Dissertation committee:
Παντερή Ειρήνη, Καθηγήτρια, τμήμα Φαρμακευτικής, ΕΚΠΑ
Βαρβαρέσου Αθανασία, Καθηγήτρια, Τμήμα Βιοϊατρικών Επιστημών, ΠΑΔΑ
Θωμαΐδης Νικόλαος, Καθηγητής, τμήμα Χημείας, ΕΚΠΑ
Οικονόμου Αναστάσιος, Καθηγητής, τμήμα Χημείας ΕΚΠΑ
Λουκάς Ιωάννης, Αναπληρωτής Καθηγητής, τμήμα Φαρμακευτικής, ΕΚΠΑ
Γκίκας Ευάγγελος, Επίκουρος Καθηγητής, τμήμα Φαρμακευτικής, ΕΚΠΑ
Ντότσικας Ιωάννης, Επίκουρος Καθηγητής, τμήμα Φαρμακευτικής, ΕΚΠΑ
Summary:
Aging of the skin, as a result of biological and environmental causes, is a process of high interest for the people of our era. The sciences of Biology, Pharmacy, Cosmetology and Medicine devote a big part of their scientific research, looking for solutions on the problem of aging, on both structure and function of the skin. Although most research concerns dermis, recent studies focus their attention on aging of the epidermis and the possible ways to deal with it, due to the effect of skin aging on both the appearance and psychology of the individual.
Bioactive peptides usage is a broad and dynamic solution in the field of Cosmetology and the treatment of skin aging.
Cosmetic products, carrying a biological action to enhance their cosmetic action, are called cosmeceuticals.
In the present thesis, we study two bioactive peptides:
-Acetyl hexapeptide-3 (Argireline®) is a commercially available synthetic peptide, component mainly designed for eye care products, according to the N-terminal model of the SNAP-25 protein.
-Titreptide-10 citrulline, (Decorinyl®) is a synthetic peptide that mimics the decorin sequences that bind to collagen fibrils.
The subject of the study was the anti-aging activity of these two peptides, as well as the possibility of their synergistic action.
Any cosmetic product containing bioactive peptides should be subjected to effectiveness and safety control in order to allow commercialization. The concentration of a peptide in cosmetic products should be supported by clinical studies. Compatibility and stability of a bioactive peptide contained in a cosmetic formulation, indicates that it can reach the skin in active form. The analytical procedure for both medicinal products and biotechnologically derived products, such as bioactive peptides, is essential. Validation of an analytical process ensures their objectives are met, and are credible, for consumer’s safety.
There is a theoretical and experimental part in this thesis.
The first chapter of the theoretical part (chapter 1) provides information on the structure of the peptides, the formation of peptide bonds and their functionality. Bioactive peptides are peptides synthesized in the laboratory but made up of modified amino acid chains that improve an already existing physiological function, such as increasing skin permeability, stability, solubility, and more. There are many applications of bioactive peptides. They act as signals, carriers, neurotransmitters and more. Also, the physicochemical characteristics of the two peptides we are studying, acetyl hexapeptide-3 and tripeptide-10 citrulline are being described.
The second chapter of the theoretical part (chapter 2) reference is made to the skin structure and physiology. There has been a description of the normal human skin and its anatomy. Changes observed in human skin aging are being described. Skin thinning, wrinkles, discoloration and elasticity are characteristics of aging skin. The quality and quantity of the skin fibers (elastin and collagen) as well as its moisture content play a decisive role in the appearance of aging.
In the third chapter of the theoretical part (chapter 3) High Performance Liquid Chromatography (HPLC) as well as Hydrophilic Interaction Liquid Chromatography (HILIC) methods are being described. Also, an extended reference to peptides analysed by HILIC method is being mentioned.
The experimental part of the thesis follows, which is divided into four sections.
In the first experimental part (chapter 4), reference was made to the different hydrophilic interaction liquid chromatography columns and their material. The chromatographic analysis of acetyl hexapeptide-3 takes into account its physicochemical characteristics. The stationary phase and the mobile phase are selected after studying the effect of salt, water content and pH on the chromatographic behavior of the peptide.
In the second experimental part of this thesis (chapter 5), an analytical method was developed and validated in order to quantify the acetyl hexapeptide – 3, in a cosmetic product. In the analytical procedure we used isocratic elution conditions, in a high-performance liquid chromatography system coupled with an ultraviolet (LC-UV) detector. For the validation of the method, we used a highperformance liquid chromatography system, coupled with a Photodiode Array Detector PDA. The analytical column used, was BEH XBridge HILIC 2,1x150mm and particle size 3,5μm. The mobile phase used was acetonitrile / 20 mM ammonium formate in a 70/30 v / v ratio. Optimal pH of the buffer solution was pH7.5 while the flow rate was 0.25 mL / min. Detection was performed at 214 nm. Elution of the peptide occurred at about 8 minutes. During the validation of the method, a study was conducted on the cream processing and the determination of the optimal chromatographic conditions. Finally, the main quality characteristics, i.e. linearity, accuracy, precision, robustness and specificity of the method, were checked.
In the third experimental part of the thesis (chapter 6), subject of the study was observation of the skin by imaging methods, following the application of a cosmetic product containing either or both of these peptides and recording their effect, individually or synergistically, on the improvement of the skin relief and its moisture. Skin microrelief is anisotropic and its changes increase with age. For 3D skin imaging, the direct skin surface imaging method used was UVA camera (Skin Visioscan VC98). The chosen parameters to study are R2 (maximum roughness) and R3 (average roughness). In both roughness parameters, the measurement of circular wrinkles (cR2, cR3) was chosen.
The transepidermal water loss (TEWL) was measured by TEWAMETER.
Finally, in the fourth experimental part of the thesis (chapter 7), skin histological observation was performed after the application of the same as above cosmetic products. Samples of biopsies were obtained, fixed and observed in the optic microscope, after staining with hematoxylin-eosin and Masson's trichrome -pigmentation. The aim was to observe the possible increase in the thickness of the epidermis, or of the dermis, as well as the morphological observation of the structure of the dermis, with respect to the collagen and elastin fibers. Intensity of the color, resulting from the amount of skin collagen, as well as the thickness of epidermis and dermis, are the factors, indicating increase in the amount of collagen and thus improvement on the skin structure.
