U.S. patent application number 16/353523 was filed with the patent office on 2019-12-05 for cytomimetic formulations and methods of manufacturing the same.
The applicant listed for this patent is MITANI GROUP, INC.. Invention is credited to Mario De Rosa, Mirela Mitan, Mose Rossi.
Application Number | 20190365637 16/353523 |
Document ID | / |
Family ID | 62556522 |
Filed Date | 2019-12-05 |
United States Patent
Application |
20190365637 |
Kind Code |
A1 |
Mitan; Mirela ; et
al. |
December 5, 2019 |
CYTOMIMETIC FORMULATIONS AND METHODS OF MANUFACTURING THE SAME
Abstract
A cytomimetic formulation is provided comprising at least two
of: (a) a fermented truffle extract; (b) a plurality of hyaluronic
acids of different molecular weight, ranging from 50 KDa up to 2000
KDa; (c) an olive leaf extract in a mineral-containing water; and
(d) a fermented grape must. The formulations mimic the skin
cytoplasmic environment and create optimal conditions for cellular
growth and skin rejuvenation. Methods of use and processes for
manufacturing thereof are also provided.
Inventors: |
Mitan; Mirela; (East Quogue,
NY) ; De Rosa; Mario; (Naples, IT) ; Rossi;
Mose; (Arco Felice, IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MITANI GROUP, INC. |
New York |
NY |
US |
|
|
Family ID: |
62556522 |
Appl. No.: |
16/353523 |
Filed: |
March 14, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15831596 |
Dec 5, 2017 |
10342757 |
|
|
16353523 |
|
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62436698 |
Dec 20, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 8/8111 20130101;
A61K 8/345 20130101; A61K 8/9728 20170801; A61K 8/735 20130101;
A61K 8/8147 20130101; A61K 8/25 20130101; A61K 2800/85 20130101;
A61K 8/9789 20170801; A61K 8/553 20130101; A61K 8/63 20130101; A61K
8/8152 20130101; A61Q 19/08 20130101; A61K 8/73 20130101; A61K
8/893 20130101 |
International
Class: |
A61K 8/9789 20060101
A61K008/9789; A61K 8/9728 20060101 A61K008/9728; A61K 8/73 20060101
A61K008/73; A61Q 19/08 20060101 A61Q019/08; A61K 8/34 20060101
A61K008/34; A61K 8/81 20060101 A61K008/81; A61K 8/893 20060101
A61K008/893; A61K 8/63 20060101 A61K008/63; A61K 8/55 20060101
A61K008/55; A61K 8/25 20060101 A61K008/25 |
Claims
1. A cytomimetic formulation comprising at least two of: (a) a
fermented truffle extract; (b) a plurality of hyaluronic acids of
different molecular weight, ranging from 50 KDa up to 2000 KDa; (c)
an olive leaf extract in a mineral-containing water; and (d) a
fermented grape must.
2. The cytomimetic formulation of claim 1, further comprising: (e)
a carrier suitable for topical application to human skin.
3. The cytomimetic formulation of claim 1, wherein when applied to
human skin the formulation elicits or enhances at least one of (i)
cell growth, (ii) skin rejuvenation, (iii) counteraction of one or
more features of skin aging, and (iv) skin tissue wound repair.
4. The cytomimetic formulation of claim 1, wherein the formulation
comprises fermented truffle extract and the fermented truffle
extract is obtained by a process comprising the following steps:
(a) homogenizing a truffle tuber in a physiological solution to
form a homogenate; (b) fermenting with one or more microorganisms
the homogenate to form a fermentate; (c) filtering the fermentate
to remove particulate matters to form a filtered fermentate; (e)
drying filtered fermentate by lyophilization or spray drying so as
to obtain a dry fermented truffle extract.
5. The cytomimetic formulation of claim 1, wherein the truffle
tuber is Tuber magnatum preciosa.
6. The cytomimetic formulation of claim 4, wherein the one or more
microorganism comprises Saccharomyces cerevisiae.
7. The cytomimetic formulation of claim 1, wherein the formulation
comprises the plurality of hyaluronic acids of different molecular
weight, comprises hyaluronic acids of 1800 KDa, 800 KDa and 200
KDa.
8. The cytomimetic formulation of claim 7, wherein the hyaluronic
acids of 1800 KDa, 800 KDa and 200 KDa are present, each in a
proportion of not less than 2% of the total weight of hyaluronic
acids present.
9. The cytomimetic formulation of claim 1, wherein the formulation
comprises olive leaf extract and the olive leaf extract is obtained
by a process comprising the steps of: (a) homogenizing fresh olive
leaves in mineral-containing water solution to form a homogenate;
(b) extracting the homogenate for a predetermined period of time to
form an extract; and (c) filtering the extract to remove solid
particulate matters, so as to obtain the olive leaf extract.
10. The cytomimetic formulation of claim 1 in which the mineral
water is a thermal water obtained from Thurio Spring at Spezzano
Thermal Baths, Calabria, Italy.
11. The cytomimetic formulation of claim 9, wherein the
solid/solvent ratio during extraction is 1 to 4 ratio in weight and
the extraction time is 24-72 hours at 4.degree. C. and pH 7.
12. The cytomimetic formulation of claim 1, wherein the formulation
comprises fermented grape must and the fermented grape must is
obtained by a process comprising the steps of (a) obtaining freshly
harvested grapes; (b) recovering grape juice from the grapes by
mechanical pressure; (c) fermenting the grape juice to form a
fermentate; (d) filtering the fermentate to remove particulate so
as to obtain a clear solution; and (e) drying the clear solution by
lyophilization or spray drying, so as to obtain dry fermented grape
must.
13. The cytomimetic formulation of claim 12, wherein the grapes are
Aglianic grapes from the slopes of Mount Falernus, Italy.
14. The cytomimetic formulation of claim 12, wherein the
fermentation is performed at 10-15.degree. C. for 24-48 hours.
15. The cytomimetic formulation of claim 1, which is a cosmetic
formulation.
16. The cosmetic formulation of claim 15, wherein the cosmetic
formulation comprises from 0.1 to 50% w/w of each of following: (a)
a fermented truffle extract; (b) a plurality of hyaluronic acids of
different molecular weight, ranging from 50 KDa up to 2000 KDa; (c)
an olive leaf extract in a mineral-containing water; and (d) a
fermented grape must.
17. A cytomimetic formulation as recited in claim 1 further
comprising at least one of: (a) an aqueous phase; (b) an oil phase;
(c) one or more preservatives; and (d) one or more fragrances.
18. (canceled)
19. The cosmetic formulation of claim 17, wherein the aqueous phase
comprises at least one of: (i) water, (ii) glycerin, (iii) glyceryl
polyacrylate, (iv) acrylates copolymer, (v) butylene glycol, (vi)
carbomer, and (vii) xanthan gum.
20. (canceled)
21. The cosmetic formulation of claim 17, wherein the oil phase
comprises at least one of: (i) Olea europaea fruit oil; (ii)
stearoxymethicone/dimethicone copolymer; (iii)
polymethylsilsesquioxane; (iv) polyacrylate-13; (v) di/trimethylol
hexylactone crosspolymer; (vi) polyisobutene; (vii) cholesteryl
nonanoate; (viii) hydrogenated lecithin; (ix) polysorbate 20; (x)
cholesteryl chloride; (xi) sodium acrylates copolymer; (xii)
cholesteryl oleyl carbonate; (xiii) silica; and (xiv) methyl
methacrylate crosspolymer.
22-25. (canceled)
26. The cytomimetic or cosmetic formulation of claim 1, which is a
skincare formulation, a hair product, a scalp product, or a makeup
formulation.
27-34. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Provisional
Application No. 62/436,698, filed Dec. 20, 2016, the contents of
which are hereby incorporated by reference.
FIELD OF INVENTION
[0002] The present invention generally relates to novel products
useful for skin and hair care, and a method of manufacturing the
same.
BACKGROUND
[0003] The intracellular environment in the skin plays a key role
on the quality of this tissue, affecting the vitality, mobility and
ability to repair the local cell populations. The development of
cosmeceuticals capable of mimicking the biological properties of
the skin extracellular matrix (ECM) is a strategic objective to
combat the aging processes. Success can be ascertained by restoring
functionality of the local cell populations. Due to consumer
preferences for natural or nature-based products, a cosmeceutical
with nature-based actives that can effectively combat the
appearance of skin aging processes is a desirable goal. The current
art is limited in this respect, and alternative and more effective
products are sought.
SUMMARY OF THE INVENTION
[0004] The present invention generally relates to novel products
useful for skin and hair care, and methods of manufacturing the
same. A benefit of these products is that they can effectively
combat the appearance of skin aging processes.
[0005] Herein, the inventors provide cytomimetic formulations made
using active ingredients derived and/or modified from natural
sources, such as fermented truffle extract, hyaluronic acids of
different molecular weight (Mw), olive leaf extract in thermal
water and fermented must of Falernum grapes. These cytomimetic
formulations provide the desired cosmeceutical characteristics to
combat the appearance of skin aging processes. Moreover, the
relevant actives have different properties than when used
individually, and are characterized by an unpredictable strong
synergy of action when used together.
[0006] In embodiments, a cytomimetic formulation is provided
comprising at least two of: (a) a fermented truffle extract; (b) a
plurality of hyaluronic acids of different molecular weight,
ranging from 50 KDa up to 2000 KDa; (c) an olive leaf extract in a
mineral-667149.1 containing water; and (d) a fermented grape must.
In embodiments, the cytomimetic formulation is a cosmetic
formulation. As used herein, a cytomimetic formulation is a
formulation containing cosmetically active ingredients.
[0007] In embodiments, a cosmetic formulation is provided as
recited hereinabove further comprising at least one of: (a) an
aqueous phase; (b) an oil phase; (c) one or more preservatives; and
(d) one or more fragrances.
[0008] In embodiments, a method is provided for manufacturing a
cosmeceutic comprising admixing the cytomimetic formulation as
recited hereinabove with a carrier suitable for topical
administration.
[0009] In embodiments, a method of eliciting or enhancing one or
more of cell growth, skin rejuvenation, counteraction of one or
more features of skin aging and promotion of skin tissue wound
repair, is provided comprising applying an amount of a cytomimetic
formulation as recited herein to human skin effective to elicit or
enhance one or more of cell growth, skin rejuvenation, counteract
of one or more features of skin aging, or promote skin tissue wound
repair.
[0010] In embodiments, a product is provided comprising fermented
truffle extract obtained by a process comprising the following
steps: (a) homogenizing a truffle tuber in a physiological solution
to form a homogenate; (b) fermenting with one or more
microorganisms the homogenate to form a fermentate; (c) filtering
the fermentate to remove particulate matters to form a filtered
fermentate; (e) drying filtered fermentate by lyophilization or
spray drying so as to obtain a dry fermented truffle extract.
[0011] In embodiments, a product is provided comprising olive leaf
extract obtained by a process comprising the steps of: (a)
homogenizing fresh olive leaves in mineral-containing water form a
homogenate; (b) extracting the homogenate for a predetermined
period of time to form an extract; and (c) filtering the extract to
remove solid particulate matters, so as to obtain the olive leaf
extract.
[0012] In embodiments, a product is provided comprising fermented
grape must obtained by a process comprising the steps of: (a)
obtaining freshly harvested grapes; (b) recovering grape juice from
the grapes by mechanical pressure; (c) fermenting the grape juice
to form a fermentate; (d) filtering the fermentate to remove
particulate so as to obtain a clear solution; and (e) drying the
clear solution by lyophilization or spray drying, so as to obtain
dry fermented grape must.
[0013] In embodiments, a method is provided of inducing expression
of a heat shock protein in a skin cell comprising administering a
cytomimetic formulation as described herein comprising a fermented
truffle extract in an amount effective to induce expression of a
heat shock protein in a skin cell.
BRIEF DESCRIPTION OF THE FIGURES
[0014] Exemplary embodiments of the present invention will be
described with reference to the accompanying figures, wherein:
[0015] FIG. 1: A flowchart showing an exemplary process for
producing fermented truffle extract. Suitable microorganism include
Saccharomyces cerevisiae.
[0016] FIG. 2: A flowchart showing an exemplary process for
producing Olive leaf extract.
[0017] FIG. 3: A flowchart showing an exemplary process for
producing fermented grape must.
DETAILED DESCRIPTION
[0018] The present invention generally relates to an improved
cosmetic formulation comprising at least two of various ingredients
derived from natural sources, which when combined provide a
synergistic effect on combating aging processes in skin.
Significantly, the ingredients are modified in a manner not present
in nature and/or are combined into a product which shows
synergistic effects not seen in individual components alone. These
cosmetic formulations are cytomimetic, and provide very desirable
cosmeceutical characteristics to combat the appearance of skin
aging processes.
[0019] In embodiments, a formulation of cosmetically active
components, a cytomimetic formulation, is provided comprising two
or more of: (1) a fermented truffle extract, (2) a hyaluronic acid,
(3) an olive leaf extract in a mineral-containing water, and (4) a
fermented grape must.
[0020] In embodiments, a cytomimetic formulation is provided
comprising at least two of: (a) a fermented truffle extract; (b) a
plurality of hyaluronic acids of different molecular weight,
ranging from 50 KDa up to 2000 KDa; (c) an olive leaf extract in a
mineral-containing water; and (d) a fermented grape must.
[0021] In embodiments, a cytomimetic formulation is provided
comprising: (a) a fermented truffle extract comprising 1-30% total
weight of active ingredients; (b) a plurality of hyaluronic acids
of different molecular weight, ranging from 50 KDa up to 2000 Kda,
comprising 20-60% of total weight of active ingredients; (c) an
olive leaf extract in a mineral-containing water, comprising 10-30%
total weight of active ingredients; and (d) a fermented grape must,
comprising 10-60% total weight of active ingredients.
[0022] In embodiments, a cosmetic formulation is provided
comprising: (a) a fermented truffle extract comprising 1-30% total
weight of the cosmetic formulation; (b) a plurality of hyaluronic
acids of different molecular weight, ranging from 50 KDa up to 2000
Kda, comprising 20-60% of total weight of the cosmetic formulation;
(c) an olive leaf extract in a mineral-containing water, comprising
10-30% total weight of the cosmetic formulation; and (d) a
fermented grape must, comprising 10-60% total weight of the
cosmetic formulation.
[0023] In embodiments, the cytomimetic formulation further
comprises (e) a carrier suitable for topical application to human
skin. In embodiments, the cytomimetic formulation further
comprising (e) a carrier suitable for topical application to human
skin is a cosmetic formulation. Examples of carriers suitable for
topical administration include, creams, ointments, pastes, gels,
solutions, lotions, suspension concentrates, suspoemulsions
niosomes, liposomes, microemulsions, and lipospheres, to name a
few.
[0024] In embodiments, the cosmetic formulation, when applied to
human skin, elicits or enhances at least one of: (i) cell growth,
(ii) skin rejuvenation, (iii) counteraction of one or more features
of skin aging, and (iv) skin tissue wound repair.
[0025] In embodiments, the cosmetic formulation when applied to
human skin elicits or enhances skin rejuvenation in reducing
scaliness (and/or improves skin hydration) versus skin not treated
with the actives.
[0026] In embodiments, the cosmetic formulation when applied to
human skin elicits or enhances skin rejuvenation by decreasing
sebum secretion of oily skin relative to untreated oily skin.
[0027] In embodiments, the cosmetic formulation when applied to
human skin elicits or enhances skin rejuvenation by increasing
sebum secretion of dry skin relative to untreated dry skin.
[0028] In embodiments, the cosmetic formulation when applied to
human skin elicits or enhances counteraction of one or more
features of skin aging by improving skin elasticity relative to
untreated skin.
[0029] In embodiments, the cosmetic formulation when applied to
human skin elicits or enhances counteraction of one or more
features of skin aging by reducing skin wrinkles relative to
untreated skin. In embodiments, the cosmetic formulation when
applied to human skin elicits or enhances counteraction of one or
more features of skin aging by reducing roughness of the skin
relative to untreated skin.
[0030] In embodiments, the cosmetic formulation when applied to
human skin elicits or enhances skin tissue wound repair by reducing
the skin wound repair time relative to untreated skin.
[0031] In embodiments, the cytomimetic formulation comprises
fermented truffle extract and the fermented truffle extract is
obtained by a process comprising the following steps: (a)
homogenizing a truffle tuber in a physiological solution to form a
homogenate; (b) fermenting with one or more microorganisms the
homogenate to form a fermentate; (c) filtering the fermentate to
remove particulate matters to form a filtered fermentate; (e)
drying filtered fermentate by lyophilization or spray drying so as
to obtain a dry fermented truffle extract.
[0032] In embodiments, the truffle tuber is homogenized in 0.1M
sodium phosphate buffer, at pH 5-7 for 1 h, and at 3000-6000
rpm.
[0033] In embodiments, the homogenate is fermented for about 24 h
at a temperature of 25-30.degree. C., and at a pH 5-7, with a flux
of sterile air of 1.5-2.5 L/min. In embodiments, the fermentate is
filtered through a 1 micron filter, then followed by 0.45 micron
filter, then followed by a 0.22 micron filter, so as to produce the
filtered fermentate.
[0034] In embodiments, the truffle tuber is a Tuber magnatum. In
embodiments, the truffle tuber is a Tuber magnatum preciosa.
[0035] In embodiments, the one or more microorganism comprises
Saccharomyces cerevisiae.
[0036] In embodiments, the cytomimetic formulation comprises the
plurality of hyaluronic acids of different molecular weight,
comprises hyaluronic acids of 1800 KDa, 800 KDa and 200 KDa.
[0037] In embodiments, the hyaluronic acids of 1800 KDa, 800 KDa
and 200 KDa are present, each in a proportion of not less than 2%
of the total weight of hyaluronic acids present.
[0038] In embodiments, the hyaluronic acids of 1800 KDa, 800 KDa
and 200 KDa are present at a 33.3% by weight relative ratio of the
total weight of hyaluronic acids present.
[0039] In embodiments, the cytomimetic formulation comprises olive
leaf extract and the olive leaf extract is obtained by a process
comprising the steps of: (a) homogenizing fresh olive leaves in
mineral-containing water solution to form a homogenate; (b)
extracting the homogenate for a predetermined period of time to
form an extract; and (c) filtering the extract to remove solid
particulate matters, so as to obtain the olive leaf extract.
[0040] In embodiments, the mineral water is a thermal water
obtained from Thurio Spring at Spezzano Thermal Baths, Calabria,
Italy.
[0041] In embodiments, the solid/solvent ratio during extraction is
1 to 4 ratio in weight and the extraction time is 24-72 hours at
4.degree. C. and pH 7.
[0042] In embodiments, the solid/solvent ratio during extraction is
1 to 4 ratio in weight and the extraction time is 24 hours at
4.degree. C. and pH 7.
[0043] In embodiments, the cytomimetic formulation comprises
fermented grape must and the fermented grape must is obtained by a
process comprising the steps of: (a) obtaining freshly harvested
grapes; (b) recovering grape juice from the grapes by mechanical
pressure; (c) fermenting the grape juice to form a fermentate; (d)
filtering the fermentate to remove particulate so as to obtain a
clear solution; and (e) drying the clear solution by lyophilization
or spray drying, so as to obtain dry fermented grape must.
[0044] In embodiments, the grapes are Aglianic grapes from the
slopes of Mount Falernus, Italy.
[0045] In embodiments, the fermentation is performed at
10-15.degree. C. for 24-48 hours. In embodiments, the fermentation
is performed at 10-15.degree. C. for 24 hours.
[0046] In embodiments, the cytomimetic formulation is a cosmetic
formulation. In embodiments, the cosmetic formulation comprises
from 0.1 to 50% w/w of the cytomimetic formulation.
[0047] In embodiments, a cosmetic formulation is provided as
recited hereinabove further comprising at least one of: (a) an
aqueous phase; (b) an oil phase; (c) one or more preservatives; and
(d) one or more fragrances.
[0048] In embodiments, the cosmetic formulation comprises an
aqueous phase. In embodiments, the aqueous phase comprises at least
one of: (i) water, (ii) glycerin, (iii) glyceryl polyacrylate, (iv)
acrylates copolymer, (v) butylene glycol, (vi) carbomer, and (vii)
xanthan gum.
[0049] In embodiments, the cosmetic formulation comprises an oil
phase. In embodiments, the oil phase comprises at least one of: (i)
Olea europaea fruit oil; (ii) stearoxymethicone/dimethicone
copolymer; (iii) polymethylsilsesquioxane; (iv) polyacrylate-13;
(v) HDI/trimethylol hexylactone crosspolymer; (vi) polyisobutene;
(vii) cholesteryl nonanoate; (viii) hydrogenated lecithin; (ix)
polysorbate 20; (x) cholesteryl chloride; (xi) sodium acrylates
copolymer; (xii) cholesteryl oleyl carbonate; (xiii) silica; and
(xiv) methyl methacrylate crosspolymer.
[0050] In embodiments, the cosmetic formulation comprises one or
more preservatives. In embodiments, the one or more preservatives
comprises at least one of: (a) phenoxyethanol; and (b)
ethylhexylglycerin.
[0051] In embodiments, the cosmetic formulation comprises one or
more fragrances.
[0052] In embodiments, the cosmetic formulation comprises each of:
(a) an aqueous phase; (b) an oil phase; (c) one or more
preservatives; and (d) one or more fragrances.
[0053] In embodiments, the cosmetic formulation is a skincare
formulation, a hair product, a scalp product, or a makeup
formulation.
[0054] In embodiments, a method is provided for manufacturing a
cosmeceutic comprising admixing the cytomimetic formulation as
recited hereinabove with a carrier suitable for topical
administration.
[0055] In embodiments, a method of eliciting or enhancing one or
more of cell growth, skin rejuvenation, counteraction of one or
more features of skin aging and promotion of skin tissue wound
repair is provided comprising applying an amount of a cytomimetic
formulation as recited herein to human skin effective to elicit or
enhance one or more of cell growth, skin rejuvenation, counteract
of one or more features of skin aging, or promote skin tissue wound
repair.
[0056] In embodiments, a product is provided comprising fermented
truffle extract obtained by a process comprising the following
steps: (a) homogenizing a truffle tuber in a physiological solution
to form a homogenate; (b) fermenting with one or more
microorganisms the homogenate to form a fermentate; (c) filtering
the fermentate to remove particulate matters to form a filtered
fermentate; (e) drying filtered fermentate by lyophilization or
spray drying so as to obtain a dry fermented truffle extract.
[0057] In embodiments, a product is provided comprising olive leaf
extract obtained by a process comprising the steps of: (a)
homogenizing fresh olive leaves in mineral-containing water form a
homogenate; (b) extracting the homogenate for a predetermined
period of time to form an extract; and (c) filtering the extract to
remove solid particulate matters, so as to obtain the olive leaf
extract.
[0058] In embodiments, a product is provided comprising fermented
grape must obtained by a process comprising the steps of: (a)
obtaining freshly harvested grapes; (b) recovering grape juice from
the grapes by mechanical pressure; (c) fermenting the grape juice
to form a fermentate; (d) filtering the fermentate to remove
particulate so as to obtain a clear solution; and (e) drying the
clear solution by lyophilization or spray drying, so as to obtain
dry fermented grape must.
[0059] In embodiments, the cosmetic formulation is in the form of
one of the following: a cream, a lotion, a gel, an ointment, a
macro-emulsion, a micro-emulsion, a nano-emulsion, a serum, a
solution, a balm, a patch, a microneedle patch, a skin delivery
enhancing system, or a mask.
[0060] In embodiments, cosmetic formulations may include those for
skin (including, in embodiments, day-cream, night cream, anti-aging
product, skin rejuvenating product, skin conditioner, moisturizer,
sun protecting gel, sun protecting cream, brightening cream,
after-sun product, mask, body lotion, shower gel, and soap).
[0061] In embodiments, cosmetic formulations may include those for
skin hair or scalp (including, in embodiments, mask, conditioner,
shampoo, and lotion).
[0062] In embodiments, cosmetic formulations may include those for
makeup (lipstick, foundations, lip gloss, lip balm, rouge).
[0063] In embodiments, the formulations can be suitable for
treatment of healthy, young, old, aged, damaged, photo-damaged,
wrinkled, irritated, acne, age spotted, or stretch marked skin.
[0064] In embodiments, the formulation may be suitable for
treatment of skin previously treated with cosmetic products, or
treated with cosmetic procedures.
[0065] In embodiments, the formulations may be suitable for
treatment of healthy, damaged, gray, or dyed hair.
[0066] In embodiments, the formulations can be suitable for
treatment of healthy or damaged scalp.
[0067] In embodiments, a cytomimetic formulation is provided
comprising one or more of a fermented truffle extract, a hyaluronic
acid, an olive leaf extract in a mineral-containing water, and a
fermented grape must.
[0068] In embodiments, a method is provided for manufacturing a
cosmeceutic comprising admixing the cytomimetic formulation as
described herein with a carrier suitable for topical
administration.
[0069] In embodiments, a method is provided for eliciting or
enhancing one or more of cell growth, skin rejuvenation,
counteraction of one or more features of skin aging and promotion
of skin tissue wound repair, comprising applying an amount of a
formulation as recited herein to human skin effective to elicit or
enhance one or more of cell growth, skin rejuvenation, counteract
of one or more features of skin aging, or promote skin tissue wound
repair.
[0070] In embodiments, a method is provided for eliciting or
enhancing one or more of cell growth, skin rejuvenation,
counteraction of one or more features of skin aging and promotion
of skin tissue wound repair, comprising applying an amount of the
formulation as recited herein to human skin effective to elicit or
enhance one or more of cell growth, skin rejuvenation, counteract
of one or more features of skin aging, or promote skin tissue wound
repair.
[0071] In embodiments, a product is provided comprising fermented
truffle extract obtained by a process comprising: a) homogenizing a
truffle tuber in a physiological solution; b) subjecting the
homogenate to a fermentation with one or more microorganisms; c)
removing particulate by filtration of fermentate; drying filtered
fermentate solution by lyophilization or spray drying so as to
obtain a dry fermented truffle extract.
[0072] In embodiments, a product is provided comprising olive leaf
extract obtained by a process comprising: a) homogenizing fresh
olive leaves in mineral-containing water; b) permitting the
homogenate to extract; c) removing solid particulate by filtration,
so as to obtain the olive leaf extract.
[0073] In embodiments, a product is provided comprising fermented
grape must obtained by a process comprising a) obtaining freshly
harvested grapes; b) recovering grape juice from the grapes by
mechanical pressure; c) fermenting grape juice from the grapes; d)
removing particulate of the fermentate by filtration so as to
obtain a clear solution; and e) drying the clear solution by
lyophilization or spray drying, so as to obtain dry fermented grape
must.
[0074] In embodiments, a method is provided of inducing expression
of a heat shock protein in a skin cell comprising administering a
cytomimetic formulation as described herein comprising a fermented
truffle extract in an amount effective to induce expression of a
heat shock protein in a skin cell. In embodiments, the heat shock
protein is HSP 70 or HSP 90. In embodiments, administering the
cytomimetic formulation induces expression of both a HSP 70 and a
HSP 90 in a skin cell.
[0075] All combinations of the various elements described herein
are within the scope of the invention unless otherwise indicated
herein or otherwise clearly contradicted by context.
[0076] Where a numerical range is provided herein, it is understood
that all numerical subsets of that range, and all the individual
integers contained therein, are provided as part of the invention.
Thus, a molecule which is 200 KDa to 800 KDa includes, unless
otherwise stated, molecules of 201 KDa, 202 KDa, 203 KDa etc. Thus,
a percentage range of 10-30% includes, unless otherwise stated,
percentage ranges of 10-25%, 12-30%, etc. as well as the individual
percentages of 11%, 15%, 20% etc.
[0077] This invention will be better understood from the
Experimental Details, which follow. However, one skilled in the art
will readily appreciate that the specific methods and results
discussed are merely illustrative of the invention as described
more fully in the claims that follow thereafter.
[0078] Active Ingredients
[0079] The development of cosmeceuticals capable of mimicking the
biological properties of the extracellular matrix (ECM) present in
the skin tissue is a strategic objective to counteract aging and
stress effects by restoring the optimal functionality of the
resident cell populations.
[0080] Cytomimetic technology, in the formulations disclosed
herein, mimics healthy, vital parts of the skin tissue environment
where skin cells thrive. Using this mimicry it effectively infuses
the skin tissue (ECM) with special, luxurious ingredients to
balance, tone and beautify. This proprietary technology enhances
biological activity by mimicking parts of the macromolecular
universe that exist in our skin.
[0081] Cytomimetic formulations, that employ the actives of
fermented truffle extracts, HA of different Mw, olive leaf extract
in thermal water, Falernum fermented grapes, have surprisingly be
shown by the inventors to possess a strong synergism amongst
themselves. The effectiveness of these naturally-based active
ingredients are enhanced when used together, by mimicking the
complex actions of bio-stimulation, signaling, repair and
protection of their own fully functional ECM. Below, the basis of
each individual actives component is discussed.
[0082] Truffle Fermented Extract--
[0083] Truffles have a number of biological activities, such as
antioxidant, antiviral, anti-microbial, hepatoprotective,
anti-mutagenic, anti-inflammatory, anti-carcinogenic, and are
anti-tuberculoid. (N. Beara et al./Food Chemistry 165 (2014)
460-466). One of the most promising truffles, from a biochemical
point of view, is Tuber magnatum preciosa. It is native to Italy,
namely the Piedmont, Tuscany and Emilia Romagna regions. The
effects of truffle oil aroma are confirmed by the ability of some
scent molecules to interact with receptors and trigger some
significant signals in human cells. The challenge has been trying
achieve the relevant effects on the skin for cosmetic purposes.
Results herein demonstrate that fermented truffle extracts contain
one or more substances, that with a synergistic action, induce the
heat shock response, eliciting quantifiable beneficial effects on
cells.
[0084] Surprisingly the inventors have found that by subjecting it
to a biotransformation/fermentation process, truffle homogenate
shows improved biological properties, as empirically demonstrated
by in vitro keratinocytes and fibroblasts scratch assay with time
lapse microscopy (TLVM) (see Table 1) and by the appearance of new
activities that are not present in the simple aqueous extract, such
as the property of stimulating a response to shock in the cell
culture.
[0085] The basis of the preservation of the chemical architecture
and of the functional properties of a cell organism under stressful
conditions is called homeostasis. A feature of homeostasis is the
rapid reaction to stressful conditions by expressing genes, whose
products and heat shock proteins are specifically dedicated to
function against the stress, while also protecting the cellular
components (Lindquist S., 1986, Ann Rev Biochem, 55, 1151-1191;
Morimoto, R. I. 1993, Science, 259: 1409-1410; Morimoto, R I., et
al., 1990, In Stress proteins in biology and medicine pp. 1-36.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
[0086] The response to shocks and, in particular, the heat shock
response (HSR), is an ancient and highly conserved cytoprotective
mechanism and the heat shock proteins (HSPs) are among the most
abundant proteins in the biosphere. The fact that they show such
remarkable evolutionary conservation, suggests they are essential
for basic cellular function.
[0087] A number of studies have shown that the expression of these
proteins has a close relationship with life history, exerting
influence on biological phenomena such as stress resistance
(Morimoto, R. I. and Santoro M. G., 1998, Nature Biotech,
16:833-838.), aging and longevity (Tao D, Lu J, et al. 2004, Acta
Biochim Biophys Sin (Shanghai), 36:618-22; Calderwood S K, et al.,
2009, Gerontology; 55:550-558).
[0088] The heat shock response mechanism for cell protection
against stress results in the down-regulation of many genes and the
activation of others, whose main function is to help the cell
survive. The production of heat shock proteins, including protein
chaperones, is essential for the folding, repair and recovery of
damaged proteins, since it promotes cellular viability under
conditions that would otherwise induce cells death (apoptosis).
[0089] During the aging process the cell reduces its capacity to
synthesize the proper amounts of heat shock proteins under
stressful conditions. It is this impairment that may be one of the
main factors that contributes to a reduced capacity to maintain
homeostasis, and is thus a culprit of damage to the cells.
[0090] Among the heat shock proteins, chaperonins are one of the
most important classes, since they prevent the incorrect
association within and between polypeptide chains during the
folding of newly synthesized proteins, while also protecting the
pre-existing proteins under cellular stresses. Chaperonins also
function in the absence of stress, namely under normal
physiological conditions, helping cellular proteins to fold
correctly during synthesis on the ribosome. HSP 70 is one of the
most studied chaperonins.
[0091] It is this mechanism that allows the cell to react to
external stress. There is strong consideration that the cell
membrane bilayer has fluidity properties that permit sensing of
changes in temperature, pH, osmotic and atmospheric pressure, etc.
In this respect the cellular membrane may be considered the key
sensor of the cell in regards to external stress factors. In fact,
following a temperature change, cells compensate for stress induced
disturbances, through physiological and biochemical mechanisms of
homeoviscous adaptation (Vigh, L., et al., 1998, TIBS 40323:
369-374).
[0092] In addition, there is evidence that membrane lipids may
participate as molecular chaperones in the folding and possibly in
the unfolding of integral membrane proteins. Furthermore, the
modification of membrane's physical state influences the expression
of heat-shock genes, simulating a heat shock condition. Such an
outcome can be caused by some pathological conditions or by the
interaction of the membrane with several molecules.
[0093] With particular regard to the skin cells (Maytin, E V.,
1995, J. Invest. Dermatol 104:448-455; Edwards M J, et al., 1991, J
Invest Dermatol 96:392-6; Trautinger F, et al., 1995, Br J
Dermatol, 133:194-202; Roh B H, 2008, Ann Dermatol 20:184-9) both
dermis and epidermis, expression of HSPs confers resistance to
damage caused by stressors, for instance, sunlight exposure. Maytin
(Maytin, E V., 1995, J. Invest. Dermatol, 104:448-455) has reported
that heat shock proteins play a general role in the protection of
the skin from environmental stressors, but also participate in the
prevention and repair of the damages caused by exposure to light,
heat, chemical injuries, and other traumas.
[0094] An important feature of heat shock proteins is their role in
the cytoprotection and repair of cells and tissues against the
deleterious effects of stress and trauma. Overexpression of one or
more heat shock protein genes is sufficient to protect against
otherwise lethal exposures to heat, cytotoxic drugs, toxins, and
tumor necrosis factor-.alpha. (Parsell, D. A. and Lindquist, S.,
1994, in The biology of heat shock proteins and molecular
chaperones. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.).
[0095] Yeast cells, engineered to overexpress HSP 70 or HSP 104
cross-protect against lethal heat shock, H.sub.2O.sub.2 heavy
metals, arsenite, anoxia, and ethanol toxicity. In vertebrates,
modulation of the heat shock response or the expression of specific
heat shock proteins can either limit or prevent the pathology
associated with certain chronic diseases.
[0096] It is desirable, from a practical point of view, to obtain
naturally derived compounds able to induce the stress response in
the cells in the absence of stress. These compounds could create a
preventive/defensive strategy in the cells, by mimicking the
effects of stress, in turn helping the cells throughout the aging
process.
[0097] In embodiments, an objective of the present invention is to
develop new classes of molecules able to mimic or activate some
fundamental process of life. Attention has been spent on repair
mechanisms, which make it possible for the cells to survive, since
these biological strategies have represented the optimal results of
the evolution process for billions of years. There are many
compounds capable of increasing the expression of HSPs but the
majority of these inducers exhibit significant cytotoxicity.
[0098] To cope with increased demand of multifunctional cosmetic
products, the induction of HSPs by natural, not toxic compounds,
come with excellent prospects.
[0099] Herein, the inventors have identified as most representative
species of HSP, the 90 and 70, representing the most ubiquitously
expressed heat shock proteins, have been taken into consideration.
These proteins represent 1-2% of the proteins present in the
eukaryotic cells, and are capable of reaching concentrations of
4-6% in the presence of stress.
[0100] The studies conducted by inventors on the effects of truffle
fermented extracts concentration on cell culture (see Table 2) show
a progressive increase of HSP 70 and HSP 90 level of expression
with no toxicity. This suggests that truffle fermented extracts
contain one or more substances, that with a synergic action, induce
the heat shock response, eliciting all the above described
beneficial effects on cells.
[0101] The discovery of new molecules that are able to activate the
stress response, by mimicking the effect of stressors, either in
the complete absence of stress or at a lower threshold of stressful
conditions, as is the case of fermented truffle extract, represents
an important target in this innovative approach, since the
knowledge accumulated on this mechanism makes for the possibility
of developing innovative strategies for its use to solve cosmetic
problems in regards to skin aging (Cf. Jindal S, 1996, Trends
Bioch. Sci 14:17-20; Magalhaes W V et al. 2012, Eur J Dermat, 22(1)
8-13).
[0102] An in vitro test was specifically used by the inventors to
assess the antioxidant activity on fermented and non-fermented
truffle extract (Table 3). Human keratinocytes and fibroblasts
treated with H.sub.2O.sub.2 or exposed to UV-A radiation show an
increased lipid peroxidation that was significantly reduced either
by treating the cells with fermented truffle extract after the
stress or before it.
[0103] Hyaluronic Acid--
[0104] In the development of innovative cosmeceutics for skin
biorevitalization there is growing interest in the use of mixtures
of HAs with different Mw, with optimized rheological and biological
properties. Results herein show the efficacy of different sized HA
to stimulate the migration of keratinocytes and fibroblasts. In
particular, the data showed that HA 1800 KDa, its intermediate
fragments (800 and 200 KDa) and their mixtures increased the
repair/remodeling process and fastened the cell migration,
confirming in particular the efficacy of HA mixtures in
biorevitalizing effect.
[0105] Hyaluronic acid, also generically indicated together with
its salts as hyaluronans (HA), is a polysaccharide with a linear
chain, negatively charged, composed by the repetition of n
disaccharide units (-4GlcUA.beta.1-3GlcNAc.beta.1-), wherein the
D-glucuronic acid (GlcUA) and N-acetyl-D-glucosamine (GlcNAc), are
linked by alternating .beta.-1,3 and .beta.-1,4 glycosidic bonds.
HA is a polysaccharide highly soluble in water and solutions of HA
show a visco-elastic behavior of non-newtonian type. These
properties are function of: Mw, (and being HA a linear polymer, the
chain length), concentration and external environmental conditions,
such as pH and ionic strength. HA has a number of unique
properties, shown below, making it one of the most versatile and
interesting biomaterials: [0106] a) Hydrophilicity and rheological
properties of HA: In aqueous solution the molecules of HA entrap
large quantities of water (about 1000 times the weight of the HA)
taking the form of extended spirals, stabilized by hydrogen bonding
between the hydroxyl groups along the chain. These chains will
envelop between them, even at very low concentrations, for this,
even very dilute, HA solutions have high viscosity, but depend on
sliding speed. [0107] b) Lubricating properties of HA:
Extraordinary rheological properties of HA solutions make this
compound an ideal candidate as a lubricant in the biological field.
This is, in fact, one of the actions to which the HA performs in
organisms. The joints of our body are an example of this type of
behavior.
[0108] More than 50% of HA is present in the skin tissue (Laurent T
C and Fraser J R., 1992, Faseb J; Oh E J, et al., 2010, J Control
Release 141:2; Juhlin L. 1997, J Intern Med, 242:61). Despite the
high molecular weight and hydrophilicity of HA, it is known to be
delivered through the skin tissue in both mouse and human (Brown T
J, et al., 1999 J Invest Dermatol, 113:740; Brown M B and Jones S
A. 2005 J Eur Acad Dermatol Venerol, 19:308). The mechanism for
transdermal transport of HA has not been clearly verified yet, but
there are some possible reasons for the positive effect of HA on
transdermal delivery. First, HA is very hygroscopic and can hydrate
the stratum corneum enhancing the permeability of the skin. Second,
the hydrophobic patch domain in HA chain can enhance the
permeability of HA across the stratum corneum. Third, HA receptors
distributed in the skin tissue may facilitate the localization of
HA in the skin tissue (Brown M B and Jones S A, 2005, J Eur Acad
Dermatol Venerol; 19:308; Wang C, et al., 1992 Histochemistry,
98:105; Tammi R et al., 1991, J Invest Dermatol 1991; 97:126).
Moreover, it is reported that HA can induce the proliferation,
migration, adhesion, and differentiation of keratinocyte (Lokeshwar
V B et al., 1996, J Biol Chem, 271:23853; Masellis-Smith A et al.,
1996 Blood, 87:1891). HA can also enhance the proliferation of
fibroblast through CD44 receptors on the cell membrane (Yoneda M et
al., 2004, J Cell Sci, 90:265). For all these reasons HA, despite
its size, when applied to the skin, is absorbed and operates as a
carrier of other molecules (U.S. Pat. No. 9,220,784 B2, hereby
incorporated by reference).
[0109] HA is widely distributed in nature. It has been identified
in various soft tissues (synovial fluid, skin, umbilical cord,
cockscomb, vitreous humor of the eye) and in some prokaryotic
cells, in which it creates a mucoid capsule surrounding the cell.
In vertebrates the HA has a wide variety of functions: in the skin
it ensures tissue hydration; in the cartilage it binds to
proteoglycans to adjust the content of water and ions, to stabilize
tissue physical properties and cell-substrate interactions. The
average molecular weight of the HA of synovial fluid and the
umbilical cord is 3,000-4,000 KDa.
[0110] The biological responses elicited by the HA depend strongly
on its Mw, in particular the accumulation of low Mw HA is an early
signal of alteration of the ECM, which activates the shelter
responses at tissue level, while a preponderance of high Mw HA
signifies a situation of good homeostasis. As a result of its
properties and biological functions, HA has a high added value (its
market value far exceeds that of other natural polysaccharides),
with applications ranging from the medical sector to the cosmetics
sector.
[0111] In many HA applications, the performance depends on its Mw.
For this, the mean molecular weight of the HA and a polydispersion
index Mw/Mn (measuring the breadth of the molecular weight
distribution curve, where Mn is the number average Mw, defined as
the total weight of all the polymer molecules of a sample divided
by the total number of molecules, and Mw is the weight average
molecular weight, which takes account of the different mass of
these molecules) needs to be the gold standard considered during
the development and production processes (Camenisch T D et al.,
2000, American J. Respiratory Cell and Molecular Biology 23,
431-433, hereby incorporated by reference).
[0112] In recent years, scientists have been studying the
correlation between HA Mw and physiological functions (Raoudi D. et
al., 2008, Wound Repair and Regen, 16(2 Suppl), 274-87; Ke, Sun
Qiao, et al., 2011, Food Chem Toxicol., 49(10), 2670-5; Cowman, L
et al., 2015 Frontiers in Immunology, 6, 261; Ferguson, R. et al.,
2011, Int J Pharm, 420 (1 Suppl), 84-92). Generally, native HAs (Mw
ranging from 2000 to 800 KDa) are space-filling molecules with
anti-inflammatory and anti-angiogenic effects, while lower Mw HA
(Mw<50 KDa) may be involved in a proinflammatory process
(Frenkel J. S., 2014, Int Wound J, 11, 159-163). In particular,
there has been an increasing effort to clarify the role of HAs in
the interaction with the epidermis/dermis tissues (Ghosh, P., &
Guidolin, D., 2002, Current Abstracts Seminars in Arthritis and
Rheumatism, 32(1), A2-A4). HA has also been reported to be a free
radical scavenger, presenting an antioxidant function. It enhances
the wound healing process and presents both angiogenic (Gao F. et
al., 2010, Matrix Biol., 29(2), 107-16) and immunostimulatory
activity (Ke et al., 2011 Food Chem Toxicol., 58, 401-7).
[0113] Nevertheless, given HA turnover, all HA fragments have a
physiological function, as could be expected, that is often very
important in the healing processes, biological tissue homeostasis
and biosynthesis of ECM. Native HA is reported to be fragmented in
smaller molecules during ECM degradation after acute tissue injury,
in order to activate the host innate immune response by recruiting
macrophages and other specific cells, to produce chemokines
required to begin repair/restoration of tissue integrity.
[0114] Due to water-attracting characteristics in tissue repair,
for example, long chain HA has cushioning and visco-elastic
properties, that create a porous scaffold onto which the cell might
migrate; on the contrary, medium-size HA fragments (100-250 kDa)
have been found to promote cell migration and contemporarily to
stimulate and modulate pro-inflammatory cytokines production;
finally very small fragments (4 saccharides) have been found to
induce chemotaxis (Frenkel, J. S., 2014, Int Wound J, 11,
159-163.).
[0115] Vigetti and collaborators have reported that small HA
fragments, ranging from 3 to 25 disaccharides (1.2-10 kDa), have
inflammatory effects and show pro-angiogenic activity in human cell
models (Vigetti, D., et al., 2014, Biochim Biophys Acta., 1840(8),
2452-9). Relative to this, a recent report has shown that HA
fragments stimulate chemokine and cytokine gene expression
interacting with TLR-4 (Jiang D et al., Physiological Reviews, Vol.
91 no. 1, 221-264). RHAMM receptor was mainly involved in the cell
migration. As a matter of fact, RHAMM-HA interaction does play an
important function in tissue injury and repair (Viola et al., 2015,
Glycoconj J., 32(3-4), 93-103).
[0116] Although sometimes contradictory, the set of knowledge on
the differentiated effects of HA as a function of its Mw, is an
important premise for the construction of innovative
cosmeceuticals, in which the optimization of mixtures of HAs with
different Mw allows for the obtainment of effective biological
responses in biostimulation processes, to contrast skin aging. In
addition, although many reports have confirmed the differences
relating to HA Mw, there have been no experimental highlights at
the biological level of these different compounds.
[0117] Prompted by this knowledge and shared experience of diffused
confusion in assessment and in the relationship between HA size and
function, the inventors addressed the question through rigorous
methods. By identifying an optimal range of HA fragments and their
biochemical activities, and by using in vitro models and specific
biochemical test, the inventors have elucidated the effect of
specific HA fragments and their mixture when they interact with
membrane receptors, in modulating cellular biostimulation, wound
repair, cell migration, and cytokine expression. Results herein
show the efficacy of different sized HA to stimulate the migration
of keratinocytes and fibroblasts. In particular, the data showed
that HA 1800 KDa, its intermediate fragments (800 and 200 KDa) and
their mixtures increased the repair/remodeling process and fastened
the cell migration, confirming in particular the efficacy of HA
mixtures in biorevitalizing effect.
[0118] With acidic hydrolysis in a heterogeneous phase (Melander C.
and Tommeraas K., 2010, Carbohydrate Polymers 82, 874-879; hereby
incorporated by reference), a full array of HA fragments of
different size (Table 4) was obtained, with identical structural
disaccharides units, thus ensuring that biochemical and biological
outcomes are not ascribed to a HA modified chemical structure.
.sup.1H-NMR analysis confirmed these results. The structural and
rheological data (Viscotek analysis) confirmed that products are
similar to the ones obtained by hyaluronidase in vivo and, for this
purpose, suitable to be tested for in vitro biological
response.
[0119] To elucidate the biological roles of HA fragments by
studying specific biomarkers from the preliminary phases of wound
healing process, an in vitro scratch test has been used with TLVM
(D'Agostino et al., 2015, BMC cell biology, 16:19, hereby
incorporated by reference) on human keratinocytes and fibroblasts.
The highest Mw HA samples led to complete repair in a shorter time;
however, all the HA fragments tested enhanced the scratch repair
rate compared to the control (Table 5).
[0120] Interaction between different sized HA and relative specific
receptors was investigated to improve knowledge of the biochemical
basis in the activation/silencing of pathways relative to HA and
its degraded products in vivo. Results have shown the efficacy of
different sized HA to stimulate the migration of keratinocytes and
fibroblasts. In particular, the data showed that HA 1800 KDa, its
intermediate fragments (800 and 200 KDa) and their mixtures
increased the repair/remodeling process and fastened the cell
migration, confirming in particular the efficacy of HA mixtures in
biorevitalizing effect.
[0121] Molecular analysis at gene and protein level corroborated
the data derived from time-lapse experiments. HA exerts biological
activity thought interaction with its receptors on cell surface; in
particular CD44, the main HA receptor, interacting with HA,
triggers different biological responses, ranging from cell
proliferation and ECM degradation to angiogenesis and
inflammation.
[0122] All HA analyzed herein activated CD44, but the major
responses were found in presence of HA 1800-800 KDa (Table 7). All
HA samples increased RHAMM expression, however results were
significantly higher for 200 KDa (Table 7).
[0123] TLRs are receptors of innate immunity and TLR-2 and TLR-4
may bind HA fragments, inducing signaling. Differently from CD44
response, TLR-4 activity, correlated to inflammatory process, is
significantly down-regulated in all HA tested (Table 7). Gene
expression data of HA receptors were confirmed by
immunostaining.
[0124] In order to follow-up the biochemical cascade turned on by
HA-receptor interaction, the expression profile of key cytokines
possibly involved have been evaluated. The results herein show an
up-regulation of TGF .beta.-1 and TNF-.alpha. in presence of all HA
treatments (Table 7). Therefore, all hyaluronan fragments tested
were both safe and biologically active. Furthermore they may
support cell activation, therefore helping in skin repairer
procedures and biological remodeling.
[0125] In consideration of the differentiated biological effects
that characterize the HA as a function of its Mw, and of the
heterogeneity of the HA molecular population naturally present in
the ECM, a cytomimetic formulation was synthesized by mixing HA of
different Mw.
[0126] The experimental data (Table 5), using the test model of
wound healing time lapse microscopy of human chondrocytes and
fibroblasts, showed that the best results were obtained using
mixtures formed by comparable amounts of HA 1800, 800 and 200 KDa.
In fact, the synergism between the biological properties of these
different HAs reduced the healing time by 50-70% in both
experimental models. An in vitro test was specifically developed to
assess the antioxidant activity of the mixture of HAs 1800+800+200
KDa (Table 8). Human keratinocytes and fibroblasts treated with
H.sub.2O.sub.2 or exposed to UV-A radiation show an increased lipid
peroxidation, that was reduced either by treating the cells with
HAs 1800+800+200 KDa after the stress or before it.
[0127] Olive Leaf Extract in Thermal Water--
[0128] Among the active ingredients that have a strategic role to
draw cosmeceuticals effective in protecting the skin and preventing
skin aging, compounds with antioxidant activity are considered
elective. Of particular interest are the natural antioxidants
obtained generally by extraction from plant sources. In order to
help achieve optimal protective effects as a radical scavenger, it
is preferable that there be a mixture of compounds with different
spectrums of antioxidant activity. A particularly interesting
source of active ingredients with antioxidant activity are Olive
leaves (Olea europaea). Only fresh leaves were used in the
extracts, instead of dried leaves or reconstituted powders.
Experimental data herein shows that liquid Olive leaf extract made
directly from fresh leaves has a broader spectrum potency than has
previously been the case, including a synergistic action with
thermal mineral water on wound closure time.
[0129] The extract of olive leaves is characterized by the presence
of a significant quantity of oleoeuropein (IUPAC name
4S,5E,6S)-4-[2-[2-(3,4-dihydroxyphenyl)
ethoxy]-2-oxoethyl]-5-ethylidene-6-[[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6--
(hydroxymethyl)-2-tetrahydropyranyl]oxy]-4H-pyran-3-carboxylic
acid, methyl ester). This composition is accompanied by minor
amounts of structurally related polyphenols with antioxidant
activity as 10-hydroxyoleuropein, ligstroside, and
10-hydroxyligstroside. Oleuropein had activity as an agonist of the
G-protein estrogen receptor (Eric R. et al., 2014, Molecular and
Cellular Endocrinology 389 (1-2): 71-83).
[0130] The `natural balance` of antioxidant actives in a fresh
Olive leaf extract provides a more powerful free radical scavenging
capacity as opposed to when the individual components are isolated.
This involves a synergistic action between flavonoids,
oleuropeosides and phenols.
[0131] Aqueous systems are used when preparing the extract of Olive
leaf, leaving the homogenized leaf to extract for 24-48 hours.
[0132] A strong innovation in the field of phytoextracts has been
the use of thermal water with this special composition and these
therapeutic properties as an extraction system. This helps create
important synergies between the biological effects of active
extracts and those of the extracting system. In this respect, the
thermal spring water is of particular interest, including that
obtained from the Thurio spring at the Spezzano Thermal Baths in
Italy. Thanks to the abundance of peculiar microelements, it is an
active principle of choice. These microelements provide outstanding
soothing properties. As reported in Table 9, using the in vitro
keratinocytes and fibroblasts scratch assay with TLVM, a
significant synergism of action between the thermal water and the
active present in the olive leaves extract. Its almost half the
time of the closure wound.
[0133] In vitro testing was used by the inventors to assess the
antioxidant activity accomplished from the Olive leaf extract in
thermal water (Table 10). Human keratinocytes and fibroblasts
treated with H.sub.2O.sub.2, or exposed to UV-A radiation, have
shown an increased lipid peroxidation. This was reduced by either
treating the cells before or after the stress, using Olive leaf
extract in thermal water.
[0134] Fermented Falernum Grapes--
[0135] Falernian wine (Latin Falernum) was historically produced
from Aglianico grapes on the slopes of the Falernus mountains, near
the border of Latium and Campania region of Italy. Falernian
fermented grapes are particularly rich in polyphenols and
anthocyanins and provide strong general antioxidant and free
radical scavenger activity. However, its antioxidant effects
directly on skin cells are not determined. Herein it is shown that
Falernian fermented grape must has an important biorevitalizing
action (Table 11) and significant antioxidant activity in human
skin cells as tested, namely, human keratinocytes and
fibroblasts.
[0136] The Falernus mountains area is now occupied by the modern
day vineyards of Rocca di Mondragone and Mount Massico. Grapes of
Falciano del Massico were collected in late August, when they are
not yet fully mature. The grapes, after baling and homogenisation,
are left to ferment for 48 hours at 10.degree. C., clarified by
filtration under pressure and then pasteurized. The ruby liquor
that is obtained can be defined as fermented grapes of Falernum.
The inventors have determined that this liquor has an important
biorevitalizing action (Table 11) and significant antioxidant
activity in human skin cells as tested, namely, human keratinocytes
and fibroblasts (Table 12). In fact, human keratinocytes and
fibroblasts treated with H.sub.2O.sub.2, or exposed to UV-A
radiation have shown an increased lipid peroxidation that was
reduced by either treating the cells before or after the stress
with fermented Falernum grape must.
[0137] Cytomimetic Formulas and Synergism of their Actives--
[0138] The skin extracellular matrix (ECM) develops at the tissue
level a series of complex functions indispensable for the correct
functionality of the cell population. The realization of a
cytomimetic formula for the skin tissue, able to mimic the ECM
complex functions, must ensure the tissue biorevitalization, an
effective anti-inflammatory/radical scavenger activity and the
activation of repair molecular mechanisms.
[0139] The inventors have found that by using the natural actives
together, a cytomimetic formula was obtained capable of developing
the above-described complex functions. The strong synergistic
effect between the active employed is absolutely unpredictable.
(Fermented truffle extract, mix of HA 1800, 800 and 200 KDa,
extract of olive lives in thermal water and fermented Falernum
grape must). These are more effective even at extremely low
concentrations, rather than employing the various actives
individually.
[0140] In vitro wound healing experiments (Table 13) have shown
that cytomimetic formulas were superior in prompting wound closure.
The potent synergism between actives of cytomimetic formula is
evident when comparing Examples 1, 5, 9 and 11, with Example 13 in
which the actives are present up to three orders of magnitude or
less.
[0141] In vitro testing was specifically developed to assess the
antioxidant activity accomplished by the cytomimetic formulas
(Table 14). Human keratinocytes and fibroblasts treated with
H.sub.2O.sub.2 or exposed to UV-A radiation have shown an increased
lipid peroxidation. This was significantly reduced either by
treating the cells before or after the stress with cytomimetic
formulas.
[0142] The potent synergism between actives in the cytomimetic
formula is also evident in the production of heath shock proteins,
in particular HSP 70 and HSP 90, which gradually increase their
expression when the cells are treated with increasing amounts of
cytomimetic formula. Table 15, in fact, demonstrates the potent
synergistic effect of actives present in the cytomimetic formulate
that, as reported in the Example 15 are present up to three orders
of magnitude in less respect to that reported in Example 3 for the
fermented truffle extract alone.
[0143] Also analysis of gene expression elicited by Cytomimetic
formula in stressful conditions, performed at 0.1 and 0.5%, (Table
16) demonstrated the potent synergistic effect of actives present
in this formulae. CD44, RHAMM, TGF.beta.-1, TNF-.alpha. and IL-6
were all up-regulated increasing with cytomimetic formulae
concentration, and only TLR-4 activity, correlated to inflammatory
process, was significantly down-regulated.
[0144] In embodiments of the cosmeceutic products, cytomimetic
formulas/formulations can be used at concentrations ranging from
0.1 to 50% w/w.
[0145] To assess the efficacy of a cosmetic composition using
cytomimetic formulations, a clinical study was performed on 10
subjects (women), aged between 30 and 54 years, for 28 days. The
ingredients of the base composition, and the base composition with
a cytomimetic formula are described in Examples 20 and 21.
[0146] The efficacy has been proved using instrumental,
non-invasive methods: moisture of comeum layer (Corneometry),
sebumetry, elasticity and firmness, SELS (Surface Evaluation of
Living Skin) parameters: roughness, smoothness, the scaliness
degree, wrinkles.
[0147] Determination of profilometry (micro-relief). Profilometry
was determined instrumentally, by Visioscan VC 98 (Courage+Khazaka
electronic GmbH). This testing method is called SELS (Surface
Evaluation of the Living Skin) which is based on the graphical
illustration of the skin surface under conditions of special
illumination and electronic processing of the image, quantified in
pixels. "Skin smoothness" (SEsm), can be quantified by finding the
average between the depth and width of the skin wrinkles. The
smooth skin has a low variation of grey, making the histogram of
the level of grey distribution narrow, resulting in a very small
SEsm value. "Skin roughness" (SEr), can be demonstrated by using
the levels of grey in comparison with the roughness of the entire
image. An increased value of this parameter expresses a reduction
of the roughness of the analyzed area. "Scaliness" (SEsc) is
expressed as the number of pixels whose level of grey is over the
threshold limit; the reduction of this parameter is correlated with
a low exfoliation of the skin. "Wrinkles" (SEw)-skin wrinkles are
calculated from the proportion of horizontal to vertical wrinkles.
The value of SEw is higher the more visible the wrinkles are from
the point of view of the width and the depth.
[0148] Skin elasticity was determined instrumentally (Cutometer MPA
580--Courage+Khazaka electronic GmbH). The elasticity curve
obtained by a suction/elongation cycle of the Cutometer,
respectively 10 repetitive cycles for the assessment of the area
parameter: Among these parameters, in this study the relevant and
correlative ones for elasticity and firmness were selected: R2
(Ualufl--gross elasticity, represented by the ratio between the
ability of redeformation and final distension, the closer this
value is to 1, the more elastic the skin becomes. R5 (Ur/Ue)--net
elasticity, the ratio between the immediate retraction and the
immediate distension, expresses the ability of the skin recovery
after the deformation.
[0149] Skin hydration was performed instrumentally, through
corneometry, measuring the capacitance. The variations in the
dielectric constant is measured by using the water content in the
epidermal superficial layer and the level of hydration.
[0150] Skin lipid levels in the skin have been quantified using a
sebumeter, by measuring the absorbance of a plastic film
impregnated with sebum. The film becomes more transparent in the
presence of the lipids. The sebum values can be measured between
50-300 .mu.g/cm.sup.3.
[0151] The clinical results obtained as described in Example 22,
show significant improvement of skin physiological parameters after
using basic composition with cytomimetic formulate as compared with
the base compositions demonstrated in vivo biological effect found
in vitro. The results show improvement in all profilometric
parameters: reduced scaliness with 116%, improved roughness and
smoothness, with 76.6% and, accordingly 47.5% and wrinkle reduction
(73.9%). What is very interesting is the balancing effect on lipid
content in the stratum corneum. This can be explained by the
restoring effect at intercellular level. In the oily skin, the
sebum level decreased with 35.67%. The dry skin level increased by
74.30%. In the same time, the base formula using cytomimetic
formulate performed a better moisturization (33.33% higher) and an
improvement of skin elasticity (R2 40%, R5 50). The comparative
performance in vivo (clinical results) of base cream and base cream
using cytomimetic formulate are presented in Table 19.
[0152] The body of results obtained, as shown through in vitro and
in vivo experiments, demonstrate the superiority of the aesthetic
treatments through the cytomimetic formulas of the invention.
Non-limiting examples are given below, describing the production,
characteristics and use of the formulate of the invention.
Example 1
Preparation of Fermented Truffle Extract
[0153] 1 Kg of Tuber magnatum preciosa, a white truffle collected
from Alba, Italy, were washed first with water many times, then
three times with 0.1 M sodium phosphate buffer at pH 6 and minced
into small pieces. The truffle pieces were suspended with 0.1 M
sodium phosphate buffer at pH 6 in the ratio 1/10 w/v and
homogenized for 1 hour with an ultra Turrax blender.
[0154] The slurry was transferred into a 20 L fermenter and, after
addition of 10 g of lyophilized Saccharomyces cerevisiae inoculum,
was fermented for 24 h at 30.degree. C. with a flux of sterile air
of 2 L/min, which automatically controlled the pH at 6. During
fermentation its evident the partial clarification of the slurry.
At the end of fermentation process the slurry was clarified by
continuous centrifugation on alfa Lavall centrifuge and the clear
supernatant was lyophilised, obtaining 110 g of a pale yellow fine
powder of fermented truffle extract.
[0155] Simple truffle extract was obtained by homogenizing washed
truffle and maintaining under agitation of the slurry at 4.degree.
C. for 24 h, before clarification and lyophilization.
[0156] Fermented yeast extract was obtained by suspending 10 g of
lyophilized Saccharomyces cerevisiae in 10 L of 0.1 M sodium
phosphate buffer at pH 6. The suspension was transferred in a 20 L
fermenter, and fermented for 24 h at 30.degree. C. with a flux of
sterile air of 2 L/min, automatically controlling the pH at 6. At
the end of fermentation process the suspension was clarified by
continuous centrifugation on Alfa Lavall centrifuge and the clear
supernatant was lyophilised.
Example 2
Fermented Truffle Extract: In Vitro Keratinocytes and Fibroblast
Scratch Assay, Using TLVM
[0157] HaCaT, a spontaneously transformed non-tumorigenic human
keratinocytes cell line was provided by Istituto Zooprofilattico,
Brescia, Italy and the cells were cultured in Dulbecco's Modified
Eagle Medium (DMEM), supplemented with 10% (v/v) heat inactivated
Fetal Bovine Serum (FBS), penicillin 100 U/ml and streptomycin 100
.mu.g/ml. DMEM, FBS, Pen-Strep PBS and Trypsin were provided by
Gibco Invitrogen (Milan, Italy).
[0158] A human dermal fibroblasts cell line immortalized with hTERT
(HDF cells, BJ-5ta, ATCC CRL-4001), was cultured in a 4:1 mixture
of DMEM and Medium 199 supplemented with 0.01 mg/ml hygromycin B
and 10% (v/v) FBS. All materials for HDF culture were purchased
from ATCC (USA). The cells were grown on tissue culture plates (BD
Falcon, Italy), using an incubator with a humidified atmosphere
(95% air/5% CO.sub.2 v/v) at 37.degree. C.
[0159] Briefly 12-wells (pre-coated with collagen) were seeded with
HaCat or HDF cells until complete confluence was reached. Scratch
wounds were created mechanically with a sterile pipette tip (O=0.1
mm). Uniformly sized scratches were carefully obtained
approximately 0.7.+-.0.2 mm in width. Detached cells and debris
were washed away with PBS solution before placing the multiwell in
the stage incubator.
[0160] In both models, the effect of truffle extract, fermented
truffle extract and fermented yeast on the rate of wound closure
were tested by incubating the scratched monolayer with the
following solution: truffle extract 1% w/v, fermented truffle
extract 0.5 and 1% w/v and fermented yeast 1% w/v. The samples were
prepared by dissolving the lyophilized powders directly in the
medium. pH and osmolality (7.2-7.4 and 300 mosm) of the medium
containing the treatments were verified to ensure physiological
conditions.
[0161] The `wound closure` phenomenon was monitored using a TLVM
station, to observe the migration of cells to repair the wound. In
the presence of different treatments, this allowed simultaneous
observation of the repair of different cells and successive
performance of qualitative and quantitative analyses of the
experiment (D'Agostino et al., 2015, BMC cell biology, 16:19,
hereby incorporated by reference).
[0162] The fermented truffle extract samples led to complete repair
in shorter time compared with truffle extract, fermented yeast and
control (Table 1).
Example 3
Effect of Fermented Truffle Extract on HSP 70 and HSP 90
Induction
[0163] 20 mg of powder of fermented truffle extract or truffle
extract or yeast lysate were resuspended in 1 ml of PBS
(physiological conditions) in order to obtain a final concentration
of 20 mg/ml.
[0164] 2.5 millions of HEK-293t cells were treated with: 1, 2, 5
and 10 mg of fermented truffle extract in a final volume of 10 ml
(DMEM medium). The cells were incubated for 18 hrs at 37.degree. C.
and then harvested. The cells were lysated in RIPA buffer (50 mM
Tris pH 8, 1% nonidet p40 0.25% Sodium Deoxycholate and 1 mM EDTA)
and equal amounts (calculated using the Bradford protein assay) of
soluble fractions were loaded on SDS-PAGE (10% polyacrylamide). The
gel was blotted on PVDF membrane and HSP 70/90 were detected using
commercial antibodies. Anti-.beta.-actin was used as control. The
signals corresponding to HSP 70 and HSP 90 were quantified using
Image J software and the results obtained were plotted.
[0165] Analyzing the cells treated as described above, it was
observed that HSP 70 and HSP 90 gradually increased their
expression when cells where treated with increasing amounts of
fermented truffle extract (mg of fermented truffle extract/ml of
medium). The filters were quantified and the results obtained were
blotted on a graph that clearly indicates a peak of HSP 70/90
expression levels at 1 mg/ml (Table 2)
[0166] Data indicate that HEK-293t cells treated with fermented
truffle extract show with a progressive increase, concentration
dependent, of HSP 70 and HSP 90 level of expression, this effect is
in minor amount present with fermented yeast and absent with non
fermented truffle extract.
Example 4
Antioxidant In Vitro Activity of Fermented Truffle Extract Using
T-BARS (Thiobarbituric Acid Reactive Substances) Assay
[0167] Generation of reactive aldehydes were assessed by measuring
thiobarbituric acid-reactive substances (TBARS), as described
previously by Stiuso et al., (Stiuso P., et al., 2014, Oxidative
Medicine and Cellular Longevity, hereby incorporated by reference).
The effect of fermented truffle extract on HaCaT cells
(2.0.times.10.sup.5) was tested in three different experimental
setups: (1) cells were pre-treated for 30 minutes with 50 .mu.M
H.sub.2O.sub.2 or with exposure to a UVA radiation (.lamda..sub.max
365 nm), and then incubated with fermented truffle extract (0.32%
w/v) for 24 h, to test the protection effect on post-stress
process; (2-3) fermented truffle extract was applied simultaneously
with 50 .mu.M H.sub.2O.sub.2 or with exposure to a UVA radiation
365 nm), to test its antioxidant activity.
[0168] The protein concentrations were determined using the Bio-Rad
protein assay reagent (Bio-Rad Laboratories, Milan Italy). Lipid
peroxidation was evaluated using an analytical quantitative
methodology. It relies upon the formation of a colored adduct
produced by the stoichiometric reaction of aldehydes
(malondialdehydes MDA) with thiobarbituric acid (TBA).
[0169] TBARs assay were performed on aliquots of membranes
extracted (10 .mu.l) added to 2 ml of TBA-TCA (TCA 15% w/v, TBA
0.3% w/v In HCl 0.12 N) solution at 100.degree. C. for 30 min. The
chromogen was quantified by spectrophotometric reading at a
wavelength of 532 nm and the amount of TBARs were expressed as a
percentage of lipid peroxidation and then normalized respect to
control. Data reported in Table 3 shows that fermented truffle
extract is effective in all oxidative stress conditions.
Example 5
Production of HA of Different Mw
[0170] The strategy used to obtain HA of different Mw is based on
an heterogeneous acid hydrolysis of 1800 KDa HA according to
Toommeras et al., (Melander C. and Tommeraas K., 2010, Carbohydrate
Polymers 82, 874-879, hereby incorporated by reference). In
particular, HA powder (1800 kDa) hydrolysis in ethanol (EtOH) (93%
v/v) was carried out using a HCl-EtOH vs. HA ratio 10/1 v/w. The
slurry was pre-warmed at 65.degree. C. in a thermostatic bath, then
a few drops of HCl 37% v/v were added under vigorous stirring, in
order to have a final concentration of 0.4M HCl. The hydrolysis was
carried out for 60 and 100 min to obtain HA 800 and 200 KDa
respectively. Each sample was then cooled in an ice-bath and
neutralized with an equimolar quantity of NH.sub.3 25%. Samples
were washed with ethanolic solution (93% v/v), recovered using a
Buchner funnel under vacuum, lyophilized and then stored at
-20.degree. C. until characterization was obtained.
[0171] HA fragments were characterized by SEC-TDA (Size Exclusion
Chromatography-Triple Detector Array) equipment by Viscotek (Lab
Service Analytica, Italy). A detailed description of the system and
its analytical conditions are reported by La Gatta et al. (La Gatta
et al., J Biomed Mater Res Part B: Appl Biomater, 104B, 9-18,
hereby incorporated by reference).
[0172] Samples' molecular weight (Mw, Mn, Mw/Mn) and molecular size
(hydrodynamic radius-Rh) are reported in Table 4. The poly
dispersity index ranged from 1.4 to 1.7, which was comparable to
the one calculated for the HA substrate, confirming an efficient
and a simple approach for degrading HA without further complex
purification steps. The decrease of intrinsic viscosity and
hydrodynamic radius were in accordance with the reduction of chain
lengths.
[0173] In order to confirm if HA, degraded by heterogeneous
hydrolysis, maintained its structural integrity, the hydrolysed HA
fragments were analyzed by .sup.1H-NMR spectroscopy. As expected,
the .sup.1H-NMR spectra showed the presence of peaks corresponding
to acetamide protons at 1.9 ppm, 2', 3', 4', 5', and 6'-protons of
HA disaccharide unit at 3.2-4.0 ppm, as well as anomeric l'-protons
at 4.4 ppm. No indication of suspected by-products such as
de-N-acetylation or ethanolysis at the reducing-end were
observed.
[0174] Endotoxin amount determination--For endotoxin content
determination, HA powders were dissolved in pyrogen free water. The
amount of pyrogens (bacterial endotoxins) in the solution were
measured by using Limulus Amebocyte Lysate (LAL) testing
(chromogenic kinetic method) according to European Pharmacopoeia
01/2005:20614. Specifically, ENDOSAFE.RTM.-PTS cartridge US License
N.1197 by Charles River Endosafe were used. All operations were
performed under conditions avoiding endotoxin contamination.
Results were reported as endotoxin units (EU/mg) of HA powder. The
low endotoxin content, crucial in pharma grade requirements, is of
key importance to better highlight the HA fragment function itself.
The endotoxins amount for all hyaluronan powders produced, resulted
in less than 0.05 EU/mg. This data proved that LPS and/or endotoxin
are below a guard level and therefore cellular phenomenon should be
driven by HAs rather than impurities.
Example 6
HAs: In Vitro Keratinocytes and Fibroblasts Scratch Assay, Using
TLVM
[0175] HaCaT and HDF cell line and their growth conditions are
described herein above in Example 2. In both models, the effect of
HA gels on the rate of wound closure was tested by incubating the
scratched monolayer with the following solutions: HA1800 KDa, HA800
KDa, HA200 KDa, HA1800 KDa+HA800 KDa (50% w/w each), and HA1800
KDa+HA800 KDa+HA200 KDa (33.3% w/w each) at final concentration of
1% w/w in the incubation medium. The samples were prepared by
dissolving the lyophilized powder directly in the medium. pH and
osmolarity (7.2-7.4 and 300 mosm) of the medium containing the
treatments were verified to ensure physiological conditions.
[0176] The `wound closure` phenomenon was monitored using a TLVM
station to observe the migration of cells to repair the wound. In
the presence of different treatments, this allowed simultaneous
observation of the repair of different wells and successive
performance of qualitative and quantitative analyses of the
experiment (D'Agostino et al., 2015 BMC cell biology, 16:19).
[0177] The highest Mw HA samples led to complete repair in shorter
time; however, all the HA fragments tested enhanced the scratch
repair rate compared to the control (Table 5).
[0178] Mixtures formed by equivalent amounts in weight of
HA1800+HA800 KDa and HA1800+HA800+HA200 KDa are characterized by a
significant reduction in the repair time with respect to HA
separately (Table 5).
Example 7
HAs: Gene Expression Analysis in Stressed Cells
[0179] For gene expression analyses in stressful conditions, human
keratinocytes and fibroblasts were grown in different cell
cultures. 3.75.times.10.sup.4 cells/cm.sup.2 were seeded in a
standard 24-well culture plate. To reproduce skin inflammation in
vitro, the mechanical injury induced to the cultures was very
extensive. With a sterile tip, parallel scratches were inflicted
upon the monolayers, estimating damage to be at least 40% of the
cells.
[0180] After addition of HAs and incubation for 16 h the cells were
directly lysed with TRIzol.RTM. (Invitrogen, Milan, Italy). Total
RNA was extracted from HA (Mw=1800, 800 and 200 KDa) treated
keratinocytes or fibroblasts. Following precipitation with
isopropyl alcohol and washing with 75% ethanol, the RNA pellets
were resuspended in nuclease-free water. The concentration of the
extracted RNA was determined through a Nanodrop spectrophotometer
(Celbio, Milan, Italy) and 1 .mu.g of DNase-digested total RNA was
retro-transcripted in the cDNA using Reverse Transcription System
Kit (Promega, Milan).
[0181] Quantitative real time PCR was obtained by iQ.TM. SYBR.RTM.
Green Supermix (Bio-Rad Laboratories Srl) in order to analyze the
gene expression of some HA key receptors such as CD44 and RHAMM,
TLR4 and alert inflammation biomarkers such as TGF-.beta.,
TNF.alpha., IL-6. The primer sequences (Table 6) were designed by
Beacon Designer.TM. software. The final melting curve was performed
from 55-95.degree. C.
[0182] Samples were run in triplicate, and the expression of
specific mRNA relative to the control was determined after
normalization with respect to HPRT housekeeping gene (internal
control). The fold-change of mRNA expression of the genes under
evaluation was calculated by using the 2-.DELTA..DELTA.Ct
comparative threshold method (.DELTA..DELTA.Ct=difference of
.DELTA.Ct between treated cells and non-treated cells used as
controls). The results were expressed as normalized fold
expression, calculated by the ratio of crossing points of
amplification curves of several genes and internal standard, by
using the Bio-Rad iQ.TM.5 software (Bio-Rad Laboratories Srl). Gene
expression data analyses for the main HA receptors are reported in
Table 7. CD44 was to be over expressed for all HA evaluated. All HA
samples increased RHAMM expression, however results were
significantly higher for 200 KDa.
[0183] Inflammation biomarkers (TGF.beta.-1, TNF-.alpha. and IL-6)
involved in epithelial cell migration were evaluated by
quantitative RT-PCR. Specifically, TGF.beta.-1 was up-regulated for
all HA fragments investigated. HA ranging from 800 to 200 KDa,
showed a significant increase in TGF.beta.-1 with respect to HA1800
KDa.
[0184] In this case the increase prompted a "positive" activation
toward the repair. Cell repair activation mechanism, implicate also
IL-6 that is regulated by HA/receptor interaction. Results showed
that both TNF-.alpha. and IL-6 present similar trend during
re-epithelisation process. In particular the expression levels
increased with the HA molecular size decreasing.
Example 8
HAs: Antioxidant In Vitro Activity Using T-BARS (Thiobarbituric
Acid Reactive Substances) Assay
[0185] The experiment was conducted as reported in the Example 4.
The effect of HAs 1800+800+200 KDa mixture (33.3% w/w each) at
final concentration of 0.5% w/v in the incubation medium, on HaCaT
cells (2.0.times.10.sup.5) was tested in three different
experimental setups: (1) cells were pre-treated for 30 min with 50
.mu.M H.sub.2O.sub.2 or with exposure to a UVA radiation
(.lamda..sub.max 365 nm) and then incubated with HAs mixture (0.5%
w/v) for 24 h, to test the protection effect on post-stress
process; (2-3) HAs mixture were applied simultaneously with 50
.mu.M H.sub.2O.sub.2 or with exposure to UVA radiation
(.lamda..sub.max 365 nm) to test antioxidant activity.
[0186] Data reported in Table 8 show that HAs 1800+800+200 KDa
mixture (33.3% w/w each) is effective in the oxidative stress
conditions.
Example 9
Preparation of Olive Leaf Extract in Thermal Water and
Characterization of Actives
[0187] 10 Kg of fresh Olive leaves, collected from centuries-old
olive trees of Puglia, Italy, were washed first with water a
plurality of times, then suspended in 40 L of thermal water from
the Thurio spring at the Spezzano Thermal Baths, Italy. Then they
were homogenized for 1 h with an ultra Turrax blender. At the end
of extraction process (24 h, 4.degree. C., pH 5.5-6.5) the slurry
was clarified by continuous centrifugation on Alfa Lavall
centrifuge and the clear pale green supernatant was directly used
in the cosmetic formulate.
[0188] To characterize the extract the solution was lyophilised.
Quantitative chemical characterization of actives present in the
extract indicates a solid residue of 50 g/L of extract containing
per g of powder 50 mg of oleuropein, 6 mg of minor olive polyphenol
and 1 mg of flavonoids.
Example 10
Olive Leaf Extract in Thermal Water: In Vitro Keratinocytes and
Fibroblasts Scratch Assay, Using TLVM
[0189] HaCaT and HDF cell line and their growth conditions are
described hereinabove in Example 2. In both models, the effect of
Olive leaf extract in thermal water on the rate of wound closure
was tested by incubating the scratched monolayer with a final
concentration of 20% v/v in the cell growth medium.
[0190] The `wound closure` phenomenon was monitored using TLVM
station, to observe the migration of cells to repair the wound
(D'Agostino et al., 2015 BMC cell biology, 16:19, hereby
incorporated by reference).
[0191] Results (Table 9) indicate a significant synergism of action
between the thermal water and the active present in the olive leaf
extract. It was almost half the time of closure of the wound.
Example 11
Olive Leaf Extract in Thermal Water: Antioxidant In Vitro Activity
Using T-BARS (Thiobarbituric Acid Reactive Substances) Assay
[0192] The experiment was conducted as reported in Example 4. The
effect of Olive leaf extract in thermal water and in water at final
concentration of 10% v/v in the incubation medium, on HaCaT cells
(2.0.times.10.sup.5) was tested in three different experimental
setups: (1) cells were pre-treated for 30 min with 50 .mu.M
H.sub.2O.sub.2 or with exposure to a UVA radiation (.lamda..sub.max
365 nm) and then incubated with Olive leaf extract in thermal water
(10% v/v) for 24 h, to test the protection effect post-stress
process; (2-3) Olive leaf extract in thermal water was applied
simultaneously with 50 .mu.M H.sub.2O.sub.2 or with exposure to UVA
radiation (.lamda..sub.max 365 nm) to test antioxidant
activity.
[0193] Data reported in Table 10 show that Olive leaf extract in
thermal water is effective in but the oxidative stress
conditions.
Example 12
Production of Fermented Grapes of Falernum
[0194] 50 Kg of fresh grapes collected from Falciano del Massico,
(Italy) vineyards (the same used by ancient Romans to produce the
most expensive and famous Faustian Falernian wine) were washed
twofold with water, and after premixing were homogenized for 1 h
with an ultra Turrax blender. At the end of fermentation process
(48 h, 10.degree. C., pH 5) the slurry was clarified by under
pressure filtration and pasteurised.
[0195] 25 L of a stable solution of a brilliant ruby was obtained,
rich in polyphenols with antioxidant activity and a complex mixture
of carbohydrate and peptide compounds, which confer a strong
biorevitalizing action.
Example 13
Fermented Grapes of Falernum: In Vitro Keratinocytes and
Fibroblasts Scratch Assay, Using TLVM
[0196] HaCaT and HDF cell line and their growth conditions are
described herein above in Example 2. In both models, the effect of
the fermented grapes of Falernum on the rate of wound closure was
tested by incubating the scratched monolayer with a final
concentration of 10% and 20% v/v in the cell growth medium.
[0197] The `wound closure` phenomenon was monitored using TLVM
station, to observe the migration of cells to repair the wound
(D'Agostino et al., 2015 BMC cell biology, 16:19, hereby
incorporated by reference). Results (Table 11) indicate a reduction
of the wound closure time in presence of fermented grapes of
Falernum to about half of the control for both the cellular systems
used.
Example 14
Fermented Grapes of Falernum: Antioxidant In Vitro Activity Using
T-BARS (Thiobarbituric Acid Reactive Substances) Assay
[0198] The experiment was conducted as reported in Example 4. The
effect of fermented grapes of Falernum at final concentration of
10% v/v in the incubation medium, on HaCaT cells
(2.0.times.10.sup.5) was tested in three different experimental
setups: 1) cells were pre-treated for 30 min with 50 .mu.M
H.sub.2O.sub.2 or with exposure to a UVA radiation (.lamda..sub.max
365 nm) and then incubated with fermented grapes of Falernum (10%
v/v) for 24 h, to test the protection effect post-stress process;
(2-3) fermented grapes of Falernum were applied simultaneously with
50 .mu.M H.sub.2O.sub.2 or with exposure to UVA radiation
(.lamda..sub.max 365 nm) to test antioxidant activity. Data
reported in Table 12 show that fermented grapes of Falernum is
effective in all the oxidative stress conditions.
Example 15
Cytomimetic Formula Preparation
[0199] 1 L of fermented grapes of Falernum, prepared as reported in
Example 10, were mixed with 0.5 L of Olive leaf extract in thermal
water, prepared as reported in Example 8. This solution was diluted
with 3 L of deionized water and under agitation 0.3% EDTA, 0.4%
sodium benzoate, 12.5 g of 1800 KDa HA, 12.5 g of 800 KDa HA and
12.5 g of 200 KDa and 5 g of fermented truffle extract prepared as
reported in Example 1 were added.
[0200] The pH was corrected at 6.5 with 3% NaOH solution and the
solution was diluted with deionized water up to 5 L. A clear ruby
solution was obtained.
[0201] The resulting composition of the cytomimetic formula is:
fermented truffle extract powder 0.1% (w/v); fermented grapes of
Falernum 20% v/v; Olive leaf extract in thermal water 10% v/v; and
HA 1880, 800, 200 KDa each 0.25% w/v.
Example 16
Synergistic Action of Actives of the Cytomimetic Formulas: In Vitro
Scratch Wound-Healing Assay Using TLVM
[0202] HaCaT and HDF cell line and their growth conditions are
described hereinabove in Example 2. In both models, the effect of
the cytomimetic formula on the rate of wound closure was tested by
incubating the scratched monolayer with a final concentration of 1%
and 2% v/v in the cell growth medium.
[0203] The `wound closure` phenomenon was monitored using a TLVM
station to observe the migration of cells to repair the wound
(D'Agostino et al., 2015 BMC cell biology, 16:19). Results (Table
9) indicate a reduction of the wound closure time in presence of
the cytomimetic formula to about 1/4 of the control for both the
cellular systems used. The data in Table 13 demonstrate a potent
synergistic effect of the actives present in the cytomimetic
formula, which in this experiment are present up to three orders of
magnitude in respect to that reported in Examples 2, 6, 10 and 13
for the single actives.
Example 17
Synergistic Action of Actives of the Cytomimetic Formula:
Antioxidant In Vitro Activity of the Cytomimetic Formula Using
T-BARS (Thiobarbituric Acid Reactive Substances) Assay
[0204] Generation of reactive aldehydes was assessed by measuring
thiobarbituric acid-reactive substances (TBARS), as described
previously by Stiuso et al., (Stiuso P., et al., 2014, Oxidative
Medicine and Cellular Longevity). The effect of the cytomimetic
formula on HaCaT cells (2.0.times.10.sup.5) were tested in three
different experimental setups: 1) cells were pre-treated for 30 min
with 50 .mu.M H.sub.2O.sub.2 or with exposure to a UVA radiation
(.lamda..sub.max 365 nm) and then incubated with cytomimetic
formula (0.1 or 0.5% v/v) for 24 h, to test the protection effect
post-stress process; (2-3) cytomimetic formula (0.32% w/w) was
applied simultaneously with 50 .mu.M H.sub.2O.sub.2 or with
exposure to UVA radiation (.lamda..sub.max 365 nm) to test
antioxidant activity.
[0205] The protein concentrations were determined using the Bio-Rad
protein assay reagent (Bio-Rad Laboratories, Milan Italy). Lipid
peroxidation was evaluated using an analytical quantitative
methodology. It relies upon the formation of a colored adduct
produced by the stoichiometric reaction of aldehydes
(malondialdehydes MDA) with thiobarbituric acid (TBA).
[0206] TBARs assay were performed on aliquots of membranes
extracted (10 .mu.l) and added to 2 ml of TBA-TCA (TCA 15% w/v, TBA
0.3% w/v in. HCl 0.12 N) solution at 100.degree. C. for 30 min. The
chromogen was quantified by spectrophotometric reading at a
wavelength of 532 nm and the amount of TBARs were expressed as a
percentage of lipid peroxidation and then normalized with respect
to control.
[0207] Data reported in Table 14 show whether oxidative stress
occurred before or with the addition of the cytomimetic formula was
effective.
Example 18
Synergistic Action of Cytomimetic Formulate: Effect on HSP 70 and
HSP 90 Induction
[0208] 2.5 millions of HEK-293t cells were treated with a
cytomimetic formulation at 0.1 and 0.5% v/v in a final volume of 10
ml (DMEM medium). The cells were incubated for 18 h at 37.degree.
C. and then harvested. The cells were lysated in RIPA buffer (50 mM
Tris pH 8, 1% nonidet p40 0.25% Sodium Deoxycholate and 1 mM EDTA)
and equal amounts (calculated using the Bradford protein assay) of
soluble fractions were loaded on SDS-PAGE (10% polyacrylamide). The
gel was blotted on PVDF membrane and HSP 70/90 were detected using
commercial antibodies. Anti-.beta.-actin was used as control. The
signals corresponding to HSP 70 and HSP 90 were quantified using
Image J software and the results obtained were plotted.
[0209] Analyzing the cells treated as described above, it was
observed that HSP 70 and HSP 90 gradually increased their
expression when cells where treated with increasing amounts of the
cytomimetic formulation.
[0210] Data from Table 15 indicate the potent synergistic effect of
actives present in the cytomimetic formulation, that in this
experiment are present at an average two orders of magnitude with
respect than that reported in Example 3 for the fermented truffle
extract alone.
Example 19
Cytomimetic Formulate: Gene Expression Analysis in Stressed
Cells
[0211] The gene expression analyses of the cytomimetic formulation
in stressful conditions was performed at 0.1 and 0.5% v/v as
described hereinabove in Example 7. Gene expression data analyses
are reported in Table 16. CD44, RHAMM, TGF.beta.-1, TNF-.alpha. and
IL-6 are all up-regulated increasing Cytomimetic formulate
concentration, only TLR-4 activity, correlated to inflammatory
process, is significantly down-regulated.
Example 20
Base Cosmetic Formulations
[0212] An example of composition used as base to test the
cytomimetic formulation described in Example 12 as reported in
Table 17. Below is the modus operandi of this cosmetic product. In
the main vessel, water is heated to 60.degree. C. Ingredients A2
through A6 are added with medium agitation. Then mixed until
uniform. In the secondary vessel phase B is prepared: ingredients
B1-21 are added and temperature increased to 60.degree. C. Each
ingredient is blended in before adding the next one. The
ingredients are mixed until homogenous. Phase B (oil phase) is
added onto water phase (phase A) and homogenized using low speed.
Phase C (preservatives) is added and cooled down to 25.degree. C.
Then the fragrance is added.
Example 21
Cosmetic Treatment
[0213] An example of composition using Cytomimetic formulation
described in Example 12 is reported in Table 18. Below is the modus
operandi of this cosmetic product. In the main vessel water is
heated to 60.degree. C. Ingredients A1 through A6 are added with
medium agitation, then mixed until uniform. Into the secondary
vessel phase B is prepared: ingredients B1-B21 are added and
temperature increased to 60.degree. C. Each ingredient is blended
in before adding the next one and then mixed until homogenous.
Phase B (oil phase) is added onto the water phase (phase A) and
homogenized using low speed. Phase C (cytomimetic formulation) is
added, followed by phase D (preservatives). It is then cooled down
to 25.degree. C. and then the fragrance added.
Example 22
Clinical Testing Methodology
[0214] The testing application of the base composition and base
composition using a cytomimetic formulation is made by subjects
morning and night, through a gentle massage, so as to have
full-absorption of the product. The measurements will be done in
the right and left zygomatic arch (1 cm. extension of the external
angle), on the cleansed skin, having not used make-up in the
previous 24 hours. This is done after acclimatization of the
subjects for at least 10 minutes in constant temperature/humidity
conditions. Each of the two products will be evaluated against
baseline initial measurements.
[0215] The efficacy will be appreciated through instrumental,
non-invasiveness methods: moisture of corneum layer (Corneometry),
sebumetry, elasticity and firmness, SELS (Surface Evaluation of
Living Skin) parameters: roughness, smoothness, the scaliness
degree, wrinkles). Successive measurements will be done, as
follows: for moisture (triplicate in each point: left/right), for
sebumetry, elasticity, firmness and profilometry an unique
measurement right/left. as follows: for the hydration (Comeometer
MPA 580, Courage+Khazaka electronic GmbH), sebumetry (Sebumeter SM
815, Courage Khazaka electronic GmbH), elasticity (cutometer MPA
580, Courage+Khazaka electronic GmbH), profilometry (Visioscan
VC80, Courage+Khazaka electronic GmbH).
[0216] Skin Hydration is determined instrumentally, through
Corneometry. Measurement principle: measuring the capacitance.
Water increases the capacitance of the capacitor as compared with a
vacuum capacitor (C=.epsilon.S/d). The water dielectric constant c
is 81, as compared with other substances <7 and the vacuum
dielectric constant is 1. The changes in capacitance due to
variations in the dielectric constant give the level of hydration.
The corneometer measures the water content in the surface epidermal
layer up to a depth of 0.1 mm.
[0217] The sebumetry is determined instrumentally, with the help of
a sebumeter. The measurement is based on the absorbance of a
plastic film impregnated with sebum is determined through
photometry, the film becoming transparent in the presence of the
lipids. The sebum values that can me measured are between 50-300
.mu.g/cm.sup.2. Values exceeding 300 .mu.g/cm.sup.2 indicate excess
sebum supersaturation and may be subject to measurement errors,
while values below 50 .mu.g/cm.sup.2 show that there is no
linearity between the values and the sebum content.
[0218] Measurements for skin elasticity are made with a Cutometer.
The measurement principle is based on suction and elongation. The
device generates a negative pressure between 20 and 500 mbar which
"sucks up" a defined skin area it is applied to. In order to
measure the mechanical characteristics of the epidermis 2 mm
diameter probes are necessary while for the dermis and
hypodermis--10 mm diameter probes. When measuring the elasticity
parameters it is important to set the suction pressure according to
the characteristics of the skin (e.g.: the skin around the eyes is
thinner--the suction pressure is lower). The measurements will be
done in Module 1, with a 2 mm probe, negative constant pressure 350
mbars, 2 seconds time of the suction and relaxation. The number of
repetitions will be 10, in order to evaluate the skin firmness and
the tiredness resistance.
[0219] The testing method is called SELS (Surface Evaluation of the
Living Skin) and it is based on the graphic illustration of the
skin surface in special lighting conditions. This image is
processed electronically taking into account four clinical
parameters which correspond to the physiological conditions of the
skin surface both from the quantitative and from the qualitative
point of view and which derive from the roughness conventional
parameters.
TABLE-US-00001 TABLE 1 In vitro scratch wound-healing assay using
TLVM and as cellular model HaCaT chondrocytes and HDF fibroblasts.
Cells were stimulated with truffle extract and two different
concentrations of fermented truffle extract. Cellular model HaCaT
HDF % of wound surface repaired 50 80 100 50 80 100 Sample Repair
time (h) Control 22 33 56 21 40 63 Truffle extract 0.1% w/v* 18 25
40 20 26 42 Fermented truffle extract 10 15 30 12 16 32 0.5** w/v
Fermented truffle extract 8 11 18 8 12 20 1% w/v** Fermented yeast
0.1% w/v*** 16 21 36 18 23 38 *Extracted as described in Example 1
**Fermented truffle extract with Saccharomyces cerevisiae prepared
and described in Example 1 ***Fermented Saccharomyces cerevisiae
prepared as described in Example 1
TABLE-US-00002 TABLE 2 Effect of fermented/non-fermented truffle
extract and of fermented yeast on HSP 70 and HSP 90 induction
Concentration (% w/v) in the growth medium Samples 0.0 01 02 05 10
HSP 70 expression (respect to the control = 100) Truffle extract*
100 98 95 97 101 Fermented truffle 100 105 130 138 180 extract**
Fermented yeast*** 100 101 105 110 125 HSP 90 expression (respect
to the control = 100) Truffle extract* 100 96 97 100 102 Fermented
truffle 100 100 135 141 200 extract** Fermented yeast*** 100 98 108
115 125 *Extracted as described in Example 1 **Fermented truffle
extract with Saccharomyces cerevisiae prepared as described in
Example 1 ***Fermented Saccharomyces cerevisiae prepared as
described in Example 1
TABLE-US-00003 TABLE 3 Antioxidant in vitro activity of fermented
and non- fermented truffle extract using T-BARS (thiobarbituric
acid reactive substances) assay (see Example 3). HaCaT HDF
Protection Protection Lipid peroxidation Sample (w/v during stress
after stress % respect the in DMEM) treatment* treatment** control
Control 100 100 No extract added UV-A 460 438 Fermented truffle
UV-A 101 108 extract 0.5% Truffle extract 0.5% UV-A 218 190 No
extract added H.sub.2O.sub.2 525 538 Fermented truffle
H.sub.2O.sub.2 112 120 extract 0.5% Truffle extract 0.5%
H.sub.2O.sub.2 198 205 Fermented truffle UV-A 111 121 extract 0.5%
Truffle extract 0.5% UV-A 230 211 Fermented truffle H.sub.2O.sub.2
109 119 extract 0.5% Truffle extract 0.5% H.sub.2O.sub.2 233 214
*Effect on lipid peroxidation of 0.5% w/v fermented truffle extract
or non-fermented truffle extract present during stress conditions.
**Effect on lipid peroxidation of 0.5% w/v fermented truffle
extract or non-fermented truffle extract applied after stress
conditions.
TABLE-US-00004 TABLE 4 The table represented the extrapolated data
of Viscotek analysis: molecular weight (Mw), polydispersion index
Mw/Mn, intrinsic viscosity (IV) and hydrodynamic radius. Sample Mw
IV Rh (KDa) (KDa) Mw/Mn (dl/g) (nm) HHA 1800 1835 .+-. 7 1.65 .+-.
0.12 24.91 .+-. 0.32 87.06 .+-. 0.43 HHA 1400 1398 .+-. 9 1.50 .+-.
0.14 22.11 .+-. 0.91 67.09 .+-. 0.10 LHA 800 788 .+-. 5 1.62 .+-.
0.11 17.01 .+-. 0.23 48.11 .+-. 0.32 LHA 200 198 .+-. 3 1.42 .+-.
0.22 3.78 .+-. 0.44 20.15 .+-. 0.60 LHA 100 97 .+-. 5 1.52 .+-.
0.24 2.88 .+-. 0.33 15.19 .+-. 0.55 LHA 50 51 .+-. 5 1.66 .+-. 0.20
1.71 .+-. 0.31 11.71 .+-. 0.33
TABLE-US-00005 TABLE 5 In vitro scratch wound-healing assay using
TLVM and as cellular model HaCaT chondrocytes and HDF fibroblast.
Cells vere stimulated with HA 1800, 800, 200 KDa and their mixture
(1% w/w respect to growth medium). Cellular model HaCaT HDF % of
wound surface repaired 50 80 100 50 80 100 Sample (KDa) Repair time
(h) Control 20 31 55 22 38 60 HA1800 4 12 21 6 16 28 HA800 10 15 35
12 18 39 HA200 12 20 40 16 26 45 HA1800 + HA800 3 10 18 5 14 20
HA1800 + HA800 + HA200 3 8 15 4 10 17
TABLE-US-00006 TABLE 6 Gene Forward primer Reverse primer Primer
Cycles Transforming 5'TgCggCAgCTgTACATTgA3' 5'TggTTgTACAgggCCAggA3'
95.degree.C. 10 s, 55.degree. C. growth factor, 30 s, 72.degree. C.
3 min, beta 1 (TGF.beta.-1) 40 cycles Tumor necrosis
5'CgAgTgACAAgCCTgTAg3' 5'ggTgTgggTgAggAgCACAT3' 94.degree. C. 1
min, 55.degree. C. factor alpha 2 min, 72.degree. C. 3 min,
(TNF.alpha.) 40 cycles Interleukin 5'gCCgCCTTTAACTggAgCAA'3
5'TTCCAggCATCTgCgATgAg3' 95.degree. C. 10 s, 55.degree. C. (IL-6)
30s, 72.degree. C. 3 min, 40 cycles Cluster of
5'gCgCCACCACAgCCAACTATg'3 5'TggATGCCgTCTATgTCgTC 94.degree. C. 1
min, 60.degree. C. differentiation TTTA3' for 2 min, 72.degree. C.
3 44 min, 40 cycles (CD44) Toll-like 5'TCCCAggAATTggTgATAAAgT
5'CTggCATgACgCgAACAAT 95.degree. C. 10 s, 60.degree. C. receptors 4
AgA'3 A'3 30 s, 72.degree. C. 3 min, (TLR4) 40 cycles Receptor for
5'gATAATCCgCATTCAgTTgTC- 5'TAACATCATAAGCACCTG 95.degree. C. 10 s,
60.degree. C. Hyaluronan 3' GAG-3' 30 s, 72.degree. C. 3 min,
Mediated 40 cycles Motility (RHAMM) Oligonucleotide sequences
relative to biomarkers used. Forward primers are, top to bottom
respectively, SEQ ID NOS: 1-6. Reverse primers are, top to bottom
respectively, SEQ ID NOS: 7-12.
TABLE-US-00007 TABLE 7 Gene expression induced by HA of different
Mw. Cellular model HaCaT HDF Normalized fold expression HA Mw (KDa)
1800 800 200 1800 800 200 CD44 5.1 4.8 4.0 6.8 5.3 3.1 RHAMM 4.8
2.3 12.2 5.7 2.9 14.0 TLR-4 0.8 0.6 0.5 1.1 0.9 1.2 TGF-.beta.1 4.6
15.2 15.0 5.8 14.6 18.3 TNF.alpha. 3.1 6.2 4.1 2.9 7.5 5.6 IL-6 1.7
3.8 3.4 2.5 4.5 4.6
TABLE-US-00008 TABLE 8 Antioxidant in vitro activity of HA1800 +
HA800 + HA200 using T-BARS (thiobarbituric acid reactive
substances) assay. HaCaT HDF Protection Protection Lipid
peroxidation Sample (v/v during stress after stress % respect the
in DMEM) treatment* treatment** control Control 100 100 No
additions UV-A 450 460 reference HA1800 + HA800 + UV-A 132 139
HA200 No additions H.sub.2O.sub.2 533 540 reference HA1800 + HA800
+ H.sub.2O.sub.2 118 122 HA200 HA1800 + HA800 + UV-A 121 118 HA200
HA1800 + HA800 + H.sub.2O.sub.2 131 127 HA200 *Effect on lipid
peroxidation of 0.5% w/v HA1800 + HA800 + HA200 present during
stress conditions. **Effect on lipid peroxidation of 0.5% w/v
HA1800 + HA800 + HA200 applied after stress conditions.
TABLE-US-00009 TABLE 9 In vitro scratch wound-healing assay using
TLVM and as cellular model HaCaT chondrocytes and HDF fibroblasts.
Cells were stimulated with Olive leaf extract in thermal water
prepared as described in Example 10. Cellular model HaCaT HDF % of
wound surface repaired 50 80 100 50 80 100 Sample Repair time (h)
Control 23 30 51 23 38 60 Olive leaf extract in thermal water 12 16
30 12 15 31 10% v/v* Olive leaf extract in water 10% v/v* 16 20 39
18 21 38 Thermal water 20 25 45 21 30 50 *% respect to growth
medium
TABLE-US-00010 TABLE 10 Antioxidant in vitro activity of Olive leaf
extract in thermal water using T-BARS (thiobarbituric acid reactive
substances) assay. HaCaT HDF Protection Protection Lipid
peroxidation Sample (v/v during stress after stress % respect the
in DMEM) treatment* treatment.degree. control Control 100 100 No
extract added UV-A 435 440 Olive leaf extract in UV-A 138 144
thermal water 10% v/v* No extract added H.sub.2O.sub.2 540 530
Olive leaf extract in H.sub.2O.sub.2 129 131 thermal water 10% v/v*
Olive leaf extract in UV-A 133 142 thermal water 10% v/v* Olive
leaf extract in H.sub.2O.sub.2 123 131 thermal water 10% v/v*
*Effect on lipid peroxidation of 10% v/v Olive leaf extract in
thermal water present during stress conditions. **Effect on lipid
peroxidation of 10% v/v Olive leaf extract in thermal water applied
after stress conditions.
TABLE-US-00011 TABLE 11 In vitro scratch wound-healing assay using
TLVM and as cellular model HaCaT chondrocytes and HDF fibroblasts.
Cells were stimulated with fermented Falernum grapes prepared as
described in Example 13. Cellular model HaCaT HDF % of wound
surface repaired 50 80 100 50 80 100 Sample Repair time (h) Control
22 33 50 22 36 58 Fermented Falernum grapes 10% v/v* 18 24 40 19 23
45 Fermented Falernum grapes 20% v/v* 13 19 29 14 19 28 *% respect
to growth medium
TABLE-US-00012 TABLE 12 Antioxidant in vitro activity of fermented
Falernum grapes 10% v/v* using T-BARS (thiobarbituric acid reactive
substances) assay. HaCaT HDF Protection Protection Lipid
peroxidation Sample (v/v during stress after stress % respect the
in DMEM) treatment* treatment.degree. control Control 100 100 No
fermentate added 453 455 Fermented Falernum UV-A 131 135 grapes 10%
v/v* No fermentate added 545 560 Fermented Falernum H.sub.2O.sub.2
133 145 grapes 10% v/v* Fermented Falernum UV-A 124 131 grapes 10%
v/v* Fermented Falernum H.sub.2O.sub.2 127 135 grapes 10% v/v*
*Effect on lipid peroxidation of 10% v/v Fermented Falernum grapes
present during stress conditions. **Effect on lipid peroxidation of
10% v/v Fermented Falernum grapes 10% v/v applied after stress
conditions.
TABLE-US-00013 TABLE 13 In vitro scratch wound-healing assay using
TLVM and as cellular model HaCaT chondrocytes and HDF fibroblasts.
Cells were stimulated with Cytomimetic formulations as described in
Example 16. Cellular model HaCaT HDF % of wound surface repaired 50
80 100 50 80 100 Sample Repair time (h) Control 22 33 50 22 36 58
Cytomimetic formulation 1% v/v* 10 16 20 13 17 21 Cytomimetic
formulation 2% v/v* 7 10 14 8 12 15 *% respect to growth medium
TABLE-US-00014 TABLE 14 Antioxidant in vitro activity of
Cytomimetic formulation using T-BARS (thiobarbituric acid reactive
substances) assay. HaCaT HDF Protection Protection Lipid
peroxidation Sample (w/v during stress after stress % respect the
in DMEM) treatment* treatment.degree. control Control 100 100 No
Cytomimetic 450 460 Cytomimetic UV-A 130 134 formulation 0.1%
Cytomimetic UV-A 98 100 formulation 0.5% No Cytomimetic 540 530
Cytomimetic H.sub.2O.sub.2 120 115 formulation 0.1% Cytomimetic
H.sub.2O.sub.2 95 100 formulation 0.5% Cytomimetic UV-A 128 132
formulation 0.1% Cytomimetic UV-A 96 100 formulation 0.5%
Cytomimetic H.sub.2O.sub.2 133 135 formulation 0.1% Cytomimetic
H.sub.2O.sub.2 95 98 formulation 0.5% *Effect on lipid peroxidation
of of 0.1 and 0.5% v/v Cytomimetic formulation present during
stress conditions. **Effect on lipid peroxidation of of 0.1 and
0.5% v/v Cytomimetic formulation v/v applied after stress
conditions.
TABLE-US-00015 TABLE 15 Effect of Cytomimetic formulation on HSP 70
and HSP 90 induction. Concentration Cytomimetic formulation (% v/v)
in the growth medium Sample 0.0 0.1 0.2 0.5 1.0 HSP 70 expression
(respect to the control = 100) Cytomimetic 100 105 130 138 180
formulation HSP 90 expression (respect to the control = 100)
Cytomimetic 100 100 135 141 200 formulation
TABLE-US-00016 TABLE 16 Gene expression induced by Cytomimetic
formulation. HaCaT HDF Cellular model Normalized fold expression
Cytomimetic concentration 0.1 0.5 0.1 0.5 (% v/v) CD44 6.4 8.3 6.5
7.3 RHAMM 4.9 6.3 5.1 7.9 TLR-4 0.3 0.2 0.4 0.2 TGF-.beta.1 6.6 8.4
6.7 9.6 TNF.alpha. 4.1 6.7 3.9 6.4 IL-6 1.9 2.8 2.2 4.5
TABLE-US-00017 TABLE 17 Example of Base formula Phase A Ingredient
Percentage A Water 60-90% A1 Glycerin 3-10% A2 Glyceryl
Polyacrylate 1-15% A3 Acrylates Copolymer .sup. 5% A4 Butylene
Glycol 1-5% A5 Carbomer 0.1-1% A6 Xanthan Gum 0.1-1% Phase B OIL
PHASE INGREDIENTS Percentage B1 Olea Europaea Fruit Oil .sup. 1-10%
B2 Stearoxymethicone/Dimethicone Copolymer 0.1-10% B3
Polymethylsilsesquioxane 0.1-10% B4 Polyacrylate-13 0.1-10% B5
HDI/Trimethylol Hexyllactone Crosspolymer 0.1-10% B6 Polyisobutene
0.1-10% B7 Cholesteryl Nonanoate 0.1-10% B8 Hydrogenated Lecithin
0.1-10% B9 Polysorbate 20 0.1-10% B10 Cholesteryl Chloride 0.1-10%
B11 Sodium Acrylates Copolymer 0.1-10% B12 Cholesteryl Oleyl
Carbonate 0.1-10% B13 Silica 0.1-10% B14 Methyl Methacrylate
Crosspolymer 0.1-10% Phase C Preservatives C1 Phenoxyethanol
0.1-1.5% C2 Ethylhexylglycerin 0.1-3% D Fragrance 0.1-10%
TABLE-US-00018 TABLE 18 Example of base formula and of
cosmeceutical preparation including Cytomimetic formulations Phase
A Ingredient Percentage A Water 60-90% A1 Glycerin 3-10% A2
Glyceryl Polyacrylate 1-15% A3 Acrylates Copolymer .sup. 5% A4
Butylene Glycol 1-5% A5 Carbomer 0.1-1% A6 Xanthan Gum 0.1-1% Phase
B OIL PHASE INGREDIENTS Percentage B1 Olea Europaea Fruit Oil .sup.
1-10% B2 Stearoxymethicone/Dimethicone Copolymer 0.1-10% B3
Polymethylsilsesquioxane 0.1-10% B4 Polyacrylate-13 0.1-10% B5
HDI/Trimethylol Hexyllactone Crosspolymer 0.1-10% B6 Polyisobutene
0.1-10% B7 Cholesteryl Nonanoate 0.1-10% B8 Hydrogenated Lecithin
0.1-10% B9 Polysorbate 20 0.1-10% B10 Cholesteryl Chloride 0.1-10%
B11 Sodium Acrylates Copolymer 0.1-10% B12 Cholesteryl Oleyl
Carbonate 0.1-10% B13 Silica 0.1-10% B14 Methyl Methacrylate
Crosspolymer 0.1-10% Phase C Cytomimetic formula 0.1-10% Phase D
Preservatives D1 Phenoxy ethanol 0.1-1.5% D2 Ethylhexylglycerin
0.1-1.5% Phase E Fragrance 0.1-10%
TABLE-US-00019 TABLE 19 Clinical results. Improvement (%) Initial
Base cream relative to Methodology Parameter (average) Base Cream
and Cyto base formula Profilometry Scaliness (Secc) 0.90 .+-. 0.13
0.71 .+-. 0.08 0.65 .+-. 0.08 .sup. 116% Roughness (Ser) 3.39 .+-.
0.36 3.16 .+-. 0.35 3.09 .+-. 0.25 76.6% Smoothness (Sesm) 46.36
.+-. 2/50 43.38 .+-. 3.20 40.09 .+-. 3.5 47.5% Wrinkless (Sew)
47.23 .+-. 6/56 44.09 .+-. 2.96 42.63 .+-. 2.54 73.9% Sebumetry
Oily Skin 258.65 .+-. 31/35 238.67 .+-. 17.64 202.67 .+-. 23.02
35.67% Dry Skin 33.50 .+-. 9.38 96.57 .+-. 15.42 118.38 .+-. 18.05
74.30% Cutometry Gross Elasticity 0.58 .+-. 0.13 0.61 .+-. 0.09
0.63 .+-. 0.11 40.00% (R2) Net Elasticity 0.42 .+-. 0.09 0.44 .+-.
0.13 0.46 .+-. 0.07 50.00% (R5) Corneometry Hydration 73.28 .+-.
7.25 74.24 .+-. 3.59 76.25 .+-. 7.35 33.33%
Sequence CWU 1
1
12119DNAArtificial Sequenceprimers directed to human genes
1tgcggcagct gtacattga 19218DNAArtificial Sequenceprimers directed
to human genes 2cgagtgacaa gcctgtag 18320DNAArtificial
Sequenceprimers directed to human genes 3gccgccttta actggagcaa
20421DNAArtificial Sequenceprimers directed to human genes
4gcgccaccac agccaactat g 21525DNAArtificial Sequenceprimers
directed to human genes 5tcccaggaat tggtgataaa gtaga
25621DNAArtificial Sequenceprimers directed to human genes
6gataatccgc attcagttgt c 21719DNAArtificial Sequenceprimers
directed to human genes 7tggttgtaca gggccagga 19820DNAArtificial
Sequenceprimers directed to human genes 8ggtgtgggtg aggagcacat
20920DNAArtificial Sequenceprimers directed to human genes
9ttccaggcat ctgcgatgag 201024DNAArtificial Sequenceprimers directed
to human genes 10tggatgccgt ctatgtcgtc ttta 241120DNAArtificial
Sequenceprimers directed to human genes 11ctggcatgac gcgaacaata
201221DNAArtificial Sequenceprimers directed to human genes
12taacatcata agcacctgga g 21
* * * * *