U.S. patent application number 15/340075 was filed with the patent office on 2017-02-16 for embelia concinna extract comprising flavonoids in cosmetic and pharmaceutical compositions.
The applicant listed for this patent is BAYER CONSUMER CARE AG. Invention is credited to VIRGINIE PETIT, ERIC THERON.
Application Number | 20170042956 15/340075 |
Document ID | / |
Family ID | 48143270 |
Filed Date | 2017-02-16 |
United States Patent
Application |
20170042956 |
Kind Code |
A1 |
PETIT; VIRGINIE ; et
al. |
February 16, 2017 |
EMBELIA CONCINNA EXTRACT COMPRISING FLAVONOIDS IN COSMETIC AND
PHARMACEUTICAL COMPOSITIONS
Abstract
The present invention deals with an Embelia extract--preferably
an extract from Embelia concinna titrated in flavonoids--, its
process for preparation and its use in a cosmetical or
pharmaceutical composition for the treatment of skin sensitivity,
for the treatment of allergic reactions of the skin, for soothing
and for the treatment of itching skin and for the regulation of
inflammatory drifts in the skin
Inventors: |
PETIT; VIRGINIE; (LONS,
FR) ; THERON; ERIC; (MONTARDON, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BAYER CONSUMER CARE AG |
Basel |
|
CH |
|
|
Family ID: |
48143270 |
Appl. No.: |
15/340075 |
Filed: |
November 1, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14394268 |
Oct 14, 2014 |
9498505 |
|
|
PCT/EP2013/057477 |
Apr 10, 2013 |
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15340075 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/352 20130101;
A61P 17/04 20180101; A61K 47/10 20130101; A61P 27/02 20180101; A61P
29/00 20180101; A61K 8/345 20130101; A61K 8/9789 20170801; A61P
17/08 20180101; A61P 31/04 20180101; A61P 11/02 20180101; A61K
45/06 20130101; A61P 31/10 20180101; A61K 8/602 20130101; A61K
8/9794 20170801; A61K 2800/75 20130101; A61P 17/06 20180101; A61K
31/7048 20130101; A61Q 19/00 20130101; A61Q 19/005 20130101; A61P
15/00 20180101; A61P 17/00 20180101; A61P 37/08 20180101; A61K
8/498 20130101; A61K 2236/333 20130101; A61K 2800/805 20130101;
A61K 36/185 20130101 |
International
Class: |
A61K 36/185 20060101
A61K036/185; A61K 8/49 20060101 A61K008/49; A61K 8/60 20060101
A61K008/60; A61Q 19/00 20060101 A61Q019/00; A61K 31/7048 20060101
A61K031/7048; A61K 31/352 20060101 A61K031/352; A61K 47/10 20060101
A61K047/10; A61K 8/97 20060101 A61K008/97; A61K 8/34 20060101
A61K008/34 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 11, 2012 |
EP |
12290128.3 |
Claims
1. A process for the preparation of an Embelia leaf extract
characterized in that it is obtained by the following process
steps: a. Extraction of the leaves with a polar solvent mixture of
water and alcohol, b. removal of the alcohol, c. filtration, and d.
partial or entire removal of the solvent from the aqueous
filtrate.
2. The process according to claim 1, wherein the Embelia leaf
extract comprises flavonoids in an amount of more than 3% by weight
with respect to the total dry plant extract.
3. The process according to claim 2, wherein the Embelia leaf
extract is a liquid extract with 1% to 8% dry matter content in a
water-glycerine blend.
4. The process according to claim 2, wherein the Embelia leaf
extract comprises quercitrin, quercetin and
Kampferol-3-O-arabinoside in an amount of 0.0001%-20%, whereby
quercitrin is present in an amount of 0.01% to 10% and quercetin in
an amount of 0.001% to 1
5. The process according to claim 2, wherein the Embelia leaf
extract is an Embelia concinna leaf extract.
6. The process according to claim 3, wherein the ratio of the
volume between water and glycerine is from 25:75 up to 75:25.
7. The process according to claim 6, wherein the ratio of the
volume between water and glycerine is 50:50.
8. The process according to claim 1, further comprises a step of
drying to form a powdered extract.
9. The process according to claim 1, wherein the polar solvent
mixture has a ratio of the volume between water and alcohol from
50:50 up to 90:10.
10. The process according to claim 1, wherein the polar solvent
mixture has a ratio of the volume between water and alcohol of
70:30.
11. The process according to claim 1, wherein the alcohol is
ethanol.
Description
[0001] The present invention deals with an Embelia
extract--preferably an extract from Embelia concinna titrated in
flavonoids--, its process for preparation and its use in a
cosmetical or pharmaceutical composition for the treatment of skin
sensitivity, for the treatment of allergic reactions of the skin,
for soothing and for the treatment of itching skin and for the
regulation of inflammatory drifts in the skin.
[0002] Embelia genus belongs to the Myrsinaceae family and consists
of about 130 species including a large number of creeping or almost
climbing tropical shrubs spread in tropical and subtropical areas
from Africa to Pacific area. In Madagascar, 8 species can be found
and all are endemic. Globally Embelia species are known in folk
medicine for antihelmintic properties (Embelia ribes and Embelia
schimperi). In terms of literature and for skin application,
Embelia ribes is documented to be used for wound healing and
Embelia officinalis for skin hydration and dryness. JP 2001302528
discloses Embelia ribes for the inhibition of 5.alpha.-reductase.
In IN 200901065, Embelia ribes is also quoted among other plants
for alleviation of various skin diseases. JP 2005289922 discloses
Embelia javanica for skin bleaching thanks to anti-tyrosinase
activity.
[0003] Embelia concinna (Baker) is endemic from Madagascar where it
is called Tanterakala, Takasina or Sirahazo. This shrub becoming
lianous at maturity can be found in forests between 1200 m and 2
000 m in altitude and is in flower from October to January. Leaves
are alternate and numerous with thin petiole and elliptic or
lanceolate limb, inflorescences is paniculate with small quadramer
or pentamer flowers. In Madagascar, aerial part of Embelia concinna
is used for antihelmintic and antihaemorraghic properties and more
specifically leaves are used against ulcers, hyperuricemia and
constipation.
[0004] The presence of the phytochemical typical markers in
Myrsinaceae family--benzoquinone and saponosides--has been reported
in Embelia concinna, with respectively embelin and pentacyclic
triterpens (primulagenin A, embeliagenin). Other substances of
interest like flavonoids were also highlighted in Embelia genus:
more specifically quercetin, quercitrin, kaempferol and
kaempferol-3-O-arabinoside were qualitatively identified in Embelia
concinna (Thesis of A. Lhuillier, L'institut National de
Polytechnique de Toulouse, 2007). However, a large number of
potential actives remain unidentified in Embelia, so it is still
unclear which actives contribute to which activity and to what
extent. Furthermore, the described extraction methods are resource
intensive and obtain only small yields rendering them unsuitable
for industrial use.
##STR00001##
[0005] Flavonoids are widely distributed in plants fulfilling many
functions including producing yellow or red/blue pigmentation in
flowers and protection from attack by microbes and insects.
Flavonoids have a widespread distribution in plants (more than 4000
natural flavonoids described to date), a great variety and a low
toxicity. Flavonoids have been referred to as "nature's biological
response modifiers" because of strong experimental evidence of
their inherent ability to modify the body's reaction to allergens,
viruses, and carcinogens. Some of them show anti-allergic,
anti-inflammatory, anti-microbial and anti-cancer activity. They
are also known for their anti-oxidant potential and potential
interaction with mitochondria.
[0006] Quercetin is the most abundant of flavonoids. It is a
plant-derived flavonol that can be found in fruits, vegetables,
leaves and grains or as an ingredient in supplements, beverages or
foods. Foods rich in quercetin include black and green tea, capers,
apples, onion, red grapes, citrus fruit, tomato, broccoli and other
leafy green vegetables and a number of berries. A large number of
studies concern oral intake of quercetin or plants containing it
for various usages, e.g. anti-oxidant, anti-cancer, anti-viral,
anti-inflammation and anti-allergic as well as for the treatment or
alleviation of heart disease, metabolic syndromes or for fibralgia
treatments. Regarding allergy, publication mention activity via
mast cells synthesis regulation, like reduction of histamine or
inflammatory mediators' synthesis. For the skin, quercetin was
mentioned in literature for various properties, such as
anti-melanogenesis, anti-microbial and anti-oxidant activities,
wound healing, anti-inflammatory activity (e.g. cyclooxygenase and
lipoxygenase inhibition), UV-mediated skin damage prevention or
anti-aging. Furthermore, in WO2004037184 quercitin has been claimed
for reduction, treatment or partial prevention of reactive and
inflammatory dermatoses and in KR20110017599 for use in pore size
reduction.
[0007] Quercitrin is a glycoside formed from the flavonoid
quercetin and the deoxy sugar rhamnose, extracted from the bark of
the oak (Quercus) as a bitter citron-yellow crystalline substance
used as a pigment. It can also be found in Tartary buckwheat
(Fagopyrum tataricum), Phyllanthus urinaria, Thodendron ponticum,
Eryhtrospermum monticolum and others. Quercitrin is known to be
active in lipid peroxidation, oxidation prevention, inflammation
modulation and hapten-induced inflammation reduction, oxidative
stress protection, allergic airways disease prevention.
JP2007217396 discloses Quercitrin or extract containing it as
anti-inflammatory agent and painkiller and it is also mentioned in
cosmetic composition for anti-aging (KR20100001167, KR20090029873).
However, the isolation and purification of quercitrin is a cost
intensive process leading to additional use of resources (solvents
and energy) and to low yields.
[0008] Reactive and inflammatory dermatoses are non-contagious
disorders of the skin whose causes, when known, are usually related
to allergic or other immune reactions. These disorders may take the
form of mild irritation; however, in more severe cases, reactive
and inflammatory dermatoses may be painful and damaging conditions
severely affecting the life quality of a patient.
[0009] Examples of reactive and inflammatory dermatoses include,
without limitation, eczema, lichens, pruritic urticarial papules
and plaques of pregnancy, allergic contact dermatitis, seborrheic
dermatitis, atopic dermatitis, and psoriasis. Besides the
widespread inflammatory status and the impairment of skin
condition, pruritus is a common manifestation of these dermatologic
conditions.
[0010] In the cosmetic field, skin reactivity and inflammation are
considered to lead to skin redness, reactive and sensitive skin as
well as skin discomfort or itch sensation. Sensitive skin or
reactive skin can be considered as a condition that exhibits a
reduced tolerance to environmental stress(es) such as UV
irradiation, cold, wind exposure, dryness, (tabacco) smoke,
irritant side effects of medicaments or cosmetics or the like.
Despite an otherwise `healthy` condition, 40% of population
experience some skin discomfort sensation up to itch corresponding
to neurosensitivity as well as redness linked to inflammation
process. These phenomena are emphasized by skin dryness that
enables enhanced penetration of external irritants, and
pathogens.
[0011] The terms skin immune system (SIS) or SALT (skin associated
lymphoid tissue) have been proposed to describe the components of
the skin, excluding the regional lymph nodes, involved in skin
defence towards external aggressions and allergen contact. The SIS
contains two major components, the cellular and humoral. The
cellular component comprises keratinocytes, epidermal dendritic
cells (Langerhans cells), dermal dendritic cells, lymphocytes,
tissue macrophages, mast cells, endothelial cells, and
granulocytes. The humoral components include Immunoglobulins,
complement components, fibrinolysins, cytokines, eicosanoids,
neuropeptides, and antimicrobial peptides.
[0012] Histamine is a mediator contained in cells such as mast
cells and basophils, which is released after degranulation during
allergic reactions of IgE-mediated hypersensitivity or
non-IgE-mediated hypersensitivity. Degranulation and histamine
release can also be stimulated by non-allergenic urticating or
stinging molecules. Such histamine release is responsible for the
appearance of allergy reactions such as anaphylaxis, edema,
pruritus (itch) or hives. Histamine has long been recognized as one
of the most important mediators of inflammation, with its
properties of vasodilatation and increased vascular
permeability.
[0013] The mediators providing from the activation of phospholipase
A2, particularly prostaglandin E2 (PGE2) and leucotrien B4 (LTB4),
play a pivoting role in cutaneous inflammatory reactions. In
intradermal injection, or in topic application, LTB4 induces the
infiltration of neutrophils in the dermis and the formation of
microabscess in the epidermis. This mediator also produces a
vascular hyperpermeability, the adhesion of leucocytes to blood and
can act in synergy with PGE2. It potentiates the release of IL17 in
mouse. LTB4 also contributes to processes linked to inflammatory
skin diseases (augmentation of the vascular permeability, formation
of edema, infiltration of inflammatory cells) such as eczema,
psoriasis, atopic dermatitis or acne.
[0014] Neuropeptides are synthetized by nerves ends and comprise a
large family of regulatory molecules including tachykinins (e.g.
Substance P), calcitonin gene-related peptide (CGRP), somatostatin,
vasoactive intestinal peptide, pituitary adenylate
cyclase-activating polypeptide and propiomelanocortin-derived
peptides. Many investigations support a role of tachykinins during
immune and inflammatory reactions via the peptide binding to
tachykinin (neurokinin) receptors (NKRs) of immune cells like skin
keratinocytes, endothelial cells, mast cells, fibroblasts, Merkel
cells, and Langerhans cells.
[0015] In inflammatory skin diseases such as atopic dermatitis, an
increased number of substance-P-expressing nerve fibers have been
observed. Substance P was also found to stimulate the release of
chemokines such as interleukin-8 (IL-8), proinflammatory cytokines
such as tumor necrosis factor .alpha. (TNF.alpha.), histamine,
leukotriene B4 and prostaglandin D2. In animal models of
inflammation, Substance P was shown to modulate immediate type skin
hypersensitivity reactions and to promote the induction of contact
hypersensitivity (CHS).
[0016] Among the calcitonin peptides family, CGRP upon stimulation
is released by sensory as well as autonomic neurons and modulates
vasodilatation, plasma extravasation as well as several biological
functions of epidermal as well as dermal cells. There is evidence
that CGRP receptors are expressed on inflammatory cells such as
monocytes, macrophages, mast cells and neutrophils as well as
epidermal cells including keratinocytes, melanocytes and Langerhans
cells. The expression of CGRP in the skin was found to be
upregulated upon UV irradiation.
[0017] Upon stimulation by exogenous or endogenous trigger factors,
besides neuropeptides release, sensitive C-fibers are capable of
transporting the itch signal to the central nervous system. For
instance, a number of substances (e g amines,
prostaglandins)--after binding on the surface of chemosensitive
nerve endings--induce firing of the axons. Burning pain, heat, and
itch are transmitted through these slow-conducting unmyelinated
C-fibers. These free nerve endings of cutaneous sensory C-nerve
fibers--part of a dense network of highly specialized afferent
sensory and efferent autonomic nerve branches present in skin--are
located in the papillary dermis and epidermis.
[0018] Bidirectional crosstalk between the nervous and the immune
systems are called neurogenic inflammation: induced neuropeptides
by inflammation mediators and neuropeptide-receptor expressing
inflammatory cells participate to intensify the inflammation
cascades.
[0019] It is desirable that the mutual intensification would be
restrained at an early stage through application of a respective
compound that is capable of affecting both the nervous and/or the
immune system.
[0020] The objective of the present invention is therefore to
provide an alternative new advantageous compound, pharmaceutical
composition or cosmetic composition for the treatment or
alleviation of skin or mucous membrane diseases or disorders
related to restraining the skin immune system as well as sensory
nerve system reactions towards external stress and as a consequence
to hinder neurogenic inflammation.
[0021] The invention thus relates to an Embelia leaf extract
characterized in that it is obtained by the following process
steps:
a. Extraction of the leaves with a polar solvent mixture of water
and alcohol, b. removal of the alcohol, c. filtration d. partial or
entire removal of the solvent from the aqueous filtrate.
[0022] The invention further relates to an Embelia leaf extract
characterized in that it is a liquid extract with 1% to 8% dry
matter content in a water-glycerine blend.
[0023] The invention further relates to an Embelia leaf extract
characterized in that it comprises flavonoids in an amount of more
than 3% by weight with respect to the total dry plant extract,
preferably more than 5%, most preferably more than 7%.
[0024] According to the invention the Embelia leaf extract is an
Embelia concinna leaf extract.
[0025] In another embodiment of the invention the Embelia leaf
extract is comprised in a cosmetic composition, preferably an
Embelia concinna leaf extract is comprised.
[0026] A further embodiment of the invention is a pharmaceutical
composition comprising the Embelia leaf extract, preferably
comprising an Embelia concinna leaf extract.
[0027] In a preferred embodiment, the inventive extract or
composition comprising the extract can be used in indications in
which the extract's activity addresses neuromediators and
pro-inflammatory mediators alleviation, more specifically histamin
degranulation lessening, prostaglandins and leucotriens synthesis
reduction and substance P and CGRP synthesis reduction.
[0028] Such indications are: [0029] dermatological or topical
indications: reactive dermatoses like eczema, exanthema, lichens,
pruritic urticarial papules and plaques of pregnancy, purpura,
allergic contact dermatitis, seborrheic dermatitis, atopic
dermatitis, psoriasis and related symptoms like pruritus, oedema of
the mucous membrane, allergic rhinitis, allergic conjunctivitis,
hay fever, vaginal inflammation in association with fungal or
bacterial infections [0030] cosmetic applications: reactive and
sensitive skin, redness issue, skin discomfort or itch sensation,
skin or mucous membrane inflammation as well as skin or mucous
membrane soothing activity.
[0031] A pharmaceutical composition according to the present
invention comprises at least one active compound or drug destined
for diagnosis or therapy. A pharmaceutical composition may
influence the state or functioning of the body or affect the state
or functioning of pathogens, parasites or xenobiotics with the aim
of their removal. A pharmaceutical composition may also aim at
substituting body compounds or fluids.
[0032] The pharmaceutical composition or cosmetic composition of
the present invention can be administered in any form by any
effective route, including, e.g., oral, parenteral, enteral,
intravenous, intraperitoneal, topical, transdermal (e.g., using any
standard patch), ophthalmic, nasally, local, non-oral, such as
aerosal, inhalation, subcutaneous, intramuscular, buccal,
sublingual, rectal, vaginal, intra-arterial, and intrathecal, etc.
The person skilled in the art will easily judge on which
administration to be either suitable for the pharmaceutical
composition or for the cosmetic composition. They can be
administered alone, or in combination with any ingredient(s),
active or inactive. Preference is given to a topical
administration.
[0033] The pharmaceutical composition or cosmetic composition of
the present invention can be converted in a known manner into the
usual formulations such as pharmaceutical or cosmetic compositions
or compositions used as food supplement (i.e. according to the
concept of "beauty from within") or medical device. These may be
liquid or solid formulations e.g. without limitation normal and
enteric coated tablets, capsules, pills, powders, granules,
elixirs, tinctures, solution, suspensions, suppositories, syrups,
solid and liquid aerosols, emulsions, pastes, creams, ointments,
milks, gels, salves, serums, foams, shampoos, sticks or
lotions.
[0034] Preference is given to a pharmaceutical or cosmetic
composition in a form of an aqueous solution, a white or coloured
cream, ointment, milk, gel, salve, serum, foam, shampoo, stick,
cream, paste, or lotion.
[0035] The pharmaceutical composition or cosmetic composition of
the present invention can be further combined with any other
suitable additive or pharmaceutically or cosmetically acceptable
carrier. Such additives include any of the substances already
mentioned, as well as any of those used conventionally, such as
those described in Remington: The Science and Practice of Pharmacy
(Gennaro and Gennaro, eds, 20th edition, Lippincott Williams &
Wilkins, 2000); Theory and Practice of Industrial Pharmacy (Lachman
et al., eds., 3rd edition, Lippincott Williams & Wilkins,
1986); Encyclopedia of Pharmaceutical Technology (Swarbrick and
Boylan, eds., 2nd edition, Marcel Dekker, 2002). These can be
referred to herein as "pharmaceutically or cosmetically acceptable
carriers" to indicate they are combined with the active drug or
compound and can be administered safely to a subject for
therapeutic or cosmetic purposes.
[0036] The dosage of the pharmaceutical composition or cosmetic
composition of the present invention can be selected with reference
to the other and/or the type of disease or disorder and/or the
disease or disorder status in order to provide the desired
therapeutic or cosmetic activity. These amounts can be determined
routinely for a particular patient or person to be cosmetically
treated, where various parameters are utilized to select the
appropriate dosage (e.g., type of disease or disorder, age of
patient, disease or disorder status, patient health, weight, etc.),
or the amounts can be relatively standard and can be easily
determined by a person skilled in the art.
[0037] The amount of the administered pharmaceutical composition or
cosmetic composition can vary widely according to such
considerations as the particular compound and dosage unit employed,
the mode and time of administration, the period of treatment, the
age, sex, and general condition of the patient or person to be
treated, the nature and extent of the condition treated, the rate
of drug or compound metabolism and excretion, the potential drug
combinations and drug-drug interactions, and the like.
[0038] The pharmaceutical composition or cosmetic composition may
comprise any amount of Embelia extract. Preference is given to a
pharmaceutical composition or cosmetic composition comprising dry
or liquid extract of Embelia concinna leaves as described below.
The liquid extract may be present in an amount of from 0.01% to 5%,
preferably from 0.1 to 2.5%, more preferably from 1% to 2% by
weight of the total composition.
[0039] In another embodiment, the dry extract can be used as a
powder in an amount of 0.001 to 0.5%, preferably 0.005 to 0.1%.
[0040] A further objective of the present invention is a method of
treating skin sensitivity and skin reactivity.
[0041] Also an objective of the present invention is a method of
cosmetic treatment of disorders related to skin immune system and
skin sensory systems.
[0042] Another objective of the present invention is a method of
treating diseases or disorders, especially skin or mucous membrane
diseases or disorders, related to inflammation, neuromediators
release, neurogenic inflammation, and preferably histamine
degranulation, substance P and CGRP releases, PGE-2 and LTB-4
releases.
[0043] The pharmaceutical composition or cosmetic composition
according to the invention is administered once or more, preferably
up to three, more preferably up to two times per day. Preference is
given to a topical administration.
[0044] Nevertheless, it may in some cases be advantageous to
deviate from the amounts specified, depending on body weight,
individual behaviour toward the active ingredient, type of
pharmaceutical preparation and time or interval over which the
administration is affected. For instance, less than the
aforementioned minimum amounts may be sufficient in some cases,
while the upper limit specified has to be exceeded in other cases.
In the case of administration of relatively large amounts, it may
be advisable to divide these into several individual doses over the
day.
[0045] The pharmaceutical composition or cosmetic composition of
the present invention can also be combined with at least one
further active substance or plant extract e.g. substances or plant
extracts usually employed for pharmaceutical, dermatological or
cosmetic use.
[0046] Further active substances include but are not limited to
desquamating and/or moisturizing agents, UV filtering or blocking
agents, depigmenting or propigmenting agents, antiglycation agents,
anti-inflammatory agents, anti-microbial agents, agents stimulating
the synthesis of dermal, epidermal, hair or nail macromolecules
and/or preventing the degradation thereof, agents stimulating the
differentiation of keratinocytes, muscle relaxants, antipollution
and/or anti-free radical agents, slimming agents, agents acting on
the microcirculation, agents acting on the energy metabolism of the
cells, tightening agents, agents preventing the loss or stimulating
the growth of hair, agents preventing grey or white hair, or a
mixture thereof. Preferably that combination is contained in a
topically dermatological composition.
[0047] The pharmaceutical composition or cosmetic composition can
also contain at least one additional agent effective in skin immune
system and sensory system modulation.
[0048] In a preferred embodiment of the invention the
pharmaceutical composition or cosmetic composition can also contain
at least one additional topical anti-inflammatory agent selected
from the group of ibuprofen, acetaminophen or aspirin, capsaicin,
or cortisone.
[0049] In an embodiment of the invention the inventive extract or a
pharmaceutical or cosmetic composition comprising the same is
advantageously combined with at least one antihistaminic.
Antihistaminics according to the invention may be selected from the
group of ketotifene, thonzylamine, mepyramine, thenalidine,
tripelennamine, chlorpyramine, promethazine, tolpropamine,
dimetindene, clemastine, bamipine, loratadine, isothipendyle,
diphenhydramine, diphenhydraminmethylbromide, chlorphenoxamine,
pheniramine, diphenylpyraline, dioxopromethazine, dimenhydrinate,
thiethylperazine, meclozine, azelastine, levocabastine, astemizole,
mebhydroline, terfenadine, mequitazine, cetirizine, emedastine,
mizolastine, olopatadine, epinastine and antazoline. Especially
preferred antihistaminiks are bamipine, clemastine,
chlorphenoxamine, azelastine, terfenadine, and loratadine.
[0050] In another embodiment of the invention the inventive extract
or a pharmaceutical or cosmetic composition comprising the same is
advantageously combined with at least one glucocorticoid.
Glucocorticoids according to the invention can be selected from the
group of triamcinolone, dexamethasone, hydrocortisone,
hydrocortisonacetate, hydrocortisonbutyrate,
hydrocortisonbuteprate, prednisolone, betamethasone,
methylprednisolone, clobetasone, flumetasone, fluocortine,
fluperolone, fluorometholone, fluprednidene, desonide,
triamcinolone, alclometasone, dexamethasone, clocortolone,
betamethasone, fluclorolone, desoximetasone, fluocinolonacetonide,
fluocortolone, diflucortolone, fludroxycortide, fluocinonide,
budesonide, diflorasone, amcinonide, halometasone, mometasone,
methylprednisolonaceponate, beclometasone, hydrocortisonaceponate,
fluticasone, prednicarbate, difluprednate, ulobetasole,
clobetasole, halcinonide, medrysone, desonide, formocortale,
rimexolone, mazipredone, flunisolide and tixocortole. Especially
preferred are the glucocorticoids hydrocortisone,
beclometasone-dipropionate, and dexamethasone.
[0051] In another embodiment of the invention the inventive extract
or a pharmaceutical or cosmetic composition comprising the same is
advantageously combined with at least one mast cell granulation
inhibitor. Inhibitors of mast cell granulation, also called mast
cell stabilizers, are substances that inhibit the release of
histamins from the mast cells. Exemplary and preferred species of
this substance class are cromoglicinic acid, spagluminic acid,
nedocromile and lodoxamide.
[0052] In another embodiment of the invention the inventive extract
or a pharmaceutical or cosmetic composition comprising the same is
advantageously combined with at least one leukotrienereceptor
antagonist. Leukotriene-receptor-antagonists are substances that
inhibit the synthesis of leukotrienes and/or the interfere with the
activity of the leukotrienes. Exemplary and preferred species of
their substance class are montelukast, pranlukast, ibudilast and
zafirlukast.
[0053] In another embodiment of the invention the embelia extract
or the embelia concinna leave extract or a pharmaceutical or
cosmetic composition comprising the same is combined with at least
one further active ingredient which is selected from the group of
phyto actives or plant extracts (e.g. evening primrose oil, borage
oil, St. John's wort extract, aloe-vera extract, hamamelis extract,
calendula extract, chamomille extract), Vitamines (e.g., vitamine E
and its salts, tritinoine), dermatika (e.g. urea, heparine,
hyaluronic acid, allantoine, lactic acid and its salts,
pyroglutaminic acid, salicylic acid, tannins, coal tar solution,
bisabolole, glycyrrhetinic acid and its salts, bitumino sulfates
like e.g. Na-bitumino sulfonate or ammonium bitumino sulfonate,
antibiotics (e.g. sulfonamide, erythromycin), antiseptics (e.g.
clorhexidine or octenidine), local anaesthetics (e.g. lidocaine,
polidocanole), decongestants (e.g. sympathomimetics such as
xylometazoline), skin care substances (e.g. jojoba oil, aloe vera,
lanoline, vegetable oils and fats) and light protectants (UV-A and
-B filters, broad filters). Especially preferred is the combination
with an anti-inflammatory compound or compound mix such as e.g.
coal tar solution, Na-bitumino sulfonate, ammonium bitumino
sulfonate, tretinoine, evening primrose oil, borage oil, St. John's
wort extract, aloe-vera extract, hamamelis extract, calendula
extract, chamomille extract, vitamine E and ist salts, bisabolole,
allantoine, glycyrrhetinic acid and its salts as well as
polidocanole.
[0054] In the cosmetic field, skin reactivity and inflammation are
considered to lead to skin redness, reactive and sensitive skin as
well as skin discomfort, itch sensation, overheating, or tingling
sensations of tightness. Sensitive skin or reactive skin can be
considered as a condition that exhibits a reduced tolerance to
environmental stress(es) such as UV irradiation, temperature,
climate, air pollution or (tabacco) smoke, irritant side effects of
medicaments, cosmetics or chemicals, xenobiotics, compounds capable
of peeling, or a skin reaction induced by friction on the skin,
mucous membranes or scalp. Compounds that are able to alleviate
said conditions are called soothing.
[0055] In an embodiment of the invention, the inventive extract or
a cosmetic composition comprising the same is used as a cosmetic
soothing agent.
[0056] In another embodiment of the invention, the inventive
extract or a cosmetic composition comprising the same is used in a
method of cosmetic treatment of skin redness, reactive and
sensitive skin as well as skin discomfort, itch sensation,
overheating, or tingling sensations of tightness by applying the
same topically onto the respective skin, mucous membrane or
scalp.
[0057] According to another embodiment of the invention the
inventive extract or a pharmaceutical or cosmetic composition
comprising the same are used to alleviate the action of active
ingredients with an irritant side effect that are thus likely to
cause skin irritation, especially in persons with sensitive skin.
As active ingredients that are likely to have an irritant side
effect, the following may be cited, for example: keratolytic agents
such as .alpha.-hydroxy-acids like glycolic, lactic, malic, citric,
tartaric, mandelic acids and their derivatives;
.alpha.-hydroxy-acids like salicylic acid and its derivatives;
.alpha.-keto-acids like ascorbic acid or vitamin C and its
derivatives; retinoids like retinol and its esters, retinal,
retinoic acid and its derivatives; minoxidil and its derivatives;
lithium salts; hair tints or dyes like para-phenylenediamine
(p-PDA) and some of its derivatives such as N-phenyl p-PDA and
toluene 2,5-diamine sulfate; meta-phenylene diamine (m-PDA) and
some of its derivatives such as toluene 3,4-diamine;
ortho-phenylene diamine (o-PDA); alcoholic fragrancing solutions
(perfume, eau de toilette, after shave, deodorant); anti-perspirant
agents (certain aluminum salts); depilatory or permanent active
ingredients (thiols, ammonium hydroxide); depigmenting agents
(hydroquinone): anti-lice active ingredients; detergent (ionic and
non-ionic) agents; and their mixtures.
[0058] For the cosmetic treatment of sensitive skin, so called
soothing agents may be combined with the inventive extract or a
pharmaceutical or cosmetic composition comprising the same.
Soothing agents according to this invention are selected from the
group of bisabolol, allantoin, menthol, sucrose, centella extract,
especially glycosylated molecules, glycryrrhiza extract, farnesol,
achillea millefolium extract, ruscus extract, malva extract,
jasminum extract, rosmarinic acid and plant extract containing it,
hamamelis extract and others as known by the person skilled in the
art.
[0059] The invention further comprises a process for preparing
Embelia extracts. According to the invention these extracts are of
plants of the Embelia genus which include but are not limited to
Embelia concinna.
[0060] The extraction can be performed on all parts of the
plant(s). Preferably the leaves of a plant of the genus embelia is
extracted. In another preferred embodiment Embelia concinna are
extracted especially preferred the leaves of the Embelia concinna
are extracted.
[0061] The extraction can be done by standard extraction methods.
Preferably the extraction is carried out with a polar solvent
applicable for extraction. Leaves are first extracted with a polar
solvent, e.g. mixtures of water and alcohol, optionally several
times. The obtained solution is then mixed, the alcohol is removed
and the precipitate is removed by filtration. In another embodiment
of the invention the whole polar solvent is removed and the residue
is extracted with water. The obtained polar phase can optionally be
further extracted with a non-polar solvent e.g. ethyl acetate or
heptane to remove the waxes, essential oils, pigments and most of
the non-polar molecules. After phase separation the solvent of the
remaining polar phase is removed in order to obtain an extract--in
powder or liquid form--containing flavonoids. Optionally the
extract can be dried by adding water and conducting a freeze-drying
or adjusted by solvents added to consider a specific final medium
and specific dry matter content. A process for preparing a Embelia
leaf extract, preferably an Embelia concinna leaf extract,
comprises the steps of
a. Extraction of leaves with a polar solvent mixture of water and
alcohol, b. removal of the alcohol, c. filtration d. partial or
entire removal of the solvent from the aqueous filtrate.
[0062] The polar solvent used for extraction is preferably alcohol
or a mixture of water and alcohol wherein the alcohol is preferably
ethanol. The ratio of the volume between water and alcohol can be
from 50:50 up to 90:10, preferably 70:30.
[0063] The final step is the partial or entire removal of the
solvent from the aqueous filtrate in order to either obtain a
liquid form or a powder form. Preference is given to a liquid form
with targeted dry matter content between 1% and 10% in a solvent,
preferably 2 to 8%, most preferably 6 to 8% by weight. Solvent is
preferably a water-glycerine blend: the ratio of the volume between
water and glycerine can be from 25:75 up to 75:25, preferably
50:50.
[0064] The invention further comprises a Embelia concinna leaf
extract. The extract according to the invention can be prepared as
described above or as disclosed in Example 1.
[0065] An extract according to the invention is normally a liquid
extract. Nevertheless the extract can also be used as powder, i.e.
that the final adjustment step of the described extraction process
is omitted or further proceeded, for instance by freeze-drying or
atomisation or any method to get powder form, or is mixed with
other active compounds or being incorporated in an optimized
carrier like encapsulation.
[0066] A powder extract according to the invention can also be
redissolved to obtain a liquid extract with predetermined dry
matter content. In an embodiment of the invention the powder is
redissolved in a water-glycerine blend: the ratio of the volume
between water and glycerine can be from 25:75 up to 75:25,
preferably 50:50.
[0067] Preference is given to a liquid plant extract containing
flavonoids in an amount of more than 3% by weight with respect to
the total dry plant extract, preferably more than 5%, most
preferably between 7%. In a preferred embodiment of the invention
the dry plant extract comprises the molecules quercitrin, quercetin
and Kampferol-3-O-arabinoside in an amount of 0.0001%-20%, whereby
quercitrin may be present in an amount of 0.01% to 10%, preferably
0.05 to 8%, most preferably 2 to 5% by weight, quercetin in an
amount of 0.001% to 1%, preferably 0.005 to 0.1%.
[0068] The invention further comprises the use of the Embelia
extracts, especially Embelia concinna extracts, preferably Embelia
concinna leaf extract in the modulation of sensory system and
immune system reactions, especially in the inhibition and/or
reduction of histamine and/or substance P and/or CGRO and/or
prostaglandins and/or leucotrienes synthesis. It further comprises
the use of the Embelia plant extracts for the treatment of skin
diseases or disorders or mucous membrane diseases or disorder
related to an enhanced level of inflammatory and neuropeptides
mediators. It further comprises the use of the Embelia plant
extracts as anti-inflammatory agent.
[0069] The invention is further described by the following
examples.
[0070] FIG. 1: Evaluation by Elisa assay of histamine release after
induced basophils degranulation. Protocol: in vitro test on human
basophils (KU812 cell line), pre-treatment with Embelia extract for
20 min, stimulation with CP48/80 (10 .mu.g/ml) for 20 min, tested
vs cromoglycate (10 nM) as standard reference.
[0071] FIG. 2: Evaluation by Elisa assay of histamine release after
induced mast cells degranulation. Protocol: in vitro test on mast
murine cells, peritoneal cells from Wistar rat including .about.50%
mast cells, pre-treatment with Embelia extract for 15 min,
stimulation with CP48/80 (10 .mu.g/ml) for 20 min, tested vs
cromoglycate (10 nM) as standard reference.
[0072] FIG. 3: Evaluation by Elisa assay of substance P release.
Protocol: in vitro test on sensory neurons, sensory neurons from
dorsal root ganglia of newborn Wistar rat, pre-treatment with
Embelia extract for 30 min, stimulation with capsaicin (0.3 .mu.M)
for 30 min, tested vs capsazepine (30 .mu.M) as standard
reference.
[0073] FIG. 4: Evaluation by Elisa assay of CGRP release. Protocol:
in vitro test on sensory neurons, Sensory neurons from dorsal root
ganglia of newborn Wistar rat, pre-treatment with Embelia extract
for 30 min, stimulation with capsaicin (0.3 .mu.M) for 30 min,
tested vs capsazepine (30 .mu.M) as standard reference.
EXAMPLES
Example 1
Extraction Process
[0074] Crushed dry leaves of Embelia concinna were extracted with a
mixture of ethanol and water 70:30. The solution was stirred and
heated below 60.degree. C. during the extraction step. Extraction
step was performed twice. After plant removal by filtration,
ethanol was evaporated to obtain an aqueous solution.
Version A: Dry Extract
[0075] After settling, extract was filtered and was finally
freeze-dried to get a powder form.
[0076] The final extract was characterized by thin layer
chromatography (TLC) and spectrophotometry for flavonoids assay.
The final composition showed by thin layer chromatography
(solvents: ethyl acetate, acetic acid, formic acid, water
(100:11:11:20 V/V/V/V)) the presence of quercetin, quercitrin &
kampferol-3-O-arabinoside and a content of 7.5% of flavonoids.
Additional investigation by HPLC led to titrate quercitrin and
quercetin with respective quantity of 3.4% and 0.06%.
Version B: Liquid Extract/7% Solution
[0077] Same protocol can be proceeded with adapting the last step
to get a liquid form. After extract filtration, adjustment was
possible by adding glycerin to target a 7% extract in a
water-glycerin blend 50/50 (v/v). Test in this protocol enabled to
get a dry matter content of 7.3% extract (73 g/L).
Example 2
Cosmetic Composition
TABLE-US-00001 [0078] TABLE 1 Emulsion comprising Embelia extract
Ingredients/INCI % (w/w) A Beheneth-10 1.5% Beheneth-25 1.5%
Dycaprylyl Carbonate 5.0% Hexyl Laurate 5.0% Isohexadecane 5.0%
Cetearyl Isononanoate 5.0% Dimethicone 1.0% Behenyl Alcohol 2.0%
Hydrogenated Vegetable Glycerides 2.0% Tocopheryl Acetate 0.5%
Preservative qs B Water qsp 100 Glycerin 3.0% Xanthan gum 0.1%
Carbomer 0.2% C Embelia extract (Liquid extract according to
example 1) 2%
[0079] Ingredients A and ingredients B are heated separately to
80.degree. C. They are then mixed under vigorous stirring. The
stirring is reduced to gentle stirring while cooling to room
temperature. Ingredient C is added below 40.degree. C.
TABLE-US-00002 TABLE 2 Cream comprising Embelia extract
Ingredients/INCI % (w/w) A Water 72.12 Acrylate/C10-30 Alkyl
Acrylate Crosspolymer 0.20 Glycerin 6.00 B Xanthan gum 0.3
Cyclopentasiloxane 2.00 C Isostearyl Alcohol, Butylene Glycol
Cocoate, 4.00 Ethylcellulose Propylene Glycol Dipelargonate 3.00
Caprylic/Capric Triglyceride 3.00 Cyclopentasiloxane,
Cyclohexasiloxane 2.00 D Sodium hydroxide (10% solution) 0.63 E
Embelia extract (Liquid extract according to example 1) 0.30
[0080] Ingredients A are mixed at room temperature. The ingredients
of B and C are mixed separately at room temperature. The mixture of
B is then added to that of A and vigorously stirred for 10 min
before the mixture of C is added and further stirred for another 10
min. The mixture of D is added and as is finally E and the cream is
stirred for another 5 min.
Example 3
Inhibition of Histamine Degranulation by Basophiles
[0081] Test was performed on liquid Embelia extract according to
example 1 to measure its activity on degranulation of human
basophils (KU812 cell line) stimulated with CP48/80. Effects on
degranulation were evaluated by measuring histamine release after
stimulation. Cells were cultured at 37.degree. C., 5% CO.sub.2 and
culture medium concerned EBSS supplemented with CaCl.sub.2 1.8 mM,
MgCl.sub.2 0.8 mM and Bovine serum albumine (BSA) 0.1%.
[0082] A preliminary cytotoxicity test to select doses for further
assay was performed with viability test (MMT reduction assay and
morphological observations) after 1 hour incubation.
[0083] KU812 cells were then seeded in culture medium containing or
not (control) the test compound or the reference (cromoglycate at
10 mM) and cells were pre-incubated for 20 minutes. Cells were then
stimulated or not (non-stimulated control) with histamine release
inducer CP48/80 at 10 .mu.g/ml and incubated for 20 minutes. All
experimental conditions were performed in n=3. At the end of
incubation, the quantity of histamine in culture supernatants was
measured using an ELISA kit according to the supplier's
instructions. The inter-group comparisons were performed by
Student's t-test for data management.
[0084] Results are expressed in % according to the following
formulas:
Viability (%)=(OD sample/OD control).times.100
Inhibition ( % ) = Stimulated Control ' s Mean - Value Stimulated
Controls Mean Non - stimulated Controls Mean .times. 100
##EQU00001##
TABLE-US-00003 TABLE 3 Effect of extract on the viability of KU812
cells Embelia Extract Composition-Liquid extract according to
example 1 with 7.3% of dry matter (% calculated as to refer to dry
matter) Control 1.92E-05 0.000096 0.00048 0.0024 0.012 0.06 0.3 1.5
Viability (%) 102 102 98 98 96 94 87 92 28 33 5 100 96 94 95 92 90
85 92 24 23 98 100 90 84 85 83 83 89 33 54 Mean 100 94 92 91 89 85
91 28 36 sem 1 2 4 3 3 1 1 2 9 Morphological + + + + + + + op 10 op
observations Legend: +: normal population; +/-: growth reduction;
-: toxicity; 0: cells mortality g: grains of compound; op: opacity
of the compound; *: morphological modification; ag: agglutinated
cells sem: Standard error of the mean (standard deviation divided
by sample size square root)
TABLE-US-00004 TABLE 4 Effect of extract on histamine release from
KU 812 cells Treatment Concentration Basic data (calculated as Mean
Stimulated Normalized data Test to refer to dry Histamine Histamine
Sem Control Sem Inhibition Sem compound matter) (nM) (nM) (nM) (%)
(%) p.sup.(1) (%) (%) p.sup.(1) Non-stimulated -- 12.9 control 12.4
12.7 0.1 31 0 *** 100 0 *** 12.7 Stimulation Stimulated -- 40.7
with CP48/80 control 44.2 41.4 1.4 100 3 -- 0 5 -- at 10 .mu.g/ml
39.5 Cromoglycate 10 mM 32.3 27.0 29.5 1.6 71 4 ** 42 5 ** 29.0
Embelia 0.0024% 18.6 Extract 12.5 17.4 2.5 42 6 ** 84 9 **
according to 20.9 example 1 0.012% 21.7 18.4 22.3 2.4 54 6 ** 67 8
** 26.7 0.06% 29.8 52.4 37.6 7.4 91 18 ns 13 26 ns 30.6 Legend:
.sup.(1): Threshold for statistical significance ns: >0.05, Not
significant *: 0.01 to 0.05, Significant **: 0.001 to 0.01, Very
significant ***: <0.001 , Extremely significant
[0085] The results are visualized in FIG. 1. The treatment with
compound 48/80, tested at 10 .mu.g/ml, markedly stimulated
histamine release from KU812 basophils and cromoglycate as a
reference standard inhibitor, tested at 10 mM significantly
inhibited this effect. These expected results validated this
assay.
[0086] Under the experimental conditions of this assay, Embelia
extract according to example 1 inhibited the histamine release by
degranulation of basophils.
Example 4
Inhibition of Histamine Degranulation by Mast Cells
[0087] Test was performed with liquid Embelia extract according to
example 1 comprising 7.3% dry matter to measure its activity on
degranulation of mast cells (Peritoneal cells from Wistar rat
including .about.50% mast cells) stimulated with CP48/80. Effects
on degranulation were evaluated by measuring histamine release
after stimulation. Cells were cultured at 37.degree. C., 5%
CO.sub.2 and culture medium concerned EBSS supplemented with
CaCl.sub.2 1.8 mM, MgCl.sub.2 0.8 mM and Bovine serum albumine
(BSA) 0.1%.
[0088] Mast cells were seeded in culture medium containing or not
containing (control) the test compound or the reference
(cromoglycate at 10 mM) and cells were pre-incubated for 20
minutes. Cells were then stimulated or not stimulated
(non-stimulation control) with histamine release inducer CP48/80 at
10 .mu.g/ml and incubated for 20 minutes. All experimental
conditions were performed in n=3. At the end of incubation, the
quantity of histamine in culture supernatants was measured using an
ELISA kit according to the supplier's instructions. The inter-group
comparisons were performed by Student's t-test for data
management.
[0089] Results are expressed in % according to the following
formulas:
Inhibition ( % ) = Stimulated Control ' s Mean - Value Stimulated
Controls Mean Non - stimulated Controls Mean .times. 100
##EQU00002##
TABLE-US-00005 TABLE 5 Effect of extract on histamine release from
mast cells Concentration (calculated as Mean Stimulated Normalized
data Test to refer to dry Histamine Control Inhibition compound
matter) (.mu.M) (%) p.sup.(1) (%) p.sup.(1) Control -- 0.8 1 ***
100 *** Stimulation Control -- 58.3 100 -- 0 -- with CP48/80
Embelia 0.0001% 61.3 105 ns -5 ns at 10.mu.g/ml Extract- 0.00048%
40.0 69 ns 32 ns according to 0.0024% 33.0 57 nc 44 nc example 1
0.012% 22.4 38 ** 63 ** 0.03% 3.0 5 *** 96 *** 0.06% 0.4 1 *** 101
*** Cromoglycate 10 mM 11.7 20 *** 81 *** Legend: .sup.(1):
Threshold for statistical significance ns: >0.05, Not
significant *: 0.01 to 0.05, Significant **: 0.001 to 0.01, Very
significant ***: <0.001 , Extremely significant Nc: not
calculable
[0090] The treatment with compound 48/80, tested at 10 .mu.g/ml,
markedly stimulated histamine release from KU812 basophils and
cromoglycate, tested at 10 mM significantly inhibited this effect.
These expected results validated this assay.
[0091] Under the experimental conditions of this assay, Embelia
extract according to example 1 showed a concentration-dependent
inhibition of induced histamine release by mast cells. The results
of table 5 are visualized in FIG. 2.
Example 5
Reduction of Substance P and CGRP Release by Nerves
[0092] Test was performed with liquid Embelia extract according to
example 1 to measure its activity on release of substance P or CGRP
induced by capsaicin in sensory neurons models. Cells were isolated
from dorsal root ganglia of newborn Wistar rat. Cells were cultured
at 37.degree. C., 5% CO.sub.2 and culture medium concerned DMEM-Ham
F12 supplemented with N2 complement 1%, antibiotics, L-glutamine 2
mM, nerve growth factor beta 20 ng/ml, neutrophin 5 ng/ml.
[0093] A preliminary cytotoxicity test to select doses for further
assay was performed with viability test (MMT assay).
[0094] Sensory neurons were then seeded in culture medium
containing or not containing (control) the test compound or the
reference (capsazepine at 30 .mu.M) and cells were pre-incubated
for 30 minutes. Cells were then stimulated or not stimulated
(non-stimulation control) with capsaicin (a pungent and irritating
agent from hot chilli pepper) at 0.3 .mu.M and incubated for 30
minutes. All experimental conditions were performed in n=3. At the
end of incubation, the quantity of neuromediators (Substance P or
CGRP) in culture supernatants was measured using respective ELISA
kit according to the supplier's instructions. The inter-group
comparisons were performed by Student's t-test for data
management.
[0095] Results are expressed in % according to the following
formulas:
Viability (%)=(OD sample/OD control).times.100
Inhibition ( % ) = Stimulated Control ' s Mean - Value Stimulated
Controls Mean Non - stimulated Controls Mean .times. 100
##EQU00003##
TABLE-US-00006 TABLE 6 Effect of Embelia liquid extract on the
substance P release + Viability results Treatment Basic data
Viability Concentration % (MTT) (calculated Mean Stimulated
Normalized data % as to refer to Substance P Substance P Sem
Control Sem Inhibition Sem Stimulated Test compound dry matter)
(pg/ml) (pg/ml) (nM) (%) (%) p.sup.(1) (%) (%) p.sup.(1) Control
Non-stimulated -- 13 control 24 19 3 8 1 *** 100 1 *** 118 21
Stimulation Stimulated -- 275 with capsaicin control 244 254 11 100
4 -- 0 5 -- 100 (0.3 .mu.M) 243 Capsazepine 30 .mu.M 23 23 23 0 9 0
*** 99 0 *** 96 23 Embelia Extract- 0.0024% 141 according to 155
136 13 54 5 ** 50 5 ** 109 example 1 112 0.012% 47 60 56 5 22 2 ***
44 2 *** 110 61 0.06% 41 43 44 2 18 1 *** 89 1 *** 132 49 Legend:
.sup.(1): Threshold for statistical significance ns: >0.05, Not
significant *: 0.01 to 0.05, Significant **: 0.001 to 0.01, Very
significant ***: <0.001 , Extremely significant Nc: not
calculable
TABLE-US-00007 TABLE 7 Effect of Embelia liquid extract on CGRP
release + Viability results Treatment Viability Concentration Basic
data (MTT) (calculated as Mean Normalized data Stimulated Test to
refer to dry CGRP CGRP Sem Stimulated Sem Inhibition Sem Control
compound matter) (pg/ml) (pg/ml) (nM) Control (%) (%) p.sup.(1) (%)
(%) p.sup.(1) (%) Non- -- 105 stimulated 15 80 33 5 2 *** 100 2 ***
116 control 120 Stimulation Stimulated -- 1741 with capsaicin
control 1365 1636 137 100 8 -- 0 9 -- 100 (0.3 .mu.M) 1802
Capsazepine 30 .mu.M 671 698 642 43 39 3 ** 64 3 ** 114 557 Embelia
0.0024% 2091 Extract- 2153 1915 208 117 13 ns -18 13 ns 104
according to 1502 example 1 0.012% 2172 2186 2034 145 124 9 ns -26
9 ns 100 1745 0.06% 361 292 325 20 20 1 *** 84 1 *** 110 322
Legend: .sup.(1): Threshold for statistical significance ns:
>0.05, Not significant *: 0.01 to 0.05, Significant **: 0.001 to
0.01, Very significant ***: <0.001 , Extremely significant
[0096] The treatment with the inducer, capsaicin tested at 0.3
.mu.g, significantly stimulated both substance P and CGRP release
in neuron supernatants and capsazepine, tested at 30 .mu.M
significantly inhibited this effect. These expected results
validated this assay.
[0097] Under the experimental conditions of this assay, Embelia
extract according to example 1 inhibited significantly and
dose-dependently the substance P released by sensory neurons and
inhibited at the highest concentration tested the CGRP release. The
tests with the two lowest Embelia extract concentrations did not
yield any significant results and must be considered to be
subcritical. The results of table 6 are visualized in FIG. 3 and
those of table 7 in FIG. 4.
Example 6
Inhibition of Prostaglandin & Leucotrien Synthesis
[0098] The purpose of this study is the evaluation of the dry
Embelia extract on the production of LTB4 (leucotriens B4) and
PGE-2 (prostaglandin E-2) in human neutrophils.
[0099] Venous blood from healthy donors was collected into tubes
containing anticoagulant and sedimented with Dextran (Dextran
T500). The supernatant was layered on Ficoll-Hypaque (density
1,077) and centrifuged at 400 g for 20 min. The cell pellet was
washed in Tyrode (in mM: NaCl 137; KCl 2.6; glucose 5.6; HEPES 4.2;
pH 7.4). Contaminating red blood corpuscules were lyzed. The cells
were resuspended at 2,106 cells/ml in the Tyrode buffer
supplemented with CaCl.sub.2 and MgCl.sub.2 (1.3 and 1 mM,
respectively).
[0100] Cells were treated with ECC at 50 .mu.g/ml and 10 .mu.g/ml
during 25 minutes and cell viability was evaluated with the test of
exclusion of trypan blue.
[0101] Cells (2,106 cells/ml) were incubated at 37.degree. C. in
the presence or absence of the products to be tested during 15 min
and then stimulated with opsonised zymosan (1 mg/ml) during 10
min.
[0102] LTB4 assay was performed using a kit ELISA (Thermo Fisher
ref. EHL LTB4). The quantity of LTB4 was expressed in pg/ml. PGE2
assay was performed using a kit ELISA (Assay designs ref 900-001
and SPIBIO ref 514010). The quantity of PGE2 was expressed in
pg/ml.
[0103] Data represent the means of values measured in one
supernatant (non stimulated cells) or three supernatants
(stimulated cells). Each measurement was assessed in duplicate.
[0104] The means of the data were compared to the control condition
(Student's t test--comparison of means--statistical significance at
95% if p<0.05* and at 99% if p<0.01**).
[0105] The percentage of inhibition was calculated as follows:
% inhibition=100.times.[(LTB4 treated with ECC (stimulated)/LTB4
control (stimulated).times.100]
[0106] Embelia dry extract was not cytotoxic up to 50 .mu.g/ml.
Thus, the effects of 50 .mu.g/ml, 5 .mu.g/ml and 0.5 .mu.g/ml on
LTB4 release were investigated.
TABLE-US-00008 TABLE 8 Effect of Embelia dry extract on LTB.sub.4
release LTB.sub.4 (pg/ml) SD Control (NS) 16-ND (S) 963.3 118.9 50
.mu.g/ml (NS) 24 0 (S) >12000 5 .mu.g/ml (NS) ND-ND (S) 643.3
40.8 0.5 .mu.g/ml (NS) 16 4.9 (S) 1086.7 325.6 Metronidazole (10
.mu.g/ml) (S) 530 46.9 Legend: (NS): Not stimulated, (S):
Stimulated, *: p < 0.05, **: p < 0.01
[0107] At the concentration of 5 .mu.g/ml, Embelia dry extract
inhibited this release by 33% as compared with the untreated cells,
p<0.05. Boswellic acid (10 .mu.g/ml) and metronidazole (5
.mu.g/ml) which are the positive controls of this experiment were
inhibitory by 45% and 100% respectively.
TABLE-US-00009 TABLE 9 Effect of Embelia dry extract on PGE.sub.2
release PGE.sub.2 (pg/ml) SD Control (NS) 89 15.6 (S) 158.7 16.9 5
.mu.g/ml (NS) 76.5 19.1 (S) 112.5 12.1 2.5 .mu.g/ml (NS) 76 2.8 (S)
127.7 17.6 1 .mu.g/ml (NS) 89.1 15.5 (S) 134.3 11.5 Indomethacin 1
.mu.M (NS) 60 8.5 (S) 77.0 10.4 Legend: (NS): Not stimulated, (S):
Stimulated, *: p < 0.05, **: p < 0.01
[0108] Embelia dry extract inhibited the production of PGE2,
dose-dependently and the inhibitory effect was significant at 5
.mu.g/ml, 2.5 .mu.g/ml and 1 .mu.g/ml (% of inhibition: 29%, 20%
and 15%, respectively). PGE2 release was inhibited by 51% with
Indometacin 1 .mu.M (positive control).
* * * * *