U.S. patent application number 16/064312 was filed with the patent office on 2019-01-03 for passion flower seed extract, and cosmetic, pharmaceutical or dermatological compositions containing same.
This patent application is currently assigned to LABORATOIRES EXPANSCIENCE. The applicant listed for this patent is LABORATOIRES EXPANSCIENCE. Invention is credited to Stephanie BREDIF, Sophie LECLERE-BIENFAIT.
Application Number | 20190000902 16/064312 |
Document ID | / |
Family ID | 55361825 |
Filed Date | 2019-01-03 |
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United States Patent
Application |
20190000902 |
Kind Code |
A1 |
LECLERE-BIENFAIT; Sophie ;
et al. |
January 3, 2019 |
PASSION FLOWER SEED EXTRACT, AND COSMETIC, PHARMACEUTICAL OR
DERMATOLOGICAL COMPOSITIONS CONTAINING SAME
Abstract
The invention relates to a polyphenolic extract of passion
flower seeds, in particular Passiflora incarnata or Passiflora
edulis seeds, comprising at least 30 percent by weight polyphenols,
expressed as gallic acid equivalent, relative to the weight of the
dry extract. The invention also relates to a method for preparing
an extract of said type, a composition containing same, and the
cosmetic, dermatological or therapeutic use thereof.
Inventors: |
LECLERE-BIENFAIT; Sophie;
(Dreux, FR) ; BREDIF; Stephanie; (Croisilles,
FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LABORATOIRES EXPANSCIENCE |
Paris La Defense Cedex |
|
FR |
|
|
Assignee: |
LABORATOIRES EXPANSCIENCE
Paris La Defense Cedex
FR
|
Family ID: |
55361825 |
Appl. No.: |
16/064312 |
Filed: |
December 21, 2016 |
PCT Filed: |
December 21, 2016 |
PCT NO: |
PCT/EP2016/082216 |
371 Date: |
June 20, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2236/55 20130101;
A61Q 17/00 20130101; A61P 29/00 20180101; A61Q 19/004 20130101;
A61K 2236/15 20130101; A61K 2236/00 20130101; A61Q 19/06 20130101;
A61K 8/368 20130101; A61K 8/498 20130101; A61Q 19/08 20130101; A61K
8/9789 20170801; A61K 2236/53 20130101; A61K 31/353 20130101; A61K
36/185 20130101; A61P 17/16 20180101; A61K 31/192 20130101; A61Q
17/04 20130101 |
International
Class: |
A61K 36/185 20060101
A61K036/185; A61K 8/9789 20060101 A61K008/9789; A61K 8/49 20060101
A61K008/49; A61K 8/368 20060101 A61K008/368; A61Q 19/08 20060101
A61Q019/08; A61Q 17/04 20060101 A61Q017/04; A61P 17/16 20060101
A61P017/16; A61P 29/00 20060101 A61P029/00; A61K 31/353 20060101
A61K031/353; A61K 31/192 20060101 A61K031/192 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 21, 2015 |
FR |
1562949 |
Claims
1-12. (canceled)
13. A polyphenolic extract of seeds of passion flower, in
particular of Passiflora incarnata or Passiflora edulis, comprising
at least 30 wt % polyphenols, expressed as gallic acid equivalents,
relative to the weight of the dry extract, and at least 10 wt %
organic acids, relative to the weight of the dry extract.
14. The extract of claim 13, comprising at least 35 wt %
polyphenols, expressed as gallic acid equivalents, relative to the
weight of the dry extract.
15. The extract of claim 13, wherein at least 50 wt % of said
polyphenols are catechin derivatives, expressed as gallic acid
equivalents, relative to the weight of polyphenols in the dry
extract.
16. The extract of claim 13, wherein said organic acids are acetic
acid, malic acid, citric acid or mixtures thereof.
17. The extract of claim 13, characterized in that it is obtained
by solid/liquid extraction of passion flower seeds in a solvent
selected from water, glycerols, glycols, and mixtures thereof.
18. The extract of claim 13, characterized in that it is obtained
by solid/liquid extraction of passion flower seeds in a solvent
selected from the binary mixtures water/glycerol, water/glycol, and
mixtures thereof.
19. A method for preparing a polyphenolic extract of passion flower
seeds as defined in claim 13, said method comprising at least one
step of solid/liquid extraction in a solvent selected from water,
glycerols, glycols, and mixtures thereof.
20. The method of claim 19, characterized in that it comprises the
following successive steps: a) grinding the seeds; b) optionally
defatting the seeds, preferably by pressing, by ethanolic
extraction or by supercritical CO.sub.2 extraction; c) solid/liquid
extraction of the ground and optionally defatted seeds in a solvent
selected from water, glycerols, glycols, and mixtures thereof; d)
separating the solid phase and the liquid phase by decantation,
and/or centrifugation and/or successive filtrations; and e)
optionally drying the extract obtained in step d).
21. The method of claim 19, characterized in that said solid/liquid
extraction solvent is selected from the binary mixtures
water/glycerol, water/glycol, and mixtures thereof.
22. A composition comprising as active principle a polyphenolic
extract of passion flower seeds comprising at least 30 wt %
polyphenols, expressed as gallic acid equivalents, relative to the
weight of the dry extract, and at least 10 wt % organic acids,
relative to the weight of the dry extractor an extract obtainable
by the method of claim 19, and a suitable excipient.
23. The composition of claim 22, comprising from 0.001 to 10 wt %,
advantageously from 0.01 to 5 wt %, of said polyphenolic extract of
passion flower seeds, the weight of the extract being expressed as
dry extract, relative to the total weight of the composition.
24. A method for preventing and/or treating: disorders or
pathologies of the skin and/or of the mucous membranes and/or of
the skin appendages, advantageously inflammatory reactions,
oxidation reactions, disorders relating to radical attacks
optionally linked to pollution, disorders of the barrier or of
homeostasis, of ageing, notably of chronological and/or actinic
ageing, of the skin and/or of the mucous membranes and/or of the
skin appendages, and/or vascular disorders, and/or damaged adipose
tissue, said method comprising the administration of a polyphenolic
extract of seeds of passion flower comprising at least 30 wt %
polyphenols, expressed as gallic acid equivalents, relative to the
weight of the dry extract, and at least 10 wt % organic acids,
relative to the weight of the dry extract or an extract obtainable
by the method of claim 19 to a subject in need thereof.
25. A method for cosmetic care of the skin and/or of the skin
appendages and/or of the mucous membranes, for the purpose of
improving the condition and/or the appearance thereof, consisting
in administering a polyphenolic extract of seeds of passion flower
comprising at least 30 wt % polyphenols, expressed as gallic acid
equivalents, relative to the weight of the dry extract, and at
least 10 wt % organic acids, relative to the weight of the dry
extractor an extract obtainable by the method of claim 19.
26. The extract of claim 13, comprising at least 40 wt %,
polyphenols, expressed as gallic acid equivalents, relative to the
weight of the dry extract.
27. The extract of claim 13, characterized in that it is obtained
by solid/liquid extraction of passion flower seeds in a solvent
selected from the binary mixtures water/glycerol, water/glycol, and
mixtures thereof, in a proportion of 30% to 90% of glycerol and/or
of glycol in water.
28. The method of claim 19, characterized in that said solid/liquid
extraction solvent is selected from the binary mixtures
water/glycerol, water/glycol, and mixtures thereof in a proportion
of 30% to 90% of glycerol and/or of glycol in water.
29. The method of claim 19, characterized in that said solid/liquid
extraction solvent is selected from the binary mixtures
water/glycerol, water/glycol, and mixtures thereof in a proportion
of 50% to 90% of glycerol and/or of glycol in water.
30. The method of claim 19, characterized in that said solid/liquid
extraction solvent is selected from the binary mixtures
water/glycerol, water/glycol, and mixtures thereof in a proportion
of 60% to 80% of glycerol and/or of glycol in water.
31. The method of claim 19, characterized in that said solid/liquid
extraction solvent is selected from the binary mixtures
water/glycerol, water/glycol, and mixtures thereof in a proportion
of 70% of glycerol and/or of glycol in water.
32. The composition of claim 22, comprising from 0.01 to 5 wt %, of
said polyphenolic extract of passion flower seeds, the weight of
the extract being expressed as dry extract, relative to the total
weight of the composition.
Description
[0001] The invention relates to an extract of seeds of passion
flower, Passiflora incarnata or edulis, preferentially edulis,
particularly polyphenol-rich, and to a method for preparing such an
extract. The present invention further relates to the cosmetic,
dermatological or therapeutic use of such a composition or such an
extract. Finally, the invention relates to a method for cosmetic
care of the skin, the skin appendages or the mucous membranes,
consisting in administering such a composition or such an
extract.
Passion Flowers
[0002] There are roughly 500 species of passion flowers (genus
Passiflora). These species are often distributed in hot, temperate
and tropical regions, particularly in the Americas, but they are
rather rare in Asia, Australia and tropical Africa.
Botany
[0003] The plants are in the form of shrubs or vines. The leaves
are alternate, sometimes simple, lobed or palmate. The flowers can
reach 9 cm in diameter and are bisexual or unisexual and regular.
They are white and purple with thin petals trimmed with filiform
appendices resembling Christ's crown of thorns. The 4- to 5-cm-long
fruit is oval and often yellow to orange in colour.
[0004] The most widespread species are notably Passiflora incarnata
(P. incarnata) and Passiflora edulis (P. edulis).
Phytochemical Aspects
[0005] P. incarnata: the major constituents are flavonoids, which
are present in large amounts in the leaves. The leaves contain a
high isovitexin content in particular. P. incarnata leaves also
contain a small amount of simple indole alkaloids (harmane,
harmine, etc.), sugars such as raffinose, sucrose, fructose and
glucose, essential oils, and maltol, which is described as the
molecule responsible for the sedative and anticonvulsive effects
attributed to this plant.
[0006] P. edulis: a specific compound, passiflorine (cyclopropane
triterpene glycoside), has been identified from a methanolic
extract of dried leaves (E. Bombardelli et al., 1975). P. edulis
leaves contain isoorientin in particular, a flavonoid not found in
the species P. incarnata. They also contain traces of essential oil
and of alkaloids identical to the species P. incarnata.
[0007] The fruit's flesh contains flavonoids (schaftoside,
isoschaftoside, isoorientin, orientin, isovitexin), luteolin
derivatives (M. L. Zeraik, J. H. Yariwake--2010), and ascorbic acid
(roughly 60 mg/100 g).
[0008] The flesh also contains glycosylated cyanogenic derivatives:
prunasin, sambunigrin and amygdalin, and two recently-identified
mandelonitrile .beta.-rutinosides (D. Chassagne and J. Crouzet,
1998; D. S. Seigler, 2002).
Toxicology
[0009] Cyanogenic constituents are present chiefly in the aerial
parts of various passion flower varieties.
Seed Characteristics
[0010] The seeds make up 6% to 12% of the P. Edulis fruit and
contain: [0011] polyphenols, including piceatannol (structure
similar to resveratrol) and its dimer scirpusin B (S. Sano; K.
Sugiyama; T. Ito, 2011), substances having vasorelaxant and
antioxidant effects. [0012] oil (18% yield after solvent
extraction) containing phytosterols (0.2%, including campesterol,
stigmasterol, sitosterol, avenasterol); 60% to 73% linoleic acid
(omega 6), 14% to 20% oleic acid and 465 ppm tocopherol (G. Piobom,
N. Barou et al., 2006; R. de V. V. Lopes et al.). [0013] sugars and
proteins.
PRIOR ART
Dietary Use
[0014] The fruit is believed to have been consumed since
prehistoric times. In 16.sup.th century Peru the magnificent
passion flowers were already regarded as a remedy, and numerous
passion flower species are still used in many countries in common
therapeutic practices.
Medical Use
[0015] Passion flowers (often the aerial parts and sometimes the
fruit) are often used throughout the world as anxiolytic, sedative,
diuretic or analgesic ("Passiflora: review update. K. Dhawan, S.
Dhawan, A. Sharma, 2004"). Maltol and certain maltol derivatives
are responsible for this sedative effect.
[0016] This activity is more constant and more significant for the
species P. incarnata.
[0017] P. incarnata extracts are capable of reversing morphine
dependence.
[0018] An antihypotensive effect of a methanolic extract of P.
edulis fruit peel and a hypocholesterolaemia effect of a fibre-rich
extract of defatted seeds have also been shown.
[0019] An antitumour effect of a fruit decoction via inhibition of
matrix metalloproteinases (MMP2 and MMP9) involved in tumour
invasion, metastases and angiogenesis, has also been shown (S. S.
Patel, 2009).
Dermo-Cosmetic Uses
[0020] In Brazil, P. foetida leaves are used topically to treat
inflammatory skin disorders, in particular by virtue of the
presence of isoorientin. In Mauritius and Rodrigues, decoctions of
P. suberosa leaves are also used in the bath to treat skin
conditions.
DESCRIPTION OF THE INVENTION
[0021] The Applicant has discovered that extracts of seeds of
passion flower, in particular Passiflora incarnata or Passiflora
edulis, and advantageously Passiflora edulis, have cosmetic,
pharmaceutical and dermatological properties that have hitherto
never been disclosed. In particular, it is the first time that such
passion flower seed extracts are used as such, for their specific
properties.
[0022] The invention thus relates to a polyphenolic extract of
seeds of passion flower, in particular of seeds of Passiflora
incarnata or of Passiflora edulis, more particularly of Passiflora
edulis, comprising at least 30 wt % polyphenols, expressed as
gallic acid equivalents, relative to the weight of the dry extract.
This content is equivalent to at least 3 mg of polyphenols per
millilitre of liquid extract.
[0023] The polyphenol content is expressed as gallic acid
equivalents, relative to the weight of the dry extract. These
percentages are obtained by a Folin-Ciocalteu assay.
[0024] In a Folin-Ciocalteu assay, all phenolic compounds are
oxidized by the Folin-Ciocalteu reagent (commercially available).
The latter comprises a mixture of phosphotungstic acid (H3PW12O40)
and phosphomolybdic acid (H3PMo12O40) which is reduced, during
oxidation of the phenolic substances, to a mixture of blue oxides
of tungsten (W8O23) and molybdenum (Mo8O23). The resultant blue
colouring has a maximum absorption around 750-760 nm. It is
proportional to the amount of oxidized phenolic compounds. The
reference phenol used in this method is gallic acid (see, e.g.,
Singleton et al., Colorimetry of total phenolics with
phosphomolybdic-phosphotungstic acid reagents). The results
obtained by this assay are thus expressed as "wt % polyphenols,
expressed as gallic acid equivalents, relative to the total weight
of the dry extract".
[0025] The polyphenol content is thus an easily measurable
parameter for persons skilled in the art.
[0026] The extract of the present invention advantageously
comprises at least 35 wt % polyphenols, expressed as gallic acid
equivalents, relative to the total weight of said dry extract,
i.e., at least 3.5 mg of polyphenols per millilitre of liquid
extract.
[0027] The extract of the present invention more advantageously
comprises at least 40 wt % polyphenols, expressed as gallic acid
equivalents, relative to the total weight of said dry extract,
i.e., at least 4 mg of polyphenols per ml of liquid extract.
[0028] The majority of the polyphenols present in the extract of
the invention are catechin derivatives.
[0029] The term "catechin derivatives", within the meaning of the
present invention, refers to the flavonoids of the catechin family,
also known as catechol. Catechin derivatives are more particularly
compounds of the following general formula (I):
##STR00001##
wherein: is a single bond of R or S configuration; R.sub.1 is OH or
a galloyl group of the following formula (II):
##STR00002##
and
R.sub.2 is H or OH.
[0030] Catechin derivatives are more particularly compounds of the
general formula (I) selected from the group consisting of:
TABLE-US-00001 Name Configuration R.sub.1 R.sub.2 (+)-catechin 2R,
3S OH H (-)-epicatechin 2R, 3R OH H (-)-catechin 2S, 3R OH H
(+)-epicatechin 2S, 3S OH H (-)-epigallocatechin 2R, 3R OH OH
(-)-epicatechin gallate 2R, 3R Galloyl group H (-)-epigallocatechin
gallate 2R, 3R Galloyl group OH (+)-gallocatechin 2R, 3S OH OH
(+)-gallocatechin gallate 2R, 3S Galloyl group OH
[0031] The extract of the invention advantageously comprises at
least 20 wt %, in particular at least 24 wt %, catechin
derivatives, expressed as gallic acid equivalents, relative to the
weight of the dry extract. Thus, in the extract of the invention,
at least 50 wt %, in particular at least 60 wt %, of the
polyphenols are catechin derivatives, expressed as gallic acid
equivalents, relative to the weight of polyphenols in the dry
extract.
[0032] Advantageously, the extract of the invention further
comprises at least 10 wt % organic acids, notably acetic acid,
malic acid, citric acid or mixtures thereof, relative to the weight
of the dry extract.
[0033] Particularly advantageously, the extract of the invention
comprises at least 30 wt % polyphenols, expressed as gallic acid
equivalents, relative to the weight of the dry extract, and at
least 10 wt % organic acids, notably acetic acid, malic acid,
citric acid or mixtures thereof, relative to the weight of the dry
extract.
[0034] According to this aspect, the extract of the invention has
the advantage of being rich in organic acids, which imparts to it
high antioxidant, anti-chelating and/or hydrating activity.
[0035] The extract of the invention is advantageously obtained by
solid/liquid extraction of passion flower seeds in a solvent
selected from water, glycerols, glycols, and mixtures thereof.
[0036] The solvent is more particularly selected from the binary
mixtures water/glycerol, water/glycol, and mixtures thereof,
advantageously in a proportion of 30% to 90%, in particular of 40%
to 90%, preferably of 50% to 90%, more preferentially of 60% to
80%, in particular of 70%, of glycerol and/or of glycol in
water.
[0037] Preferably, the solvent used is selected from the binary
mixtures water/glycerol or water/propanediol, in particular
water/propanediol, more particularly water/1,3-propanediol.
[0038] In particular, the extract of the invention advantageously
contains, by weight relative to the dry extract obtained: [0039]
roughly 40% polyphenols; [0040] roughly 10% sugars; [0041] roughly
11% fruit acids; and [0042] roughly 5% proteins.
[0043] Particularly, the extract of the invention does not comprise
isoorientin, orientin, vitexin or isovitexin.
[0044] The invention also relates to a method for preparing a
polyphenolic extract of passion flower, in particular of seeds of
Passiflora incarnata or of Passiflora edulis, advantageously of
Passiflora edulis, comprising at least 30 wt % polyphenols,
expressed as gallic acid equivalents, relative to the weight of the
dry extract, said method comprising at least one step of
solid/liquid extraction in a solvent selected from water,
glycerols, glycols, and mixtures thereof.
[0045] Advantageously, said method for preparing a polyphenolic
extract of passion flower seeds of the invention comprises the
following successive steps: [0046] a) grinding the seeds; [0047] b)
optionally defatting the seeds, preferably by pressing, ethanolic
extraction or CO.sub.2 extraction; [0048] c) solid/liquid
extraction of the ground and optionally defatted seeds in a solvent
selected from water, glycerols, glycols, and mixtures thereof;
[0049] d) separating the solid phase and the liquid phase by
decantation, and/or centrifugation and/or successive filtrations;
[0050] e) optionally drying the extract obtained in step d).
[0051] Step a) of grinding the seeds can be performed by methods
known to persons skilled in the art, notably using a knife mill, a
hammer mill, etc.
[0052] In step c), the solid/liquid extraction phase is performed
preferably at a temperature of 20.degree. C. to 90.degree. C., in
particular of 30.degree. C. to 80.degree. C., more particularly of
45.degree. C. to 75.degree. C., typically of 70.degree. C.
[0053] The extraction is performed for 30 minutes to 4 hours, in
particular for 1 hour to 3 hours, advantageously for roughly 2
hours.
[0054] Advantageously, the extraction solvent used in step c) is
selected from the binary mixtures water/glycerol, water/glycol, and
mixtures thereof, advantageously in a proportion of 30% to 90%, in
particular of 40% to 90%, preferably of 50% to 90%, more
preferentially of 60% to 80%, in particular of 70%, of glycerol
and/or of glycol in water. In particular, the extraction solvent is
selected from the binary mixtures water/glycerol or
water/propanediol, in particular water/propanediol, more
particularly water/1,3-propanediol.
[0055] In an advantageous variant of the method, prior to step c),
the passion flower seeds are defatted. Before being dispersed, the
ground seeds can be defatted, notably in ethanol. Removal of the
lipids allows better efficacy of the extraction and filtration
steps. It is also and preferentially possible to use oil-cakes of
these seeds, i.e., the residue resulting from preliminary
extraction of the oil by solvent, using the supercritical CO.sub.2
technique, for example, and preferentially by mechanical
pressing.
[0056] Step d) of separating the solid phase and the liquid phase
is performed by methods known to persons skilled in the art,
notably by decantation, centrifugation and/or successive
filtrations until perfect clarity and microbiological cleanliness
are achieved.
[0057] Advantageously, the polyphenolic extract of the invention
can be stabilized by the drying step e), by methods known to
persons skilled in the art.
[0058] For example, the drying step can be performed in the
presence of an additive of type maltodextrin or acacia fibre
(Fibregum.RTM., CNI), for example. The additive content typically
varies from 0% to 80% additive relative to the percentage of dry
matter obtained in the liquid form of the extract.
[0059] The extract is preferentially dried by lyophilization so as
to obtain a final powder. The final powder advantageously comprises
30 to 70 wt % dry matter of the extract, the remainder to 100%
being the lyophilization additive. More advantageously, the final
powder comprises 50% dry matter derived from the extract and 50%
lyophilization additive.
[0060] Alternatively, the starting raw material of the method of
the invention can be an oil cake of defatted passion flower seeds,
in particular defatted by pressing. Within this context and by way
of non-limiting example, the polyphenolic extract of the invention
can be obtained according to the following method: [0061] a')
preparation of a solution of oil-cake of passion flower seeds
defatted by pressing, at a concentration of 10% dry matter in
water; [0062] b') solid/liquid extraction, with stirring, for 2
hours at a temperature of 70.degree. C.; [0063] c') purification by
successive filtrations; and [0064] d') sterile filtration.
[0065] The extract obtained by the method of the invention, as
described in the preceding paragraphs, advantageously comprises at
least 35 wt %, more advantageously at least 40 wt %, polyphenols,
expressed as gallic acid equivalents, relative to the weight of the
dry extract.
[0066] Advantageously, the extract obtained by the method of the
invention also comprises at least 10 wt % organic acids, notably
acetic acid, malic acid, citric acid or mixtures thereof, relative
to the weight of the dry extract.
[0067] Advantageously, the extract obtained by the method of the
invention comprises at least 20 wt %, in particular at least 24 wt
%, catechin derivatives, expressed as gallic acid equivalents,
relative to the weight of the dry extract. Thus, in the extract
obtained by the method of the invention, at least 50 wt %, in
particular at least 60 wt %, of the polyphenols are catechin
derivatives, expressed as gallic acid equivalents, relative to the
weight of polyphenols in the dry extract.
[0068] The present invention thus also relates to an extract of
seeds of passion flower, in particular of seeds of Passiflora
incarnata or of Passiflora edulis, advantageously of Passiflora
edulis, obtainable by the above-mentioned method. Such an extract
meets the specifications defined above concerning the extract of
the invention.
[0069] In the description below, the expression "extract of the
invention" refers to the extract as such, as defined above, or the
extract obtainable by the method of the invention as described
above.
[0070] The invention further relates to a composition comprising a
polyphenol-rich extract of passion flower seeds of the invention,
as active principle, and if necessary a suitable excipient. The
extract of the invention is as defined in the paragraphs above
concerning the extract as such and those concerning the extract
obtainable by the method of the invention.
[0071] The composition of the invention advantageously comprises
from 0.001 to 10 wt %, advantageously from 0.01 to 5 wt %, of said
polyphenolic extract of passion flower seeds of the invention, the
weight of the extract being expressed as dry extract, relative to
the total weight of the composition.
[0072] The composition is advantageously cosmetic, pharmaceutical
or dermatological. Said composition is preferably formulated to be
administered via the external topical route.
[0073] The composition of the invention can further comprise one or
more other active principles.
[0074] The composition of the invention can be formulated as
various preparations suitable for topical administration.
[0075] In particular, the topical compositions can be notably
creams, emulsions, milks, ointments, lotions, oils, aqueous or
hydro-alcoholic or glycolic solutions, powders, patches, sprays,
shampoos, varnishes or any other product for external
application.
[0076] Depending on its nature (cosmetic, dermatological or
pharmaceutical), the composition of the invention can further
comprise at least one cosmetically, pharmaceutically or
dermatologically acceptable excipient. Notably, the composition of
the present invention can further comprise at least one
cosmetically, pharmaceutically or dermatologically acceptable
adjuvant known to persons skilled in the art, selected from
surfactants, thickeners, preservatives, fragrances, dyes, chemical
or mineral filters, hydrating agents, thermal spring water, etc.
Persons skilled in the art can adapt the formulation of the
composition of the invention based on their general knowledge.
[0077] The optimal modes of administration, dosing schedules and
dosage forms of the compositions of the invention can be determined
according to the criteria generally taken into account in the
establishment of a pharmaceutical, dermatological or cosmetic
treatment adapted to a patient or to an animal, such as, for
example, the patient's or the animal's age or body weight, general
state of health, tolerance to the treatment and skin type, and the
side effects observed.
[0078] The invention also relates to an extract of the invention or
a composition of the invention for use in preventing and/or
treating conditions or diseases of the skin and/or of the mucous
membranes and/or of the skin appendages, advantageously
inflammatory reactions, oxidation reactions, disorders relating to
radical attacks optionally linked to pollution, disorders of the
barrier or of homeostasis, of ageing, notably of chronological
and/or actinic ageing, of the skin and/or of the mucous membranes
and/or of the skin appendages.
[0079] The invention also relates to an extract of the invention or
a composition of the invention for use in preventing and/or
treating vascular disorders and/or damaged adipose tissue.
[0080] The invention also relates to the use of a passion flower
seed extract of the invention, or of a composition of the
invention, in the manufacture of a cosmetic, pharmaceutical or
dermatological composition for preventing and/or treating disorders
or pathologies of the skin and/or of the mucous membranes and/or of
the skin appendages, advantageously inflammatory reactions,
oxidation reactions, disorders relating to radical attacks
optionally linked to pollution, disorders of the barrier or of
homeostasis, of ageing, notably of chronological and/or actinic
ageing, of the skin and/or of the mucous membranes and/or of the
skin appendages.
[0081] The invention also relates to the use of a passion flower
seed extract of the invention, or of a composition of the
invention, in the manufacture of a cosmetic, pharmaceutical or
dermatological composition for preventing and/or treating vascular
disorders and/or damaged adipose tissue.
[0082] The invention further relates to a method for preventing
and/or treating disorders or pathologies of the skin and/or of the
mucous membranes and/or of the skin appendages, advantageously
inflammatory reactions, oxidation reactions, disorders relating to
radical attacks optionally linked to pollution, disorders of the
barrier or of homeostasis, of ageing, notably of chronological
and/or actinic ageing, of the skin and/or of the mucous membranes
and/or of the skin appendages, comprising the administration, in
particular the topical administration, of an effective amount of a
passion flower seed extract of the invention, or of a composition
of the invention, to a subject in need thereof.
[0083] The invention further relates to a method for preventing
and/or treating vascular disorders and/or damaged adipose tissue,
comprising the administration, in particular the topical
administration, of an effective amount of a passion flower seed
extract of the invention, or of a composition of the invention, to
a subject in need thereof.
[0084] In particular, the composition or the extract of the
invention is intended for the prevention and/or treatment of
inflammatory reactions, oxidation reactions, disorders related to
radical attacks linked to environmental stress, such as pollution,
UV radiation, cigarettes, etc., disorders of the barrier or of
homeostasis, of ageing, notably of chronological and/or actinic
ageing, of the skin, of the skin appendages (hair and nails) and/or
of the mucous membranes (gums, periodontium, genital mucosa)
whether immature, normal or mature/aged.
[0085] Notably, the composition or the extract of the invention is
intended for the prevention and/or treatment of disorders related
to inflammatory and/or radical reactions caused by exposure to UV
radiation and/or to pollutants such as heavy metals, or related to
intrinsic reactions, and thus generating accelerated ageing,
disorders of the barrier, vascular disorders, blotches, etc.
[0086] Notably, the composition or the extract of the invention is
intended for combatting skin ageing, notably chronological and/or
actinic ageing.
[0087] In particular, the extract of the invention is intended to
be used as antipollution cosmetic agent.
[0088] The expression "antipollution cosmetic agent" refers to an
agent which protects the skin and the keratinous material so as to
prevent, attenuate and/or eliminate the disorders or pathologies
generated by toxic gases such as ozone and organic combustion
residues. In particular, an antipollution cosmetic agent has an
antioxidant and antiradical activity. The pollution concerned
herein is in particular atmospheric pollution (such as ozone),
outdoor pollutants (e.g., nitrogen dioxides, carbon monoxide,
sulphur dioxide, ammonia, volatile organic compounds such as
polycyclic aromatic hydrocarbons (e.g., benzo-.alpha.-pyrene)),
indoor pollutants (e.g., volatile organic compounds, paint
residues, biocontaminants, cigarette smoke, cooking smoke,
construction materials, domestic cleaning or wood treatment
products).
[0089] The above-mentioned skin disorders or pathologies are more
particularly vascular disorders, atopic dermatitis, eczema,
irritative dermatitis, sensitive skin, reactive skin, blotched
skin, cutaneous erythema, aged or photoaged skin, photosensitive
skin, sunburns and inflammations due to rays of any kind.
[0090] The invention also relates to a method for cosmetic care of
the skin and/or of the skin appendages and/or of the mucous
membranes, for the purpose of improving the condition and/or the
appearance thereof, consisting in administering a composition or an
extract of the present invention, advantageously via the external
topical route.
[0091] The invention relates to a method for cosmetic care of the
skin, for the purpose of preventing the ageing thereof, consisting
in applying to the skin a composition or an extract of the present
invention.
[0092] The invention also relates to a cosmetic treatment method
for obtaining a protection of the organism against the effects of
pollution, consisting in applying to the skin and to the skin
appendages an extract or a composition of the invention, notably in
a cosmetically effective amount.
[0093] The following examples illustrate the invention.
DESCRIPTION OF THE FIGURES
[0094] FIG. 1 shows the percentage of Nrf2 activation by an extract
of the invention.
[0095] FIG. 2 shows claudin-4 immunostaining in skin explants
treated with BaP (Protocol 1)
[0096] FIG. 3 shows filaggrin immunostaining in skin explants
treated with BaP+nicotine (Protocol 2)
[0097] FIG. 4 shows loricrin immunostaining in skin explants
treated with BaP+nicotine (Protocol 2)
[0098] FIG. 5 shows collagen-I immunostaining in skin explants
treated with BaP+nicotine (Protocol 2)
[0099] FIG. 6 shows elastin immunostaining in skin explants treated
with BaP (Protocol 1)
[0100] FIG. 7 shows fibronectin immunostaining in skin explants
treated with BaP (Protocol 1)
[0101] FIG. 8 shows the visual appearance of the comet assay after
electrophoresis of treated normal human keratinocytes. FIG. 8A
corresponds to a negative control sample, FIG. 8B to a UV control
sample and FIG. 8C to a 0.001% extract of the invention.
[0102] FIG. 9 shows the change in heavy metal contents at T0 and at
T28 in the studied subjects.
[0103] FIG. 10 shows the change in MDA (FIG. 10A), catalase and SOD
(FIG. 10B) contents, expressed as U/mg of proteins, at T0 and at
T28 in subjects having received the placebo or having received the
active agent.
[0104] FIG. 11 shows the change in carbonylated protein content at
T0 and at T28 in subjects having received the placebo or having
received the active agent. FIG. 11A is a bar chart and FIG. 11B
shows photographs of cell layers with fluorescent staining of
carbonylated proteins. Quantification is then performed by image
analysis.
EXAMPLES
Example 1: Extract of the Invention
[0105] A polyphenolic extract is obtained according to the
following method: [0106] a) preparation of a solution of oil cake
of Passiflora edulis seeds, defatted by pressing and ground (10%
dry matter), in a 70:30 (w/w) 1,3-propanediol/water mixture; [0107]
b) extraction with stirring for 2 hours at 70.degree. C.; [0108] c)
removal of the residual plant by coarse filtration; and [0109] d)
purification of the extract obtained by filtrations including an
additional sterile filtration.
[0110] The liquid polyphenolic extract thus obtained has the
following characteristics (% of dry extract): [0111] Dry extract
(drying chamber): 0.9% (m/m) [0112] Total polyphenol content (by
spectrophotometry--in gallic acid equivalents): 43% [0113] Catechin
derivative content (by HPLC--in gallic acid equivalents): 22.3%
[0114] Malic acid content (by enzymatic kit): 5.4% [0115] Citric
acid content (by enzymatic kit): 3.7% [0116] Protein content (by
Kjeldahl.times.6.25): 4.5%
Example 2: Extract of the Invention
[0117] A polyphenolic extract is obtained according to the
following method: [0118] a) preparation of a solution of 13.3%
Passiflora edulis seeds, non-defatted and ground, in a 70:30
1,3-propanediol/water mixture [0119] b) extraction with stirring
for 2 hours at 70.degree. C. [0120] c) removal of the residual
plant by coarse filtration [0121] d) purification of the extract
obtained by filtrations, including an additional sterile
filtration.
[0122] The liquid polyphenolic extract thus obtained has the
following characteristics (% of dry extract): [0123] Dry extract
(drying chamber): 1.07% (m/m) [0124] Total polyphenol content (by
spectrophotometry--gallic acid equivalents): 40% [0125] Protein
content (Kjeldahl.times.6.25): 5.7%
Example 3: Biological Activity of the Extract of the Invention
[0126] 1. Biological Potential
[0127] The biological potential of an extract of the invention was
investigated using a gene expression modulation test on normal
human fibroblasts (NHF) and on reconstructed and melanized human
epidermises.
[0128] Thus, the expression of 46 genes involved in various
physiological pathways of the epidermis (barrier, pigmentation,
inflammation, etc.) and of the dermis (scarring, elasticity,
firmness, etc.) was studied by PCR-array.
Materials and Methods:
[0129] Normal human fibroblasts (NHF) and melanized reconstructed
epidermises were incubated for 24 hours in the presence of an
extract of the invention, as obtained in Example 1, at 0.002% and
0.05% (w/v) for the NHF or at 0.002% and 0.005% (w/v) for the
reconstructed epidermises, and in the presence of 20 ng/ml
TGF-.beta.1 on the NHF or in the presence of 1 nM vitamin D3 on the
reconstructed epidermises (controls for validating the tests).
[0130] At the end of the treatment, RNA was extracted and gene
expression was analysed by qRT-PCR using the TaqMan array targeting
the key functions of the dermis and of the epidermis.
Results and Conclusion:
[0131] The results are presented in Tables 2 and 3 below and show
in particular that the extract of the invention, while varying the
gene expression of certain markers, is of particular interest in
the following activities: [0132] the homeostasis and structure of
the dermal extracellular matrix ( MMP3); and [0133] the
dermo-epidermal junction ( LAMC2).
[0134] More particularly, the polyphenols of the extract of the
invention enabled modulation of the expression of genes involved in
antioxidant defences and the hormesis phenomenon ( HMOX1, FTL and
G6PD).
[0135] This shows that the extract of the invention has
antioxidant, antiradical and antiaging activity.
TABLE-US-00002 TABLE 2 Screening on NHF Extract of Extract of the
invention the invention (0.002%) (0.05%) RQ* p-value RQ* p-value
Haem oxygenase (HMOX1) -- -- 4.0641 0.0025 Ferritin, light
polypeptide (FTL) -- -- 2.3263 0.0221 Glucose-6-phosphate -- --
1.7197 0.0218 dehydrogenase (G6PD) Matrix metalloproteinase 1 -- --
0.6052 0.0282 (MMP1) Matrix metalloproteinase 3 0.5513 0.0173 -- --
(MMP3) *Level of gene expression expressed as relative quantity
(RQ) in relation to the untreated control = 1
TABLE-US-00003 TABLE 3 Screening on reconstructed epidermises
Extract of Extract of the invention the invention (0.002%) (0.05%)
RQ* p-value RQ* p-value Laminin subunit gamma-2 2.2309 0.0387
4.1885 0.0165 (LAMC2) Proopiomelanocortin (POMC) 2.3447 0.0398
1.7019 0.0183 Melanocyte-stimulating hormone 0.7109 0.0175 0.6188
0.0127 receptor (MC1R) Cytosolic phospholipase A2 0.6829 0.0391 --
-- (PLA2G4A) L-Dopachrome tautomerase (DCT) -- -- 0.4637 0.0296
*Level of gene expression expressed as relative quantity (RQ) in
relation to the untreated control = 1
[0136] 2. Activity on Induction of Hormesis and Cellular
Detoxification
[0137] A hormetic molecule (or hormetin) is a substance having a
biphasic effect, namely a beneficial effect at a low dose and the
opposite effect at a high dose (e.g., prooxidant or antioxidant). A
hormetin is also described as being a molecule which reproduces the
effects of mild stress on the organism but which in return enables
the cell to protect itself against future attacks and thus to
protect the organism against various age-related diseases (cancers)
or physical phenomena (skin ageing, poor healing, etc.) or harmful
environmental effects (UV radiation, pollution, etc.).
[0138] The following analyses made it possible to study the
activity on induction of hormesis and cellular detoxification of an
extract of the invention.
[0139] A--Activation of Translocation of Transcription Factor
Nrf2:
[0140] The effect of the extract of the invention was evaluated
with respect to activation of translocation of Nrf2, precursor of
the cascade responsible for the hormetic response and for certain
detoxification pathways of the organism.
Materials and Methods
[0141] ARE-luciferase-transfected HaCaT keratinocytes, containing
the antioxidant response element (ARE) plasmid NQO1, which is a
specific plasmid for activation of Nrf2 and luciferase (reporter
gene), were treated for 6 hours at 37.degree. C. with an extract of
the invention, as obtained in Example 1, at concentrations ranging
from 0.01% to 0.0005% (w/v) and with a positive reference, 20 .mu.M
tert-butylhydroquinone.
[0142] At the end of the treatment, the cell monolayers are lysed,
luciferase activity is assayed using a "Luciferase Assay Kit" from
PROMEGA, and the protein content of each lysate is assayed using
the Bradford method (BioRad).
Results and Conclusion
[0143] The results obtained are presented in Table 4 and in FIG. 1.
These results show that the extract of the invention induced an
increase in Nrf2 activity in the nuclei, and thus a translocation
of Nrf2 across the nuclear membrane, showing activation of this
transcription factor.
[0144] This shows that the extract of the invention has
antioxidant, antiradical and antiaging activity.
TABLE-US-00004 TABLE 4 Nrf2 activity in ARE-luciferase HaCaT (RLU =
relative luciferase activity) Nrf2 activity (mean normalized
Activation RLU/.mu.g of proteins) (%) Control 0 0
Tert-butylhydroquinone 23216.4 100 *** (20 .mu.M) Extract of the
invention 4149.6 18 * (0.0005%) Extract of the invention 8178.2 35
*** (0.001%) Extract of the invention 9998.6 43 *** (0.005%)
Extract of the invention 6324.6 27 *** (0.01%) * 0.01 < p <
0.05; ** 0.001 < p < 0.01; *** p < 0.001 and ns = not
significant vs control cells - one-way ANOVA followed by Dunnett's
test
[0145] B--Gene Expression of the Principal Markers of Hormesis and
of Cellular Detoxification.
[0146] The effect of an extract of the invention was studied on the
gene expression of various markers involved in the hormesis
pathways and in cellular detoxification.
Materials and Methods:
[0147] Normal human fibroblasts were treated for 6 hours, 24 hours
and 48 hours at 37.degree. C. with a 0.005% and a 0.002% (w/v)
extract of the invention, as obtained in Example 1.
[0148] At the end of the treatment, the gene expression of markers
of hormesis (HMOX1, FTL, G6PD and Nrf2) and of markers involved in
cellular detoxification (SOD1 and catalase) was analysed by
quantitative real-time RT-PCR and normalized to the housekeeping
gene HPRT (SybrGreen technology).
[0149] The results were statistically analysed by one-way ANOVA
followed by Dunnett's test (GraphPad PRISM version 5.02 software,
GraphPad Software, San Diego, Calif., USA).
Results:
[0150] The results obtained are presented in Table 5. These results
show that the extract of the invention significantly stimulated the
gene expression of HMOX1 at 6 hours and 24 hours, of FTL at three
times, of G6PD at 6 hours and 24 hours, of SOD1 at 24 hours, of
Nrf2 at 24 hours and 48 hours, and of catalase at 24 hours.
[0151] This shows that the extract of the invention has
antioxidant, antiradical and antiaging activity.
TABLE-US-00005 TABLE 5 Gene expression of hormesis markers in
normal human fibroblasts (relative quantity) HMOX1 FTL G6PD SOD1
Nrf2 Catalase 6 hours Control cells 1.00 1.00 1.00 1.00 1.00 1.00
Extract of the 3.38 0.94 1.31 1.03 1.00 0.97 invention (0.002%)
(+238% ***) .sup. (-6% ns) (+31% *).sup. (+3% ns) .sup. (+0% ns)
(-3% ns) Extract of the 23.06 1.37 1.31 1.05 1.04 1.02 invention
(0.005%) (+2206% ***) (+37% ***) (+31% *).sup. (+5% ns) .sup. (+4%
ns) (+2% ns) 24 hours Control cells 1.00 1.00 1.00 1.00 1.00 1.00
Extract of the 1.66 1.62 1.41 1.13 1.25 1.23 invention (0.002%)
(+66% ***) (+62% **) (+41% ns) (+13% ns) (+25% *) (+23% ns) Extract
of the 2.68 2.40 1.70 1.33 1.30 1.31 invention (0.005%) (+168% ***)
(+140% ***) .sup. (+70% **) (+33% *) .sup. (+30% *) (+31% *) .sup.
48 hours Control cells 1.00 1.00 1.00 1.00 1.00 1.00 Extract of the
0.98 1.10 0.88 1.39 1.34 1.06 invention (0.002%) .sup. (-2% ns)
(+10% ns).sup. (-12% ns) (+39% ns) (+34% *) (+6% ns) Extract of the
1.00 1.66 0.86 0.97 1.26 1.04 invention (0.005%) (0% ns) (+66% ***)
(-14% ns) (-3% ns) .sup. (+26% ns) (+4% ns) * 0.01 < p <
0.05; ** 0.001 < p < 0.01; *** p < 0.001 and ns = not
significant vs control cells - one-way ANOVA followed by Dunnett's
test
[0152] C--Production of Haem Oxygenase:
[0153] An extract of the invention was analysed on the protein
expression of haem oxygenase.
Materials and Methods:
[0154] Normal human fibroblasts were treated for 24 hours with a
0.002% and a 0.005% (w/v) extract of the invention, as obtained in
Example 1, and with 5 and 10 .mu.M curcumin (reference
hormetin).
[0155] At the end of the treatment, intracellular haem oxygenase 1
(or HMOX1 or HO1) was quantified using an ELISA technique.
Staining, proportional to the quantity of the marker of interest,
was measured by reading the optical density (OD) at 450 nm, and the
value obtained was related to the quantity of cells obtained by a
protein assay using the BC Assay (Interchim).
[0156] The results were statistically analysed by one-way ANOVA
followed by Dunnett's test (GraphPad PRISM version 5.02 software,
GraphPad Software, San Diego, Calif., USA).
Results:
[0157] The results obtained are presented in Table 6. These results
show that the extract of the invention induces the production of
haem oxygenase 1 with the same intensity as does curcumin. This
result confirms the action of the extract of the invention which we
observed during the study of gene expression of this same
marker.
[0158] This shows that the extract of the invention has
antioxidant, antiradical and antiaging activity.
TABLE-US-00006 TABLE 6 HMOX1 production by fibroblasts HMOX1 (ng/ml
on quantity of cells) Inhibition Control cells 0.033 .+-. 0.003
Curcumin (5 .mu.M) 0.060 .+-. 0.002 82% *** Curcumin (10 .mu.M)
0.105 .+-. 0.006 218% *** Extract of the invention (0.002%) 0.056
.+-. 0.002 69% *** Extract of the invention (0.005%) 0.098 .+-.
0.004 197% *** *** p < 0.001 vs control cells - one-way ANOVA
followed by Dunnett's test D- Production of haem oxygenase under
Nrf2 siRNA conditions:
[0159] In order to verify whether the haem oxygenase activation
pathway by an extract of the invention indeed passes through
activation of Nrf2, the potential to induce production of haem
oxygenase was verified in a system where Nrf2 expression is blocked
(small interference RNA, or siRNA).
Materials and Methods:
[0160] Normal human fibroblasts were pretreated for 24 hours with
Nrf2 siRNA and scrambled siRNA (control siRNA without action) and
then, on each preceding condition, for 24 hours with a 0.005% (w/v)
extract of the invention, as obtained in Example 1.
[0161] At the end of the treatment, intracellular haem oxygenase 1
(or HMOX1) was quantified using an ELISA technique. Staining,
proportional to the quantity of the marker of interest, was
measured by reading the optical density (OD) at 450 nm.
[0162] The results were statistically analysed by one-way ANOVA
followed by Tukey's test (GraphPad PRISM version 5.02 software,
GraphPad Software, San Diego, Calif., USA).
Results:
[0163] The results obtained are presented in Table 7. These results
show that the stimulation of HMOX1 production induced by an extract
of the invention is substantially decreased (-62%, p<0.001) when
the Nrf2 pathway is partially blocked (Nrf2 siRNA).
[0164] The activity of an extract of the invention on the
production of haem oxygenase thus indeed passes through the Nrf2
pathway.
[0165] This shows that the extract of the invention has
antioxidant, antiradical and antiaging activity.
TABLE-US-00007 TABLE 7 HMOX1 production by fibroblasts -
Comparative: scrambled siRNA vs Nrf2 siRNA HMOX1 Inhibition related
(ng/ml) to Nrf2 siRNA Control cells - scrambled 3.371 .+-. 0.702 --
-- Control cells - Nrf2 siRNA 2.199 .+-. 0.089 -35% * Extract of
the invention (0.005%) - 18.821 .+-. 0.316 -- -- scrambled Extract
of the invention (0.005%) - 7.105 .+-. 0.131 -62% *** Nrf2 siRNA
*** p < 0.001 vs control cells - one-way ANOVA followed by
Tukey's test
[0166] E--Effect on Production of Reactive Oxygen Species
(ROS):
[0167] The antioxidant potential of an extract of the invention
with respect to H.sub.2O.sub.2 induction of reactive oxygen species
was studied.
Materials and Methods:
[0168] Normal human keratinocytes were incubated for 24 hours in
the presence of a 0.002%, a 0.005% and a 0.01% (w/v) extract of the
invention, as obtained in Example 1, or of 500 .mu.M vitamin C and
10 .mu.M quercetin (reference antioxidants) before incorporation of
the H2DCF-DA probe (incubation for 60 minutes).
[0169] The keratinocytes were then stimulated with 100 .mu.M
hydrogen peroxide (H.sub.2O.sub.2) for 20 minutes and the
production of reactive oxygen species (ROS) was evaluated by
measurement of fluorescence.
[0170] The results were statistically analysed by one-way ANOVA
followed by Tukey's test (GraphPad PRISM version 5.02 software,
GraphPad Software, San Diego, Calif., USA).
Results:
[0171] The results obtained are presented in Table 8. These results
show that the extract of the invention significantly inhibited the
production of ROS by keratinocytes in response to hydrogen peroxide
(H.sub.2O.sub.2)-induced oxidant stress. The level of this
antioxidant activity is equivalent to that of the two control
antioxidants (vitamin C and quercetin).
[0172] This shows that the extract of the invention has
antioxidant, antiradical and antiaging activity.
TABLE-US-00008 TABLE 8 ROS production in H.sub.2O.sub.2-treated
keratinocytes ROS (fluorescence units) Significance Control cells
30752.921 .+-. 4555.136 -- -- Stimulated cells (H.sub.2O.sub.2)
44179.976 .+-. 7445.110 +44% *** Reference (vitamin C) 21719.018
.+-. 3174.253 -51% *** Reference (quercetin) 14367.847 .+-.
1790.753 -67% *** Extract of the invention (0.002%) 18769.676 .+-.
2780.206 -58% *** Extract of the invention (0.005%) 14404.891 .+-.
2974.938 -67% *** Extract of the invention (0.01%) 16531.685 .+-.
1902.378 -63% *** *** p < 0.001 - one-way ANOVA followed by
Tukey's test
[0173] 3. Protection Against the Harmful Effects of Pollution
[0174] The preceding results showed that an extract of the
invention stimulates the production of haem oxygenase 1 via
activation of translocation of transcription factor Nrf2.
Consequently, the extract of the invention enabled antioxidant
cellular protection via reduction of ROS formation induced by
H.sub.2O.sub.2 stress.
[0175] The extract of the invention making it possible to stimulate
skin defences, we evaluated their protective effect with respect to
various environmental stresses, in this case pollution.
[0176] A--Effect on Oxidant Stress:
Materials and Methods:
[0177] Normal human keratinocytes were treated for 24 hours with a
0.002% (w/v) extract of the invention, as obtained in Example 1,
500 .mu.M vitamin C and 10 .mu.M quercetin (reference
antioxidants), with 10 .mu.M curcumin (reference hormetin) or with
10 .mu.M resveratrol before incorporation of the H2DCF-DA probe
(incubation for 45 minutes). The keratinocytes were then stimulated
with 9 .mu.g/ml benzo-.alpha.-pyrene (BaP) for 20 minutes.
[0178] ROS production was evaluated by measurement of
fluorescence.
[0179] The significance of the results was verified by Student's
t-test.
Results and Conclusion:
[0180] The results obtained are presented in Table 9. These results
show that the 0.002% extract of the invention inhibited ROS
production by keratinocytes in response to oxidant stress induced
by 6 and 9 .mu.g/ml BaP.
[0181] Therefore, the extract of the invention exerts a protective
effect with respect to pollution-induced oxidant stress. The
extract of the invention thus has antioxidant, antiradical,
antipollution and antiaging activity.
TABLE-US-00009 TABLE 9 ROS production in keratinocytes treated with
9 .mu.g/ml BaP ROS (fluorescence units) Significance Control cells
4730 .+-. 324 -- -- Stimulated cells 16777 .+-. 1755 +255% *** (9
.mu.g/ml BaP) Reference (vitamin C) 14089 .+-. 1719 -16% NS
Reference (quercetin) 17140 .+-. 930 -32% NS 10 .mu.M curcumin
14065 .+-. 1231 -16% NS 10 .mu.M resveratrol 20357 .+-. 503 -21% *
Extract of the invention 11052 .+-. 828 34% ** (0.002%) * p <
0.05; ** p < 0.01; *** p < 0.001 and ns = not significant -
Student's t-test
[0182] B--Protection of Skin Structures:
[0183] The ability of an extract of the invention to protect the
integrity of the skin (dermis and epidermis) from the harmful
effects of pollution was studied on human skin explants.
Materials and Methods:
[0184] Human skin explants from a 45-year-old woman were pretreated
for 24 hours with a topical application of a cosmetic formula
containing or not containing (placebo) 3% extract of the invention,
as obtained in Example 1.
[0185] Said explants were then treated again with the cosmetic
formulas in the presence of benzo-.alpha.-pyrene (BaP, 20 .mu.M)
for Protocol 1 or of Bap+nicotine (20 .mu.M) for Protocol 2.
[0186] Immunostainings of various skin structure markers were
performed.
Results and Conclusion:
[0187] The results obtained are presented in FIGS. 2 to 7.
[0188] The stress mimicking pollution induced by BaP.+-.nicotine
led to altered expression of the structural markers studied.
[0189] Under these conditions, the extract of the invention
protected the following epidermal markers: [0190] Claudin 4 (marker
of the tight junctions/barrier function), [0191] Filaggrin (marker
of the barrier function and precursor of natural hydration
factors), and [0192] Loricrin (marker involved in cell
differentiation/barrier function); And the dermal markers: [0193]
Collagen I (marker involved in skin firmness), [0194] Elastin
(marker involved in skin elasticity), and [0195] Fibronectin
(marker involved in dermal structure).
[0196] These results show a real protective effect of the
structural integrity of the epidermis and of its barrier function
as well as the maintenance of a normal structure of the dermis and
therefore an overall skin protective effect against environmental
pollution.
[0197] The extract of the invention thus has antioxidant,
antiradical, antipollution and antiaging activity.
[0198] C--Evaluation of Detoxifying Activity with Respect to Ozone
Stress
[0199] The protective and detoxifying potentials of an extract of
the invention with respect to ozone stress were evaluated on
reconstructed epidermises.
Materials and Methods:
[0200] Reconstructed human epidermises (RHE) were pretreated for 24
hours with a topical application of a cosmetic formula containing
or not containing (placebo) 3% extract of the invention, as
obtained in Example 1.
[0201] The epidermises were then subjected to stress with 0.5 to 1
ppm ozone.
[0202] The enzymatic activities of the detoxifying enzymes
catalase, glutathione peroxidase (GPX) and superoxide dismutase
(SOD) were quantified by a calorimetric (SOD and GPX) or
fluorescence (catalase) ELISA method.
[0203] Lipid peroxidation was evaluated by the malondialdehyde
(MDA) assay, performed by GC.
[0204] DNA oxidation was evaluated by the 8-hydroxydeoxyguanosine
(8-OHdG) assay, performed by colorimetry.
[0205] The results were statistically analysed by one-way ANOVA
followed by Tukey's test (GraphPad PRISM version 5.02 software,
GraphPad Software, San Diego, Calif., USA).
Results and Conclusion:
[0206] Ozone stress induced overactivation of catalase, SOD and GPX
enzymes; this increase attests to the ozone-induced oxidant
stress.
[0207] Under these conditions, the extract of the invention
significantly inhibited this overactivation.
[0208] In the absence of ozone stress (basal condition), the
extract of the invention induces no inhibition of the activity of
the detoxifying enzymes. This confirms that it is indeed a
protective effect with respect to the induced stress and not an
inhibition of the detoxification systems of the cell.
(Tables 10, 11 and 12).
[0209] Ozone stress induced oxidative damage expressed as an
increase in 8-OHdG and in MDA.
[0210] The extract of the invention significantly protects cell
components from the oxidative damage induced by ozone stress by
significantly inhibiting the production of 8-OHdG and of MDA.
(Tables 13 and 14).
TABLE-US-00010 [0211] TABLE 10 Catalase activity in RHE optionally
treated with ozone Mean % change Tukey Catalase assay under ozone
stress conditions Control epidermises 156.1 0 Ozone 340.1 118 $$$
Placebo 306.4 -10 ns Extract of the invention (3%) 244.2 -28 ***
Catalase assay under basal conditions Control 181.8 0 Placebo 250.3
38 $$$ Extract of the invention (3%) 231.4 27 $$ $$ 0.001 < p
< 0.01; $$$ p < 0.001 vs control ** 0.001 < p < 0.01;
*** p < 0.001 vs ozone
TABLE-US-00011 TABLE 11 GPX activity in RHE optionally treated with
ozone Mean % change Tukey GPX assay under ozone stress conditions
Control 13.0 0 Ozone 34.9 168 $$$ Placebo 31.1 -11 * Extract of the
invention (3%) 23.3 -33 *** GPX assay under basal conditions
Control 13.0 0 Placebo 12.3 -6 ns Extract of the invention (3%)
13.2 2 ns $$$ p < 0.001 vs control- * 0.01 < p < 0.05; ***
p < 0.001 vs ozone
TABLE-US-00012 TABLE 12 SOD activity in RHE optionally treated with
ozone Mean % change Tukey SOD assay under ozone stress conditions
Control 5.7 0 Ozone 13.0 130 $$$ Placebo 9.5 -27 *** Extract of the
invention (3%) 7.2 -45 *** SOD assay under basal conditions Control
- 4.7 0 Placebo formula 5.4 15 $$ Extract of the invention (3%) 4.9
4 ns $ 0.01 < p < 0.05; $$ 0.001 < p < 0.01 $$$ p <
0.001 vs control ** 0.001 < p < 0.01; *** p < 0.001 vs
ozone
TABLE-US-00013 TABLE 13 8-Oxo-dG assay in ozone-treated RHE Mean %
change Tukey Control 2.9 0 Ozone 7.4 155 $$$ Placebo 5.4 -27 **
Extract of the invention (3%) 3.7 -51 *** $$$ p < 0.001 vs
control * 0.01 < p < 0.05; ** 0.001 < p < 0.01; *** p
< 0.001 vs ozone
TABLE-US-00014 TABLE 14 MDA assay in ozone-treated RHE Mean %
change Tukey Control 96.6 0 Ozone 251.4 160 $$$ Placebo 204.0 -19
** Extract of the invention (3%) 176.6 -30 *** $$$ p < 0.001 vs
control ** 0.001 < p < 0.01; *** p < 0.001 vs ozone
[0212] 4. Protection Against Sun-Related Deleterious Effects
[0213] Ultraviolet (UV) and infrared (IR) rays penetrate the skin
to varying depths and are responsible for, amongst other things, a
decrease in skin firmness and an increase in the quantity of free
radicals released, thus leading to premature skin ageing. Such rays
are also responsible for the formation of melanomas and for skin
immunosuppression. The protective effect of an extract of the
invention with respect to UV- or IR-induced stress was studied.
[0214] A--Protection Against UV-Induced DNA Damage (Comet
Assay):
[0215] Maintenance of the nuclear integrity of the cell, with
respect to UV radiation, was tested using the comet assay.
Materials and Methods:
[0216] Normal human keratinocytes were treated for 2 hours with a
0.001% (w/v) extract of the invention, as obtained in Example
1.
[0217] A comet assay is performed according to the method described
by "Singh et al." in 1988 and by "Meo et al." in 1991, and which
consists in irradiating cells with light at 4.5 J/cm.sup.2 (0.28
J/cm.sup.2 UVA; 0.08 J/cm.sup.2 UVB and 4.14 J/cm.sup.2 visible
light) corresponding to 1 to 3 minutes of sun exposure in
mid-summer. This is followed by DNA migration using agarose gel
electrophoresis.
[0218] A relative value .chi..sup.2 OTM is calculated using the
Systat software, this value being directly proportional to the size
of the comet and thus to the degree of protection of the active
agent with respect to UV radiation.
[0219] The significance of the results was verified by analysis of
variance using the SigmaPlot software (version 11.0, Systat
Software, Chicago, Ill., USA).
Results and Conclusion:
[0220] The results are presented in Table 15 and in FIG. 8.
[0221] These results show that the 0.001% extract of the invention
protected keratinocytes with respect to UV-irradiation-induced DNA
damage (54% protection, p<0.001).
[0222] The extract of the invention thus has antioxidant,
antiradical, antipollution and antiaging activity.
TABLE-US-00015 TABLE 15 Percent protection of cells with respect to
UV radiation .chi..sup.2 OTM % protection Control without UV 2.09
.+-. 0.1 100 Control irradiated at 4.5 J/cm.sup.2 11.59 .+-. 0.41 0
*** Extract of the invention (0.001%) 6.43 .+-. 0.28 54.4 *** *** p
< 0.001 vs non-irradiated cells and vs cells irradiated at 4.4
J/cm.sup.2. Statistics from SigmaPlot
[0223] B--Inhibition of IR-Induced Production of MMP1
[0224] Infrared (IR) rays, which represents more half of the solar
spectrum, can induce degradation of the dermal matrix, contributing
to skin ageing, by stimulating the production of proteases such as
MMP1.
[0225] The ability of an extract of the invention to protect dermal
cells from the harmful effects of IR rays was studied by evaluating
MMP1 production.
Materials and Methods:
[0226] Normal human fibroblasts (NHF) were incubated in the
presence of a 0.001% extract of the invention, as obtained in
Example 1, or of 10.sup.-7 M dexamethasone (reference
anti-inflammatory) for 48 hours following 1 hour of infrared
irradiation (0.57 kJ/cm.sup.2).
[0227] After incubation, the supernatants are collected in order to
assay released MMP1 (ELISA Kit, R&D Systems).
[0228] The significance of the results was verified by Student's
t-test.
Results and Conclusion:
[0229] The results are presented in Table 16. These results show
that the 0.001% extract of the invention significantly inhibited
infrared-induced production of MMP1 in normal human
fibroblasts.
[0230] The extract of the invention thus has antioxidant,
antiradical, antipollution and antiaging activity.
TABLE-US-00016 TABLE 16 IR-induced production of MMP1 in normal
human fibroblasts MMP1 (ng/ml) % protection Control without IR 11
100 *** Control irradiated at 0.57 kJ/cm.sup.2 88.6 0 Dexamethasone
(10.sup.-7 M) 9.3 102 *** Extract of the invention (0.001%) 77.4 14
* * p < 0.05; *** p < 0.001 vs irradiated cells
[0231] C--Protection Against UV-Induced Oxidant Stress:
[0232] The antioxidant potential of an extract of the invention
with respect to UV-irradiation-induced reactive oxygen species
(ROS) was studied.
Materials and Methods:
[0233] Normal human keratinocytes were incubated for 24 hours in
the presence of a 0.001% and a 0.005% (w/v) extract of the
invention, as obtained in Example 1, or of 500 .mu.M vitamin C
(reference antioxidant), before incorporation of the H2DCF-DA probe
(incubation for 1 hour).
[0234] The keratinocytes were then stimulated with UV at 2400
J/m.sup.2 (2000 J/m.sup.2 UVB and 400 J/m.sup.2 UVA) then returned
to culture in the presence of a 0.001% and a 0.005% (w/v) extract
of the invention, as obtained in Example 1, or of 500 .mu.M vitamin
C, for 15 minutes at 37.degree. C.
[0235] The production of reactive oxygen species (ROS) was
evaluated by measurement of fluorescence and the value obtained was
normalized to the quantity of cells obtained using an MTT cell
viability assay.
[0236] The results were statistically analysed by one-way ANOVA
followed by Tukey's test (GraphPad PRISM version 5.02 software,
GraphPad Software, San Diego, Calif., USA).
Results and Conclusion:
[0237] The extract of the invention significantly inhibited ROS
production by keratinocytes in response to UV-induced (2400
J/m.sup.2) oxidant stress (Table 17).
TABLE-US-00017 TABLE 17 ROS production in keratinocytes treated by
UV at 2400 J/m.sup.2 ROS (fluorescence Signifi- intensity/MTT) %
change cance Control cells 30936 .+-. 4925 Irradiated cells (UV
2400 J/m.sup.2) 43038 .+-. 5735 39 $$$ Reference (vitamin C) 26005
.+-. 3723 -40 *** Extract of the invention (0.001%) 22564 .+-. 3849
-48 *** Extract of the invention (0.005%) 19614 .+-. 4576 -54 ***
$$$ p < 0.001 vs control without UVs *** p < 0.001 vs control
with UVs One-way ANOVA followed by Tukey's test
[0238] 5. Protection Against the Effects of Chemical Stress
[0239] Effect on PMA-Induced Production of PGE2:
[0240] The anti-inflammatory protection of an extract of the
invention with respect to a chemical molecule, PMA, was studied by
analysis of the release of prostaglandin 2 (PGE2).
Materials and Methods:
[0241] Normal human keratinocytes were pretreated with a 0.002% and
a 0.005% extract of the invention, as obtained in Example 1, and
with 10.sup.-6 M indomethacin (anti-inflammatory control of the
prostaglandin pathway) for 24 hours at 37.degree. C., in order to
be able to measure a level of protection of the active agent with
respect to inflammation by 0.1 .mu.g/ml PMA (phorbol 12-myristate
13-acetate) used on these same cell monolayers for a further 24
hours.
[0242] At the end of the treatment, the supernatants are collected
and an assay of prostaglandin E2 (PGE2; R&D Systems) is
performed. Staining, proportional to the amount of PGE2, was
measured by reading the optical density (OD) at 450 nm.
[0243] The significance of the results was verified by Student's
t-test.
Results:
[0244] The results are presented in Table 18. These results show
that the extract of the invention at both concentrations
significantly decreased PGE2 release with respect to induction by
0.1 .mu.g/mlPMA.
[0245] The extract of the invention thus has antioxidant,
antiradical, antipollution and antiaging activity.
TABLE-US-00018 TABLE 18 PGE2 production in PMA-treated
keratinocytes PGE2 (pg/ml) Inhibition Control cells 39 .+-. 0 100%
*** PMA (0.1 .mu.g/ml) 113806 .+-. 11441 0% Indomethacin (10.sup.-6
M) 52 .+-. 8 100% *** Extract of the invention 35750 .+-. 2192 69%
** (0.002%) Extract of the invention 30382 .+-. 2401 73% **
(0.005%) ** 0.001 < p < 0.01 and *** p < 0.001 vs
PMA-stimulated cells Student's t-test
[0246] 6. Protection Against the Effects of Ageing
[0247] The repeated action of environmental stresses such as
pollution, the harmful effects of the sun, chemical molecules and
all other forms of induction of oxidant stress, lead to degradation
of the dermal matrix and thus to premature skin ageing.
[0248] An extract of the invention was analysed on a model of cell
ageing in order to analyse their actions with respect to proteins
which are underexpressed or overexpressed with age.
Materials and Methods:
[0249] Keratinocytes are cultured, and trypsinized each week, for 4
weeks in culture medium inducing an ageing phenotype ("pro-age"
medium) optionally in the presence of a 0.000025% DM extract of the
invention, as obtained in Example 1.
[0250] At the end of 3 weeks of culture/passages, a proteomic
analysis is performed. The analysis grouped the proteins of the
various cellular pathways into six domains: metabolism, apoptosis,
detoxification, protein catabolism and protein synthesis.
Results and Conclusion:
[0251] The results are presented in Table 19. In this context of
induction of ageing, the extract of the invention stimulated and/or
protected the expression of several proteins involved mainly in
cellular detoxification/protection and immune defences:
Cellular Detoxification and Protection:
[0252] Stimulation of peroxiredoxin 2 (PRDX2): an antioxidant
enzyme playing a role in cellular protection against damage caused
by intracellular ROS. [0253] Stimulation of carbonyl reductase 1
(CBR1): a dehydrogenase/reductase which reduces carbonyl compounds
(medicinal products) and intervenes in detoxification during lipid
peroxidation. The latter is inhibited under the "pro-age"
conditions and is stimulated by the extract of the invention under
these conditions. [0254] Inhibition of aldehyde dehydrogenase 2
(ALDH2): a mitochondrial enzyme playing a role in cell protection
and differentiation which catalyses/detoxifies aldehyde/carbonyl
molecules (medicinal products, pollution, etc.). This protein is
stimulated under the "pro-age" conditions, and the extract of the
invention restores its expression to a basal level. [0255]
Stimulation of fatty acid-binding protein 5 (FABP5): a chaperone
protein mainly expressed in the epidermis and involved in
regulation of lipid homeostasis and thus playing a role in the
barrier function. The "pro-age" conditions inhibit its expression,
which is restored by passion fruit polyphenols. [0256] Stimulation
of proteasome .beta.2 and .beta.6 subunits (PSMB2 and PSMB6):
Proteasomes are involved in the removal of damaged/oxidized
proteins and in the replacement of intracellular proteins. The
proteasome consists of a and .beta. subunits which, amongst other
things, cleave damaged proteins on the level of glutamine (.beta.6)
and on the level of trypsin (.beta.2). Age decreases proteasome
activity, which leads to an accumulation of damaged/oxidized
proteins; this age effect is found under the "pro-age" conditions,
and under these conditions the extract of the invention stimulates
expression of these two proteasome subunits.
Immune Defence:
[0256] [0257] Inhibition of beta-2-microglobulin (B2MG): a small
surface protein (epidermis) involved in the immune response. It is
part of the major histocompatibility complex and is overexpressed
in disease conditions, thus producing interleukins, notably 6 and
8, as well as 10, which is an immunosuppressive interleukin. Here,
under conditions inducing ageing, its expression is increased. The
extract of the invention prevents this increase. [0258] Inhibition
of lectin, mannose-binding, 1 (LMAN1): a protein participating in
the immune response by enabling phagocytosis of apoptotic cells and
of pathogens. A deficiency in this gene entails an increase in cell
debris in the skin. It is scarce, if present at all, in the basal
state and is increased in inflammatory conditions (e.g., UV). Under
the "pro-age" conditions, its expression is increased and modulated
under the action of the extract of the invention.
TABLE-US-00019 [0258] TABLE 19 Protein expression (in %) of markers
involved in detoxification and in immunity % induction with respect
% induction with respect to normal medium to "pro-age" medium
Extract of Extract of "Pro-age" the invention "Pro-age" the
invention Proteins medium (0.000025%) medium (0.000025%)
Peroxiredoxin 2 11.46 16.68 0.00 45.51 (PRDX2) Carbonyl reductase
-15.87 -7.46 0.00 52.99 1 (CBR1) Aldehyde dehydro- 46.81 4.97 0.00
-89.38 genase 2 (ALDH2) Fatty acid-binding -24.82 4.68 0.00 118.87
protein 5 (FABP5) Proteasome .beta.2 -21.15 -10.96 0.00 48.21
(PSMB2) Proteasome .beta.6 -16.85 -10.92 0.00 35.21 (PSMB6)
Beta-2-micro- 31.16 -0.57 0.00 -101.83 globulin (B2MG) Lectin,
mannose- 40.13 3.71 0.00 -90.75 binding, 1 (LMAN1)
[0259] 7. Conclusion
[0260] These various tests show an anti-inflammatory, antioxidant,
antipollution and thus antiaging effect of the polyphenolic extract
of the invention.
Example 4: Evaluation of In Vivo Efficacy of the Passion Flower
Polyphenol Active Agent Versus Placebo by Measurement of the Amount
of MDA, SOD, Catalase (CAT) and Carbonylated Proteins
Study Design:
[0261] Double-blind study. [0262] Comparative, randomized study
Population:
[0263] Two groups of 30 Asian female subjects (1 active agent group
and 1 placebo group), between 30 and 50 years of age, of all skin
types, living in a polluted environment.
Use:
[0264] The products are applied to one side of the face, by the
subjects themselves, at home, twice per day (morning and evening)
for 28 days.
Study Protocol:
TABLE-US-00020 [0265] TABLE 20 Protocol of the clinical study T 28
Evaluation T0 days Hair sampling X X Biological sampling of the
cheeks for MDA, CAT, SOD X X Biological sampling of the cheeks for
carbonylated proteins X X
Mean Real Age Per Panel:
[0266] Panel total: 39.9 years (between 30 and 50 years)
Composition Administered:
Placebo Formula
TABLE-US-00021 [0267] % Material name INCI EU material PURIFIED
WATER R&D AQUA 75.12 SODIUM EDETATE DISODIUM EDTA 0.10 BUTYLENE
GLYCOL BUTYLENE GLYCOL 3.00 CARBOPOL ULTREZ 10 CARBOMER 0.60
OCTANEDIOL XI CAPRYLYL GLYCOL 0.30 EMULIUM DELTA CETYL ALCOHOL 3.50
GLYCERYL STEARATE PEG-75 STEARATE CETETH-20 STEARETH-20 ISONONYL
ISONONYL ISONONANOATE 10.00 ISONONANOATE PURE CETYL ALCOHOL CETYL
ALCOHOL 0.50 CAPRYLOYL GLYCINE CAPRYLOYL GLYCINE 0.80 SODIUM
HYDROXIDE XI AQUA 1.08 SODIUM HYDROXIDE PURIFIED WATER R&D AQUA
2.00 PURIFIED WATER R&D AQUA 3.00 100.00
Active Formula
TABLE-US-00022 [0268] % Material name INCI EU material PLACEBO 97
PASSION FRUIT POLYPHENOLS AQUA 3 1 KG PROPANEDIOL PASSIFLORA EDULIS
FRUIT EXTRACT 100
[0269] 1. Evaluation of the Pollution Received by the Subject at T0
and T28
[0270] At T0 and T28, a 1-cm lock of hair was taken from the scalp.
A biochemical analysis was performed in order to determine the
heavy-metal exposure of the subjects at each time point. The
analysis involved 10 subjects, 5 from each panel.
[0271] The results (Table 21 and FIG. 9) show a constant amount of
heavy metals or a slight increase. It can be concluded that the
subjects' exposure to pollution did not decrease during the study,
ruling out the hypothesis of a beneficial effect due only to a
variation in pollution.
TABLE-US-00023 TABLE 21 Heavy metal contents Pb Fe Ni Cu Cd (ng/g)
(ng/g) (ng/g) (ng/g) (ng/g) T0 218.5 7603.9 249.7 9283.8 25.5 T28
366 9487 328 10711 37 Significant Yes Yes No No Yes difference p =
p = p = p = p = 0.0098 0.0488 0.0840 0.2324 0.0019
[0272] 2. Evaluation of the Amount of MDA, CAT and SOD at T0 and
T28
[0273] At T0 and T28, a swab was taken from each cheek of the
subjects. A biochemical analysis was performed in order to
determine the amount of MDA, CAT and SOD. The analysis involved 30
subjects, 15 from each panel.
[0274] The amount of MDA, CAT and SOD significantly decreases
between T0 and T28 for the active agent and the placebo, with a
significantly larger decrease for the active agent (Table 22 and
FIGS. 10A and 10B).
TABLE-US-00024 TABLE 22 MDA, CAT and SOD contents at T0 and T28 MDA
CAT SOD Placebo Active agent Placebo Active agent Placebo Active
agent T0 244.0 .+-. 77.9 244.0 .+-. 77.9 34.1 .+-. 4.98 34.1 .+-.
4.98 30.6 .+-. 5.0 30.6 .+-. 5.0 T28 213.7 .+-. 65.1 185.7 .+-.
56.3 28.9 .+-. 4.7 27.8 .+-. 4.6 25.8 .+-. 3.4 24.8 .+-. 3.7 %
change -12.4% -23.9% -15.4% -18.6% -15.9% -18.9% Significance Yes
Yes Yes Yes Yes Yes T28 - T0 p < 0.01 p < 0.01 p < 0.01 p
< 0.01 p < 0.01 p < 0.01 Significance Yes Yes Yes Placebo
vs p < 0.01 p < 0.01 p < 0.01 Active agent
[0275] 3. Evaluation of the Amount of Carbonylated Proteins at T0
and T28
[0276] At T0 and T28, D-Squame sampling was performed on each cheek
of the subjects. Biochemical staining was performed in order to
determine the amount of carbonylated proteins. The analysis
involved 20 subjects, 10 from each panel.
[0277] The amount of carbonylated proteins significantly decreases
between T0 and T28 for the active agent and the placebo, with a
significantly larger decrease for the active agent (Table 23 and
FIGS. 11A and 11B).
TABLE-US-00025 TABLE 23 Carbonylated protein contents at T0 and T28
Carbonylated proteins Placebo Active agent T0 17.6 .+-. 5.6 17.6
.+-. 5.6 T28 12.6 .+-. 7.9 8.5 .+-. 5.5 % change -28.1% -51.5%
Significance T28 - T0 Yes Yes p < 0.01 p < 0.01 Significance
Placebo vs Yes Active agent p = 0.01
[0278] 4. Summary of Biochemical Measurements
[0279] The results show an antiradical/detoxifying efficacy both on
the overall panel and on the non-smoker and smoker subpanels.
[0280] The active agent substitutes for the skin's natural
defences, SOD and CAT, thus leading to a smaller amount of residues
resulting from the detoxification process (MDA). The results for
carbonylated proteins confirm this antiradical/detoxifying
action.
[0281] 5. Summary of Efficacy
[0282] The active agent (3%) showed a significant efficacy on the
following parameters: [0283] Amount of MDA in the cheek
[0284] The active agent induced the following effects on the amount
of MDA measured from the biological sample of the cheek: [0285] For
the overall panel (30 subjects), a significant decrease of 23.9%.
The effect observed is significant relative to the effect observed
with the placebo. [0286] Amount of SOD in the cheek
[0287] The active agent induced the following effects on the amount
of SOD measured from the biological sample of the cheek: [0288] For
the overall panel (30 subjects), a significant decrease of 18.6%.
The effect observed is significant relative to the effect observed
with the placebo. [0289] Amount of CAT in the cheek
[0290] The active agent induced the following effects on the amount
of CAT measured from the biological sample of the cheek: [0291] For
the overall panel (30 subjects), a significant decrease of 18.9%.
The effect observed is significant relative to the effect observed
with the placebo. [0292] Amount of carbonylated proteins in the
cheek
[0293] The active agent induced the following effects on the amount
of carbonylated proteins measured from the biological sample of the
cheek: [0294] For the overall panel (20 subjects), a significant
decrease of 51.5%. The effect observed is significant relative to
the effect observed with the placebo. [0295] .fwdarw.All of these
results show significant effects in terms of detoxification.
Example 5: Compositions for Application Via the Topical Route
[0296] Several compositions for application via the topical route
are presented below. The polyphenolic extract of passion flower
seeds, of Example 1 or 2, can be incorporated in various cosmetic
products, such as cleansing waters, oil-in-water emulsions,
water-in-oil emulsions, oils, milks, lotions, shampoos, foaming
products and sprays, the compositions of which are presented below
by way of example.
TABLE-US-00026 SENSITIVE SKIN CLEANSING WATER Raw material/Brand
name or INCI name % CAPRYLOYL GLYCINE From 0 to 1% SODIUM HYDROXIDE
From 0 to 1% SEQUESTRANT From 0 to 1% BUTYLENE GLYCOL From 1 to 5%
BETA CAROTENE From 0 to 2% POLYPHENOLIC EXTRACT OF From 0.001 to
10% PASSION FLOWER PRESERVATIVES From 0 to 1% PEG-32 From 1 to 5%
PEG-7 PALM COCOATE From 1 to 5% ZINC GLUCONATE From 0 to 1% CITRIC
ACID From 0 to 1% PURIFIED WATER QS 100% FRAGRANCE From 0 to 1%
POLOXAMER 184 From 1 to 5%
TABLE-US-00027 ANTI-AGE EMULSION Raw material/Brand name or INCI
name % LIQUID ISOPARAFFIN From 5 to 20% ISOCETYL STEARATE From 5 to
20% AL--MG HYDROXY STEARATE From 5 to 20% ABIL WE 09 From 1 to 5%
GLYCEROL From 1 to 5% VASELINE OIL From 1 to 5% MICRONIZED ZINC
OXIDE From 1 to 5% BUTYLENE GLYCOL From 1 to 5% RETINOL From 0 to
1% VITAMIN C From 0 to 5% POLYPHENOLIC EXTRACT OF From 0.01 to 10%
PASSION FLOWER ISONONYL ISONONANOATE From 1 to 5% BEESWAX From 1 to
5% SODIUM TARTRATE From 1 to 5% SODIUM CHLORIDE From 0 to 5%
GLYCINE From 1 to 5% PRESERVATIVES From 0 to 1% CHOLESTEROL From 0
to 1% PHYTOSPHINGOSINE From 0 to 1% TARTARIC ACID From 0 to 1%
PURIFIED WATER QS 100%
* * * * *