COSMETIC DETAILED DESCRIPTION & MEDICAL EXPERIMENTS

Research of the essential oil extracted by hydro distillation or by means of an extraction fluid preferably at or close to the supercritical state from the plant material of the flower tops of a plant, known as the flower summit, and belonging to the Asterasceae (daisy) family: Helichrysum Italicum variety serotinum, also named Helichrysum angustifolium D.C has been conducted in order to comprehend the beneficial virtues of the different chemical compositions of the flower and its cosmetic properties.
The following discoveries of a remarkable positive result in cosmetic and dermatologic properties have been confirmed in the essential oil produced by the plant called Helichrysum Italicum. The Neryl Acetate contents have an extremely high percentage. This provides the Helichrysum Italicum with the following particularities:

The hydroxyl radical is a very reactive molecule, the production of which is activated by the different stresses to which the skin is subjected, pollution, UV, temperature changes and stressful aggressions. This radical is responsible for numerous waste products in the cell and especially the degradation of the membranes by the phenomenon of lipid peroxidation.

Sounds confusing ? In fact this radical is responsible for all the “not wanted waste” found in your cell which is activated due to stress; unfortunately it also declines the membranes of your cells due to a surge of lipid peroxidation.
The experiments and tests carried out will show you the realistic results of Helichrysum Italicum in skin care and its effectiveness upon wrinkles. This method, used by the cosmetic technicians, makes it possible to test the protective action of the essential oil on liposomes, cell membranes for the rest of us, subjected to an attack by hydroxyl radicals (OH-).
The action of free radicals on the polyunsaturated fatty acids of the membranes is a “peroxidation” which leads to the formation of conjugated dienes absorbing at 233 nm. The activity of the antioxidants is, therefore, determined by their capacity to prevent peroxidation of polyunsaturated fatty acids contained in liposomes. This peroxidation is characterized by an absorption peak at 233 nm. Cosmetic technicians found that the essential oil of Helichrysum italicum inhibits lipid peroxidation by 82%+2% under the experimental conditions. This result is remarkably positive. Tested under the same operating conditions, an essential oil of lavender inhibited lipid peroxidation by 19%+2%. This is a great explanation as to why Helichrysum Italicum is as efficient as an anti ageing in cosmetic skin care.
This antiradical activity is essential in the battle against accelerated aging. The preferential targets of the free radicals are the lipids of the cell membranes. By attacking them, they disturb the meticulous organization. The membrane loses its fluidity and its cohesion. In the long term, it breaks up and can no longer fulfil its role as a protective barrier. The connecting tissue, constituted essentially of collagen, is also affected. This accelerates the aging process. That explains that then! We all need to protect our lipids with Helichrysum Italicum .

Two simple concepts should be kept in mind to better comprehend the phenomenon of accelerated aging:
These two main reasons will help you to understand better the phenomenon of accelerated ageing. Just a small comment, it’s a bit of paradox that we spend much of our youth trying to look older and the best part of adulthood trying to look younger.
The first is an atom, which has an electrically neutral set of elements in equilibrium.
The second is a free radical, which is an atom possessing a single electron which confers upon the radical a very high degree of reactivity in the search for another electron to restore a stable state.

I’m sure the amount of research and hard work over a long period of time of such quality by the cosmetic technicians can only, and I’m sure you’ll agree readers, be immensely thanked.
In the search for a stable state, the free radicals are very active and induce noteworthy reactional modifications within the cell, modifications that can reach the DNA and the amino acids. They depolymerize the glycoprotein constituents of the fundamental substance such as hyaluronic acid. This is the stuff that re pulps your lips, naturally, or other parts of, more commonly, your face. I think that most of us recognize the term “free radicals”, and no, it’s not a political movement just the little monsters responsible for wrinkles.

The free radicals have a profoundly negative impact on the lipids, proteins and other components of the cell membrane. The attacks on the cell membranes, due to the peroxidation process, increase with age. Peroxides and free radicals are directly linked with this latter mentioned process. These studies also showed the importance of antioxidants in the longevity process. In fact, the animal or plant cell protects itself against oxidation agents by means of enzyme systems and natural antioxidants.
Helichrysum italicum essential oil has been found to be an effective natural antioxidant.

A Composition for the Face

The following formulation of a product for the face containing 0.7% of essential oil of Helichrysum italicum was prepared to demonstrate antiwrinkle activity:

The study of the cutaneous relief was performed. The test involved 20 volunteer subjects. Application of cosmetics or other topical products was discontinued 48 hours prior to the beginning of the experiment. The product was applied each morning for four weeks and the following parameters were evaluated:

Total Wrinkled Surface:
This parameter provides information on the total surface area of the wrinkles (mm<2> )

Average at Time 0=4.571 mm<2 >
Average at Time 4 weeks =2.843 mm<2 >
Thus, a decrease of 37.8%.
Number of Wrinkles:
Average at Time 0=61.3
Average at Time 4 weeks =52.7
Thus, a decrease of 14%.
Average Depth of the Wrinkles (Taking into account the number of wrinkles that had disappeared after treatment):
A decrease of 19.8%.

The results for these three parameters were very satisfactory and demonstrated the remarkable antiwrinkle property of the compositions according to the invention. Biomechanical properties of the skin with the composition for the face lead to the recruitment of twenty users for daily application over a four week period.
Result: UR/UB=R5: firmness under stress: +14.7%+0.11.
The composition for the face above provides a good cutaneous firmness. Evaluation of the hydration by the prints method with the composition for the face
Twenty users were recruited for a daily application over four weeks.

Result: The parameter AR=Average roughness decreased by 8.9%, which was manifested by cutaneous smoothness, i.e., a hydrating effect. Encapsulation of Helichrysum italicum essential oil in nanospheres.
The oil was incorporated in nanospheres to optimize the activity of Helichrysum italicum essential oil. This method has the advantage of increasing the bio availability of the active ingredients. Their controlled release augments the chrono availability at the level of the cutaneous cells. The Helichrysum italicum essential oil was encapsulated in spherulites. Spherulites are multilamellar vesicles of approximately one micron which imitate the structure of an onion. The hundreds of membranes of which they are composed form an alternation of bilayers containing sucroester and an optimized level of essential oils. Because of the slow and controlled release of the active ingredient, the spherulites make it possible to reduce the percentage of essential oil while still retaining the same level of efficacy. The benefit is a very improved tolerance, hypoallergenic and an activation of the microcirculation.
This is the example used to demonstrate the cosmetic virtues on the skin.

This was done in the form of a facial cream.

A study of the cutaneous relief was performed using the prints method. The experiment involved 20 volunteer subjects. Applications of cosmetics or other topical products were suspended 48 hours prior to the beginning of the experiment. The product was applied every morning over four weeks and the following parameters were evaluated:
Total Wrinkled Surface:
This parameter provides information on the total surface area of the wrinkles (mm<2>)
Average at Time 0=4.571 mm<2 >
Average at Time 4 weeks=2.843 mm<2 >
A decrease of 37.8%.
Number of Wrinkles:
Average at Time 0=61.3
Average at Time 4 weeks =52.7
Thus, a decrease of 14%/ which is pretty good.
Average Depth of the Wrinkles (Taking into Account the Number of Wrinkles That Had Disappeared after Treatment):
A decrease of 19.8%. Now that’s even better.
The results for these three parameters were very satisfactory and demonstrated the remarkable antiwrinkle property of the compositions according to the invention. Study of the biomechanical properties of the skin with the composition in nanospheres.
Twenty users were recruited for daily administration over four weeks.
Result: UR/UB=R5: firmness under stress: +14.7%+0.11.
The composition of the composition in nanosphere above provides good cutaneous firmness.
Result: The parameter AR=Average roughness decreased by 8.9%, which was manifested by cutaneous smoothness, i.e., a hydrating effect.
Introduction
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. Anthony Bozzi mentioned to me the effects of Helichrysum Italicum on the 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.