USE OF SILYMARIN AND/OR CONSTITUENTS THEREOF AS SKIN OR
HAIR PIGMENTATION PROMOTERS
The colour of human skin depends on many factors and
especially on race and sex, but also on environmental
factors (season, exposure to sunlight, etc.); it is mainly
dependent on the nature and concentration of melanin
produced by the melanocytes. Melanocytes are specialized
cells that synthesize melanin, using particular organelles,
the melanosomes. Certain individuals naturally or
accidentally have more or less localized pigmentation
defects, requiring palliative local treatments, or demand
more general treatments, for stimulating natural
pigmentation.
Pigmentation is a natural effective protection against the
harmful effects of ultraviolet radiation and against light-
induced ageing of the skin in general. Skin pigmentation is
also a protection against the onset of skin cancers; for
the same magnitude of exposure to sunlight, dark-skinned
individuals and ethnic groups develop far fewer skin
cancers than individuals with pale skin.
Similarly, the colour of body hair and head hair is due to
melanin. At different periods in their life, especially
during ageing, certain people develop gradual
depigmentation of their head hair, with a reduction in or
even the stoppage of the melanogenesis process in the
melanocytes associated with the hair bulbs. It would be
very advantageous to be able to propose preventive or
curative treatments capable of maintaining the process of
pigmentation of the hair or of stimulating melanogenesis
and pigmentation of hair with a tendency towards greying.
2

Exposure to sunlight and UV radiation have harmful effects
on the hair, not only on the hair stem (oxidation and
bleaching) , but also, and more destructively, on the hair
bulb, which may lead to loss of the hair. The recovery or
stimulation of hair follicle pigmentation is capable of
limiting the loss of the hair or of stimulating its
regrowth. The use of harmless pro-pigmenting substances, by
topical or systemic application in compositions, and which
show good efficacy, is most particularly sought in order to
treat natural regional hypopigmentations (of genetic
origin, leukodermias such as vitiligo, and ageing) or
accidental regional hypopigmentations (post-lesional scars
or fungal mycoses), and also the stress-related loss of
hair pigmentation or loss in the course of ageing.
The use of harmless substances, by topical or systemic
application in compositions, and which protect the hair,
limit its loss and/or stimulate its regrowth, under normal
conditions, stress conditions, conditions of exposure to
sunlight and/or in the course of ageing, is also of major
interest.
The mechanism of formation of skin pigmentation is complex
and schematically involves the following main steps:
tyrosine → dopa → dopaquinone → dopachrome → melanin.
Melanin is stored in organites or melanosomes, and then
transferred to the neighbouring keratinocytes.
Each of these steps is essential to pigmentation.
Tyrosinase (monophenol dihydroxyl phenylalanine: oxygen
oxidoreductase EC 1.14.18.1) is the first enzyme involved
in this sequence of reactions. It especially catalyses the
reaction for transformation of tyrosine to dopa
(dihydroxyphenylalanine) by virtue of its hydroxylase
3

activity, and the reaction for transformation of dopa to
dopaquinone via its oxidase activity. This tyrosinase acts
only when it is in the mature state, under the action of
certain biological factors; signalling via specific
receptors such as the melanocortin receptors (MCR) is
involved for induction of the melanin synthesis process by
the melanocytes, especially the receptor MC1R.
In the epidermis, the melanocyte is involved in the
epidermal melanic unit, which comprises a melanocyte
surrounded by about 36 neighbouring keratinocytes. All
individuals, without distinction as to phototype, have
approximately the same number of melanocytes for a given
area of skin. The ethnic differences, in terms of
pigmentation, are not due to the number of melanocytes, but
to the properties of their melanosomes. The melanosomes are
aggregated as complexes and are of small size. They are
highly specialized organelles whose sole function is to
produce melanin. Gradually, as melanin is synthesized in
the melanosomes, they move from the perinuclear region to
the extremity of the melanocytes' dendrites. Via
phagocytosis, the extremity of the dendrites is captured by
the keratinocytes, and the melanosomes are redistributed in
the keratinocytes. The dendritic extensions of the
melanocytes, and the phagocytic activity of the
keratinocytes, thus play an essential role in the transfer
of melanin. Melanosome transfer is a phagocytic phenomenon
considered as standard, which involves receptors known as
the "protease-activated receptor 2" (PAR-2).
Although the level of melanin varies from one population to
another, the amount of tyrosinase does not vary
significantly and the level of tyrosinase messenger RNA is

identical in white or black skin. The variations in
melanogenesis are thus due to variations either in
tyrosinase activity or in the capacity of the keratinocytes
to phagocytose the melanosomes. This indicates that the
keratinocyte is a major player in pigmentation; 1) it is
quantitatively the major representative of the melanic
unit, and is also the agent that influences, via
information molecules (cytokines and hormones), a large
proportion of the melanogenic activity; 2) it is its
capacity for phagocytosis, combined with an adequate
presentation of the melanosomes, in a dense dendritic
network, which allows optimum distribution of melanin in
the epidermis and pigmentation.
A substance is recognized as being pro-pigmenting if it
acts directly or indirectly on activation of the melanin
synthesis process, and/or if it stimulates the melanosome
phagocytosis capacity by the keratinocytes.
Substances such as -melanotropin (-melanocyte-
stimulating hormone, -MSH) and corticotropin
(adrenocorticotropic hormone, ACTH) stimulate melanin
proliferation and synthesis by the melanocytes, via binding
to specific receptors, especially the receptor MC1-R.
Few natural inducers are currently available and used for
natural melanic pigmentation of the skin or the hair.
There is thus a need for a novel agent for promoting the
pigmentation of and/or for pigmenting human skin, body hair
and/or head hair with activity that is more effective than
the known agents, and which has a reinforced action so as
to be able to be used in low amount without any side
effects on the skin.
5

There is also a need for a novel agent for protecting the
hair, limiting its loss and/or stimulating its regrowth,
under normal conditions, stress conditions, conditions of
exposure to sunlight and/or in the course of ageing.
In this regard, the Applicants have demonstrated that
silybin and the other constituents of silymarin and silybin
derivatives and/or analogues show good pro-pigmenting
activity, even at low concentration, without showing any
cytotoxicity.
These constituents and/or derivatives also have the
advantage of acting on several major components of the
pigmentation mechanism.
They stimulate 1) melanin biosynthesis by the melanocytes,
2) the formation of a dense dendritic network in the
melanocyte, and 3) the phagocytic activity of the
keratinocytes, thus increasing both the amount of melanin
produced and the efficiency of the transfer of the
melanosomes to the neighbouring keratinocytes.
Moreover, as regards the hair, it has been demonstrated
that, 4) the invention clearly stimulates pigmentation of
the follicles (hair bulbs) and that 5) the invention limits
the degeneration and increases the survival of the
follicles.
Silymarin is a mixture of various flavonolignans derived
from taxifolin (a 2,3-dihydroflavanol) and coniferyl
alcohol.
This mixture consists mainly of silybin (or 2, 3-dihydro-3-
(4-hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-6-(3,5,7-
trihydroxy-4-oxobenzopyran-2-yl)benzodioxine), a benzo-
dioxane found in two diastereoisomeric forms (7"R,8"R and
6

7"S,8"S), silybin and isosilybin or isolated enantiomers
thereof.
The other constituents of silymarin are silydianin,
silychristin, silandrin, silymonin and taxifolin or
isolated enantiomers thereof.
Semi-synthetic or synthetic derivatives may also be used.
Pharmaceutically and/or cosmetically acceptable salts may
be used. Examples of salts that will be mentioned include
the sodium salts, the pentaacetates and the tribromides.
Glycosyl derivatives, and natural or synthetic esters or
ethers may also be used. Mention will be made, for example,
of silymarin-n-methylglycamate, esters with hemisuccinic
acid, or silymarin pentamethyl or trimethyl ether.
All these compounds are listed in Chemical Abstracts:
mention will be made, for example, of silymarin indexed
under Registry Numbers 39468-33-2, 65666-07-1, 144160-53-2,
104444-08-8, 104444-07-7, the sodium salt under
RN 66-580-75-4, the N-methyl glycamate under RN 53026-30-5,
the sodium hemisuccinate under RN 52691-96-0, the
pentaacetate, 27900-74-9, the tribromide 27359-07-5, the
pentamethyl ether 27359-05-3, and the trimethyl ether 27359-
04-2.
Taxifolin or 2-( 3,4-dihydroxyphenyl)-2 , 3-
dihydro-3,5,7-trihydroxy-4H-l-benzopyran-4-one (2R,3R)-
(9CI) is indexed under RN 480-18-2.
Silybin or 2-[(2R,3R)-2,3-dihydro-3-(4-
hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-1,4-benzodioxin-
6-yl]-2,3-dihydro-3,5,7-trihydroxy-4H-l-benzopyran-4-one,
(2R,3R)- (9CI) is indexed under RN 25888-70-6.
Isosilybin or 2-[2,3-dihydro-2-(4-hydroxy-3-
methoxyphenyl)-3-(hydroxymethyl)-1,4-benzodioxin-6-yl]-2,3-
7

dihydro-3,5,7-trihydroxy-4H-l-benzopyran-4-one,(2R,3R)-
(9CI) is indexed under RN 72581-71-6.
Silydianin or 4-[(2R,3R)-3,4-dihydro-3,5,7-
trihydroxy-4-oxo-2H-l-benzopyran-2-yl]-2,3,3a,7a-tetra-
hydro-7a-hydroxy-8-(4-hydroxy-3-methoxyphenyl)-3,6-methano-
benzofuran-7(6H)-one, (3R,3aR,6R,7aR,8R)- (9CI) is indexed
under RN 29782-68-1.
Silychristin or 2-[(2R,3S)-2,3-dihydro-7-
hydroxy-2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-5-
benzofuranyl]-2,3-dihydro-3,5,7-trihydroxy-4H-l-benzopyran-
4-one, (2R,3R)- (9CI) is indexed under RN 33889-69-9.
Silandrin or 2-[(2R,3R)-2,3-dihydro-2-(4-
hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-1,4-benzodioxin-
6-yl]-2,3-dihydro-5,7-dihydroxy-4H-l-benzopyran-4-one,
(2S)- (9CI) is indexed under RN 70815-2-6.
Silymonin or 4-[(2S)-3,4-dihydro-5,7-dihydroxy-
4-oxo-2H-l-benzopyran-2-yl]-2,3,3a,7a-tetrahydro-7a-
hydroxy-8-(4-hydroxy-3-methoxyphenyl)-3,6-methanobenzo-
furan-7(6H)-one, (3R,3aR,6R,7aR,8R)- (9CI) is indexed under
RN 70815-31-5.
Most of the constituents of silymarin exist in the form of
diastereoisomers and/or enantiomers.
These isomers may be separated via techniques known to those
skilled in the art, to be used in optically pure form as
active agent in compositions according to the invention.
Mention will be made in this respect of the publication by
David Y.W. Lee, J. Nat.Prod. 2003, 66, 1171-1174, which
describes the separation of silybin and isosilybin isomers
into silybin A, silybin B, isosilybin A and isosilybin B.
Silymarin is conventionally obtained by extraction,
especially of St Mary thistle, the compounds mentioned
9

above being the main compounds responsible for the
therapeutic action of the plant.
One subject of the present invention is the cosmetic or
dermatological use of at least one taxifolin-based compound
of natural, semi-synthetic or synthetic origin.
Structure of taxifolin
It may be an extract from plants containing this
phytochemical family, for instance the genus Silybum
(especially Silybum marianum (L.) Gaertn), of the Asteracea
family. It may be silymarin or silibinin, a mixture of
molecules initially isolated in the form of a mixture of
adducts of a phenylpropanyl alcohol, coniferyl alcohol,
with a 2,3-dihydroflavonol, taxifolin. This mixture
consists mainly of silybin (or 2,3-dihydro-3-(4-hydroxy-3-
methoxyphenyl)-2-(hydroxymethyl)-6-(3,5,7-trihydroxy-4-oxo-
benzopyran-2-yl)benzodioxine), a benzodioxane found in two
diastereoisomeric forms (7"R,8"R and 7"S,8"S). The other
constituents of silymarin are silydianin, silychristin,
silandrin, silymonin and taxifolin. All the glycosyl or
non-glycosyl precursors of silybin, its isomers and its
derivatives are of. interest for the invention. Semi-
synthetic or synthetic derivatives may also be used.
One subject of the present invention is the use for
cosmetic and dermatological preparations of at least one
source of flavonolignan in pure form or in the form of a
mixture as a pigmenting and/or colouring agent for the skin
and/or body hair and/or head hair.
9

The present invention relates to the use of glycosyl or
non-glycosyl flavonolignan derivatives of the silymarin
family, or 3-hydroxyflavones, with silybin as prototype,
but also silydianin, silychristin or isosilybin, in a
composition, especially a cosmetic composition, the said
derivatives and/or the said composition being intended to
induce pigmentation of human skin, body hair or head hair.
The invention also relates to the use of f lavonoglycan
derivatives of the silymarin family, or 3-hydroxyflavones,
with silybin as prototype, but also silydianin,
silychristin or isosilybin, for the preparation of a
composition, especially a dermatological composition, for
inducing pigmentation of human skin, body hair or head
hair.
The compounds may be identified as belonging to the family
of flavonolignans of structure:
silybin
or more generally of structure (I):

in which:
10

Rl, R2, R3, R4 and R5, which may be
identical or different, each represent
either a hydrogen atom,
or a halogen atom,
- or a nitro, (C1-C6) alkyl, (C1-C6) alkylCOOH, (C1-C6) alkyl-
COONa, trifluoro(C1-C6) alkyl, (C3-C6) cycloalkyl, acyl,
(C2-C6) alkenyl, (C2-C6) alkynyl, (C6-C18) aryl,
(C6-C18)arylCOOH, (C6-C18) arylCOONa, (C6-C18) aryl-
(C1-C4) alkyl, (C1-C6) alkyl (C6-C18) aryl, (C5-C18) heteroaryl
containing from 1 to 3 heteroatoms, CH(OH) (C6-C18) aryl,
CO (C6-C18) aryl, (CH2)nCONH- (CH2)m- (C6-C18) aryl,
(CH2)nSO2NH-(CH2)m-(C6-C18)aryl or (CH2)nCONH-CH (COOH) -
(CH2)P-(C6-C18) aryl group with n = 1 to 4, m = 0 to 3
and p = 0 to 2,
or a group 0Rx, SRX or NRxRy in which (i) Rx and Ry,
independently of each other, are chosen from a hydrogen
atom and (C1-C6) alkyl, (C3-C6) cycloalkyl, (C6-C18) aryl,
(C6-C18) aryl (C1-C4) alkyl, (C1-C12) alkyl (C6-C18) aryl,
(C3-C6) cycloalkyl (C6-C12) aryl, (C5-C12) heteroaryl
containing 1 to 3 heteroatoms, NR'R" and NHCOR'R"
groups, R' and R", independently of each other, being
chosen from a hydrogen atom and (C1-C6) alkyl,
(C3-C6) cycloalkyl and (C6-C12)aryl groups, and aromatic
or non-aromatic (C5-C12) heterocycles, containing 1 to 3
heteroatoms, or (ii) Rx and Ry together form a linear or
branched hydrocarbon-based chain containing from 2 to 6
carbon atoms, optionally comprising one or more double
bonds and/or optionally interrupted with an oxygen,
sulfur or nitrogen atom.
11

More specifically, R4 and R5 may correspond to
a phenylpropanyl alcohol, fused with the general structure
(I) in order to give rise to a flavonolignan.
The invention thus relates to their use in a
composition, as an agent for pigmenting the skin and/or
body hair and/or head hair, or as a hair loss
counteractant. They may thus be dermatological or cosmetic
compositions comprising as active agent at least one
compound of general structure (I) or a derivative or
analogue thereof or alternatively at least one plant
extract containing at least one compound of general
structure (I), the said active agent possibly being
advantageously combined in the composition with a vehicle
that is compatible with and suitable for the chosen mode of
administration.
St Mary thistle (Silybum marianum (L.) Gaertner) from the
Asteracea family is a Mediterranean plant, which also grows
in North America, South America and Australia, which is
used in traditional pharmacopoeias, especially for
combating various liver complaints.
Silymarin and/or one of the constituents thereof in pure
form or as a mixture have antioxidant properties that are
used in the treatment of various toxic complaints
(especially as an anti-hepatotoxic agent) and for promoting
cell regeneration.
Patent application WO 99/55326 discloses the use of
silymarin for restoring the level of glutathione in
mammalian cells.
EP 180 505 discloses the use of silymarin in cosmetic
preparations for retarding ageing of the skin.

WO 01/13879 discloses the cosmetic use of Silybum marianum
oils for promoting the cutaneous absorption of compositions
also comprising cynarin with a free-radical effect.
Carcinogenesis, Vol. 25, No. 8, 1459-1465,
2004, also discloses the use of silybin for protecting the
skin against damage caused by UV radiation.
The present invention thus concerns the use of silymarin,
or of one of the main constituents thereof alone or as a
mixture, chosen from silybin (or 2,3-dihydro-3-(4-hydroxy-
3-methoxyphenyl)-2-(hydroxymethyl)-6-(3,5,7-trihydroxy-4-
oxobenzopyran-2-yl)benzodioxine), isosilybin, silydianin,
silychristin, silandrin, silymonin and taxifolin or
isolated enantiomers thereof and also salts thereof, for
the manufacture of compositions for inducing, restoring or
stimulating pigmentation of the skin, body hair or head
hair.
The present invention also relates to the use of silymarin,
or one of the main constituents thereof alone or as a
mixture, chosen from silybin (or 2,3-dihydro-3-(4-hydroxy-
3-methoxyphenyl)-2-(hydroxymethyl)-6-(3,5,7-trihydroxy-4-
oxobenzopyran-2-yl)benzodioxine), isosilybin, silydianin,
silychristin, silandrin, silymonin and taxifolin or
isolated enantiomers thereof and also salts thereof, for
the manufacture of compositions for preserving the
integrity of the hair, for limiting its loss and for
stimulating hair regrowth.
In one embodiment, the silymarin constituent is
enantiomerically pure silybin or isosilybin.
In this embodiment, the constituents are chosen from silybin
A, silybin B, isosilybin A and isosilybin B, or salts
thereof, or as a mixture.
13

According to one embodiment, silymarin or one of the
constituents thereof alone or as a mixture is obtained by
extraction of a plant of the genus Silybum.
According to one embodiment, silymarin or one of the
constituents thereof alone or as a mixture is obtained by
semi-synthesis or synthesis.
The compositions will be dermatological or
cosmetic compositions comprising as active agent silymarin,
or one of the main constituents thereof alone or as a
mixture, chosen from silybin (or 2,3-dihydro-3-(4-hydroxy-
3-methoxyphenyl)-2-(hydroxymethyl)-6-(3, 5, 7-trihydroxy-4-
oxobenzopyran-2-yl)benzodioxine), isosilybin, silydianin,
silychristin, silandrin, silymonin and taxifolin, and also
salts thereof, the said active agent possibly being
advantageously combined in the composition with a vehicle
that is compatible with and suitable for the chosen mode of
administration.
When the active agent is obtained by extraction, the
production of a Silybum extract is most particularly
intended. It is more especially an extract of Silybum cells
and most specifically an extract of cells of at least one
plant of the genus Silybum of the Asteracea family. This
cell material may be obtained by in vitro or in vivo
culturing. The term "in vitro culturing" means any
technique known to those skilled in the art for
artificially obtaining a plant or part of a plant. The term
"in vivo culturing" means any culture technique for
obtaining a plant or part of a plant. Thus, the extract may
be an extract of an organ (root, stem, leaf or bark), or of
organ cells, of at least one plant of the genus Silybum of
the Asteracea family, or alternatively an extract of

undifferentiated cells of at least one such plant. These
extracts are enriched in flavonolignans in variable
proportions depending on the type of extract. These
purified extracts thus have the advantage of being free of
any problem of toxicity compared with the crude extract.
More particularly, three extraction/purification formulae
may be envisaged: (i) a total extract of the plant, (ii) an
extract intended for concentrating the taxifolin-based
flavonolignans, and finally (iii) the production of
taxifolin and pure derivatives.
Any extraction or purification method known to those
skilled in the art may be used according to the invention.
Mention may be made in particular of alcoholic (especially
methanolic or ethanolic) or aqueous extracts or extracts
using solvents such as ketones, esters, ethers, polyols or
chlorinated solvents, and mixtures of at least two of the
abovementioned solvents, for instance aqueous-alcoholic
extracts.
A compound that is particularly suitable for use in the
present invention is 2, 3-dihydro-3-(4-hydroxy-3-
methoxyphenyl)-2-(hydroxymethyl)-6-(3,5,7-trihydroxy-4-oxo-
benzopyran-2-yl)benzodioxine or silybin, which may be used,
however, as a mixture in the complex known as silymarin or
else purified, or else may even be enantiomerically pure
(use of only one isomer).
This compound has the advantage of being already used
therapeutically as a hepato-protective agent. Numerous data
concerning its harmlessness are already known and
available. Furthermore, its industrialization is also
already operational and inexpensive.
)5

The main constituents of silymarin, alone or as a mixture,
chosen from silybin (or 2,3-dihydro-3-(4-hydroxy-3-
methoxyphenyl)-2-(hydroxymethyl)-6-(3,5,7-trihydroxy-4-oxo-
benzopyran-2-yl)benzodioxine), isosilybin, silydianin,
silychristin, silandrin, silymonin and taxifolin and also
salts thereof, may be obtained by a person skilled in the
art via synthesis according to usual methods.
The present invention also relates to the cosmetic use of
silymarin, or one of the main constituents thereof alone or
as a mixture, chosen from silybin (or 2,3-dihydro-3-(4-
hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-6-(3,5,7-tri-
hydroxy-4-oxobenzopyran-2-yl)benzodioxine), isosilybin,
silydianin, silychristin, silandrin, silymonin and
taxifolin and salts thereof, in a composition, as an agent
for tanning the skin and for maintaining, inducing and/or
restoring the pigmentation of the hair, and/or as a hair-
protecting agent and/or as a hair-loss counteractant and/or
as a hair regrowth stimulant.
The invention also relates to the use of silymarin, or one
of the main constituents thereof alone or as a mixture,
chosen from silybin (or 2,3-dihydro-3-(4-hydroxy-3-
methoxyphenyl)-2-(hydroxymethyl)-6-(3,5,7-trihydroxy-4-oxo-
benzopyran-2-yl)benzodioxine), isosilybin, silydianin,
silychristin, silandrin, silymonin and taxifolin and salts
thereof, for the manufacture of a topical or systemic
dermatological composition for pigmenting the skin and/or
body hair and/or head hair via a natural biosynthetic
process, and/or as a hair-protecting agent and/or as a
hair-loss counteractant and/or as a hair regrowth
stimulant.
16

For cosmetic and dermatological use, the compositions of
the invention may be in the form of creams, gels, lotions,
milks, 0/W and W/0 emulsions, solutions, ointments, sprays,
body oils, hair lotions, shampoos, after-shave lotions,
soaps, lip-protecting sticks and makeup sticks and pencils.
In gel form, they comprise suitable excipients such as
cellulose esters or other gelling agents, such as carbopol
or guar gum.
These cosmetic and dermatological compositions may also be
in the form of a lotion or solution in which the extracts
and/or molecules are in encapsulated form, for example in
microspheres. These microspheres may consist, for example,
of fatty substances, agar and water. The active agents may
also be incorporated into vectors such as liposomes,
glycospheres, cyclodextrins, into chylomicrons, macro-,
micro- or nanoparticles and also macro-, micro- and
nanocapsules, and may also be adsorbed onto pulverulent
organic polymers, talcs, bentonites and other mineral
supports. These emulsions show good stability and may be
kept for the time required for use at temperatures of
between 0 and 50°C without any sedimentation of the
constituents or phase separation taking place.
The cosmetic compositions of the invention comprise from
about 0.01% to 10% by weight and preferentially between
0.1% and 2.5% of active agents when they are in powder form
and from about 0.01% to 2.5% and preferentially between
0.5% and 10% when they are in encapsulated form.
For the preparation of these compositions, silymarin, or
one of the main constituents thereof alone or as a mixture
chosen from silybin, (or 2,3-dihydro-3-(4-hydroxy-3-
methoxyphenyl)-2-(hydroxymethyl)-6-(3,5,7-trihydroxy-4-oxo-
17

benzopyran-2-yl)benzodioxine), isosilybin, silydianin,
silychristin, silandrin, silymonin and taxifolin, and also
salts thereof or a plant extract, are mixed with the
excipients generally used in cosmetics.
The cosmetic compositions of the invention may also contain
additives or adjuvants that are common in cosmetology, for
instance antibacterial agents or fragrances, but also
extracted and/or synthetic lipids, gelling and viscosity-
increasing polymers, surfactants and emulsifiers, water-
soluble or liposoluble active principles, plant extracts,
tissue extracts, marine extracts or synthetic active
agents.
The cosmetic compositions of the present invention may also
comprise other additional active principles chosen for
their action, for example for antisun protection, the anti-
wrinkle effect, the free-radical-scavenging and antioxidant
activity, the anti-irritant activity, cell nutrition, cell
respiration, cell hydration and regeneration, anti-
seborrhoeic treatments, and also other active principles
with action on skin tonicity or hair protection.
The cosmetic compositions of the present invention are
preferably to be used daily by applying them one or more
times a day.
The cosmetic compositions of the present invention are very
well tolerated, show no phototoxicity and their application
to the skin, for prolonged periods of time, involves no
systemic effect.
Oral application may also be envisaged. It is thus also
worthwhile discussing compositions comprising at least
silymarin, or one of the main constituents thereof alone or
as a mixture, chosen from silybin (or 2,3-dihydro-3-(4-
18

hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-6-(3,5,7-tri-
hydroxy-4-oxobenzopyran-2-yl)benzodioxine), isosilybin,
silydianin, silychristin, silandrin, silymonin and
taxifolin and also salts thereof, combined with a
pharmaceutically, cosmetically and dermatologically
acceptable vehicle or excipient.
The dermocosmetic compositions are in the form of liquid,
powder, paste or emulsion, alone or in combination with
other substances. They comprise from about 0.01% to 25% by
weight of flavonolignans, and more particularly of silybin,
of the biosynthetic precursor thereof, taxifolin and
derivatives, of silychristin or of a plant extract
containing them.
For preparations of these dermocosmetic compositions, the
extracts and/or pure compounds mentioned above, alone or as
a mixture and/or in the form of salts, are mixed with
excipients.
The examples that follow illustrate the invention without
limiting its scope.
Example 1: Demonstration of the activity on melanogenesis
in melanocyte cultures.
A biological test demonstrated the stimulatory activity of
silybin on melanin synthesis.
The melanogenesis-stimulating effect of silybin was
measured on normal human melanocyte cultures.
For silybin, the following were determined:
- after culturing for 10 days under standard conditions, in
24-well plates, in Promocell medium free of "phorbol
myristate acetate" (PMA):
the cytotoxicity, by estimating the reduction
of "methyl thiazolyl tetrazolium" (MTT), the amount of
19

proteins, by assay according to the Bradford method and
observation of the cell lawns,
the amount of melanin present in the cultures,
by spectrophotometric measurement of the melanin produced,
after alkaline extraction, relative to 100% of the control
(the control corresponds to the test performed without test
compound);
- after culturing for 3 days under standard conditions:
the effects on the proliferation of normal
human melanocytes by incorporation of tritiated thymidine
into DNA (labelling for the last 24 hours),
the effects on the metabolism of normal human
melanocytes by incorporation of tritiated leucine into
neosynthesized proteins (labelling for the last 24 hours).
The results are collated in the following tables:
Melanin synthesis by normal human melanocytes:

In all cases: concentrations in % (w/v); sd: standard
deviation; p: statistical significance.
Effects on the proliferation and on protein neosynthesis of
normal human melanocytes:
20

Proliferation (3H-thymidine), normal human melanocytes

Protein synthesis (3H-leucine), normal human melanocytes

cpm: counts per minute.
Silybin thus caused a significant increase in melanin
production in the treated human melanocyte cultures. This
stimulation is accompanied by an apparent moderate increase
in melanocyte metabolism (MTT, proteins). This effect is
not due to an increase in melanocyte proliferation
(incorporation of thymidine), on the contrary, under these
artificial conditions, the product has a tendency to limit
proliferation, without any cytotoxic activity. Moreover,
stimulation of the metabolic activity over shorter tests
(protein neosynthesis over 72 hours) confirms a very weak
stimulatory effect on melanocyte metabolism (protein
synthesis increased by only 20%).
The product did not otherwise show any effect on the
proliferation/protein synthesis of keratinocytes.
The melanogenesis-stimulating effect of silybin was
measured on B16F10-line mouse melanoma cells.
For silybin, the following were determined:
- after culturing for 7 days under standard conditions, in
24-well plates, DMEM medium containing 10% calf serum:

the cytotoxicity, by estimating the hydrolysis
of MTT, the amount of proteins, by assay according to the
Bradford method and observation of the cell lawns,
the amount of melanin present in the cultures,
by spectrophotometric measurement of the melanin produced,
after alkaline extraction, relative to 100% of the control
(the control corresponds to the test performed without test
compound).
The results are collated in the following table:
Melanin synthesis by B16F10-line melanocytes:

Silybin thus caused a very marked increase in melanin
production in the B16FlO-line melanocyte cultures (250% of
the control at 0.001%), which confirms the observed pro-
pigmenting effect, on a second cell model. In this case,
the stimulation is observed without modification of the
cell metabolism, according to MTT and protein synthesis
parameters, which confirms the specificity of the
stimulation of melanin synthesis.
The binding of silybin to the receptor MCl-R (melanocortin-
1 receptor) was studied. Structural data suggested a
possibility of binding of these compounds to the receptor
MCl-R (melanocortin-1 receptor), which is strongly involved
in melanogenesis.
22

Silybin was tested in a test of displacement of an
extremely powerful MC1-R ligand: displacement of
[125I]NDP-alpha-MSH, (according to Siegrist et al, 1988, J.
Recept. Res., 8: 323-343).
The ligand has an affinity of the order of 10-10 M. At
concentrations of 100 M (0.005%) and higher, silybin
significantly displaces this ligand (10% at 100 (M; 30% at
500 (M; higher concentrations were not tested, for reasons
of solubility), indicating a specific impact on the
receptor MC1-R. An effect of these compounds on the
inhibition of phosphodiesterases, especially of type 4:
increased cAMP, should also be noted.
Example 2: Demonstration of the activity on melanocyte
dendricity:
The aim of this test is to show the effect of the compounds
used according to the invention on the morphological
modulation of melanocytes.
Method: Normal human melanocytes are treated at the time of
inoculation with the compounds according to the invention
for 2 days, under the conditions of the melanogenesis tests
on human melanocytes, and then labelled with CFDA
(carboxyfluorescein succinimidyl ester diacetate) and
observed by fluorescence microscopy (green colouration).
Observations; see Figure 1.
Results: In the absence of the compounds according to the
invention, the melanocytes in culture are sparingly
dendritic, or even bipolar. In the presence of the
compounds according to the invention, the melanocytes show
markedly greater dendricity.
23

Example 3: Demonstration of the phagocytosis activity of
keratinocytes:
The aim of this test is to show the effect of the compounds
used according to the invention on modulation of the
phagocytosis of particles by the keratinocytes.
Method: Normal human keratinocytes are cultured in KSFM
medium and treated for 24 hours with the compounds
according to the invention, in the presence of calibrated
fluorescent beads (Molecular Probes) of the size of
melanosomes. The phagocytosis of the particles is
visualized by fluorescence microscopy, and the cells are
then harvested after trypsination and analysed by flow
cytometry, to determine the number of cells that have
phagocytozed a threshold number of particles and the
intensity of the phagocytozed overall fluorescence (10 000
cells analysed per condition, in triplicate).
Observations: The effects of silybin are reported in the
following table and Figure 2:

* cells which have phagocytozed more than twice the basal
phagocytosis
24

Results: Silybin induces a significant and reproducible
stimulation of phagocytosis of fluorescent particles that
may be likened to melanosomes; this dose-dependent
stimulation was measured by flow cytometry and by direct
observation of the cell lawns via fluorescence microscopy.
Example 4: Demonstration of the pro-pigmenting activity on
the hair: The aim of this test is to show the effect of the
compounds used according to the invention on melanin
synthesis in the follicles and their pigmentation.
Method: Normal human hair follicles are isolated by
microdissection of human scalp (lifting) and cultured
individually in vitro, in 24-well plates, according to
Philpott et al, 1990, J. Cell. Sci., 3: 463-471, with a
minimum of 15 homogeneous pigmentation hairs per condition.
The follicles are cultured for 7 days in the presence of
the compounds according to the invention. The follicles are
photographed on DO and D7 and the pigmentation is evaluated
visually.
Observations: The effects of silybin are reported in Figure
3.
Results: Silybin is not toxic to the hair and allows
lengthening of the hair stem; it induces very marked
pigmentation of the bulb and of the newly formed hair stem
compared with untreated controls. The compounds used
according to the invention thus stimulate melanin synthesis
in the follicles and their pigmentation.
Example 5: Demonstration of protection against degeneration
of the follicles (hair bulbs): The aim of this test is to
show the effect of the compounds used according to the
25

invention on degeneration of the follicles in in vitro
cultures of human hair follicles.
Method: Normal human hair follicles are isolated by
microdissection of human scalp (lifting) and cultured as in
Example 4. The bulbs are all precultured ("aged", without
treatment) for 7 days (D7), and then left untreated or
treated by the invention for a further 7 days (D14) . The
follicles are photographed and the morphology is analysed.
Observations: The effects of silybin are reported in Figure
4.
Results: After culturing for 17 days, a majority of control
follicles show signs of advanced degeneration, generally
with a bulb in budding state. This follicular degeneration
in culture is characterized by gradual budding of the bulb,
followed by expulsion. The follicles treated with silybin
have an apparently normal morphology, without visual
budding of the bulb, and display pigmentation of all of the
hair root.
Certain other taxifolin derivatives show activities
analogous to those presented above. Mention is made, for
example and in a non-limiting manner, of silydianin,
rhodiolin, silyhermin, silandrin, hydnocarpin or
silychristin.
The invention thus relates to the use of at
least one compound of formula (I):

26

in which:
Rl, R2, R3, R4 and R5, which may be
identical or different, each represent
either a hydrogen atom,
or a halogen atom,
- or a nitro, (C1-C6) alkyl, (C1-C6) alkylCOOH, (C1-C6) alkyl-
COONa, trifluoro (C1-C6) alkyl, (C3-C6) cycloalkyl, acyl,
(C2-C6)alkenyl, (C2-C6) alkynyl, (C6-C18) aryl,
(C6-C18) arylCOOH, (C6-C18) arylCOONa, (C6-C18) aryl-
(C1-C4) alkyl, (C1-C6) alkyl (C6-C18) aryl, (C5-C18) heteroaryl
containing from 1 to 3 heteroatoms, CH(OH) (C6-C18) aryl,
CO(C6-C18)aryl, (CH2)nCONH- (CH2)m- (C6-C18) aryl,
(CH2)nSO2NH-(CH2)m-(C6-C18)aryl or (CH2)nCONH-CH (COOH) -
(CH2) p- (C6-C18) aryl group with n = 1 to 4, m = 0 to 3
and p = 0 to 2,
or a group 0Rx, SRX or NRxRy in which (i) Rx and Ry,
independently of each other, are chosen from a hydrogen
atom and (C1-C6) alkyl, (C3-C6) cycloalkyl, (C6-C18) aryl,
(C6-C18) aryl (C1-C4) alkyl, (C1-C12) alkyl (C6-C18) aryl,
(C3-C6) cycloalkyl (C6-C12) aryl, (C5-C12) heteroaryl
containing 1 to 3 heteroatoms, NR'R" and NHCOR'R"
groups, R' and R", independently of each other, being
chosen from a hydrogen atom and (C1-C6) alkyl,
(C3-C6) cycloalkyl and (C6-C12)aryl groups, and aromatic
or non-aromatic (C5-C12) heterocycles, containing 1 to 3
heteroatoms, or (ii) Rx and Ry together form a linear or
branched hydrocarbon-based chain containing from 2 to 6
carbon atoms, optionally comprising one or more double
bonds and/or optionally interrupted with an oxygen,
sulfur or nitrogen atom,

and more specifically, R4 and R5 may correspond
to a phenylpropanyl alcohol, fused with the general
structure (I) to give rise to a flavonolignan,
in a cosmetic, pharmaceutical, especially
dermatological or nutritional composition for inducing,
restoring or stimulating pigmentation of the skin, body
hair or head hair.
The invention also relates to the use of at
least one pure isomer of silybin, a mixture of silybin
isomers, a derivative or analogue thereof, silymarin or one
of the constituents thereof such as silydianin,
silychristin or isosilybin, or alternatively a plant
extract containing them, especially of the genus Silybum,
for inducing, restoring or stimulating pigmentation of the
skin, body hair or head hair.
The invention relates to a composition
comprising a compound as defined above, characterized in
that the said derivative, which is synthetic or a plant
extract, is capable of modulating the MC1R and PAR-2
activities simultaneously or independently of each other.
The invention relates to a cosmetic,
dermatological, pharmaceutical or nutraceutical
composition, characterized in that it comprises a pure
silybin isomer, a mixture of silybin isomers, a derivative
or analogue thereof, silymarin, or alternatively a plant
extract containing it, especially of the genus Silybum.
The invention relates to the use of at least
one compound or extract as defined above, in a cosmetic,
pharmaceutical, especially dermatological or nutritional
composition for protecting the skin or the hair from the
harmful effects of exposure to sunlight, including light-

induced ageing of the skin, skin inflammation and erythema,
hair loss related to exposure to sunlight, skin cancers and
photo-induced pathologies.
The invention relates to the use of at least
one compound or extract as defined above, in a cosmetic,
pharmaceutical, especially dermatological or nutritional
composition for preserving the integrity of the hair, for
limiting hair loss and for stimulating hair regrowth.
The invention relates to the use of at least
one compound or extract as defined above, in a cosmetic,
pharmaceutical, especially dermatological or nutritional
composition for inducing, restoring or stimulating
pigmentation of the skin, body hair or head hair.
The invention relates to a cosmetic,
pharmaceutical or dermatological, topical or systemic
composition as defined above, characterized in that it is
combined with one or more other active agents that
reinforce the desired main effect or that induce a
complementarity of effects.
The invention relates to the use of lignans,
especially of flavonolignans, for inducing, restoring or
stimulating pigmentation of the skin, body hair or head
hair.
The present invention relates to a
pharmaceutical, dermatological or cosmetic composition
comprising as active agent at least one taxifolin
derivative or at least one plant extract containing at
least one taxifolin derivative, for inducing, restoring or
stimulating pigmentation of the skin, body hair or head
hair, or for slowing down hair loss. Advantageously, the
29

active agent (s) will be flavonolignans derived from
silymarin and more specifically from silybin.
30

We claim:
1. Use of silymarin, or of the main constituents thereof
alone or as a mixture, chosen from silybin (or 2,3-dihydro-
3-(4-hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-6-(3,5,7-
trihydroxy-4-oxobenzopyran-2-yl)benzodioxine), isosilybin,
silydianin, silychristin, silandrin, silymonin and
taxifolin, isolated enantiomers thereof and also salts
thereof, for the manufacture of compositions for inducing,
restoring or stimulating pigmentation of the skin, body
hair or head hair.
2. Use of silymarin, or of the main constituents thereof
alone or as a mixture, chosen from silybin (or 2,3-dihydro-
3-(4-hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-6-(3,5,7-
trihydroxy-4-oxobenzopyran-2-yl)benzodioxine), isosilybin,
silydianin, silychristin, silandrin, silymonin and
taxifolin, isolated enantiomers thereof and also salts
thereof, for the manufacture of compositions for preserving
the integrity of the hair, for limiting its loss and for
stimulating hair regrowth.
3. Use according to either of the preceding claims,
characterized in that silymarin or one of the constituents
thereof alone or as a mixture is obtained by extraction of
a plant of the genus Silybum.
4. Use according to any one of the preceding claims,
characterized in that silymarin or one of the constituents
thereof alone or as a mixture is obtained by semi-synthesis
or synthesis.
31

5. Use according to one of the preceding claims,
characterized in that the silymarin constituent is 2,3-
dihydro-3-(4-hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-6-
(3,5,7-trihydroxy-4-oxobenzopyran-2-yl)benzodioxine or
silybin.
6. Use according to one of the preceding claims,
characterized in that the silymarin constituent is
isosilybin.
7. Use according to one of the preceding claims,
characterized in that the silymarin constituent is
enantiomerically pure silybin or isosilybin.
8. Use according to any one of the preceding claims,
characterized in that the compositions comprise from about
0.01% to 10% by weight of active agents.
9. Use according to any one of the preceding claims,
characterized in that the compositions comprise from about
0.1% to 2.5% by weight of active agents.
10. Use according to any one of the preceding claims,
characterized in that the compositions comprise from about
0.01% to 2.5% by weight of active agents.

The present invention relates to the use of silymarin, or
of the main constituents thereof alone or as a mixture,
chosen from silybin (or 2,3-dihydro-3-(4-hydroxy-3-
methoxyphenyl)-2-(hydroxymethyl)-6-(3,5,7-trihydroxy-4-oxo-
benzopyran-2-yl)benzodioxine), isosilybin, silydianin,
silychristin, silandrin, silymonin and taxifolin, isolated
enantiomers thereof and also salts thereof, for the
manufacture of compositions for inducing, restoring or
stimulating pigmentation of the skin, body hair or head
hair. The invention also relates to the use of these agents
for the manufacture of compositions for preserving the
integrity of the hair, for limiting its loss and for
stimulating hair regrowth. Silymarin or the constituents
thereof alone or as a mixture are obtained by extraction of
a plant of the genus Silybum.