U.S. patent application number 10/331277 was filed with the patent office on 2003-05-22 for tyrosinase inhibitors from plants.
Invention is credited to Steck, Warren.
Application Number | 20030096023 10/331277 |
Document ID | / |
Family ID | 22211643 |
Filed Date | 2003-05-22 |
United States Patent
Application |
20030096023 |
Kind Code |
A1 |
Steck, Warren |
May 22, 2003 |
Tyrosinase inhibitors from plants
Abstract
The present invention provides tyrosinase inhibiting extracts
from dictotyledonous plant species indigenous to Canada and
compositions containing the extracts for the treatment of skin,
particularly skin lightening. The present invention also provides a
method of detecting tyrosinase activity in an extract of a
dicotyledonous plant species indigenous to Canada.
Inventors: |
Steck, Warren; (Saskatoon,
CA) |
Correspondence
Address: |
FROMMER LAWRENCE & HAUG
745 FIFTH AVENUE- 10TH FL.
NEW YORK
NY
10151
US
|
Family ID: |
22211643 |
Appl. No.: |
10/331277 |
Filed: |
December 30, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10331277 |
Dec 30, 2002 |
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09719205 |
Apr 9, 2001 |
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6521267 |
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09719205 |
Apr 9, 2001 |
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PCT/CA99/00530 |
Jun 8, 1999 |
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60088484 |
Jun 8, 1998 |
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Current U.S.
Class: |
424/725 ;
424/740; 424/750; 424/757; 424/764; 424/765; 424/769 |
Current CPC
Class: |
A61P 17/00 20180101;
A61K 36/48 20130101; A61K 36/282 20130101; A61K 36/70 20130101;
A61K 36/40 20130101; A61K 36/185 20130101; A61Q 19/02 20130101;
A61P 17/16 20180101; A61K 2800/782 20130101; A61K 36/73 20130101;
A61K 36/47 20130101; A61K 8/9789 20170801; A61K 36/704 20130101;
A61K 36/185 20130101; A61K 2300/00 20130101; A61K 36/282 20130101;
A61K 2300/00 20130101; A61K 36/40 20130101; A61K 2300/00 20130101;
A61K 36/47 20130101; A61K 2300/00 20130101; A61K 36/48 20130101;
A61K 2300/00 20130101; A61K 36/70 20130101; A61K 2300/00 20130101;
A61K 36/704 20130101; A61K 2300/00 20130101; A61K 36/73 20130101;
A61K 2300/00 20130101 |
Class at
Publication: |
424/725 ;
424/765; 424/764; 424/750; 424/757; 424/769; 424/740 |
International
Class: |
A61K 035/78 |
Claims
1. A product comprising an extract derived from a dicotyledonous
plant species indigenous to Canada, wherein the extract inhibits
tyrosinase.
2. The product according to claim 1, wherein the extract is derived
from a plant species selected from Polygonaceae, Rosaceae and
Onagraceae.
3. The product according to claim 1, wherein the extract is derived
from one or more parts of the plant selected from leaves, twigs,
flowers, flowering aerials, fruiting aerials, seeding aerials,
roots and fruits.
4. The product according to claim 1, wherein the extract is derived
from the group consisting of: flowering aerials of Artemisia
campestris (plains wormwood); flowering aerials of Aster ericoides
(white prairie aster); flowering aerials of Aster hesperius (willow
aster); leaves, twigs or flowers of Cornus stolonifera (red-osier
dogwood); leaves and twigs of Cotoneaster acutifolia (cotoneaster);
flowering aerials of Epilobium angustifolium (fireweed); seeding
aerials of Euphorbia esula (leafy spurge); fruiting aerials of
Fragaria americana (wild strawberry); fruiting aerials of Fragaria
glauca (Wild strawberry); flowering aerials of Geranium bicknelli
(Bicknell's geranium); flowering aerials of Geum aleppicum (yellow
avens); flowering aerials of Geum triflorum (3-flowered avens);
flowering aerials of Glycyrrhiza lepidota (wild licorice);
flowering aerials of Hedysarum americanum (American hedysarum);
roots of Heuchera richardsonii (alumroot); flowering aerials of
Oenothera biennis (yellow evening-primrose); flowering aerials of
Polygonum persicaria (lady's-thumb); flowering aerials of
Potentilla fruticosa (shrubby cinquefoil); flowering aerials of
Potentilla norvegica (rough cinquefoil); flowering aerials of Rosa
acicularis (prairie rose); fruiting aerials of Rosa arkansana (low
rose); fruiting aerials of Rumex maritimus (golden dock); fruiting
aerials and flowering aerials of Rumex occidentalis (western field
dock); fruiting aerials and fruits of Rumex pseudonatronatus (field
dock); and fruits of Rumex stenophyllus (narrow-leaved dock).
5. A composition comprising the product as defined in claim 1,
together with a cosmetically or pharmaceutically acceptable, or
edible, diluent or carrier.
6. A composition comprising the product as defined in claim 4,
together with a cosmetically or pharmaceutically acceptable, or
edible, diluent or carrier.
7. The composition according to claim 5, wherein the diluent or
carrier is cosmetically acceptable and the composition is for
cosmetic treatment of skin.
8. The composition according to claim 7, wherein the extract is
derived from the group consisting of: fruiting aerials of Rumex
maritimus (golden dock); fruiting aerials of Rumex occidentalis
(western field dock); flowering aerials of Rumex occidentalis
(western field dock); fruiting aerials of Rumex pseudonatronatus
(field dock); fruits of Rumex pseudonatronatus (field dock); and
fruits of Rumex stenophyllus (narrow-leaved dock).
9. The composition according to claim 8, which further comprises
ascorbic acid.
10. Use of the composition according to claim 7 as a skin
lightener.
11. Use of the composition according to claim 7 to reduce melanin
and/or melanogenesis in skin.
12. The composition according to claim 5, wherein the diluent or
carrier is edible and the composition is for treating edible
products to inhibit browning.
13. A method for detecting tyrosinase inhibiting activity in an
extract derived from a dicotyledonous plant species indigenous to
Canada, which method comprises: a) preparing a first solution
comprising an amount of tyrosinase and a suitable substrate; b)
preparing a second solution comprising the same amount of
tyrosinase and the suitable substrate and further comprising an
amount of the extract; c) measuring a tyrosinase activity of each
of the first and second solutions; d) comparing the tyrosinase
activities of the first and second solutions; and e) detecting
tyrosinase inhibiting activity, present when the tyrosinase
activity of the second solution is less than the tyrosinase
activity of the first solution.
14. The method according to claim 13, wherein tyrosinase activity
is measured spectrophotometrically.
15. The method according to claim 13, wherein the substrate is
L-DOPA.
16. The method according to claim 13, wherein the tyrosinase
inhibiting activity is equal to, or greater than, that of
hydroquinone (1,4-dihydroxybenzene).
17. The method according to claim 13, wherein the extract is
derived from the group consisting of: flowering aerials of
Artemisia campestris (plains wormwood); flowering aerials of Aster
ericoides (white prairie aster); flowering aerials of Aster
hesperius (willow aster); leaves, twigs and flowers of Cornus
stolonifera (red-osier dogwood); leaves or twigs of Cotoneaster
acutifolia (cotoneaster); flowering aerials of Epilobium
angustifolium (fireweed); seeding aerials of Euphorbia esula (leafy
spurge); fruiting aerials of Fragaria americana (wild strawberry);
fruiting aerials of Fragaria glauca (Wild strawberry); flowering
aerials of Geranium bicknelli (Bicknell's geranium); flowering
aerials of Geum aleppicum (yellow avens); flowering aerials of Geum
triflorum (3-flowered avens); flowering aerials of Glycyrrhiza
lepidota (wild licorice); flowering aerials of Hedysarum americanum
(American hedysarum); roots of Heuchera richardsonii (alumroot);
flowering aerials of Oenothera biennis (yellow evening-primrose);
flowering aerials of Polygonum persicaria (lady's-thumb); flowering
aerials of Potentilla fruticosa (shrubby cinquefoil); flowering
aerials of Potentilla norvegica (rough cinquefoil); flowering
aerials of Rosa acicularis (prairie rose); fruiting aerials of Rosa
arkansana (low rose); fruiting aerials of Rumex maritimus (golden
dock); fruiting aerials and flowering aerials of Rumex occidentalis
(western field dock); fruiting aerials and fruits of Rumex
pseudonatronatus (field dock); and fruits of Rumex stenophyllus
(narrow-leaved dock).
18. The method according to claim 17, wherein the extract is
derived from the group consisting of: fruiting aerials of Rumex
maritimus (golden dock); fruiting aerials of Rumex occidentalis
(western field dock); flowering aerials of Rumex occidentalis
(western field dock); fruiting aerials of Rumex pseudonatronatus
(field dock); fruits of Rumex pseudonatronatus (field dock); and
fruits of Rumex stenophyllus (narrow-leaved dock).
19. A tyrosinase inhibiting plant extract having tyrosinase
inhibiting potency at least equal to hydroquinone.
Description
FIELD OF THE INVENTION
[0001] The invention relates to a product comprising a tyrosinase
inhibiting extract derived from a dicotyledonous plant species
indigenous to Canada. The present invention also relates to
compositions comprising the product and a suitable diluent or
carrier for the treatment of skin or of fruit, vegetables or
beverages. The present invention further relates to a method for
identifying plant extracts that are capable of inhibiting the
enzyme tyrosinase.
BACKGROUND OF THE INVENTION
[0002] Tyrosinase (tyrosine oxidase, EC 1.10.3.1) is an enzyme or
closely homologous group of enzymes of universal occurrence in
microbes, plants and animals. Its primary metabolic function is to
catalyze the oxidative degradation of the amino acid tyrosine. This
degradation takes slightly different routes in animals, plants and
microbes, but the rate-controlling first steps those catalyzed by
tyrosinase--are the same in virtually all living species. In
animals, including man, tyrosinase first transforms tyrosine into
3,4-dihydroxyphenylalanine (DOPA), thence to the corresponding
quinone (DOPAquinone), and finally to
2-carboxy-2,3-dihydroindole-5,6-quinone (DOPAchrome) which is
further converted by other enzymes to still more highly oxidized
materials which include the melanin substances responsible for skin
pigmentation.
[0003] Skin pigmentation thus depends upon the action of tyrosinase
If this enzyme is not active, normal pigmentation does not occur,
and skin loses or fails to acquire its normal tan-to-brown
coloration. The effect is independent of racial or environmental
factors. Pigmentation loss manifests itself in lentigo senile,
so-called "age spots", a small-scale, patchy color loss often seen
in skin of people over 50 years of age; typically the de-pigmented
patches will be 1-3 mm across and can be very numerous. A more
serious effect is leucoderma, a group of diseases in which large
areas of skin lose their melanin and appear pink. An extreme effect
is albinism, a condition in which tyrosinase enzyme is entirely
dysfunctional and no pigmentation of skin (or eyes or hair)
occurs.
[0004] If on the other hand tyrosinase activity is accelerated, as
in sun-tanning or in some pathological conditions, the amount of
melanin formed increases and skin color darkens. When the
distribution of the new melanin is even and controlled, the result
is a "glorious bronzed body"; but if the new melanin is patchy or
produced uncontrollably, the result is pathological. Skin melanomas
are sites of localized hyper-activity by tyrosinase; they are often
associated with cancerous cell modification. The causal
relationship of tyrosinase action to skin pigmentation was
established many years ago (Mason, H. S. (1948) J. Biol. Chem. 172,
83-86; Balin, A. K. and Kligman, A. M. (1989) Aging and the Skin
372 pp, Raven Press, New York) and is now well-understood.
[0005] Materials which suppress the action of tyrosine oxidase are
presumed to slow the build-up of skin spots over time, and this
presumption is vindicated by scientific evidence. Thus, the
correlation between tyrosinase inhibition and protection of skin
against unwanted pigmentation is accepted by the medical profession
and by the cosmetic industry.
[0006] In addition to its skin functions, tyrosinase is active in
other types of living tissue in the turnover metabolism of tyrosine
and in the production of pigmented materials in those tissues.
[0007] With respect to cosmetics, skin lightening and skin
darkening have been practiced since earliest times, and today these
are mainstays of some sectors of the world cosmetics industry. Skin
lightening has traditionally been accomplished by rigorously
excluding sunlight from skin, or by the use of chemical lightening
agents. One such agent is synthetic hydroquinone
(1,4-dihydroxybenzene). This substance is considered the cosmetic
industry standard for tyrosinase inhibition potency.
SUMMARY OF THE INVENTION
[0008] Prior to the present invention, the possibility of finding
effective tyrosinase inhibitors in dicotyledonous plant species
indigenous to Canada had not been appreciated.
[0009] Accordingly, the present invention provides a product
comprising an extract derived from a dicotyledonous plant species
indigenous to Canada, wherein the extract inhibits tyrosinase.
Preferably, the extract is derived from a plant species selected
from Polygonaceae, Rosaceae and Onagraceae. The extract is
preferably derived from one or more parts of the plant selected
from leaves, twigs, flowers, flowering aerials, fruiting aerials,
seeding aerials, roots and fruits.
[0010] Preferably, the extract is derived from the group consisting
of:
[0011] flowering aerials of Artemisia campestris (plains
wormwood);
[0012] flowering aerials of Aster ericoides (white prairie
aster);
[0013] flowering aerials of Aster hesperius (willow aster);
[0014] leaves, twigs and flowers of Cornus stolonifera (red-osier
dogwood);
[0015] leaves and twigs of Cotoneaster acutifolia
(cotoneaster);
[0016] flowering aerials of Epilobium angustifolium (fireweed);
[0017] seeding aerials of Euphorbia esula (leafy spurge);
[0018] fruiting aerials of Fragaria americana (wild
strawberry);
[0019] fruiting aerials of Fragaria glauca (Wild strawberry);
[0020] flowering aerials of Geranium bicknelli (Bicknell's
geranium);
[0021] flowering aerials of Geum aleppicum (yellow avens);
[0022] flowering aerials of Geum triflorum (3-flowered avens);
[0023] flowering aerials of Glycyrrhiza lepidota (wild
licorice);
[0024] flowering aerials of Hedysarum americanum (American
hedysarum);
[0025] roots of Heuchera richardsonii (alumroot);
[0026] flowering aerials of Oenothera biennis (yellow
evening-primrose); flowering aerials of Polygonum persicaria
(lady's-thumb);
[0027] flowering aerials of Potentilla fruticosa (shrubby
cinquefoil);
[0028] flowering aerials of Potentilla norvegica (rough
cinquefoil);
[0029] flowering aerials of Rosa acicularis (prairie rose);
[0030] fruiting aerials of Rosa arkansana (low rose);
[0031] fruiting aerials of Rumex maritimus (golden dock);
[0032] fruiting aerials and flowering aerials of Rumex occidentalis
(western field dock);
[0033] fruiting aerials and fruits of Rumex pseudonatronatus (field
dock); and
[0034] fruits of Rumex stenophyllus (narrow-leaved dock).
[0035] The present invention also provides a composition comprising
the product as defined herein, together with a cosmetically or
pharmaceutically acceptable, or edible, diluent or carrier. The
composition is preferably for cosmetic treatment of skin or for
inhibiting browning of edible products. The composition is more
preferably for skin lightening, even more preferably to reduce
melanin and/or melanogenesis. The at least one plant extract in the
composition is preferably selected from the group consisting
of:
[0036] fruiting aerials of Rumex maritimus (golden dock);
[0037] fruiting aerials of Rumex occidentalis (western field
dock);
[0038] flowering aerials of Rumex occidentalis (western field
dock);
[0039] fruiting aerials of Rumex pseudonatronatus (field dock);
[0040] fruits of Rumex pseudonatronatus (field dock); and
[0041] fruits of Rumex stenophyllus (narrow-leaved dock).
[0042] The present invention also provides a method for detecting
tyrosinase inhibiting activity in an extract derived from a
dicotyledonous plant species indigenous to Canada. This method
comprises:
[0043] a) preparing a first solution comprising an amount of
tyrosinase and a suitable substrate;
[0044] b) preparing a second solution comprising the same amount of
tyrosinase and the suitable substrate and further comprising an
amount of the extract;
[0045] c) measuring tyrosinase activities of the first and second
solutions by suitable methods;
[0046] d) comparing the tyrosinase activities of the first and
second solutions; and
[0047] e) detecting tyrosinase inhibiting activity, present when
the tyrosinase activity of the second solution is less than the
tyrosinase activity of the first solution.
[0048] It is preferable that the tyrosinase inhibiting activity is
comparable to, or greater than, that of hydroquinone.
BRIEF DESCRIPTION OF THE DRAWINGS
[0049] FIG. 1 is summary of the main steps in the biogenesis of
melanin from tyrosine.
[0050] FIG. 2 is a plot of "percent inhibition of enzyme action"
vs. log "percent extract in assay reaction mixture" indicating the
effectiveness of extracts of Rumex pseudonatronatus, Rumex
occidentalis and Oenothera biennis as tyrosinase inhibitors.
Tyrosinase 5 mg. Standard hydroquinone.
[0051] FIG. 3 is a plot of "percent inhibition of enzyme action"
vs. log "percent extract in assay reaction mixture" indicating the
effectiveness of extracts of Hedysarum americanum and Rosa
acicularis as tyrosinase inhibitors. Tyrosinase--5 mg.
Standard--hydroquinone.
[0052] FIG. 4 shows the elution profile of whole extract of Rumex
pseudonatronatus from a polyvinylpyrrolidone chromatographic column
in terms of percent inhibition of tyrosinase in the assay. Elution
was with water (ff 1-4) and water ethanol gradient (ff 5-10).
[0053] FIG. 5 shows the effect of alcohol in the extraction solvent
on tyrosinase inhibiting activity from Epilobium angustifolium.
Epilobium angustifolium (=WH) was extracted using water (WH-00),
25% ethanol (WH-25), 50% ethanol (WH-50), 75% ethanol (WH-75) and
95% ethanol (WH-95). Each extract was assayed for tyrosinase
inhibitory power at three concentrations. The carrier was
butanediol and water.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0054] The definitions of various terms used in this specification
appear below.
[0055] "Plant extracts" are substances obtained from plants by
physical separation processes such as solvent extraction, steam
distillation, or pressing. Broadly, the term "extract" may
sometimes be extended to include the solvent as well as the plant
substance dissolved in it.
[0056] The term "screening test" refers to any simple and
standardized test whose purpose is to separate or screen out, from
a large number of materials, those unable to perform the function
upon which the test is based, from those able to perform that
function.
[0057] A typical pilot scale plant extraction method for Rumex
species is provided below.
[0058] Ripe, dry (post-season) Rumex species, mostly R.
Occidentalis, were field collected and dry-milled into small
fragments. These (250 kg) were extracted at 70.degree. C. for one
hour with 2000 L of approximately 70% ethanol in water. An orange
extract was separated from the mash by decantation and the latter
was washed with a further 1000 L of 70% alcohol. The extract
liquids were combined, filtered and concentrated to 50 L, by which
point the ethanol had virtually all been removed and extract solids
(content 18-20%) were then beginning to precipitate out.
Alternatively, 50 L of glycerol or 1,3-butanediol was added to the
partly concentrated solution to provide a final concentration
end-point volume of 100 L. This approach avoids precipitation of
extract solids.
[0059] Carrier glycerol (or 1,3-butanediol) and water were added to
the concentrate to make a red-coloured solution having 5% solids
content. Then a further amount of 1:1 glycerol (or
1,3-butanediol)-water carrier liquid was added, amounting to 25% of
the weight of the 5% solution and itself containing 5% of
L-ascorbic acid (Vitamin C) as an anti-colorant and anti-oxidant.
Finally, an anti-microbial preservative, such as phenoxyethanol,
was added to a concentration of 0.3 to 0.4%.
[0060] The above method provides the basis for the method used to
produce the other extracts of the invention. A person skilled in
the art will understand that variation in certain parameters and
substances used in the method will still produce an extract of the
invention.
[0061] The tyrosinase assay was based on methods previously
published (Mason, H. S., (1948) J. Biol. Chem. 172, 83-86; Dawson,
C. R. and Magee, R. J. (1955) Methods in Enzymology II 817-827
(Editors-in-chief S. P. Colowick and N. O. Caplan); Lerner, A. B.
(1955) ibid 827-831; Bergmeyer, U. H. (1983) Methods of Enzymatic
Analysis (Published by Verlag Chemie, Weinheim)). The protocol was
as follows, scaled for the approximately twenty sample
determinations:
[0062] (a) A buffer solution of pH 6.5 was prepared by dissolving
1.4 g disodium monohydrogen phosphate (Na.sub.2HPO.sub.4) and 1.4 g
monopotassium dihydrogen phosphate (KH2PO4) in pure water to make
200 mL.
[0063] (b) An enzyme solution of either 1.0 mg or 3.0 mg tyrosinase
(Sigma T-7755) was prepared in 30 mL buffer and stored at 2.degree.
C. until use.
[0064] (c) A substrate solution was prepared by dissolving 0.109
L-DOPA (L-3,4-dihydroxyphenyl-alanine, Sigma D-9661) in 100 mL
buffer.
[0065] (d) The unfettered enzyme reaction rate was measured by
mixing together 0.5 mL buffer solution and 0.5 mL substrate
solution in twin acrylate mini-cuvettes (path 2.0 mm), and after
observing the stability of the mixture, mixing in 0.25 mL enzyme
solution to one cuvette. The formation of red DOPAquinone was
monitored spectrophotometrically at 475 nm, at times 0, 1, 2 and 3
minutes.
[0066] (e) The effect of added substances on the enzyme reaction
rate was measured by mixing together 0.5 mL buffer solution, 0.5 mL
substrate solution and 0.25 mL test substance (at 1% strength in
50:50 butyleneglycol:water) in twin acrylate mini-cuvette. The
reaction was tracked spectrophotometrically at 475 nm, reading at
0, 1, 2 and 3 minutes.
[0067] (f) The effect of test substances on reaction rate was
expressed as inhibition (I) where I=100-[100 (trt absorbance
change, 3 min)/100(control absorbance change, 3 min)].
[0068] Concentrations of substrate, enzyme or test substance can be
varied as appropriate.
[0069] Plant extracts screened by the method of the invention were
selected which exhibited tyrosinase inhibiting potency equal to, or
better than, hydroquinone. Those extracts are listed in Table 1.
The concentration dependence of the potency of various extracts is
summarized in FIGS. 2 and 3 in comparison to the concentration
dependence of the potency of hydroquinone.
[0070] Table 2 shows the effect of varying the extraction solvent
on the tyrosinase inhibiting activity of extracts from three
different plant species. The effect is also shown by the results in
FIG. 5.
1TABLE 1 Plant extracts and phytochemicals with tyrosinastatic
power equal to or greater than that of hydroquinone Enzymes
reaction rate reduction Plant botanical name Common name Parts used
@ 5 .mu.g enzyme @ 15 .mu.g enzyme Standard: pure HYDROQUINONE @ 1%
65% 55% PLANT EXTRACTS OF Amalanchier alnifolia Saskatoonberry
flwrg, leaves & twigs 83%, 85% nd Artemisia campestris plains
wormwood flowering aerials nd 65% Aster ericoides white prairie
aster flowering aerials nd 78% Aster hesperius willow aster
flowering aerials 66% nd Cornus stolonifera red-osier dogwood
leaves, twigs, flowers 79% 60% Cotoneaster acutifolia cotoneaster
leaves & twigs 65% nd Epilobium angustifolium A firewood
flowering aerials 70% 61% Epilobium angustifolium B firewood
flowering aerials 82% nd Euphorbia esula leafy spurge seeding
aerials nd 58% Fragaria americana wild strawberry fruiting aerials
nd 61% Fragaria glauca wild strawberry fruiting aerials 77%, 68%
72% Geranium bicknelli Bicknell's geranium flowering aerials 64%
61% Geum aleppicum yellow avens flowering aerials 78% 67% Geum
triflorum 3-flowered avens flowering aerials 78% 71% Glycyrrhiza
lepidota A wild licorice flowering aerials 76% nd Hedysarum
americanum B American hedysarum flowering aerials 93%, 87%, 90% 88%
Standard: pure HYDROQUINONE @ 1% 65% 55% PLANT EXTRACTS OF Heuchera
richardsonii alumroot roots 90% 72% Oenothera biennis yellow
evening-primrose flowering aerials 82% 67% Polygonum persicaria
lady's-thumb flowering aerials 90% 73% Potentilla fruticosa shrubby
cinquefoil flowering aerials 83%, 78% Potentilla norvegica rough
cinquefoil flowering aerials 78% 74% Rosa acicularis prairie rose
flowering aerials 83% 83% Rosa arkansana low rose fruiting aerials
81% 61% Rumex maritimus golden dock fruiting aerials 90% 81% Rumex
occidentalis A western field dock fruiting aerials 87% 71% Rumex
occidentalis B western field dock flowering aerials nd 82% Rumex
pseudonatronatus A field dock fruiting aerials 92% 77% Rumex
pseudonatronatus B field dock fruits 88% 70% Rumex stenophyllus
narrow-leaved dock fruits 88% 69%
[0071]
2TABLE 2 Extractability of tyrosinastatic activity from plants The
grid numbers are the percent inhibitions of the enzyme function
obtained using each extracted solution in two standard tyrosinase
assays (5 .mu.g enzyme). Data are corrected for ethanol effects.
EXTRACTION SOLVENT 1:1 Water- Ethyl SPECIES Ethanol ethanol Water
acetate Epilobium angustifolium 45%, 39% 65%, 62% 80%, 75% <10%
(aerials) Oenothera biennis <10% 58%, 67% 66%, 63% <101%
(aerials) Rumex occidentalis 54%, 48% 79%, 79% 81%, 84% <10%
(ripe aerials)
[0072] There is a very large market for consumer goods containing
natural products, such as plant extracts. Compositions for
treatment of skin may be prepared comprising a tyrosinase
inhibiting plant extract as disclosed herein, together with an
acceptable diluent or carrier. Acceptable diluent or carriers for
cosmetic applications can include such substances as water, aqueous
alcohols, glycerol, propanediol and butanediol, as well as certain
silicones and waxes. Acceptable diluents or carriers for
pharmaceutical applications are well known in the art.
[0073] The tyrosinase inhibiting plant extract can also be used to
treat fruit, vegetables or beverages to prevent or reduce browning,
since tyrosinase activity is a contributor to the browning process.
Compositions may be prepared to treat edible products such as
fruit, vegetables or beverages to inhibit browning, which
composition comprises a tyrosinase inhibiting plant extract as
disclosed herein, together with a diluent or carrier that is
suitable for consumption. The diluent or carrier suitable for
consumption can include such substances as water, aqueous alcohols,
propanediol and butanediol, as well as any other suitable substance
acceptable by law.
EXAMPLE 1
[0074] A composition known as TYROSTAT-10 (T-10) has the following
ingredients:
3 Purified water 45-50% 1,3-butanediol 45-50% Plant extract solids
(Canadian Rumex species) 4.0-4.3% [Rumex occidentals, R,
pseudonatronatus, R. stenophyllus and R. martimus] L-Ascorbic acid
0.9-1.1% Phenoxyethanol (anti-fungal) 0.2-0.3%
[0075] The composition contains no added minerals or other organic
materials. It is a readily pourable transparent solution,
orange-red coloured at pH 4.0-7.5. Upon dilution, the colour moves
toward pastel yellow. This colour darkens perceptibly but
reversibly at pH values much above 8, and lightens perceptibly on
long product storage when sealed from air.
[0076] The specific gravity is 1.05 at 21.degree. C.
[0077] This material contains natural plant-sourced inhibitors of
tyrosinase enzymes. The potency has been measured using in vitro
assays of the conversion of DOPA (3,4-dihydroxyphenylalanine) to
the corresponding quinone by the enzyme, in presence and in absence
of the TYROSTAT product and the results are summarized in Table 3.
This assay duplicates rate-controlling steps in the human metabolic
pathway to skin pigmentation. Pure hydroquinone in 1% solution was
employed as a comparison standard.
4TABLE 3 INHIBITION TESTED MATERIAL SOLIDS PRESENT OF ENZYME
Undiluted TYROSTAT-1O 5% 100% TYROSTAT-10 diluted 10.times. 0.5
100% TYROSTAT-10 diluted 100.times. 0.05 80-85% Hydroquinone 1.0
58-62%
[0078] Rumex extracts appear to inhibit tyrosinase by binding or
deforming the enzyme molecule around the active site. Ascorbic acid
contributes to the overall inhibition by maintaining the enzyme's
copper atoms in their reduced Cu.sup.+ state. Other constituents of
TYROSTAT-10 have no significant effect on tyrosinase in standard
assays, although the carrier component butanediol is slightly
inhibitory. The data in Table 4 indicate the relative capabilities
of the active ingredients for tyrosinase inhibition, at various
concentrations of the ingredients.
5 TABLE 4 Inhibition of tyrosinase enzyme step 2 Ingredient 1% conc
0.1% conc 0.01% conc 0.001% conc Rumex extract 81% 69% 53% 38%
Ascorbic acid 100% 100% 82% 23% [cf.hydroquinone] 60% 31% <10%
<10%
EXAMPLE 2
[0079] A composition known as TYROSTAT-11 (T-11) has the following
ingredients:
6 Purified Water 45-50% Glycerol 45-50% Plant extract solids
(Canadian Rumex 4.0-4.3% species) [Rumex occidentalis, R.
pseudonatronatus, R. stenophyllus and R. martimus] L-Ascorbic acid
0.9-1.1% Phenoxyethanol (anti-fungal) 0.2-0.3%
[0080] The composition contains no added minerals or other
inorganic materials, and no synthetics other than phenoxyethanol
preservative. It is a readily pourable transparent solution,
orange-red coloured at pH 4.0-7.5. Upon dilution, the colour moves
toward pastel yellow. This colour darkens perceptibly but
reversibly at pH values much above 8, and lightens perceptibly on
long product storage when sealed from air.
[0081] The specific gravity is 1.15 at 21.degree. C.
[0082] This material contains natural plant-sourced inhibitors of
tyrosinase enzymes. The potency has been measured using in vitro
assays of the conversion of DOPA (3,4-dihydroxyphenylalanine) to
the corresponding quinone by the enzyme, in the presence and in
absence of the TYROSTAT product and the results are summarized in
Table 5. This assay duplicates rate-controlling steps in the human
metabolic pathway to skin pigmentation. Pure hydroquinone in 1%
solution was employed as a comparison standard.
7TABLE 5 INHIBITION TESTED MATERIAL SOLIDS PRESENT OF ENZYME
Undiluted TYROSTAT-11 5% 100% TYROSTAT-11 diluted 10.times. 0.5
100% TYROSTAT-11 diluted 100.times. 0.05 80-83% Hydroquinone 1.0
38-62%
[0083] Rumex extracts appear to inhibit tyrosinase by binding or
deforming the enzyme molecule around the active site. Ascorbic acid
contributes to the overall inhibition by maintaining the enzyme's
copper atoms in their reduced Cu.sup.+ state. Other constituents of
TYROSTAT-11 have no significant effect on tyrosinase in standard
assays. The data in Table 6 indicate the relative capabilities of
the active ingredients for tyrosinase inhibition, at various
concentrations of those ingredients.
8 TABLE 6 Inhibition of tyrosinase enzyme step 2 Ingredient 1% conc
0.1% conc 0.01% conc 0.001% conc Rumex extract 81% 69% 53% 38%
Ascorbic acid 100% 100% 82% 23% [cf.hydroquinone] 60% 31% <10%
<10%
[0084] Numerous modifications and variations of the present
invention are possible in light of the above teachings. It is
therefore to be understood that within the scope of the appended
claims, the invention may be practised otherwise than as
specifically described herein.
* * * * *