLE activity in a dose dependent manner as
shown in Table 7.
Table 7
damage and degradation.
0
0.00001%
0.001%
0.005%
0.01%
0.02%
Example 11
Inhibition of UV-Induced MMP inductior~
The ability of Paulo~niiuto rr~eritosae xtract (E3) to inhibit UV induced matrix
metalloproteinases-1 and -9 (MMP-I and -9) was evaluated in epidermal equivalents derived
from normal human epidermal keratinocytes. MMPs are a family of enzymes that play a major
role in physiological remodeling and pathological destructiot~o f extracellular matrix. It is well
established that suberythemal doses of UV light induce MMP secretion in human skin, which in
turn degrades the extracellular matrix and plays a significant role in photoaging wrinkle
formation and loss of firmness and elasticity. See G. J. Fisher, et al., J lnvestig Dermatol.
Symposium Proceedings. 14(1): 20-24 (2009).
In order to evaluate the ability of Patilolvnia tomeritosa extracts to inhibit UV induced
MMPs, epidermal equivalents (EPI 200 HCF), multilayer and differentiated epidermis consisting
of normal human epidermal keratinocytes, were purchased from MatTek (Ashland, MA). Upon
receipt, epidermal equivalents were incubated for 24 hours at 37OC in maintenance medium
without hydrocortisone. Equivalents were topically treated (2mglcm2 ) with Paulo~niia
tori~e~tfoesxatr act (E3) in 70% ethanol/30% propylene glycol vehicle 2 hours before exposure to
solar ultraviolet light (1000~-oriels olar simulator equipped with a 1-mtn Schott WG 320 filter;
UV dose applied: 70 kJIm2 as measured at 360nm). Equivalents were incubated for 48 hours at
37°C with maintenance medium then supernatants were analyzed for MMP-1 and -9 using
0.0
23.1%
30.2%
66.6%
75.5%
99.4%
This example demonstrates that Paulo~eriialo r~~eritoesxat racts can protect elastin fibers from
commercially available kits (R&D Systems, Minneapolis, MN). Data in Table 8 represents the
mean of 2 independent experiments, each experimental cot~ditionis conducted using duplicate
tissues.
Table 8
Table 9
Percent Inl~ibitiono f MMP-1
Production
0%
53.9%
59.7%
65.2%
Treatment (Dose, as % wlv)
UV + Vehicle (70:30 Ethanol:
Propylene Glycol)
UV + Patrlo~sniaf orttertfosa 0.1%
UV + Patrlo~cwiato r~ienfosa1 %
UV + Paztlo~srtiaf onte~tfosa5 %
Based on the example topical application of Patrloivrtia fontenfosa extract was able to
significantly reduce the UV-stimulated release of MMP-1 and -9. Therefore when applied to
skin, Paulo111niufo rttertfosae xtracts would be expected to provide protection against induction of
MMP-1 and -9 following solar irradiation.
Mean of MMP-1 Release
(nglml)
12046.2
5555.9
4851.4
4186.4
Treatment (Dose, as % wlv)
UV + Vehicle (70:30 Ethanol:
Propylene Glycol)
UV + Pazilo~~~rfotirart ertfosa 0.1%
UV + Patrlo~sniaf ontentosa 5%
Mean of MMP-9 Release
(nglml)
20795.5
4585.9
7077.2
Percent I~lllibitiono f MMP-9
Production
0%
77.9%
65.9%
Example 12
Inhibition of TNF-a - Iuduced MMP induction
hl order to evaluate the ability of Pazrlo~cwiafo irreiifosae xtract (E3) to inhibit TNF-a induced
MMPs, epidermal equivalents (EPI 200 HCF), multilayer and differentiated epidermis consisting
of normal human epidermal keratinocytes, were purchased from MatTek (Ashland, MA). Upon
receipt, epidermal equivaletlts were incubated for 24 hours at 37°C in maintenance tnedium
without hydrocot-tisone. Equivalents were topically treated (2mg/cm2) with Parrlonotia
torirer7fosa extract (E3) in 70% ethanol/30% propylene glycol vehicle 2 hours before treatment
with TNF-a (100ngImL). Equivalents were incubated for 48 hours at 37'C with maintenance
medium then supernatants were analyzed for MMP-1 and -9 using commercially available kits
(R&D Systems, Minneapolis, MN).
Table 10
Treatment (Dose, as % wlv)
Untreated
Table 11
I I
Treatment (Dose, as % tdv) I Mean of MMP-9 Release I Percent Inhibition of MMP-9
Meall of MMP-1 Release
(nglml)
4848.4
Percent I~thibitioor~f MMP-1
Production
-
TNF-a induced 7867.2 0%
(nglml) Production
I I
TNF-a induced 0%
Untreated -
42958.6
13217.6
Based on the example topical application of Pazrloisnia toiiieiifosa extract was able to
significantly reduce the TNF-u stimulated release of MMP-1 and -9. Therefore when applied to
skin, Paztloivriia torrtei?tosa extracts would be expected to provide protection against induction of
MMP-I and -9.
Example 13
The TROPOELASTIN PROMOTER ASSAY was performed using Tatiacetirt~t
partlietiitmt (parthenolide-free feverfew extract from Integrated Botanical Technologies of
Ossining, NY).
Tatiacetiittt partlietiiritt~w as diluted in cell culture media (DMEM Media of Invitrogen, San
Diego CA) and Palrlo~stiiaf oltletitosaw as diluted in DMSO to the concentration of "active"
indicated in Table 12 below. The cornpounds were added to the transfected H9c2 cells and were
incubated for 24 hours. Test samples were compared to respective controls. The results are
shown in Table 12 below.
Table 12
Untreated control
Respective
Concentrations of
Actives
(on active basis)
Normalized
Tropoelastin
Promoter
Activity (RLU)
Percent
change over
respective
controls
Inhibitor:
Tropoelastin
Promoter
Tmacetintt partlieiiizrttt
' Vehicle control (DMSO)
0.005%
0.005%
2.73
2.25
2%
-
As can be seen from the results shown in Table 12, Pairloistiiu totnetitosa and Tanacet~rtn
partl~enizrrtd~e monstrated percent changes in tropoelastin promotion over the respective controls
of 32% and 2%, respectively. In contrast, the combination of both Pazrloivnia fontentosa and
Tatiacetzrt11parthetiizittr demonstrated a 55% improvement in tropoelastin promotion over the
vehicle control. This was much greater than a mere additive effect in performance.
A similar synergistic effect was observed when the concentration of Ta~iucetztrit
pal.tlietiitrt11w as raised from 0.002% to 0.005%. Tatiacet~rtt~parthetii~iatt~ tth e higher
concentration also showed a percent change in tropoelastin promotion over the control of 2%,
whereas the combination of Paulo~!aliato tnentosa and Tariacei~rpt~ar~t l~enitmta chieved a percent
change in tropoelastin promotion over the vehicle control of 62%.
The data demonstrates that the combination of Pattlo~c~titioat neiifosaa nd a tropoelastin
promoter (Tutiacefzit~~partl?etlpiurond~ uces a surprising and synergistic increase in tropoelastin
promotion activity.
Example 14: Isolation and identification of Paulownin
Isolation of major component (Paulownin) from Pa1rloistiiu tonleiltosa was accomplished
with a combination of fractionation and prep HPLC steps. A sample of 170mg was obtained
with identical retention times and UV spectra as of the major signal of the extract. Spectroscopic
studies confirmed its identity as of Paulownin. The purity of the isolated Paulownin was
determined to be 98%.
Example 15
The anti-oxidant activity of Paulownin was established via the Inhibition of Reactive
Oxygen Species Formation assay described above. In this study primary human keratinocytes
were substituted for human epithelial ( KB ) cells. The keratinocytes were plated in 96-well
tissue culture treated plates at a density of 5000 cells/~veliln Dulbecco's modified Eagle's
medium (DMEM) supplemented with 10% fetal bovine serum (Invitrogen Corp., San Diego ,
CA). After 48 hours, cells were incubated for 30 minutes with 5 pM of the hydrogen peroxidesensitive
fluorescent probe 5-(and-6)-chlorometl1yl-2',7'-dichlorodihydro-fluorescei1 diacetate,
acetyl ester (CM-H2DCFDA) (Invitrogen Corp., Carlsbad, CA). After incubation, the plate was
rinsed to remove excess probe and Panlo\vnin was added at the indicated concentrations. The
plate was immediately read on a fluorescent plate reader set at wavelengths 485ntn excitation1
530nm emission to detect basal peroxide formation. The plate was then exposed to UV (1000WOriel
solar simulator equipped with a 1 mm Schott WG 320 filter; UV dose applied 4.2 k.J1m2 as
measured at 360nm). The plate was read 60 minutes post UV exposure and the data reported as
mean fluorescent units (MFU). The results are shown in Table 13 below.
Table 13
I Treatment ' I Reactive Oxygen Species I Percent Inhibition of UV I
Untreated
UV Treated
UV +
Paulownin
(50ug/ml)
** Indicates significant difference from UV treated using a student's t-Test with
significance set at Pi0.05.
Pauiowuin
(2OOug/ml)
UV +
Paulownin
Based on the example, treatment with Paulownin was able to significantly reduce the UVinduced
production of ROS in human epithelial cells. Therefore when applied to skin, Paulownin
would be expected to provide protection against induction of ROS from solar irradiation and as
such would be expected to provide sttbstantial activity to reduce the signs of aging such as
improving the firmness of the skin, improving the texture of the skin, improving the appearance
of wrinkles in the skin, and treating the external aggressions in skin.
produced ( MFU) ---
13.8 + 2.4
133.2 f 16.7
37.2 f 3.4**
Example 16
The following skin care composition was prepared using the ingredients shown in Table 14
in accord with the present invention.
Induced ROS Formation
-
-
72.1%
40.5 f 6.1** 69.5%
TABLE 14
I
Ultrez 10
I I
Carbomer 0.60
I I I
0.20
4
6
7
I I
9 I PENRECO SNOW WHITE I Petrolatum
3 Disodium EDTA
Brij 72 Steareth-2
I I I
VERSENE NA
0.50
Finsolv TN
Miglyol 8 12 Neutral Oil
I I
Methylparaben, ethyparaben,
8
10
I l1 I Phenonip XB I propylparaben, I 1.00
C12-15 Alkyl Benzoate
Caprylic/Capric triglyceride
Emery 917 Glycerin
2.00
2.50
3.00
Dimethicone
12
13
Dow Corning Q7-9120
Silicone Fluide (20 cst)
I I
I I I I SODIUMHYDROXIDE I
2.00
Transcutol CG
1 .O% Citric Acid
14
I
1 16 1 PELLETS (7680-88) NP / Sodium Hydroxide / As needed
Princess Tree Extract I Pa~rlo~sni~al iperialiEs xtract
phenoxyethanol
EthoxyDiclycol
Citric acid
15
The above composition was prepared as follows: The purified water was added to a main
5.00
0.01
Butylene Glycol Botylene Glycol
FCC Pellets
tank at a temperature of20-40 'C with smooth agitation. The Versene NA (disodium EDTA) was
20.00
then added to the main tank. Agitation on the tank was stopped and Ultrez 10 (Carbotner) was
Total
added by evenly coating the top of the water mixture. The mixture was allowed to soak and
100.00
agitatiotl and heating was started. The mixture was heated and maintained at 55-60°C, and further
mixed for 15 minutes or until hotnogeneous.
An oil phase was prepared by adding Fiusolv TN ((212-15 alkyl benzoate) to a clean,
suitable phase container with agitation and heating to achieve 55-60 "C. After such temperahwe
was achieved Brij 72 & 721 (Steareth-2, -21 resp.), Miglyol (CaprylicICapric triglyceride), Emery
91 7 (glycerin), and Penreco snow white (Petrolahlm) were added and mixed at 55-60 "C until
addition to main tank.
The oil phase was added to the main tank with increased agitation and heating was stopped.
The resulting tnixh~rew as mixed at high speed for 10-20 minutes. At 50°C or lowel; the
Dimethicone (Dow Corning Silicone Fluid) was added. The batch mixture was then cooled to 40°C
and Phenonip XB (preservative mix) was added. The mixture was further mixed for 10 min or until
uniform. Sodium hydroxide was added quickly (target pH = 5.4) with further mixing for 10 minutes
or until uniform pH is achieved.
An actives premix was made by adding Transcutol CG, Butylene glycol, Citric acid, and
Pritlcess Tree extract to a separate beaker and mixingwell until uniform.
The final formulation was made by adding the actives premix to the main phase of the main
tank, and mixing for an additional 10- 20 minutes to dissolve completely or until uniform. The final
volumes were made up with water, the formulation mixed for I0 minutes, and pH recorded.
The samples of composition were placed in 50°C oveu for 2 week and showed primary
good stability.
Example 17
The following skin care composition was prepared using the in~edientsh own in Table 15
in accord with the present invention.
TABLE 15
. .. -.
CTFA / INCI N~IIIC Percel~t;~gine liorntul;ttiol~( lvhv)
.~ .. . . .- L... .. I 1 Purified Water 75.55
2 Disodium EDTA 0.15
1 3 1 Ammonium Ac~~~loyldin~etl~yltat~CraotpeoNlyP~ nerI 0.30 I
I I
I I
4 0.20
6
Chlorphenesin C
5 6.00
Cetearyl OlivateISorbitan Olivate
I
Butylene glycol
7
I I
I I
10 I Cyclopentasiloxane & Dimethicone Crosspolymer 1 3.00
8
I I
Stearic Acid 0.50
Ethylhexylglycerin
9 5.00
1.00
Cyclopentasiloxne & Cyclohexasiloxane
11 2.00
12
13
14
The above composition was prepared as follows: The purified water was added to a main
tank followed by addition of disodit~mE DTA and mixed until the EDTA dissolved. Ammonium
ac~yloyldimethyltaurateNP Copolymer was sprinkled in and the resulting mixture heated to 70-
75OC. After set temperature is achieved, Cetea~yOl livate/Sorbitan Olivate and Stearic acid were
added while agitating the mixture for 5 minutes at set temperature.
An actives premix was prepared by dissolving Priucess tree extract into butylene glycol at
40-50°C in a separate container. Polyacrylate 13 and polyisobutene and polysorbate 20 were added
to the Princess tree mix and then mixed until uniform. The temperahlre was lowered to 35-40°C
atld set aside until ready to add to the main tank.
A Chlorophenesin premix was prepared by adding Chlorophehesin to butylenes glycol in a
separate container and heating to 35'C, followed by addition of Methylisothiazolinone. The
Dimethiconol & Dimethicone
15
16
17
18
Sodium hydroxide
Polyacrylate 13 & Polyisobutene & Polysorbate 20
Methylisothiazolinone
2.40
1 .OO
0.15
Fragrance
FD&C Red
D&C Yellow
Paulowriia iniperialis (Princess Tree) Extract
Total
0.01
0.12
0.12
2.00
100.00
resulting mixture was heated to 50-55'C and the temnperature maintained until ready to mix into
main tank.
An oil phase was prepared in a separate container by adding Cyclopentasiloxane and
Dimethicone Crosspolymer to Cyclopentasiloxai~ea nd Cyclohexasiloxane while mixing and
heating to 55-60'' C uutil uniform. Then Etliylhexylglycerin was added and mixed until uniform,
followed by addition of Stearic acid with the temperature maintained between 55-60°C.
The oil phase was then added to the main tank slowly with vigorous agitation at a
temperature of 70-75°C. Diinethiconol and dimethicone were added to the main batch and the
resulting mixture stirred for 20 minutes or until uniform, after which the heating was stopped. The
main pH was adjusted to between 5.0-5.5 with sodium hydroxide. The Chlorophenesin phase was
added slowly at 50-55'C while maintaining agitation. The mixture was cooled to 35-40°C and the
fiagance, FD&C red and D&C Yellow added and mixed well while maintaining the temperature.
The final formulation was achieved by adding the active premix to the main tank slowly
with gentle mixing. The mixture was mixed for an additional 10-20 until uniform. The final
volumes were made up with water, the formulation mixed for 10 minutes, and pH recorded.
The samples of composition were placed in 50°C oven for 2 week and showed primary
good stability.
CLAIMS
WHAT IS CLAIMED IS:
1. A method of improving a sign of aging in skin selected from the group consisting of
improving the firmness of the skin, improving the texture of the skin, improving the
appearance of wrinkles in the skin, treating the external aggressions in skin, and
co~nbinationso f two or more thereof, comprising applying a therapeutically effective
amount of Paulownin to human skin in an amount effective to improve the firmness of
the skin, improve the texture of the skin, improve the appearance ofwrinkles in the skin,
treat the external aggressions in skin, or to improve combinations of two or more thereof.
2. The method of claim 1, wherein said applying step comprises applying from greater than
zero to about 20% of the Panlownin to skin in need of improving a sign of aging.
3. The method of claim 1, wherein said applying step comprises applying at least about 20%
by weight of the Paulownin to skin in need of improving a sign of aging.
4. The method of claim 1, wherein said applying step comprises applying at least about 50%
by weight of the Paulownin to skin in need of improving a sign of aging.
5. The method of claim 1, wherein said applying step comprises applying at least about 90%
by weight of the Paulownin to skin in need of improving a sign of aging.
6. The method of claim 1, wllerein said applying step comprises applying from about 0.01
to about 1% of the Paulownin to skin in need of improving a sign of aging.
7. The method of claim 1, wherein said applying step comprises applying from about 0.1 to
about 5% of the Paulownin to skin in need of improving a sign of aging.
8. The method of claim 1, wherein said applying step comprises applying a cornposition
comprising Paulownin and a carrier to skin in need of improving asign of aging, wherein
said composition is in the form of a solution, suspension, lotion, cream, serum, gel, stick,
spray, ointment, liquid wash, soap bar, shampoo, hair conditioner, paste, foam, powder,
mousse, shaving cream, hydrogel, or film-forming product.
The method of claim 8, wherein said composition further comprises a tropoelastin
promoter.
The method of claim 9, wherein said tropoelastin promoter is selected from the group
consisting of blackberry extracts, cotinus extracts, feverfew extracts, extracts of
Phyllanthus niruri and combinations of two or more thereof.
The method of claim8, wherein said composition further cotnprises a collagen promoter.
The method of claim 1 I, wherein the collagen promoter is a non-retinoid collagen
promoter.
The method of claim 12, wherein the non-retinoid collagen promoter is selected from the
group consisting of extracts of Tanacetum parthenium, extracts of Centella asiatica,
extracts of Siegesbeckia orientalis, extracts of soy, and combinations of two or more
thereof.
The method of claim 11, wherein the collagen promoter is a retinoid.
The method of claim 14, wherein the retinoid is selected from the group consisting
retinol, retinal, retinyl acetate, retinyl propionate, retinyl linoleate, retinyl palmitate, and
combinations of two or more thereof.
The method of claim 1, wherein improving a sign of aging comprises improving the
firmness of the skin and said applying step comprises applying to human skin in need of
improving the firmness of the skin an effective amount of Paulownin.
The method of claim 16, wherein said human skin in need of improving the firmness of
the skin comprises skin that is sagging, loose, lax, rough, wrinkly, thinned, uneven, or a
combination of two or more thereof.
18. The metliod jf claim 1, whctein itnproving a sign of aging corn
appearance of wrinkles in the skin and said applying step cotnp
skin in need of itill~rovingth e appearance ofwrinkles in the skit
Paulownin.
19. The incthod of claim 18, wherein said human skin in need of in
of wrinkles in She skin cotnprises skiti having wrinkles, fine linc
thereof.
20. The method of claim 8, wherein said composition further comprises a sunscreen.