H₂OBioEV® BioActive is a multifunctional cosmetic ingredient that moisturizes by replenishing essential humectants, providing an optimal environment for epidermal proteins to form and maintain a strong barrier, thus restoring a smooth and radiant appearance.
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Technical product information
Unique ingredient designed to help revitalize skin, making it look healthier, more radiant and smoother.
- Revitalizes skin by moisturizing so effectively that skin roughness, and the appearance of lines and wrinkles are visibly diminished (in vivo data).
- Helps to improve barrier structure, by increasing Involucrin expression (ex vivo data).
- Increases the expression of Filaggrin, thus increasing the source of natural moisturizing factor (NMF), the skin´s own moisturization solution (ex vivo data).
- Increases E-cadherin expression, contributing to improved cell-cell cohesion (ex vivo data).
- Decreases expression of IL-8 in keratinocytes; lessening the discomfort of dry skin (in vivo data).
Nature and Technology
H₂OBioEV® BioActive contains a unique combination of naturally sourced ingredients including Aphanothece sacrum extract and Galactoarabinan.
Aphanothece sacrum is a scarce edible freshwater unicellular cyanobacterium from Suizenji nori, aqua-cultured in Japanese rivers. Aphanothece sacrum extract is rich in an extremely high molecular weight (1.6x107 Da) ampholytic macromolecular polysaccharide which is highly hydrophilic and absorbs more water than other typical polysaccharides.
Galactoarabinan, is also a polysaccharide and is sourced sustainably from the Larch tree and extracted via a patented extraction technology. The galactoarabinan has a narrow molecular weight distribution (~20,000 Daltons) and is known to reduce Transepidermal Water Loss (TEWL) on the skin; providing long lasting moisturization and hydration.
As a result of extensive trials, the ratio of ingredients found in H₂OBioEV® BioActive has been optimized to create a unique ingredient that offers multiple biophysical and biochemical approaches to address dry skin as an epidermal barrier enhancer.
Normal Skin Vs Dry Skin - Structural Changes
The outermost layer of the epidermis is the stratum corneum, which is the first line of protection against external stress factors, and helps to retain water in the skin thereby keeping the skin hydrated.
The skin maintains its moisture balance by using caspase 14 to transform profilaggrin into filaggrin, which is then processed into a mixture of small molecules collectively known as natural moisturizing factors (NMF’s). Involucrin binds hydrophilic lipids around the cornified envelope of fully differentiated keratinocytes, (known as corneocytes), whilst Ecadherin supports the strength and cohesion of the viable epidermis by forming junctions between keratinocytes. If this process is not working properly then the skin will be dry, and there can be an increase in the expression of Interleukin-8 (IL-8) (a pro-inflammatory cytokine), which ultimately leads to skin discomfort.
H2OBioEV® is unique in that it offers a number of different biophysical and biochemical approaches to address dry skin from a single ingredient:
- By depositing an invisible film on the skin that locks moisture inside the skin.
- By replenishing essential humectants and providing a stabilizing environment for functional skin proteins.
- By stimulating the expression of proteins essential to forming and maintaining an optimal skin barrier function.
The skin has evolved to maintain its own moisture balance by processing the protein filaggrin into a mixture of small molecules collectively known as natural moisturizing factor (NMF). Low levels of filaggrin, and/or NMF, have a noticeable impact on barrier integrity and function.
In addition to filaggrin, the protein involucrin also participates in the formation of the epidermal barrier. At the same time that filaggrin is present in the hydrophilic area of the SC, involucrin binds hydrophilic lipids around the cornified envelope of fully differentiated keratinocytes, corneocytes. Involucrin is therefore a key component in the formation of a strong, cohesive barrier.
Also supporting the strength and cohesion of the viable epidermis by forming junctions between keratinocytes, is e-cadherin. It partners with catenins, and other proteins, to form adherens junctions. Experiments where e-cadherin expression was disrupted, resulted in tissues with various integrity defects.
Given the importance of having both the appropriate water content and the highly ordered structure of the epidermis required to provide a functional barrier, multiple targets were selected for screening H₂OBioEV® Bioactive and controls: filaggrin, involucrin, e-cadherin and IL-8.
Emulsions both with and without 3% H₂OBioEV® Bioactive were applied topically at a dose of 12 mg/cm2 to ex vivo human skin explants and incubated at 37°C for 72 hours. Explants that received no topical application of any kind served as a control. All samples were rinsed, fixed with 4% paraformaldehyde (PFA), sectioned, stained for either filaggrin, involucrin, or e-cadherin protein expression, and counterstained with DAPI. The resulting fluorescence signal was imaged and quantified using ImageJ software, and results were probed for statistical significance using ANOVA. Tukey-Kramer Multiple Comparisons or Dunnett’s test were used when the analysis of variance detected significant differences between groups. Results with p ≤ 0.05 were considered significant.
The expression of several epidermal and barrier proteins was increased following topical application of 3% H₂OBioEV® Bioactive formulated in an emulsion, when compared to application of the control emulsion, to ex vivo human skin explants.
Filaggrin expression was upregulated by 125% following topical application of an emulsion containing 3% H₂OBioEV® Bioactive, when compared to the fluorescence emitted following application of the control emulsion. The upregulation is clearly evident from the increased green fluorescence of representative sections (Fig. 2a).
Figure 2: Filaggrin protein expression in ex vivo human skin. (a) Fluorescence staining for filaggrin (green) and cell nuclei (blue) at 400X magnification of 10 µm sections. (b) Quantification of fluorescence signal (green) was performed using ImageJ software. Six images were quantified for each experimental condition. Significance was *p<0.001 for the 3% H₂OBioEV® sample compared to control emulsion (ANOVA, Tukey-Kramer).
Another protein expressed in the upper layers of the epidermis that is partly responsible for the formation of a strong and cohesive barrier is involucrin. When 3% H₂OBioEV® Bioactive was applied topically to ex vivo skin samples, as described above, a slight, but significant, upregulation of 24% in the fluorescence signal due to involucrin protein was observed, compared to levels observed in the presence of a control emulsion (Fig. 3).
Figure 3: Involucrin protein expression in ex vivo human skin. Quantification of involucrin fluorescence signal was performed using Leica Application Suite software. Six images were quantified for each experimental condition. Significance was *p<0.05 for the 3% H₂OBioEV® sample compared to control emulsion (ANOVA, Tukey-Kramer).
Further, e-cadherin expression also increased significantly in the presence of H₂OBioEV® Bioactive. Following topical application of 3% H₂OBioEV® Bioactive to ex vivo skin explants for 72 hours, resulted in an increase of 70% as compared to baseline expression (Fig. 4). This upregulation of a key junction protein may serve to strengthen cell-cell cohesion in the viable epidermis.
Figure 4: E-cadherin protein expression in ex vivo human skin. (a) Immunofluorescence staining for e-cadherin (green) and cell nuclei (blue) at 400X magnification of 10 µm sections. (b) Quantification of the green fluorescence signal was performed using Leica Application Suite software. Six images were quantified for each experimental condition. Significance was **p<0.01 for the 3% H₂OBioEV® sample compared to baseline expression (ANOVA, Dunnett)
It has been previously shown that dermatological conditions where the epidermal barrier may be weakened, and skin appears dry and scaly, expression of pro-inflammatory cytokines increases. We therefore examined the effect of H₂OBioEV® Bioactive on one of the cytokines, IL-8, in cultured keratinocytes. A significant, dose-dependent decrease in IL-8 expression was observed following incubation of H₂OBioEV® Bioactive with HaCaT cells in vitro for 48 hours, compared to that observed with cells stimulated with bacterial lipopolysaccharide (LPS). H₂OBioEV® Bioactive was able to reduce the expression of IL-8 by 20% - 59%, compared to LPS control. At the highest concentration tested, 3.1%, the observed IL-8 level was even lower than baseline expression (Fig. 5).
Figure 5: Quantification of IL-8 protein expression in cultured HaCaT keratinocytes. Significance was **p<0.01 for the H₂OBioEV® sample compared to baseline expression or control emulsion (ANOVA, Dunnett).
Given the results observed on skin explants and cell culture, a clinical study was carried out to evaluate the efficacy of an emulsion containing 3% H₂OBioEV® Bioactive , versus a control emulsion, on skin appearance.
A double-blind monadic study was carried out with 46 subjects (41-65 years old) presenting mild to moderate facial skin photoaging and sensitive skin as determined by the lactic acid sting test. The subjects were divided in two groups, and given either Cream A (control emulsion) or Cream B (3% H₂OBioEV® Bioactive) to apply twice daily on the face for 28 ± 2 days. All were asked to maintain a diary and answered a questionnaire on perceived efficacy of the product after 14 or 28 days of use. In addition, subjects were evaluated using a PRIMOS 3D optical measuring device (GFMesstechnik GmbH) and facial images were taken using a Visia CR imaging system (Canfield Scientific, Inc.) at days T0, T14 and T28. Facial images and topography profiles were analyzed for wrinkles and skin texture. The comparison between products was performed using Student t-test with unilateral hypothesis. The response variable was the difference with initial time-point (T14-T0 and T28-T0). The confidence level used on the comparative analysis was 95%. The analysis software packages were MINITAB 14 and XLSTAT 2018.
After 14 and 28 days of use, there was a significant reduction in the wrinkle depth and mean roughness, as measured with the PRIMOS 3D optical measuring device. There was no such improvement observed following application of the control emulsion (Fig. 6).
Figure 6: PRIMOS 3D topographical profile of skin, with a color scale that indicates deep wrinkles (blue) and shallow surface (yellow). The effect of 3% H₂OBioEV® BioActive (right) (Subject 48) and the control emulsion (left) (Subject 31) at T28 are shown (bottom panels).
The parameters of wrinkle depth and skin roughness were quantified from the above images for 5 subjects in each group. The quantitative results reflect a reduction in both parameters following twice daily application of 3% H₂OBioEV® , and no such reduction is observed with subjects applying the control emulsion (Fig. 7 & 8).
Figure 7: Quantification of wrinkle depth from the PRIMOS 3D topographical profile. The effect of 3% H₂OBioEV® (blue bars) and the control emulsion (grey bars) are shown. There was a significant difference between the formula with 3% H₂OBioEV® and the control emulsion at T14-T0 (p=0.038) and at T28-T0 (p=0.031).
Figure 8: Quantification of skin texture - roughness from the PRIMOS 3D topographical profile. The effect of 3% H₂OBioEV® (blue bars) and the control emulsion (grey bars) are shown. There was a significant difference between the formula with 3% H₂OBioEV® and the control emulsion at T14-T0 (p=0.022) and at T28-T0 (p=0.009).
These results indicate that after 14 and 28 days of use, the formulation containing 3% H₂OBioEV® Bioactive was able to provide a skin smoothing effect in the periorbital region of the face, by both reducing the appearance of fine lines and wrinkles, and reducing skin roughness.
The effect on the appearance of lines and wrinkles was also examined using the Visia CR imaging system. There was a statistically significant reduction in the coefficient of visibility of the wrinkles (Fig. 8 & 9) after 14 and 28 days of application of 3% H₂OBioEV® Bioactive.
Figure 9: Periorbital wrinkles of individual subjects at T0, T14 and T28.
Figure 10: Quantification of periorbital wrinkle visibility. There was a significant difference between the formula with 3% H₂OBioEV® and the control emulsion at T14- T0 (p=0.006) and at T28-T0 (p<0.001).
As a further measure of skin surface irregularity and wrinkle visibility, skin texture analysis was performed using the images taken with the Visia CR system. When the images were quantified, a statistically significant decrease in surface irregularity was observed (Fig. 11), which translates to reduced visibility of wrinkles at T14 & T28, compared to T0 after application of 3% H₂OBioEV® Bioactive (as shown in Fig. 12).
Figure 11: Skin texture of individual subjects at T0, T14 and T28.
Figure 12: Quantification of surface irregularity. There was a significant difference between the formula with 3% H₂OBioEV® and the control emulsion at T14-T0 (p=0.036) and at T28-T0 (p=0.016).
The moisturization properties from the 3% H₂OBioEV® Bioactive formulation provide a measurable superiority over the control emulsion, and the resulting skin is smoother, with fewer visible lines and wrinkles. The improvement appears to increase with time (T14 vs T28), suggesting that the skin is not only immediately moisturized, but that skin condition continues to improve with consistent use.