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Biotechnological Hyaluronic Acid Enhancer for Mature Skin

By Mu00edriam Mateu, Cristina Davi, Elena Cau00f1adas, Albert Soley and Raquel Delgado | February 12, 2013

Lipotec researchers detail the benefits of applying hyaluronic acid on wrinkles.

Time plays a main role in altering and deteriorating the skin structure, so that features of mature skin differ from those younger. Some of them are frown lines in the forehead, periorbital wrinkles and nasolabial folds, which appearance is influenced by genetics and enhanced by gravity force, stress, inadequate rest, frequent and constant positional pressures of facial skin, repeated facial movements and environmental factors.
 
Some reasons for the appearance of these lines and folds are histological and physiological alterations that include dermal extracellular matrix (ECM) impairment, collagen and elastin degradation, and barrier function damage. The ECM is a 3D mesh formed by various macromolecules that comprise proteins and glycosaminoglycans (GAGs). GAGs are long linear heterogeneous polysaccharides with repeating disaccharide units.1 Due to their highly negative charges, GAGs are hydrophilic and able to attract water inside the tissue.2

Hyaluronic acid (HA) is a major compound of the ECM that belongs to the GAG family, and it is mainly found as a free molecule.3 The skin presents half of the total body content of HA, where it provides hydration (retains water up to 1000 times its own weight), support and volume, and it participates in cellular migration, proliferation and wound healing.3 This non-sulphated GAG is able to diminish epidermal water loss and raise water retention into the dermis, which results in a notable plumping effect.4

There is a dynamic equilibrium between the synthesis and degradation of HA in the skin, ensuring a stable quantity of available and functional HA. Unfortunately, this balance is altered when aging due to the increasing number of HA molecules that bind the tissues, the diminution of its synthesis and the raise in its degradation by hyaluronidases.

The HA loss leads to dehydration, volume shrink and visible wrinkles. The small zone between the nose and the upper lip, known as nasolabial area, is highly susceptible to volume changes and even small variations can easily form nasogenian folds. Such wrinkles are considered one of the clearest signs of mature skin and the aging process.

Marine ecosystems are rich in diversity and microorganisms, especially those in bays and estuaries, due to its combination of salt and fresh water. These habitats are exposed to tides and waves, and suffer gradients of temperature, oxygen and light typical between salty and fresh water. The inhabiting bacteria, algae and fungi have had to develop mechanisms to help their colony and ensure their survival.5-7

In estuaries, some bacteria produce functional Exopolysaccharides (EPSs).7 EPSs are glucidic biopolymers naturally secreted to the surrounding media as a response to environmental stress. These polysaccharides can raise microorganism survival by increasing hydration and nutrition, acting in intracellular processes, immunologic modulation, cell recognition, proliferation and migration, and assisting in favourable adhesions to solid surfaces. Thus, they are also thought to possibly interact with cell receptors promoting beneficial activities, like the production of structural compounds.5,7-9

Although most EPSs present either uronic acids like D-glucuronic acid or ketal-linked pyruvate, EPSs have many possible compositions according to their main required function implying a wide range of chemical and physical properties. Therefore, they present many potential uses and applications in the cosmetic industry, such as stabilising, film-forming, gelling, thickening, anti-aging and water retaining agents.6

The naturally found EPSs can also be obtained by biotechnology. This technology encompasses the use of living microorganisms to obtain natural molecules for a specific use, while helping to preserve the environment, as there is no harvesting or extracting from nature. The use of science and engineering in the manufacturing processes helps to achieve an optimal performance, which yields an adequate growth and maximal productivity of a microorganism, allowing to obtain complex molecules that could not be possible to get through chemical reactions due to technical or economic limitations.

Hyanify (INCI: Saccharide Isomerate) was obtained via biotechnological fermentation of a marine γ-proteobacteria strain isolated from the surface of a Laminaria alga in the Aber Wrac’h estuary in Brittany (France). This area has both the influence of salt and fresh water, so the inhabiting microorganisms need to develop special structures and mechanisms to survive, including the production of such functional EPS.

Several studies were performed to analyse the efficacy of this special EPS in mature skin and concretely on the recovery of the nasolabial area which is extremely susceptible to the aging natural process and easily loses volume.

Materials and Methods
Due to the importance of HA in skin appearance and conditions, an ELISA test was performed to observe the effect of Saccharide Isomerate on the induction of HA in human dermal fibroblasts, as these cells are its main producers .10 After cells were seeded in 24-well plates and incubated during 24 hour, deprivation medium was added and cells were incubated for additional 24 hour. Afterwards, cells were treated with 1mg/mL of the active EPS for 48 hours. Then, medium was collected and HA quantitated by an ELISA test measuring absorbance values at 405nm in a microtiter plate reader.

Non-treated cells were used as the negative control and cells treated with Platelet-Derived Growth Factor (PDGF-BB) were used as the positive control. HA concentrations were determined using a linear regression of the HA standard solutions curve, calculating the percentage of induction with respect to the negative control.

Another study was to assess the in vivo efficacy of Saccharide Isomerate on the nasolabial fold volume recovery by measuring physical parameters related to skin topography before its application and after 2 and 4 weeks of treatment. Thus, a panel of 19 volunteers between 44-56 years old, with nasolabial fold of moderate intensity and II-III Fitzpatrick phototype, applied a cream with 1% of a solution with the EPS twice a day on the face, insisting on the nasogenian fold.

Skin topography was evaluated by FOITS. Fringe projection gave 3D images where the maximum and average depth, circumference, area and volume were calculated. The maximum depth represents the distance between the skin basal height and the bottom of the cavity, and the average depth is the mean of all the possible depths of the cavity. The volume parameter refers to the volume of the cavity created in the skin, the circumference is the circumference of the cavity at the basal height and the area is the surface corresponding to the cavity.

Fig. 1. Maximum and average depth parameters.

 


















Results and Discussions
 
Results showed that Saccharide Isomerate efficiently induced HA synthesis compared to the negative control, causing a statistically significant 66.0% stimulation on human dermal fibroblasts.
 
After 14 and 28 days of treatment, Saccharide Isomerate diminished the maximum depth of the nasogenian fold by an average of 13.6% and 19.6% respectively, being statistically significant values. The maximum recorded reductions were 64.7% and 70.6% after the same periods.
 
Likewise, the average value of all the depths of the nasogenian fold was reduced by 14.7% and 18.5% due to Saccharide Isomerate after 14 and 28 days of treatment respectively, being statistically significant results. Maximal decreases of 65.8% and 71.4% were observed after the same periods.


Fig. 2 Improvement of the average value of all wrinkle depths.
 























Results showed that the nasolabial fold already experienced a diminution in its average volume, area and circumference just after 14 days of treatment with Saccharide Isomerate. At the end of the treatment, the average circumference was reduced by 15.3%, the area by 17.2% and the volume by 27.0% (statistically significant effects). The maximum reductions found were 93.5% for the volume, 77.8% for the area and 79.3% for the circumference.


Fig. 3. Average circumference, area and volume improvement.



 



















The pictures taken at the beginning and after 28 days of the treatment with the active ingredient undoubtedly demonstrated the positive evolution of the nasolabial fold, visible improving its appearance. The silicon patterns also showed that the nasolabial fold notably decreased.

Fig. 4. Images and silicon patterns at the beginning (left) and after 28 days of treatment with a cream containing Saccharide Isomerate (right).
 
Saccharide Isomerate clearly improved the nasolabial wrinkles and provided a visible replenishing effect on the area, which rejuvenated the appearance of the skin.

Conclusions
Saccharide Isomerateproved to notably act on HA synthesis, inducing it by 66.0% in human dermal fibroblasts in culture. In a panel of volunteers, it decreased the maximum and average wrinkle depth (13.6% and 14.7% respectively) in the nasogenian folds in just 14 days, showing even better mean results after 28 days (19.6% and 18.5% respectively). Additionally, the average volume, area and circumference values of this zone were also reduced after 28 days (27.0%, 17.2% and 15.3% respectively). All results were statistically significant.
 
Therefore, at the light of the results, this EPS demonstrated to improve one of the most concerning facial areas in mature skin by boosting HA synthesis. Hyanify™ is an excellent anti-aging ingredient that visibly ameliorates the appearance of nasolabial folds, replenishing them, which leads to a visible younger skin appearance.
 
 
References
1. Souza-Fernandes AB, Pelosi P, Rocco PR. Bench-to-bedside review: The role of glycosaminoglycans in respiratory disease. Crit Care. 10(6): 237, 2006.
2. House M, Kaplan DL, Socrate S. Relationships between mechanical properties and extracellular matrix constituents of the cervical stroma during pregnancy. Semin Perinatol. 33(5): 300-307, 2009.
3. Stern R. Review: Devising a pathway for hyaluronan catabolism: are we there yet? Glycobiology. 13(12): 105R-115R, 2003.
4. John HE, Price RD. Perspectives in the selection of hyaluronic acid fillers for facial wrinkles and aging skin. Patient Prefer Adherence. 3: 225-230, 2009.
5. Raguénès GHC, Peres A, Ruimy R, et al. Alteromonas infernus sp. nov., a new polysaccharide-producing bacterium isolated from a deep-sea hydrothermal vent. J App Microbiol. 82:422-430, 1997.
6. Guezennec J. Deep-sea hydrotermal vents: A new source of innovative bacterial exopolysaccharides of biotechnological interest? J Ind Microbiol Biotechnol. 29: 204-208, 2002.
7. Chi Z, Fang Y. Exopolysaccharides from Marine Bacteria. J Ocean Univ China. 4(1): 67-74, 2005.
8. Zanchetta P, Lagarde N, Guezennec J. Systemic effects on bone healing on a New Hyaluronic acid-like bacterial exopolysaccharide. Calcif Tissue Int. 73: 232-236, 2003.
9. Hsu HY, Hua KF, Lin CC, et al. Extract of reishi polysaccharides induces cytokine expression via TLR4-modulated protein kinase signaling pathways. J Immunol. 173: 5989-5999, 2004.
10.Li L, Asteriou T, Bernert B, et al. Growth factor regulation of hyaluronan synthesis and degradation in human dermal fibroblasts: importance of hyaluronan for the mitogenic response of PDGF-BB. Biochem J. 404(2): 327-336, 2007.
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