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.