The fibers of the dermis
The fibers of the dermis comprise collagen fibers and
elastic fibers. Quantitatively, they represent the more
important structural proteins of the dermis, i.e., respectively
75 and 5% of their dry weight. Their relative proportion
and their arrangement differ according to the superficial
or deep regions of the dermis.
The
fibroblasts
The fibroblasts are responsible for the synthesis and
maintenance of the extra cellular material. These are
the cells of mesenchymal origin which synthesize collagen,
elastin, the fundamental substance and the structural
glycoprotein’s.
The
collagen fibers:
The collagens form a very large family. They are molecules
with an extra cellular matrix composed of three polypeptide
chains bearing the repetition of three amino acids:
-Gly-X-Y, in which X and Y are often prolines or hydroxyprolines.
The collagen fibers of the dermis are constituted respectively
of collagen I and collagen III, around an axis composed
of collagen V. These collagens belong to the group of
fibrillary collagens. In the adult, collagen I is on
average six times more abundant than collagen III.
Collagens
and ageing:
The collagen I/collagen III ratio decreases over the
course of aging. There can be seen an exponential increase
in chemical bridges between the collagen fibers due
to Maillard's non enzymatic glycation reaction. This
chemical bridging of the collagen results in a rigidification
of the fibers. Its degradation and its renewal are thereby
slowed down.
Modification of the fibroblasts: The fibroblast is a
key cell of the connective tissue which intervenes in
the formation and stabilization of the elastic fibers,
but also in their dystrophy and their lysis. Upon aging,
the fibroblast decreases its activity and this cell
at rest is called a fibrocystic. It becomes globular
with diminution of its cytoplasm and rarefaction of
its endoplasmic reticulum the vesicles of which are
very dispersed. This cell is no longer in contact with
the collagen.
Principle
of the Study
I thought I’d mention that the following results
are fairly complicated and obviously chemistry related,
I wish you all a good read and hope that you have some
head ache pills on hand?
The modification promoting the loss of elasticity and
firmness of the skin due to the disorganization and
rarefaction of the collagen was studied to demonstrate
the efficacy of the compositions of the invention.
Thus,
the activity of the composition on the secretion of
collagen I in the culture medium by the fibroblasts
was evaluated. The experimental model employed consisted
of a culture of normal human fibroblasts up to confluence.
These cells were incubated with the composition for
the face at 0.05% (concentration determined to be non
cytotoxic for the cells in the MTT test). Using the
enzyme-linked immunosorbent assay (ELISA) method, which
has the advantage of being easy to perform, sensitive
and specific; we determined quantitatively the type-1
collagen in the culture medium.
In
a first step, the fibroblast cultures were established
by the explant method from samples of healthy Caucasian
skin (abdominal plasty). The fibroblasts were cultured
until confluence at 37[deg.] in a minimum essential
medium (MEM) containing 2 mM of L-glutamine and 10%
fetal calf serum in a moisture-saturated atmosphere
with 5% of CO2. Then, after detachment with a buffered
pH 7.2 isotonic solution of trypsin at 0.1% and EDTA
0.02%, the cells were distributed on 96-well microplates
(Falcon) at the rate of 10<4 > cells per well
in the previously specified medium. After 24 hours,
this medium was replaced for an incubation of 48 hours
in the same medium without serum containing 0.15 mM
of sodium ascorbate (incubation medium) with or without
the complex of active ingredients to be tested.
A
type of fibroblast was selected for the study, being
fibroblasts of the 4<th > passage or re-inoculation
of culture, representing cells having normal characteristics
(fusiform or star-shaped appearance, good metabolic
activity, good spreading capacity and the like).
Quantitative determination of the collagen: The quantity
of type-I collagen secreted in the medium after incubation
with or without the active complex was determined using
an ELISA test. This method detects certain non radioactive
molecules at very low levels. A direct method was selected,
consisting of the absorption of the antigen (type-I
collagen) in a solid phase (plastic of a micro titration
plate designed for ELISA).
The
incubation medium was collected and transferred into
the wells of a micro titration plate. Incubation for
24 hours at+4[deg.] C. caused adhesion of the collagen
to the plastic. The plate was then rinsed in phosphate
buffer saline (PBS). After 24 hours of incubation at+4[deg.]
C. with serum albumin (2% in PBS) to avoid non-specific
antibody-plastic binding, murine anticollagen I antibodies
(Sigma) (dilution 1: 2000) conjugated to an alkaline
phosphatase (2 hours at ambient temperature), which
in the presence of paranitrophenyl phosphate would produce
paranitrophenol absorbing at 405 nm (1 hour at ambient
temperature). The optical densities obtained were converted
into ng of collagen using a calibration curve established
under the same experimental conditions with type-I collagen.
The
composition for the face used at 0.05% in the culture
medium enabled the fibroblasts to synthesize up to six
times more type-I collagen compared to the control (culture
medium alone).
The composition for the face initiated and stimulated
synthesis programs of one of the predominant components
of the extra cellular matrix of the dermis, type-I collagen.
It is thus an excellent booster of the reorganization
of the collagen fibers. Thus, the composition for the
face improves the tonicity of the skin. The study of
the antigenic capacity of the composition or the face.
(This more than confirms the positive effects of Helichrysum
Italicum and the regeneration of collagen.)
Angiogenesis is a fundamental process by which new blood
vessels are formed. A large number of events are involved
including:
Proteolytic degradation of the extra cellular matrix;
Migration and proliferation of endothelial cells;
Formation of a new extra cellular matrix; and
Formation of tubules and anastomoses of the neoformed
vessels. These actions depend on vascular endothelial
growth factor (VEGF). VEGF is an antigenic factor and
a mitogen for endothelial cells. It is a chemo tactic
factor for the monocytes and osteoblasts. Growth factor
of the vascular endothelium, which is a vascular proliferation
factor of production of neovessels, enables revascularization.
This is relevant to all the thread veins apparent on
some people’s cheeks etc.
In
vitro experimental models take into account solely in
an individualized manner the different components of
the angiogenesis process by studying the proliferation
of endothelial cells or their migration or capacity
to associate together in tubules when they are in contact
with proteins of the extra cellular matrix. However,
none of these models reflects the global impact of angiogenesis.
The in vitro model we used was the AngioKit (Biopredic-ref
ZHA-1000-lot no. 25173T) which approaches a more global
vision of angiogenesis. In this model, human endothelial
cells are seeded in a specific medium in co-culture
with another type of human cells. In the initial phase,
the endothelial cells form small islets. They begin
to proliferate and then enter into a migration phase
which leads to formation of structures of filamentous
tubule form in the matrix. At the end of 10 to 12 days,
the connections established form a network of anastomosed
tubules. The staining of the tubules is obtained by
specific marking of the antigen of surface antigen CD31
(PECAM-1), which is expressed by the endothelial cells.
The test is validated by staining the tubules and verification
of the inhibitory and activating action of two pharmacological
compounds known to act on angiogenesis. The action on
angiogenesis of the active ingredients tested was evaluated
by quantitative image analysis.
The cells were cultured in 24-well plates in an incubator
at 37[deg.] C. under a humid atmosphere containing 5%
CO2. On the first day of the experiment, the microscopic
observation showed the cells in the initial growth stage
with a few small islets of endothelial cells.
The specific culture medium was eliminated and the cells
were placed under different experimental
Conditions:
Control:
culture medium;
Negative control: suramin, agent inhibiting formation
of tubules-final concentration: 20 [mu] M;
Positive control: VEGF, activating agent of angiogenesis-final
concentration: 2 ng/ml;
Composition for the face: Since this composition of
essential oils is not miscible in the culture medium,
it was dissolved in absolute ethanol and tested at three
concentrations (the non-cytotoxic character of which
had been verified in advance by 24-hour contact with
normal human fibroblasts): 0.005%-0.01%-0.05% (v/v);
and
Solvent control: since ethanol could have effects on
angiogenesis, it was tested at the final concentration
required for dissolving the essential oils composition:
0.1% (v/v).
Each condition was implemented in duplicate. Microscopic
observation of the cells was performed daily to monitor
formation of tubules.
The media were eliminated and the conditions were renewed
on the 3<rd>, 6<Th > and 9<Th > day
of the experiment. On the 10 <th> days, the cells
were fixed and stained: after rinsing in the PBS buffer,
the cells were fixed in a 70% ethanol solution cooled
at -20[deg.] C. for 30 minutes. The cells were then
incubated for 1 hour at 37[deg.] C. with the human anti-CD-31
ZIS -3090 antibody (TCS Cell Works). After washing,
a second incubation of one hour at 37[deg.] C. was performed
with a murine anti-IgG antibody conjugated with alkaline
phosphatase. After rinsing, addition of BCIP/NBT substrate
induced dark violet coloration of the tubules after
incubation for 10 to 15 minutes.
Evaluation of the antigenic power of the compound tested
was quantified by image analysis (Olympus IX70 Auto
Analysis System) by measuring the length of the tubules
developed (in [mu]m) and the number of junctions established
within the anastomosed network. The results correspond
to the average values obtained by analyzing 8 images
for each condition.
Evaluation of the antigenic power of the composition
for the face was performed by analysis of two criteria:
the length of the tubular network and the number of
junctions present in this network.
The quantitative analysis regarding the length of the
neoformed tubules and the results obtained for the pharmacological
compounds used as internal reference show in the presence
of suramin an inhibition of 49% regarding the length
of the tubular network compared to the control. VEGF
had a stimulator effect of 54%. The solvent ethanol
also interacted with the angiogenesis with an inhibitory
effect of 11% compared to the control. The results obtained
with the "Immortelle" elixir composition were
calculated in relation to the solvent control.
The composition of the essential oil of Helichrysum
angustifolium D.C. exhibited a slight stimulatory effect
with regard to the length of the tubular network formed
compared to the solvent condition. It was increased
respectively by 6% at the concentration of 0.005%, by
11% at the concentration of 0.01% and by 6% at the concentration
of 0.05%. These variations, however, were not statistically
significant.
The results of the quantitative analysis regarding the
number of junctions established in the tubular network
were diminished by a factor of 5.7 compared to the control
in the presence of suramin. In contrast, with VEGF,
the number of junctions was augmented by a factor of
1.7. The solvent ethanol also interacted by diminishing
by a factor of 2 the number of junctions present in
the network.
In the presence of the composition for the face, there
can be seen for each of the concentrations a marked
augmentation in the number of junctions compared to
the solvent condition: augmentation by a factor of 1.5
at the concentration of 0.005%, augmentation by a factor
of 2 at the concentration of 0.01% and augmentation
by a factor of 1.6 at the concentration of 0.05%. It
should be noted that these stimulatory effects had intensity
close to or even greater than those obtained with the
positive reference (VEGF).
The composition for the face does not inhibit angiogenesis.
To the contrary, it induces stimulation by a factor
of 2 of the number of anastomoses, an effect comparable
to or even greater than that of VEGF. It thus exerts
a stimulating role on the essential parameters of anglogenesis.
Well
that made some pretty interesting reading; it certainly
proves the medical use of Helichrysum Italicum.
