U.S. patent application number 09/737072 was filed with the patent office on 2002-04-11 for cosmetic skin conditioning compositions containing red yeast rice extract.
Invention is credited to Bajor, John Steven, Januario, Thomas Eugene, Mahajan, Manisha Narayan, Pillai, Sreekumar, Santhanam, Uma.
Application Number | 20020041883 09/737072 |
Document ID | / |
Family ID | 22620814 |
Filed Date | 2002-04-11 |
United States Patent
Application |
20020041883 |
Kind Code |
A1 |
Januario, Thomas Eugene ; et
al. |
April 11, 2002 |
Cosmetic skin conditioning compositions containing red yeast rice
extract
Abstract
Cosmetic methods and compositions for conditioning human skin by
topical application to the skin of cosmetic compositions containing
red yeast rice extract. Cosmetic compositions containing red yeast
rice extract in combination with ascorbyl palmitate, or sodium
ascorbyl phosphate, or retinol, or retinyl ester are also
disclosed.
Inventors: |
Januario, Thomas Eugene;
(Middletown, NY) ; Santhanam, Uma; (Tenafly,
NJ) ; Pillai, Sreekumar; (Wayne, NJ) ;
Mahajan, Manisha Narayan; (Westwood, NJ) ; Bajor,
John Steven; (Ramsey, NJ) |
Correspondence
Address: |
UNILEVER
PATENT DEPARTMENT
45 RIVER ROAD
EDGEWATER
NJ
07020
US
|
Family ID: |
22620814 |
Appl. No.: |
09/737072 |
Filed: |
December 14, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60170669 |
Dec 14, 1999 |
|
|
|
Current U.S.
Class: |
424/195.16 ;
424/750 |
Current CPC
Class: |
A61K 8/671 20130101;
A61P 43/00 20180101; A61K 8/9728 20170801; A61P 17/16 20180101;
A61K 8/678 20130101; A61Q 19/008 20130101; A61Q 19/00 20130101;
A61Q 19/08 20130101; A61K 8/676 20130101; A61Q 19/007 20130101 |
Class at
Publication: |
424/195.16 ;
424/750 |
International
Class: |
A61K 035/70; A61K
035/72; A61K 035/78 |
Claims
What is claimed:
1. A cosmetic skin conditioning composition comprising: (a) from
about 0.001 to about 20 wt. % of red yeast rice extract; (b) a
co-active compound selected from the group consisting of ascorbyl
palmitate, sodium ascorbyl phosphate, retinol, retinyl ester, and
mixtures thereof; and (c) a cosmetically acceptable vehicle.
2. The composition of claim 1 wherein the red yeast rice extract is
an organic solvent extract.
3. A cosmetic method of reducing, preventing or controlling sebum
secretion from sebocytes by applying to the skin the composition of
claim 1.
4. A cosmetic method of stimulating collagen synthesis by
fibroblasts in the skin by applying to the skin the composition of
claim 1.
5. A cosmetic method of improving the appearance of wrinkled,
lined, dry, flaky, aged or photodamaged skin and improving skin
thickness, elasticity, flexibility and plumpness by applying to the
skin the composition of claim 1.
6. A cosmetic method of delivering a phytoestrogen to skin by
applying to the skin a cosmetic composition comprising a red yeast
rice extract in a cosmetically acceptable vehicle.
7. A method of controlling an oily skin the method comprising
applying to the skin a cosmetic composition comprising a red yeast
rice extract in a cosmetically acceptable vehicle.
8. A cosmetic method of stimulating collagen synthesis by
fibroblasts in the skin by applying to the skin a cosmetic
composition comprising a red yeast rice extract in a cosmetically
acceptable vehicle.
9. A cosmetic method of improving the appearance of wrinkled,
lined, dry, flaky, aged or photodamaged skin and improving skin
thickness, elasticity, flexibility and plumpness by applying to the
skin a cosmetic composition comprising a red yeast rice extract in
a cosmetically acceptable vehicle.
Description
[0001] This application claims the benefit of U.S. provisional
application no. 60/170,669 filed Dec. 1, 1999.
FIELD OF THE INVENTION
[0002] Cosmetic methods and compositions for conditioning human
skin by topical application to the skin of cosmetic compositions
containing red yeast rice extract.
BACKGROUND OF THE INVENTION
[0003] Cosmetic products which improve the appearance of skin are
increasingly popular with consumers. Frequently, consumers seek to
alleviate or delay the signs of aged or photoaged skin, such as
fine lines and wrinkles, dry and sagging skin. Consumers also
frequently seek other benefits in addition to anti-aging. A
frequent, undesirable skin condition is "oily skin," the condition
which results from the excessive amount of sebum on the skin. Sebum
is skin oil which is produced by sebocytes (cells of the sebaceous
glands in the skin) and is then secreted to the skin surface. Oily
skin is associated with a shiny, undesirable appearance and a
disagreeable tactile sensation. Oily skin affects various age
groups. Cosmetic actives which are able to provide both anti-aging
benefits and sebum control are highly desirable, both from the
manufacturer's and consumer's perspective.
[0004] In recent years, phytoestrogens (i.e., natural compounds
which have estrogen-like activity and which are found in plants)
have been increasingly used for cosmetic and therapeutic purposes.
Estrogens and synthetic compounds which act like estrogens are
known to increase the thickness of the dermal layer and reduce
wrinkle formation in the aging skin. Changes in the skin such as
skin dryness, loss of skin elasticity and plumpness occurring after
menopause are attributed to the lack of estrogen production.
Estrogen therapy prevents or slows down many of the changes
associated with aging skin (Creidi et al., Effect of a conjugated
oestrogen cream (Premarin.RTM.) on aging facial skin, Maturitas,
19, p.211-23, 1994).
[0005] Red yeast rice is also known as Monascus sp. Prior art
describes a melanin-formation inhibitor in skin care comprising
pigment obtained by culturing Monascus mould (JP 6065038 A).
Monascus chitinase has been used to prepare chitosan derivatives
with tyrosinase inhibiting activity (JP 4210615 A). Cultured
extract of the monascus fungus has been claimed in cosmetic
material for skin cleansing (JP 1090109 A) and cosmetics for
make-up (JP 10194928). U.S. Pat. No. 4,996,196 mentions red yeast
rice used in a dehydration process of hydrous matter.
[0006] None of the art described above discloses anti-sebum or
estrogenic properties of red yeast rice, or any cosmetic
compositions containing red yeast rice in combination with
acorbates or retinoids presently claimed.
SUMMARY OF THE INVENTION
[0007] The present invention includes a skin conditioning
composition comprising:
[0008] (a) from about 0.001 to about 20 wt. % of red yeast rice
extract;
[0009] (b) a co-active compound selected from the group consisting
of ascorbyl palmitate; sodium ascorbyl phosphate, retinol, retinyl
ester, and mixtures thereof; and
[0010] (c) a cosmetically acceptable vehicle.
[0011] The invention also includes a cosmetic method of controlling
oily skin condition and of reducing, preventing or controlling
sebum secretion from sebocytes by applying either the red yeast
rice extract alone, or the inventive composition, to the skin.
[0012] The invention also includes a cosmetic method of stimulating
collagen synthesis by fibroblasts in the skin, by applying either
the red yeast rice extract alone, or the inventive composition, to
the skin.
[0013] The invention also includes a cosmetic method of improving
the appearance of wrinkled, lined, dry, flaky, aged or photodamaged
skin and improving skin thickness, elasticity, flexibility and
plumpness by applying either the red yeast rice extract alone, or
the inventive composition, to the skin.
[0014] The invention also includes a cosmetic method of delivering
a phytoestrogen to skin by applying to the skin a cosmetic
composition comprising a red yeast rice extract in a cosmetically
acceptable vehicle.
[0015] The inventive methods and compositions provide control of
sebum secretion from sebocytes, improved oil control and improved
skin feel, prevent shine and stickiness, while also providing
anti-aging benefits which results in reduced appearance of wrinkles
and aged skin, improved skin color, treatment of photoaged skin,
improvement in skin's radiance and clarity and finish, and an
overall healthy and youthful appearance of the skin.
DETAILED DESCRIPTION OF THE INVENTION
[0016] Except in the examples, or where otherwise explicitly
indicated, all numbers in this description indicating amounts of
material or conditions of reaction, physical properties of
materials and/or use are to be understood as modified by the word
"about." All amounts are by weight of the composition, unless
otherwise specified.
[0017] The inventive compositions contain an extract of red yeast
rice. Preferably the extract is an organic solvent extract. The
organic extracts are prepared by extracting the red yeast rice
powder with a solvent by stirring 1 part of red yeast rice with 2
to 5 parts of the solvent for from 4 to 24 hours at room
temperature. Suitable solvents are described hereinbelow. The
extracts are clarified by filtration and/or centrifugation, then
used as is or dried by evaporation (optionally, under vacuum).
[0018] Solvents suitable for the preparation of red yeast rice
extract for use herein include, but are not limited to: ethanol,
butylene glycol, ethylene glycol, propylene glycolmethanol, hexane,
chloroform, dichloromethane, DMSO (dimethylsulfoxide) and ethyl
acetate. The preferred solvents are dichloromethane, methanol, or
ethanol, or DMSO, or glycol:ethanol mixtures in order to maximize
activity. The extract may be further concentrated, fractioned,
re-extracted or purified, e.g. by organic solvent extraction or by
chromatography.
[0019] The red yeast rice extract is employed in the present
invention in an amount from 0.001 to 20 wt. %, preferably from 0.01
to 10 wt. %, most preferably from 0.01 to 5 wt. %.
[0020] Red yeast rice may be obtained from Shangai International
Greenmen Trading Company.
[0021] According to one aspect of the invention, red yeast rice
extract is employed in a variety of cosmetic methods: to deliver
estrogenic activity to the skin; to control sebum secretion or oily
skin, and to stimulate collagen production by fibroblasts.
[0022] According to another aspect of the invention, red yeast rice
extract is employed in combination with a co-active compound, in
order to obtain substantially improved performance. The co-active
compound is selected from the group consisting of ascorbyl
palmitate; sodium ascorbyl phosphate, retinol, retinyl ester, and
mixtures thereof. The preferred co-active compounds are sodium
ascorbyl phosphate (due to its chemical stability) or
trans-retinol.
[0023] The co-active compound may be obtained from Sigma Chemical
Co. or BASF.
[0024] The term "retinol" includes the following isomers of
retinol: all-trans-retinol, 13-cis-retinol, 11-cis-retinol,
9-cis-retinol, 3,4-didehydro-retinol. Preferred isomers are
all-trans-retinol, 13-cis-retinol, 3,4-didehydro-retinol,
9-cis-retinol. Most preferred is all-trans-retinol, due to its wide
commercial availability.
[0025] Retinyl ester is an ester of retinol. The term "retinol" has
been defined above. Retinyl esters suitable for use in the present
invention are C1-C30 esters of retinol, preferably C2-C20 esters,
and most preferably C2, C3, and C16 esters because they are more
commonly available. Examples of retinyl esters include but are not
limited to: retinyl palmitate, retinyl formate, retinyl acetate,
retinyl propionate, retinyl butyrate, retinyl valerate, retinyl
isovalerate, retinyl hexanoate, retinyl heptanoate, retinyl
octanoate, retinyl nonanoate, retinyl decanoate, retinyl
undecandate, retinyl laurate, retinyl tridecanoate, retinyl
myristate, retinyl pentadecanoate, retinyl heptadecanoate, retinyl
stearate, retinyl isostearate, retinyl nonadecanoate, retinyl
arachidonate, retinyl behenate, retinyl linoleate, retinyl oleate,
retinyl lactate, retinyl glycolate, retinyl hydroxy caprylate,
retinyl hydroxy laurate, retinyl tartarate.
[0026] The preferred ester for use in the present invention is
selected from retinyl palmitate, retinyl acetate, retinyl linoleate
and retinyl propionate, because these are the most efficaceous or
the most commercially available and therefore the cheapest.
[0027] The co-active compound is employed in the inventive
compositions and methods generally in an amount of from 0.001 to 20
wt %, preferably from 0.01 to 10 wt %, most preferably from 0.01 to
5 wt. % .
[0028] Cosmetically Acceptable Vehicle
[0029] The composition according to the invention also comprises a
cosmetically acceptable vehicle to act as a dilutant, dispersant or
carrier for the actives in the composition in the composition, so
as to facilitate their distribution when the composition is applied
to the skin.
[0030] Vehicles other than or in addition to water can include
liquid or solid emollients, solvents, humectants, thickeners and
powders. An especially preferred nonaqueous carrier is a
polydimethyl siloxane and/or a polydimethyl phenyl siloxane.
Silicones of this invention may be those with viscosities ranging
anywhere from about 10 to 10,000,000 mm.sup.2/s (centistokes) at
25.degree. C. Especially desirable are mixtures of low and high
viscosity silicones.
[0031] These silicones are available from the General Electric
Company under trademarks Vicasil, SE and SF and from the Dow
Corning Company under the 200 and 550 Series. Amounts of silicone
which can be utilized in the compositions of this invention range
anywhere from 5% to 95%, preferably from 25% to 90% by weight of
the composition.
[0032] The cosmetically acceptable vehicle will usually form from
5% to 99.9%, preferably from 25% to 80% by weight of the
composition, and can, in the absence of other cosmetic adjuncts,
form the balance of the composition. Preferably, the vehicle is at
least 80 wt. % water, by weight of the vehicle. Preferably, the
amount of water is at least 50 wt. % of the inventive composition,
most preferably from 60 to 80 wt. %, by weight of the composition.
The preferred compositions are oil-in-water emulsions, containing
at least 60%, preferably at least 80% water.
[0033] Optional Skin Benefit Materials and Cosmetic Adjuncts
[0034] The inventive compositions preferably include sunscreens to
lower skin's exposure to harmful UV rays.
[0035] Sunscreens include those materials commonly employed to
block ultraviolet light. Illustrative compounds are the derivatives
of PABA, cinnamate and derivatives of salicylate. For example,
octyl methoxycinnamate and 2-hydroxy4-methoxy benzophenone (also
known as oxybenzone) can be used. Octyl methoxycinnamate and
2-hydroxy4-methoxy benzophenone are commercially available under
the trademarks, Parsol MCX and Benzophenone-3, respectively. The
exact amount of sunscreen employed in the emulsions can vary
depending upon the degree of protection desired from the sun's UV
radiation.
[0036] An oil or oily material may be present, together with an
emollient to provide either a water-in-oil emulsion or an
oil-in-water emulsion, depending largely on the average
hydrophilic-lipophilic balance (HLB) of the emollient employed.
Levels of such emollients may range from about 0.5% to about 50%,
preferably between about 5% and 30% by weight of the total
composition. Emollients may be classified under such general
chemical categories as esters, fatty acids and alcohols, polyols
and hydrocarbons.
[0037] Esters may be mono- or di-esters. Acceptable examples of
fatty di-esters include dibutyl adipate, diethyl sebacate,
diisopropyl dimerate, and dioctyl succinate. Acceptable branched
chain fatty esters include 2-ethyl-hexyl myristate, isopropyl
stearate and isostearyl palmitate. Acceptable tribasic acid esters
include triisopropyl trilinoleate and trilauryl citrate. Acceptable
straight chain fatty esters include lauryl palmitate, myristyl
lactate, oleyl eurcate and stearyl oleate. Preferred esters include
coco-caprylate/caprate (a blend of coco-caprylate and
coco-caprate), propylene glycol myristyl ether acetate, diisopropyl
adipate and cetyl octanoate.
[0038] Suitable fatty alcohols and acids include those compounds
having from 10 to 20 carbon atoms. Especially preferred are such
compounds such as cetyl, myristyl, palmitic and stearyl alcohols
and acids.
[0039] Among the polyols which may serve as emollients are linear
and branched chain is alkyl polyhydroxyl compounds. For example,
propylene glycol, sorbitol and glycerin are preferred. Also useful
may be polymeric polyols such as poly-propylene glycol and
polyethylene glycol. Butylene and propylene glycol are also
especially preferred as penetration enhancers.
[0040] Exemplary hydrocarbons which may serve as emollients are
those having hydrocarbon chains anywhere from 12 to 30 carbon
atoms. Specific examples include mineral oil, petroleum jelly,
squalene and isoparaffins.
[0041] Another category of functional ingredients within the
cosmetic compositions of the present invention are thickeners. A
thickener will usually be present in amounts anywhere from 0.1 to
20% by weight, preferably from about 0.5% to 10% by weight of the
composition. Exemplary thickeners are cross-linked polyacrylate
materials available under the trademark Carbopol from the B.F.
Goodrich Company. Gums may be employed such as xanthan,
carrageenan, gelatin, karaya, pectin and locust beans gum. Under
certain circumstances the thickening function may be accomplished
by a material also serving as a silicone or emollient. For
instance, silicone gums in excess of 10 centistokes and esters such
as glycerol stearate have dual functionality.
[0042] Powders may be incorporated into the cosmetic composition of
the invention. These powders include chalk, talc, kaolin, starch,
smectite clays, chemically modified magnesium aluminum silicate,
organically modified montmorillonite clay, hydrated aluminum
silicate, fumed silica, aluminum starch octenyl succinate and
mixtures thereof.
[0043] Other adjunct minor components may also be incorporated into
the cosmetic compositions. These ingredients may include coloring
agents, opacifiers and perfumes. Amounts of these other adjunct
minor components may range anywhere from 0.001% up to 20% by weight
of the composition.
[0044] Product Use, Form, and Packaging
[0045] In use, a small quantity of the composition, for example
from 1 to 100 ml, is applied to exposed areas of the skin, from a
suitable container or applicator and, if necessary, it is then
spread over and/or rubbed into the skin using the hand or fingers
or a suitable device.
[0046] The cosmetic skin conditioning composition of the invention
can be formulated as a lotion, a cream or a gel. The composition
can be packaged in a suitable container to suit its viscosity and
intended use by the consumer. For example, a lotion or cream can be
packaged in a bottle or a roll-ball applicator, or a
propellant-driven aerosol device or a container fitted with a pump
suitable for finger operation. When the composition is a cream, it
can simply be stored in a non-deformable bottle or squeeze
container, such as a tube or a lidded jar. The composition may also
be included in capsules such as those described in U.S. Pat. No.
5,063,507 (silicone-based anhydrous composition within a gelatine
capsule), incorporated by reference herein. The invention
accordingly also provides a closed container containing a
cosmetically acceptable composition as herein defined.
[0047] The following specific examples further illustrate the
invention. Red yeast rice for the following experiments was
purchased from Shanghai Greenmen International Trading Co. Red
yeast rice extract was prepared by adding 1 ml ethanol to 1 mg of
red yeast rice powder, warming it at 37.degree. C. for 10 minutes,
letting the powder settle and testing the supernatant (Example 1).
Alternately, Red yeast rice extract was prepared by mixing 100 mg
of the dry red yeast powder with 1 ml of a mix of DMSO: ethanol
(10:90 v/v) to get a 10% solution. The solution was warmed to
40.degree. C. for 2hrs, vortexed, and then filtered and diluted for
the test (Examples 2 and 3).
EXAMPLE 1
[0048] This example measured production of procollagen I by
fibroblasts in response to treatment with various test
compounds.
[0049] Collagen is a predominant skin protein. Its synthesis
decreases with aging or photodamage. The degradation or destruction
of collagen increases the tensile strength of the skin causing
wrinkles and laxity. Many studies involving human subjects have
shown that collagen type I is decreased with increasing severity of
photodamage (See Kligman, A., JAMA, (1969), 210, pp. 2377-2380;
Lavker, R., J. Inv Derm., (1979), 73, 79-66; Smith J. et al., J.
Inv. Derm., (1962), 39, pp. 347-350; and Shuster, S. et al., Br. J.
Dermatol., (1975), 93, pp. 639-643); and some correlation in the
histology of wrinkles and J6569(C) reduction in collagen levels in
the sun-exposed skin has been reported. See Chen, S.; Kiss, I., J.
Inv. Derm., (1992), 98. pp. 248-254. Voorhees and colleagues have
supported these findings by showing the restoration of collagen
type I in photo-damaged human skin by a topical treatment with
tretinoin. See Christopher, E., et al., The New Eng. Jou. of
Medicine (1993), 329, pp. 530-535. Procollagen I is a precursor of
collagen. Increased production of procollagen I in response to a
test compound application is a marker of an increased collagen
level.
[0050] Methodology for Determination of (Pro) Collagen-I Protein
Expression Using Dot Blot Technique:
[0051] Cell Culture method:
[0052] Neonatal human dermal fibroblasts (passage 4 to 8) were
purchased from Clonetics Corp. San Diego, CA. All materials for
cell culture were purchased from Life technologies, NY and used in
passages 5-10. Cells were seeded at a density of approximately
7,500/well in the inner 48 wells of a 96-well plate in DMEM
(Dulbecco's Modified Eagle's Medium), high glucose supplemented
with 2 mM L-glutamine, 10% fetal bovine serum, and antibiotic and
antimycotic solutions. Cells were grown to confluence and washed
with serum free, phenol red free DMEM and cells dosed with 200
.mu.l actives in media. Each dosing was replicated in a total of
six to twelve wells. Test compounds were used at concentrations as
indicated in tables below. After 24 hours, the test compound
solution or the control solution was removed and cells re-dosed
with 100 .mu.l actives in media. After 24 hours incubation, remove
media and store at 4.degree. C. with protease inhibitor (Aprotinin
1:200) until further use. The test compound solution was then
diluted in 1.times.Tris buffered saline (pH=8) buffer
(approximately 20 .mu.l media in 200 .mu.l buffer).
[0053] Dot Blot Technique:
[0054] PVDF membrane soaked in methanol and 2 filter papers in 1X
TBS (pH=8). Bio-Rad dot blot apparatus was set up with filter paper
on bottom, membrane on top, tighten, added 100 .mu.l TBS per well.
Vacuum dry. Vortex or mix, then load 100 .mu.l diluted sample per
well; let it sit for 15 minutes and then vacuum dry. Remove
membrane from the apparatus, cut off excess, and notch bottom right
corner for orientation. Place membrane in blocking buffer (5% milk
powder in 1.times.TBS-Tween) for 1 hour at room temperature with
shaking. Incubate for 1.5 hours at RT or overnight at 4.degree. C.
with 3 ml Rat Anti-Human Procollagen amino terminal antibody
(Chemicon MAB1912) at 1:100 in 1% milk in TBS-T in a sealed bag
with shaking. Remove membrane, do a 10 minute wash three to five
times in TBS/0.1% Tween. Incubate for 1 hour at RT or O/N at
4.degree. C. in 5 ml 1:1000 anti-rat conjugated alkaline
phosphatase (AP) secondary antibody in 1-% milk in TBS-T in a
sealed bag with shaking. Wash membrane for 15 minutes five to eight
times with TBS-T, and final rinse with 1.times.PBS. The membrane
was developed using Chemiluminiscent substrate (Boehringer
Mannheim, Ind.) and results were analysed using a laser
densitometer (Bio-Rad instrument). Fold increase was calculated as
a ratio of densitometer reading for cells treated with a test
compound over control.
[0055] The results that were obtained are summarized in Table
1.
1 TABLE 1 P value vs Procollagen 1 P value vs Vitamin C/ P value vs
Production % of Control Control Derivative Red yeast rice Control
0.748+/-0.26 100+/-35 1 Vitamin C(50 .mu.g) 1.311+/-0.28 175+/-37
0.005 1 Ascorbyl 0.775+/-0.36 103+/-48 0.887 1 Palmitate(50 .mu.g)
Sodium ascorbyl 0.871+/-0.2 116+/-27 0.389 1 phosphate(50 .mu.g)
Red Yeast Rice 2.265+/-0.47 302+/-64 0.000 1 0.0001% Red Yeast Rice
0.172+/-0.04 22+/-5 0.000 0.000 0.000 0.0001% + Vitamin C Red Yeast
Rice 2.04+/-1.16 272+/-155 0.027 0.034 0.677 0.0001% + Ascorbyl
Palmitate Red Yeast Rice 2.53+/-1.4 338+/-187 0.014 0.018 0.671
0.0001% + Sodium Ascorbyl phosphate Red Yeast Rice 0.07+/+0.05
10+/-7 0.000 1 0.003% Red Yeast Rice 1.2+/-1.7 160+/-234 0.547 0.88
0.148 0.003% + Vitamin C Red Yeast Rice 2.83+/-1.14 379+/-152 0.001
0.002 0.000 0.003% + Acorbyl Palmitate Red Yeast Rice 1.632+/-0.28
218+/-38 0.001 0.001 0.000 0.003% + Sodium Ascorbyl phosphate
[0056] It can be seen from the results in Table 1 that red yeast
rice alone, or in combination with ascorbyl palmitate or sodium
ascorbyl phosphate, stimulated collagen production by
fibroblasts.
EXAMPLE 2
[0057] This example investigated the phytoesrogenic activity of red
yeast rice extract.
[0058] Estrogen Responsive Cell Line, ZR 75 Cells:
[0059] The ZR75 cell line is a ductal breast carcinoma cell line,
originally isolated from malignant mammary epithelium of a
sixty-three year old Caucasian female (10). The chromosome number
is triploid and the trypsin-Giemsa banding patterns indicate a
unique karyotype distinct from HeLa cells. While containing
receptors for estrogen, insulin, progesterone and other hormones,
ZR75 cells only respond through an increase in proliferation to
estrogen and insulin. The cell line contains high affinity
estrogen-specific receptors. Therefore, this cell line is used for
testing estrogen-like activity.
[0060] To grow and maintain ZR75 cells, the most effective media is
RPMI1640 media with ten percent fetal bovine serum (FBS), 100 units
penicillin per milliliter and 100 units of streptomycin per ml. The
media itself does not contain Phenol Red (a weak estrogen mimetic).
The cells are split 1:2 once every week and seeded in 24 well
plates at one hundred thousand cells per milliliter per well for
the assay.
[0061] Assay for Estrogen Like Activity:
[0062] After growing for twenty-four hours, the media was removed,
the cells were washed with PBS and one ml of RPMI 1640 without
serum was readded. Different dilutions of the red yeast rice
extract were then dosed directly into each well. After another
twenty-four hours, one microCurie of [methyl-3H] thymidine was
added to the 5 media in each well. The media was removed after
twenty-four hours to begin the thymidine uptake assay. The cells
were washed once in PBS, the PBS was removed completely and the
cells were left on ice to incubate with one milliliter per well of
10% TCA for one half hour. The plates were washed three times with
5% TCA to remove all traces of thymidine which wasn't incorporated
into the cells. Five hundred microliters of 0.1 M sodium hydroxide
was added to each well and the plates were incubated at 500C for 15
min. The samples were transferred to scintillation vials and after
adding five milliliters of counting fluid (scintiverse), the vials
were counted for five minutes each on a setting for tritium. Data
from triplicate dishes were calculated as % thymidine incorporation
into DNA compared to that of control wells which did not receive
any actives. Each assay also included 10 nM of estradiol as a
positive control. Data was calculated as % of control to normalize
for experiment-to-experiment variation.
[0063] The results that were obtained are summarized in Table
2.
2TABLE 2 Groups Concentration cpm +/-SD % of control Control 0
117579+/-7608 100+/-6.4 Estradiol 0.27 .times. 10.sup.-6 % (10 nM)
263982+/-711 224+/-6.0* Red Yeast Rice 0.01% 345313+/-3275
293+/-2.8* 0.001% 360195+/-4864 306+/-4.1* 0.0001% 147358+/-6736
125+/-5.7 0.00001% 145873+/-3304 124+/-2.8 *-p less than 0.001.
[0064] It can be seen from the results in Table 2 that, at 0.01%
and 0.001%, red yeast rice extract had estrogenic activity
equivalent to or greater than that of 10 nM estradiol. Although red
yeast rice extract is not as potent as estradiol, use of red yeast
rice is advantageous because estradiol, being a drug, cannot be
used in cosmetics whereas red yeast rice can be used in cosmetic
products.
EXAMPLE 3
[0065] This example investigated the effect of various tested
compounds on CRABP-2 production by fibroblasts.
[0066] Within the cells, retinol and retinoic acid are bound to
specific cellular binding proteins, 2 of the major proteins are
CRABP-1 and 2 (Roos et al., Pharmacological reviews: 50, 315-333,
1998). These proteins act in regulating the intracellular
concentration of retinoids by acting as both storage or shuttle
proteins in retinoid metabolism. High or low levels of retinoids
cause cell damage, including cell death, therefore regulation of
constant levels of retinoids and its binding proteins are very
critical for cell survival. The levels of this protein are
regulated by the amount of retinoic acid within the cells. Higher
cellular levels of retinoids increase the expression of CRABP-2.
Therefore, the amount of this protein in the cells, is a measure of
the retinoid activity of the cells. Skin cells contain CRABP-2 both
in the epidermis and the dermis. CRABP-2 response to retinoid
administration in fibroblasts in vitro is used as a reproducible
measure of retinoid bioactivity that predict human skin responses
(Elder et al., J. Invest. Dermatol., 106: 517-521, 1996).
[0067] Cell Culture Method for Fibroblasts:
[0068] Human adult fibroblasts obtained from sun-protected inner
arm of 25-30 year female volunteer were used in this. Cells were
grown in 1:1 DMEM/Hams F12 media containing 10% FBS, maintained at
37.degree. C. in a 5% CO2 atmosphere under normal atmospheric
oxygen tension. Third passage adult fibroblasts were grown in DMEM
media with 10% FBS in 12-well plates at a seeding density of 2500
cells/ml/well. The cells at 80% confluence were rinsed in serum
free and phenol red free (PRF) DMEM media twice. Pre-treatment with
test compound for 4 hours was conducted and then dosed with
retinoids and was incubated for 48 hours.
[0069] Detection of Cellular Retinoic Acid Binding Protein 2
(CRABP-2) in Fibroblasts:
[0070] After the incubation, the wells were washed twice with
1.times.PBS and the cell monolayer was harvested in 100 .mu.l cell
lysis buffer (contains 1.times.PBS, 1% TritonX, 0.5% sodium
deoxycholate, 0.1% SDS containing protease inhibitor (10mg/ml PMSF
in isopropanol, 10 .mu.l/ml). The suspension was spun at 1400 rpm
for 10 minutes, the supernatant collected and an aliquot of this
supernatant was used for protein quantification. Protein
concentration was determined using Pierce protein kit. The
remainder of 100 .mu.l supernatant (cell lysate) was denatured in a
mixture of 40 .mu.l sample buffer (NOVEX) and 0.5% Beta
mercaptoethanol (BME) and by boiling the sample for 5 minutes.
Equal amount of protein was then loaded onto 16% Tris-glycine gels
for protein analysis by SDS page and Western Immuno-blotting for
CRABP-2 protein expression.
[0071] The gels were run and transferred to nitrocellulose blots
and Western Blotting was carried out using monoclonal antibodies to
CRABP-2 according to standard procedures. The CRABP-2 protein band
was visualized in the Western Blots using the chemiluminescence
system obtained from Santa Cruz Biotechnology (SantaCruz, Calif.).
The bands in the film were quantitated by densitometric scanning,
the data from triplicate J6569(C) samples were calculated as % of
control and expressed in the following tables as % increase over
control (with control as 100%) +/-SD of triplicates.
[0072] The results that were obtained are summarized in Table
3.
3 TABLE 3 CRABP-2 CRABP-2 P value vs P value vs P value vs OD+/-SD
% of Control Control retinoids Red yeast Control 1.25+/-0.30
100+/-24 1 10e-6M Retinol 1.92+/-0.06 153+/-5 0.02 1 10e-6M Retinyl
linoleate 1.96+/-0.16 156+/-13 0.02 1 10e-6M Retinyl palmitate
2.00+/-0.04 178+/-33 0.045 1 10e-6M Retinyl Acetate 3.86+/-0.28
398+/-157 0.0023 1 0.1 .mu.l Red Rice Yeast 1.29+/-0.04 102+/-3
0.84 1 extract(RYR) 10e-6M Retinol + 0.001% 3.53+/-0.22 282+/-29
0.0011 0.00162 0.00045 RYR 10e-6M Retinyl linoleate + 3.39+/-0.22
270+/-17 0.000605 0.000897 9.30E-05 300.001% RYR 10e-6M Retinyl
palmitate + 3.64+/-0.42 290+/-33 0.00133 0.01377 0.000651 0.001%
RYR 10e-6M Retinyl Acetate + 6.09+/-1.15 486+/-91 0.00215 0.083
0.00195 300.001% RYR
[0073] It can be seen from the results in Table 3 that red yeast
extract by itself had no effect on CRABP-2 levels. Pretreatment for
4 hours with red yeast rice extract increased the efficacy of
retinoids (retinol, retinyl palmitate, retinyl linoleate and
retinyl acetate) to induce CRABP-2 protein levels.
EXAMPLE 4
[0074] This example investigated the effect of red yeast rice on
sebum secretion by sebocytes.
[0075] Secondary cultures of human sebocytes obtained from an adult
male were grown in 48-well tissue culture plates (Costar Corp.;
Cambridge, Mass.) until confluent. Sebocyte growth medium consisted
of Clonetics Keratinocyte Basal Medium (KBM) supplemented with 14
.mu.g/ml bovine pituitary extract, 0.4 .mu.g/ml hydrocortisone, 5
.mu.g/ml insulin, 10 ng/ml epidermal growth factor, 1.2.times.10-10
M cholera toxin, 100 units/ml penicillin, and 100 .mu.g/ml
streptomycin. All cultures were incubated at 37.degree. C. in the
presence of 7.5% CO2. Medium was changed three times per week.
[0076] On the day of experimentation, the growth medium was removed
and the sebocytes washed three times with sterile Dulbecco's
Modified Eagle Medium (DMEM; phenol red free). Fresh DMEM was added
to each sample (triplicates in Table 4 and duplicated in Table 5)
with 5 microliter of test agent solubilized in ethanol. Controls
consisted of addition of ethanol alone. Each plate was returned to
the incubator for 20 hours followed by the addition of 14C-acetate
buffer (5 mM final concentration, 56 mCi/mmol specific activity).
Sebocytes were returned to the incubator for four hours after which
each culture was rinsed three times with phosphate buffered saline
to remove unbound label. Radioactive label remaining in the
sebocytes was harvested and counted using a Beckman scintillation
counter. Phenol Red was incorporated as an internal positive
control.
[0077] The results that were obtained are summarized ion Tables 4
and 5.
4TABLE 4 Treatment % Reduction T-test 0.001% Phenol Red 83.4
7.9e10.sup.-6 0.01% Red Yeast Rice 61.4 0.0008
[0078]
5TABLE 5 Treatment % Reduction T-test .00035% Phenol Red 8.1 0.1437
.0035% Phenol Red 63.5 0.0246 0.001% Red Yeast Rice 30.7 0.0007
0.0001% Red Yeast Rice 0.5 0.7296
[0079] It can be seen from the results in Tables 4 and 5 that red
yeast rice at concentrations 0.001% or higher significantly
inhibited sebum production by sebocytes.
[0080] Example 5 illustrates topical compositions according to the
present invention. The compositions can be processed in
conventional manner. They are suitable for cosmetic use. In
particular the compositions are suitable for application to
wrinkled, rough, flaky, aged oily and/or UV-damaged skin and/or
oily skin to improve the appearance and the feel thereof as well as
for application to healthy skin to prevent or retard deterioration
thereof.
6 OIL-IN-WATER EMULSION INGREDIENT % w/w Carbomer 0.30 Disodium
EDTA 0.10 Glycerin 3.00 Polysorbate 20 2.50 Butylene Glycol 2.00
Methylparaben 0.30 Triethanolamine 99% 0.30 Isopropyl Myristate
5.00 Octyl Palmitate 3.00 Cetyl Alcohol 1.00 Red Yeast Rice Extract
0.10 Sodium Ascorbyl Phosphate 5.00 Dimethicone, 100 cst 0.50
Beeswax 0.30 Propylparaben 0.10 Germall II 0.10 Fragrance 0.10 DI
Water to 100% Total .fwdarw. 100.00
[0081]
7 OIL-IN-WATER EMULSION INGREDIENT % w/w Xanthan Gum 0.20 Disodium
EDTA 0.10 Glycerin 5.00 Butylene Glycol 2.00 Methylparaben 0.30
Isopropyl Myristate 5.00 Octyl Palmitate 3.00 Cetyl Alcohol 1.00
Dimethicone, 100 cst 0.50 Steareth-2 0.40 Steareth-21 3.00 Red
Yeast Rice Extract 1.00 Propylparaben 0.10 Germall II 0.10
Fragrance 0.10 Retinol 0.075 DI Water to 100% Total .fwdarw.
100.00
[0082]
8 WATER-IN-OIL EMULSION INGREDIENT % w/w Disodium EDTA 0.10
Glycerin 3.00 Retinyl Acetate 3.00 Red Yeast Rice Extract 3.00
Propylene Glycol 2.00 Sodium Chloride 0.70 Methylparaben 0.30
Cyclomethicone 14.00 Isopropyl Myristate 5.00 Octyl Palmitate 3.00
Dimethicone Copolyol 2.50 Dimethicone, 100 cst 0.50 Beeswax 0.30
Propylparaben 0.10 Germall II 0.10 Fragrance 0.10 DI Water to 100%
Total .fwdarw. 100.00
[0083]
9 HYDRO-GEL INGREDIENT % w/w Butylene Glycol 5.00 PPG-5-Ceteth 20
5.00 Glycerin 3.00 Ascorbyl Palmitate 2.00 Red Yeast Rice Extract
2.00 Retinyl Palmitate 2.00 Carbomer 1.20 Triethanolamine 99% 1.20
Methylparaben 0.30 Polysorbate 20 0.25 Disodium EDTA 0.10 Germall
II 0.10 DI Water to 100% Total .fwdarw. 100.00
[0084]
10 ANHYDROUS SERUM INGREDIENT % w/w Isopropyl Myristate 5.00 Octyl
Palmitate 3.00 Polyglycerol-6 Dioleate 5.00 Butylene Glycol 4.00
Dimethicone, 100 cst 5.00 Red Yeast Extract 1.00 All-Trans Retinol
0.10 Beeswax 0.30 Propylparaben 0.20 Fragrance 0.10 Cyclomethicone
to 100% Total .fwdarw. 100.00
[0085]
11 HYDRO-ALCOHOLIC GEL INGREDIENT % w/w Alcohol SDA40B 30.00 Red
Yeast Rice Extract 5.00 Butylene Glycol 5.00 PPG-5-Ceteth 20 5.00
Sodium Ascorbyl PO.sub.4 3.00 Glycerin 3.00 Carbomer 1.20
Triethanolamine 99% 1.20 Methylparaben 0.30 Polysorbate 20 0.25
Disodium EDTA 0.10 Germall II 0.10 DI Water to 100% Total --->
100.00
* * * * *