ANTI-WRINKLES. Linefill. All-in-one wrinkle filler and lip plumper.

Transcription

1 ANTI-WRINKLES All-in-one wrinkle filler and lip plumper

2 FOR A HARMONIOUS AND REJUVENATED FACE As we age, our skin undergoes a number of changes. One of the most important is the loss or redistribution of facial volume and subcutaneous fat, which modifies the face s balance and which is one of the most visible signs of aging. This loss means that, as we age, our folds and wrinkles become more accentuated, especially those that form around the mouth and eyes, as well as a loss of lip volume (Wong et al., 21; Buckingham, 213; Ilankovan, 213). Dermal fillers are currently among the most popular methods used to fill wrinkles and expression lines and replace facial volume. The American Society of Plastic Surgeons has stated that, in 212, these fillers were the second most popular non surgical anti aging treatment, after Botox, with more than 2 million applications in the USA. In addition, lip augmentation is also a common treatment for retaining a young, attractive appearance and to increase self esteem (Park, 26). However, these invasive procedures can often give rise to unexpected results and they can cause a loss of harmony between the injected features and the rest of the face, giving it an unnatural appearance. The restoration of a natural volume distribution of is one of the main objectives of facial rejuvenation, and is one that can now be obtained through the use of cosmetics. stimulates the body s natural mechanisms, allowing it to promote a more natural and attractive redistribution of facial volume in the area around the mouth and lips. V2 7/

3 ADIPOSE TISSUE AND ITS FORMATION THE COMPONENTS OF ADIPOSE TISSUE Adipose tissue or body fat is a specialized type of connective tissue. It is formed when adipocytes join together along reticular fibers (collagen), forming small clumps that make up fatty lobes. Body fat is predominantly composed of white adipose tissue, which is distributed throughout the body, mainly in the subcutaneous layer. White adipose tissue has traditionally been considered to be a passive store of energy, however, it has recently been established that it plays an active role in hormone regulation and in homeostasis, as well as determining the shape of the body's surface. One of this tissue's main characteristics is that it has a highly heterogeneous cell population, however, the majority of cells are mature adipocytes (Rodríguez, 22): Preadipocytes are cells that are predestined to differentiate only into adipocytes. They still retain a phenotype similar to that of a fibroblast, but they have already developed the enzymatic machinery of an adipocyte, although they are not able to synthesize or accumulate lipids. Adipocytes are the most common cells in adipose tissue. They have a rounded shape when they are isolated and become polyhedral when they are grouped into lobes. One of their functions is the synthesis and accumulation of lipids (in a large triglyceride filled vacuole). THE DIFFERENTIATION PROCESS During adipogenesis (Figure 1), the cells look increasingly less like fibroblasts as they become more spherical. Large changes in cellular morphology also take place at this time, both in the cytoskeleton and in the extracellular matrix (Gregoire et al., 1998). Figure 1. Process of adipogenesis. V2 7/

4 This phenomenon is a process comprised of multiple steps involving various transcription factors, which regulate the activity of more than 2, genes and allow the development and differentiation of adipocytes (Symonds, 212). Adipogenesis involves two different phases: 1. Determination phase: conversion of stem cells into preadipocytes. 2. Terminal differentiation phase: the preadipocytes develop the characteristics of mature adipocytes. These, in turn, acquire the cellular machinery necessary for the transport and synthesis of lipids. The key to this process is the level of transcription factors, especially C/EBP (CCAAT/enhancer binding protein) and PPARγ (peroxisome proliferation activated receptor γ). The differentiation phase starts with the expression of C/EBPβ, which increases the expression of PPARγ. PPARγ becomes activated when bound to a ligand, stimulating the expression of C/EBPα, which in turn also increases the expression of PPARγ, forming a positive feedback loop (Kirkland, 22). This process causes the formation of lipid droplets in the cell cytoplasm, which increase over time and fuse until one or two large lipid droplets are formed that occupy a large part of the adipocyte. During our youth, all these phases of adipogenesis take place without complications, giving rise to subcutaneous adipose tissue that is diffuse and homogenously distributed (Ilankovan, 213). However, as we age, even though the determination phase remains active and the stem cells continue their subsequent differentiation into preadipocytes in the later stages, they do not complete their final differentiation into functional adipocytes capable of synthesizing and accumulating fat (Niemela et al., 28). This results in a loss of subcutaneous fat, particularly around the eyes, on the forehead and around the mouth. activates the adipogenesis to achieve more mature adipocytes capable of synthesizing and accumulating fat V2 7/

5 s MODE OF ACTION It is possible to reverse the aging process by stimulating the first phases of the preadipocyte s final differentiation; this decreases the effects of the loss of subcutaneous fat. is able to activate PPARy by acting as a ligand or agonist, which stimulates the conversion of preadipocytes to adipocytes, resulting in more mature adipocytes that are able to synthesize and accumulate fats. restores lost facial volume, reducing lines and making lips fuller COMPOSITION is a fraction rich in sesamin, which is obtained from sesame seeds (Sesamum indicum). BOTANY Sesamum indicum is an annual shrub belonging to the Pediliaceae family, commonly known as sesame. Depending of conditions, plants grow from.5 to 2.5 m tall. The leaves are ovate, opposite and deeply veined. Its flowers are bell shaped and are white with a hint of blue, red or yellow. The fruit is about 2.5 cm in size and is an oblong capsule with small seeds (Chakraborthy et al., 28) The seeds are flat, small and can be white, grey or black. They are between 2 and 4 mm long and 1 to 2 mm wide. Sesame is widely cultivated for its seeds, which are rich in oil. It is thought that sesame originated in Ethiopia and that it was brought to America by slaves, who used the seeds to season a wide variety of dishes. The plants are currently found in tropical and subtropical temperate zones, particularly in India, China, South America and Africa. V2 7/

6 CHEMISTRY Figure 2. Structure of sesamin. Sesame seeds are rich in sesamin (Figure 2), which belongs to a group of compounds called lignans. Lignans are a widely occurring class of natural compound. The last investigations show that sesame oil is able to increase the expression of PPARγ (Periasamy et al., 214) and that sesamin itself increases the activity of transcriptional PPARγ in some cell types (Liu et al., 214).On the other hand, it has been demonstrated that sesamin has a series of effects at different levels: reduces inflammation and expression of certain cytokines (Jeng et al 25), reduces arteriosclerosis, diminishes the resistance to insulin and glycemic index and reduces the levels of cholesterol, as well as the levels of circulating triglycerides degrading them to fatty acids, that can be already stored (Hong et al., 213). All these activities can be explained by the activation of PPARγ receptor, demonstrating its direct implication in all these processes (Houseknecht et al., 22; Wen et al, 21). All these studies support the described mechanism of sesamin to stimulate adipogenesis. TRADITIONAL USES Sesame oil plays an important role in Indian Ayurvedic medicine. It is rubbed onto the skin during abhyanga, a type of Indian massage that improves energy flow and helps eliminate impurities from the body. It is also used as a base for many oils due to its antioxidant and moisturizing properties (Chakraborthy et al., 28; Hazra and Panda, 213) The oil is also used as an antibacterial agent in mouthwash preparations and in preparing Iodinol and Brominol, which are used for both external and internal use. It is also used for the production of edible oil and margarine. It is appreciated in the countries where it is consumed for its pleasant and palatable flavor, especially in the Far East (China and Japan). V2 7/

7 IN VITRO EFFICACY IN VITRO STUDY PROTOCOL The ability of to enhance adipocyte differentiation was studied in order to ascertain its effect as a lipofiller. In order to achieve this, an adipocyte cell model was chosen using the 3T3 L1 cell line, which is one of the most widely used for investigating the adipogenesis process. The protocol was designed to mimic the body s natural process and takes approximately 1 days to develop (Figure 3): Preparation of preadipocytes Initial phases of differentiation Final phases of differentiation Mature adipocytes MD1 MD2 6 days 2 days 5 days removed Samples taken Figure day In vitro study protocol. 1. Once the preadipocyte culture was ready, was added at three different concentrations (43, 143 and 43 ppm), along with the MD1 differentiation medium, and the preparation was incubated for 48 hours. 2. After this, the medium containing the active ingredient was removed and the MD2 differentiation medium was added (without active ingredient). The preparation was incubated for a further 5 days, changing the medium every 2 3 days. 3. At the end of 13 days, samples were taken in order to measure total triglycerides and to assay using the Oil Red O staining method. By adding during the early phases of differentiation, along with the MD1 culture medium, and by only incubating for two days, it was ensured that the effect of the active ingredient occurred in the initial phases of terminal differentiation (the preadipocyte to mature adipocyte step). This is a key stage in the adipogenesis process, which includes the activation of PPARy by a ligand or agonist in order to complete the adipogenesis process. 1. QUANTIFICATION OF TOTAL TRIGLYCERIDES Triglyceride quantification is a biochemical indicator of adipocyte differentiation or adipogenesis. V2 7/

8 In order to quantify triglyceride concentrations in the adipocytes, the cells were lysed and the triglycerides (TG) hydrolyzed to obtain glycerol (Figure 4), which is measured by optical density. Triglyceride Glycerol Fatty Acids STRUCTURE OF A TRYGLYCERIDE HYDROLYSIS GL YC ER OL Fatty Acid 1 Fatty Acid 2 Fatty Acid 3 Figure 4. Structure and hydrolysis of triglycerides. The results are expressed as µm of glycerol as shown on Graph 1 (Evaluation of total TG) as a percentage of triglyceride accumulation compared to the control. % Variation triglyceride (Glycerol) Evaluation of total triglycerides 3.2% 11.7% 5.6% (43ppm) (143ppm) (43ppm) Graphic 1. Quantification of total triglycerides. It can be seen that is able to increase the amount of triglyceride stored in adipocytes for all concentrations tested. Triglyceride levels increased by some 3% at a concentration of 43 ppm. is therefore a potent stimulator of the early stages of adipogenesis (preadipocyte differentiation to mature adipocytes) and increases lipid synthesis and storage capacity. V2 7/

9 2. OIL RED O STAINING Oil Red O staining was carried out to allow the variation in triglyceride accumulation obtained by stimulating adipogenesis to be seen under the microscope (Figure 5): Control culture (D) Culture with (D13) Figure 5. Photograph of adipocyte cultures; the adipocytes of the linefill group show a greater accumulation of triglycerides and more voluminous vacuoles (larger and more intense red points). The number and size of the lipid vesicles was seen to increase, showing that the preadipocytes have successfully differentiated into fully functional, mature adipocytes that are able to accumulate more triglycerides. Absorbance or optical density was then measured at 54 nm (Graph 2). This demonstrated an increase in absorbance, which represents an increase in triglyceride concentration, with respect to the control for all concentrations tested. An increase of 48% was recorded at the highest dose (43 ppm). V2 7/

10 % Absorbence variation Oil Red O 1.2% 12.7% 48.2% (43ppm) (143ppm) (43ppm) Graph 2. Quantification of total triglycerides. can clearly increase the quantity of adipose tissue, since it increases both the size and the number of adipocytes (Larger lipid vacuoles and more intense red points) IN VIVO EFFICACY IN VIVO STUDY PROTOCOL A double blind comparative study was conducted in order to evaluate the in vivo efficiency of as a wrinkle filler and lip volumizer when compared to a placebo. The study was carried out in line with the following protocol: 41 volunteers (women) with wrinkles in perioral area: nasolabial folds, supralabial and lateral wrinkles. 2 groups: 2 women applied a formulation with 2%, while the control group of 21 women applied the same formulation without (placebo). Age: years of age. Application area: perioral area, including the nasogenian folds and lips. Twice a day for 28 days. Measurements were made on Day (D), before the application, and again on Day 28 (D28). V2 7/

11 1. EVALUATION OF WRINKLE REDUCTION AND ROUGHNESS IN THE PERIORAL AREA The reduction in wrinkling and roughness was determined using the capture and comparison of 3D images by fringe projection (PRIMOS) both on D and on D28. The efficacy of was evaluated in three areas with different types of wrinkles, in order to demonstrate that it is effective against all types of wrinkles: nasolabial fold, supralabial area (barcode wrinkles) and the lateral wrinkles that appear at the ends of the lips (marionette lines). Nasolabial fold decreased the volume of the nasolabial fold by 9.2% compared to the start of the study (D). This result was 7.8 percentage points (pp) better than that obtained by the placebo (Graph 3). Nasolabial fold volume (%) 2 4 Placebo 1.4% % 7.8 Graph 3. Decrease in volume of nasolabial fold after 28 days of application. D D28 Figure 6. Visible decrease in nasolabial fold volumen obtained by. V2 7/

12 Supralabial wrinkles 1. Number of supralabial wrinkles reduced the number of supralabial wrinkles by 45.6% compared with D and with a significant difference of 19 pp compared with the placebo (Graph 4). Number of supralabial wrinkles (%) % 19. Placebo 26.6% Graph 4. Decrease in the number of supralabial wrinkles after 28 days of application. 2. Volume of supralabial wrinkles The volume of the wrinkles in this area decreased by 12.1% in the group treated with, while in the placebo group this value increased by 1.7%. There was therefore a statistically significant variation between the two groups, with a difference of 22.8 pp (Graph 5) Volume of supralabial wrinkles (%) 1.7% Placebo % Graph 5. Decrease in volume of supralabial wrinkles obtained by after 28 days of application. V2 7/

13 D D28 Figure 7. Visible decrease in supralabial wrinkles obtained by. 3. Cutaneous microrelief Shallow regular grooves form in young skin, but as the skin ages, this relief will change and deeper grooves form without actually forming a wrinkle, but giving the skin a more aged and rough appearance. The roughness of the skin can be measured to provide an estimate of cutaneous microrelief: Ra (average roughness) and Rz (an average of the 5 most pronounced peaks and valleys). An improvement for both parameters was seen in the group treated with, compared to initial values (Graph 6): Ra: was decreased by by 6.7%, while in the placebo group it increased by 3.5%, giving a difference of 1.1 pp between the active ingredient and the placebo. Rz: treatment with produced a decrease of 5.4%, while with the placebo there was an increase of 3.6%. There was therefore a difference of 8.6% compared with the placebo. Cutaneous microrelief (%) Ra Rz 3.5% 4 3.3% % 6.7% Placebo Graph 6. Decreased roughness (Ra and Rz) of supralabial wrinkles obtained by after 28 days of application. V2 7/

14 Lateral wrinkles In the case of lateral wrinkles, or marionette lines, the number and volume of the wrinkles was measured. A comparison of the decrease in number and volume of these wrinkles in the treated group when compared to the placebo group showed that the efficacy of was statistically significant. 1. Number of lateral wrinkles The number of lateral wrinkles for the group treated with decreased by 58.8% when compared with D, with a significant difference of 19.2 pp compared to the placebo (Graph 7). Number of lateral wrinkles (%) 2 Placebo % % Graph 7. Decrease in the number of lateral wrinkles after 28 days of application. 2. Volume of lateral wrinkles produced an 18.5% decrease in lateral wrinkle volume, with a significant percentage difference of 17.9 pp compared to the placebo (Graph 8). Volume of lateral wrinkles (%) 5 Placebo.6% % 17.9 Graph 8. Decrease in volume of lateral wrinkles after 28 days of application. V2 7/

15 D D28 Figure 8. Visible decrease in lateral wrinkles obtained by. visibly decreases all types of wrinkles and refines roughness of the skin, thus blurring the imperfections caused by the passage of time 2. EVALUATION OF THE ANTIAGING EFFECT THROUGH IMAGE ANALYSIS Standardized photographs were taken using the VISIA CA system under normal, polarized and ultraviolet lighting in order to provide an overall confirmation of the antiaging effect, supported by analysis of texture and redness parameters. With age, the skin s texture and appearance becomes increasingly affected by the appearance of imperfections, blemishes, wrinkles and expression lines that give a rougher and irregular texture and a more aged look to the skin. Thanks to its overall antiaging effect, reduced blemishes by 6.3%, giving a significant difference of 11 pp compared to the placebo. D D28 Figure 9. Visible decrease in redness (darkest spots) obtained by. V2 7/

16 The skin s texture provides us with general information regarding its roughness, which increases with age. reduced the number of imperfections caused by the skin s roughness by 5.2%, and this improvement was statistically significant compared to the placebo, with a difference of 5.5 pp (Graph 9) % Texture (%) 5.5.3% Placebo Graph 9. Decrease in the number of imperfections obtained by after 28 days of application. softens roughness and soothes redness of the skin, for an overall younger appearance. 3. INSTRUMENTAL EVALUATION OF THE INCREASE IN LIP VOLUME Instrumental evaluation Fringe projection (PRIMOS) was used to evaluate the increase in lip volume. As well as reducing wrinkles and redness, also increased lip volume by 23.1%, in a way that was clearly superior and statistically significant when compared to the placebo, with a difference of 24.8 pp (Graph 1) % Lip Volume (%) % Placebo Graph 1. Increase in lip volume obtained by after 28 days of application. V2 7/

17 Figure 1. Visible increase in lip volume obtained by. increased lip volume, thus achieving more voluminous and sensual lips 4. EVALUATION OF HYDRATION The skin s hydration is a parameter that has a certain influence on the condition of wrinkles and on the skin s appearance. If the skin is kept hydrated, this will optimize an active ingredient s anti wrinkle effects. A Corneometer was therefore used to analyze the amount of water in the skin s upper layers. hydrates the skin, showing a 22.5% increase, with a difference of 11.2 percentage points with respect to the placebo (Graph 11) Hydration (%) 22,5% % Placebo Graph 11. Increase in cutaneous hydration after 28 days of application. hydrates the skin, improving both the status of wrinkles and the overall appearance of the skin V2 7/

18 5. SUBJECTIVE QUESTIONNAIRE At the end of the study, the volunteers filled in a questionnaire to evaluate the efficacy of or of the placebo. A total of 95% considered that the product containing was good or very good and 85% wanted to buy it; in contrast only 52.4% thought that the placebo product was good or very good and only 47.5% intended to buy the product. In addition, the following graph (Graph 12) shows the percentage of volunteers that were satisfied with the other subjectively evaluated parameters: Subjective questionnaire Purchase intention General lip improvement 1 8 Lip volume increase Very good product 6 4 More hydrated lips 2 Beautiful skin ssofter skin Placebo Uniform skin tone More hydrated skin Nasolabial fold reduction Lateral wrinkles reduction Supralabial wrinkles reduction Graph 12. Results of subjective questionnaire on the effectiveness of after 28 days of application. is an active lipo filler that decreases wrinkles, increases lip volume and improves the overall condition of the skin V2 7/

19 CONCLUSIONS is a vegetable based active ingredient that is able to increase the accumulation of lipids in the adipose tissue, providing a greater volume to the areas that have lost their shape over the years. Thanks to its action, is able to perceptibly refill wrinkles and cutaneous folds, as well as having a potent volumizing effect on the lips, giving a rejuvenated, attractive and relaxed face. COSMETIC APPLICATIONS Anti aging lines. Lip and eye contour creams. Lip fillers. Targeted body volumizers (breasts, buttocks, cheeks etc.). Anti aging hand treatments. Firming treatments. RECOMMENDED DOSE The recommended dose is between 1 and 3%. BIBLIOGRAPHY Buckingham ED. Poly L Lactic Acid Facial Rejuvenation: An Alternative to Autologous Fat? Facial Plast Surg Clin North Am. 213, 21(2): Chakraborthy GS, Sharma G, Kaushik, KN. Sesamum Indicum:A Review. Journal of Herbal Medicine and Toxicology. 28, 2(2): V2 7/

20 Gregoire, FM, Smas CM, Sul HS. Understanding Adipocyte Differentiation. Physiological Reviews. 1998, 78(3): Hazra J, Panda AK. Concept of Beauty and Ayurveda Medicine. J Clin Exp Dermatol Res. 213, 4:3. Ilankovan V. Anatomy of ageing face. Br J Oral Maxillofac Surg. 213, 1 8. Kirkland JL, Tchkonia T, Pirtskhalava T, Han J, Karagiannides I. Adipogenesis and aging: does aging make fat go MAD? Exp Gerontol. 22, 37(6): Niemelä S, Miettinen S, Sarkanen JR, Ashammakhi N. Adipose Tissue and Adipocyte Differentiation: Molecular and Cellular Aspects and Tissue Engineering Applications Topics in Tissue Engineering. 28, 4:1 26. Park, JI. Asian Facial Cosmetic Surgery. Philadelphia: Saunders Elsevier, p. ISBN 13: ; ISBN 1: Periasamy S, Hsu DZ, Chang PC, Liu MY. Sesame oil attenuates nutritional fibrosing steatohepatitis by modulating matrix metalloproteinases 2, 9 and PPAR γ. J Nutr Biochem. 214, 25(3): Rodríguez, ML. Diferenciación Adipocitaria y Factores Reguladores de la Biogénesis Mitocondrial. Efectos de los Fármacos Antiretrovirales. Barcelona: Universidad de Barcelona. 22, 93 p. Symonds ME (Ed). Adipose Tissue Biology. London: Springer, 212, VI. 414 p. ISBN: ; eisbn Wong WW, Davis DG, Camp MC, Gupta SC. Contribution of lip proportions to facial aesthetics in different ethnicities: A three dimensional analysis. Journal of Plastic, Reconstructive & Aesthetic Surgery. 21, 63: V2 7/