Inhibiting Cathepsin G to Protect Against Photoaging and Infra’aging

Inhibiting Cathepsin G to Protect Against Photoaging and Infra’aging

September 27, 2013 | Contact Author | By: Estelle Loing, PhD; Elisabeth Lamarque; Carine Bezivin; and Magali Borel, Lucas Meyer Cosmetics
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Title: Inhibiting Cathepsin G to Protect Against Photoaging and Infra’aging
photoagingx infra’agingx wrinklesx cathepsin Gx MMP-1x
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Keywords: photoaging | infra’aging | wrinkles | cathepsin G | MMP-1

Abstract: Ultraviolet and infrared exposure are sources of premature skin aging, characterized by solar elastosis and extracellular matrix degradation. Polygonum aviculare extract is a cathepsin G inhibitor, and is shown here to oppose photoaging and infra’aging by improving skin firmness and elasticity, in turn reducing the appearance of wrinkles.

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E Loing, E Lamarque, C Bezivin and M Borel, Inhibiting Cathepsin G to Protect Against Photoaging and Infra’aging, Cosm & Toil 128(9) 658 (2013)

Photoaging refers to premature skin aging that results from chronic exposure to solar radiation or other sources that mimick solar effects. Until recently, photoaging essentially was linked to the ultraviolet (UV) portion of the solar spectrum; specifically, to UVB and UVA rays with wavelengths in the 290–400 nm range. UVB is absorbed by chromophores in the epidermis and is responsible for sunburn, whereas the less energetic UVA penetrates more deeply into the dermis and is associated with premature aging. Both UVB and UVA may also cause cancer and immunosuppression.1, 2

However, the solar spectrum is much broader, and attention is now turning toward infrared radiation (IR), i.e., the 760 nm–1 mm end of the rainbow. IR can be divided into IRA, IRB and IRC, and while all three have low energy and may seem inoffensive, IRA penetrates deeply into the skin, even reaching the hypodermis (see Figure 1).3, 4 Moreover, nearly half of solar rays fall into the IR range, of which 30% is IRA, versus around just 5% for UV.3 As a consequence, any effect of IRA on skin physiology deserves a closer look.

The hallmark of photoaged skin is an accumulation of elastolytic material in the upper and middle dermis, which occurs through a process known as solar elastosis.5 Together with collagen, elastic fibers normally form a support for cell attachment, since dermal fibroblasts need such anchoring to function properly. Chronic sun exposure activates various proteases that dismantle the extracellular matrix (ECM) scaffold, leaving amorphous material behind.

Cathepsin G has recently emerged as an important mediator of proteolytic ECM degradation in photoaging. This serine protease is secreted by inflammatory neutrophils and dermal fibroblasts. It is present in higher amounts in aged human skin than in younger skin, and its expression and/or activity can be further increased with UVA exposure.6, 7 Further, in one animal model of photoaging, a cathepsin G inhibitor prevented UVB-induced ECM degradation, suggesting activation of the enzyme by UVB as well.8

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Photoaging refers to premature skin aging that results from chronic exposure to solar radiation or other sources that mimick solar effects. Until recently, photoaging essentially was linked to the ultraviolet (UV) portion of the solar spectrum; specifically, to UVB and UVA rays with wavelengths in the 290–400 nm range. UVB is absorbed by chromophores in the epidermis and is responsible for sunburn, whereas the less energetic UVA penetrates more deeply into the dermis and is associated with premature aging. Both UVB and UVA may also cause cancer and immunosuppression.1, 2

However, the solar spectrum is much broader, and attention is now turning toward infrared radiation (IR), i.e., the 760 nm–1 mm end of the rainbow. IR can be divided into IRA, IRB and IRC, and while all three have low energy and may seem inoffensive, IRA penetrates deeply into the skin, even reaching the hypodermis (see Figure 1).3, 4 Moreover, nearly half of solar rays fall into the IR range, of which 30% is IRA, versus around just 5% for UV.3 As a consequence, any effect of IRA on skin physiology deserves a closer look.

The hallmark of photoaged skin is an accumulation of elastolytic material in the upper and middle dermis, which occurs through a process known as solar elastosis.5 Together with collagen, elastic fibers normally form a support for cell attachment, since dermal fibroblasts need such anchoring to function properly. Chronic sun exposure activates various proteases that dismantle the extracellular matrix (ECM) scaffold, leaving amorphous material behind.

Cathepsin G has recently emerged as an important mediator of proteolytic ECM degradation in photoaging. This serine protease is secreted by inflammatory neutrophils and dermal fibroblasts. It is present in higher amounts in aged human skin than in younger skin, and its expression and/or activity can be further increased with UVA exposure.6, 7 Further, in one animal model of photoaging, a cathepsin G inhibitor prevented UVB-induced ECM degradation, suggesting activation of the enzyme by UVB as well.8

Once activated, cathepsin G degrades fibronectin (FN) into fragments that induce and up-regulate the expression and activity of matrix metalloproteinase (MMP-1 and MMP-2) while simultaneously inhibiting the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1)—the natural inhibitor of MMPs.6, 8 Such double jeopardy significantly compromises collagen integrity. In addition, cathepsin G has been shown in vitro to degrade elastin into fragments that can be further processed by elastase.9 Cathepsin G thus appears to be an important mediator of UV-induced photoaging.

IR radiation is also a mediator of photodamage to the skin. Within dermal fibroblasts, IRA can directly affect mitochondrial functions, lowering cellular energy production, generating ROS, and activating pathways that culminate into increased MMP-1 expression.10 IRA, IRB and IRC together may additionally affect the skin through the generation of heat. The activation of heat receptors at the surface of skin cells promotes inward calcium influx and induces MMP-1 expression, resulting in collagen degradation and wrinkle formation.11

Since IR appears to participate in the development of photoaging, this effect will is referred to as infra’aging— a term recently coined by the authors’ company. The aim of the present study was to develop and document the anti-photoaging and anti-infra’aging potential of a new cosmetic ingredient derived from the Polygonum aviculare plant. The study also provides insights into the mode of action of this extract.

Polygonum aviculare Extract

Fifty plants initially were screened for activity against the cathepsin G enzyme to find the best botanical extract to inhibit its activity (data not shown). Polygonum aviculare was among the best candidates. Polygonum aviculare (knotgrass) is one of the most common wild plants; it is able to grow on almost any soil and in any climate. Generally considered to be a weed, the plant grows about one foot tall (~30.5 cm). The plant is used in phytotherapy and traditional Chinese medicine as a diuretic, anti-rheumatic, hypoglycemic, anti-bacterial, antiphlogistic and a cardiotonic, among other things. Applied externally, it stops bleeding and has wound-healing properties. The plant extract is rich in flavonoids, which are natural chemicals with well-recognized antioxidant properties.12

Materials and Methods

Cathepsin G activity: The effect of Polygonum aviculare extracta on cathepsin G activity was evaluated using an enzymatic assay. The enzyme cathepsin G (1:50) was pre-incubated with increasing concentrations of the extract, from 0–1.0%. The reaction was initiated by adding fluorescein-labeled casein (1 mg/mL) as a substrate. Enzymatic activity was followed over time through the release of quenched fluorescence, as measured with a spectrofluorometer. The cathepsin G inhibitor phosphonic acid served as a positive control. Results presented in Figure 2 show a neat inhibition of cathepsin G activity with increasing concentrations of the extract, with an IC50 value of 0.014%.

UVA and UVB radiation, ex vivo studies: Skin specimens were obtained from a 65-year-old woman of the skin type II/III having undergone plastic surgery. The skin biopsies were maintained at 37°C, in a specific media and under properly humidified atmosphere with 5% CO2. For six days, 2 µL of an aqueous gel containing 2% Polygonum aviculare extract and carbomer was applied to the surface of the skin explants. The control was no treatment. On day five, 30 min post-product application, half of the skin biopsies were irradiated with UVA and UVB—29 J/cm2 and 2 J/cm2, respectively—using a solar simulatorb. On day six, skin explants were recovered and frozen prior to immunolabeling.

UVA and UVB radiation, inhibiting MMP-1: The expression of MMP-1 was detected on frozen skin sections using a mouse rabbit polyclonal anti-MMP-1 antibody (1:100). Staining was enhanced with a streptavidin/avidin system and revealed using green fluorescence, i.e., fluorescein isothiocyanate (FITC). Results from MMP-1 immunolabeling are presented in Figure 3. In untreated control samples, a moderate and diffuse green staining of MMP-1 can be observed throughout the epidermis, while staining remained weak within the dermis. UV radiation significantly increased MMP-1 expression at the epidermis level and also, to some extent, within the upper dermis. However, pre-treatment with the extract prevented this UV-induced increase in MMP-1 expression.

UVA and UVB radiation, protection of fibrillin-1: A similar immunolabeling system was used to detect the presence of fibrillin-1 on frozen skin sections. As can be seen in Figure 4, immunostaining of fibrillin-1 revealed a strong expression of the protein in control samples, with regular distribution at the dermal-epidermal junction (DEJ) and in the upper dermis. UV exposure resulted in a clear reduction in fibrillin-1 expression throughout the skin. Again, pre-treatment with the extract reversed this UV-induced effect, thus preserving the level and pattern of fibrillin-1 expression normally seen in control samples.

Protection from IR, ex vivo studies: In continuity with the protocol described, on day five of Polygonum aviculare extract application, the remaining skin biopsies were irradiated or not with 720 J/cm2 radiation, delivered for 60 min by a specific IRA lampc. Interestingly, a significant increase in skin temperature was noted at the surface of the explants following IR exposure. Skin explants were recovered and frozen on days six or nine for MMP-1 or tropoelastin immunolabeling, respectively.

Protection from IR, inhibiting MMP-1: The expression of MMP-1 was detected on day six frozen skin sections using a mouse rabbit polyclonal anti-MMP-1 antibody (1:100). Staining was enhanced with a streptavidin/avidin system and revealed using green fluorescence, i.e., FITC. Results from MMP-1 immunolabeling are presented in Figure 5

.

In non-irradiated control samples, a moderate and diffuse green staining of MMP-1 was observed throughout the epidermis, while staining remained weak within the dermis. MMP-1 expression was dramatically increased post-IR exposure, particularly at the upper dermis level. Pre-treatment with the extract prevented this IR-induced increase in MMP-1 expression.

Protecting tropoelastin under IR exposure: The same technique was used for immunostaining of tropoelastin on day nine frozen skin sections. Results are illustrated in Figure 6Figure 6. The presence of tropoelastin was detected clearly, with a well-organized distribution in the dermis of non-irradiated control samples. Importantly, tropoelastin expression was reduced throughout the skin following IR exposure, an effect that was alleviated when pre-treating with Polygonum aviculare extract.

Clinical Studies

The potential of Polygonum aviculare extract to effectively protect the skin from UV and IR-induced photoaging was next assessed under real use conditions. The study mobilized 20 healthy volunteers, ages 35–65 (mean = 49), during high summer season in southern Europe. All subjects were asked to blindly apply a test and control cream (see Formula 1) containing, or not, 2% Polygonum aviculare extract. Subjects applied and massaged the test cream on one forearm and the placebo on the other twice daily for 28 days, in the absence of any sunscreen. Quantities applied varied but corresponded to subjects’ normal use. At the end of the test period, skin firmness and elasticity were evaluated with a cutometerd. Volunteers also blindly applied the extract to one side of their face and the placebo to the other side, twice daily for 28 days, in the absence of sunscreen. Facial skin topography was then evaluated using an imaging systeme.

Cutometer readings: The cutometer used for this study measures the stretch capacity and residual deformation of the skin when sucked into the opening of a measuring probe. The resistance of the skin to suction-induced deformation, R0, represents skin firmness, while its capacity to return to its initial position post-deformation, R2, is taken as a measure of skin elasticity. The 2-mm-diameter measuring probe was positioned vertically inside the marked area of skin to be tested. Then a negative pressure gradient equal to 450 mbar was applied—i.e., suction, provoking skin penetration inside the probe opening; range = 1–10 mm.

The suction time was set to 2 sec, as was the release time; these standards were suggested by the device manufacturer. Each measurement was performed throughout three consecutive cycles of suction/release. For statistical analysis, the distribution of the values obtained during measurements at the various experimental times were compared with intra-group analyses, D0 versus D28, using the Student’s t-test; p values < 0.05 were considered significant.

Results from viscoelastic measurements are shown in Figure 7. Skin firmness was increased significantly (p < 0.001) by a mean value of 11.9%, following 28 days of twice daily application of the extract; the placebo treatment resulted in a non-significant increase of only 1.3%. At the same time, skin elasticity improved significantly (p < 0.001) by a mean value of 4.8% with extract application, compared to only 0.5% for the placebo.

Topography readings: The topography system used for this study is based on a patented fringe projection unit using blue light combined with imaging techniques, allowing for non-contact local measurement of skin topography. Measurements were performed on the crow’s feet area for each volunteer initially, at D0, and compared with values obtained at D28 for both the extract and placebo-treated areas. Scanning software was used for viewing, capturing and analyzing results. The parameters used for this technique included surface roughness, wrinkle volume, mean wrinkle depth and maximum wrinkle depth. For statistical analysis, the distribution of the values obtained during measurements at the various experimental times were compared with intra-group analysis, D0 versus D28, using the Student’s t-test; p values < 0.05 were considered significant.

Results from skin topography measurements in the crow’s feet area are shown in Figure 8. Upon treatment with the extract, all measured parameters were significantly (p < 0.0001) improved for all (100%) subjects. Skin roughness was reduced by 11.2%, wrinkle volume by 11.5%, mean wrinkle depth by 11.2% and maximum wrinkle depth by 11.5%. In contrast, placebo treatment resulted in slight, insignificant increases of all parameters. A selection of photographs, representative of the clinical results described, are presented in Figure 9. In these photographs, wrinkle reduction and a smoothing effect are clearly observed in the crow’s feet area, after only one month of treatment.

Discussion

The present study identifies Polygonum aviculare extract as an inhibitor of cathepsin G activity—a serine protease, whose production by neutrophils and fibroblasts under UV exposure has been recently linked to photoaging via activation of MMPs. This is corroborated by the present results showing that UV-induced overexpression of MMP-1 in the epidermis of irradiated skin explants is strongly reduced by pre-treatment with an inhibitor of cathepsin G, i.e., Polygonum aviculare extract. MMPs have long been associated with collagen degradation and wrinkle formation in photoaging.13

Polygonum aviculare extract also had an effect on fibrillin-1 under the same conditions. Fibrillins are large, 350–kDa glycoproteins that make up the major structural element of microfibrils. The latter serve as scaffolds for the assembly of elastin fibers.14, 15 During photoaging, fibrillins can be degraded,16 thus dismantling the protective support of elastic fibers and exposing insoluble elastin. Because elastin is resistant to proteolysis, it tends to accumulate in a disorganized fashion, typical of solar elastosis.17 Destruction of the fibrillin network at the DEJ and in the upper dermis following UV exposure was observed ex vivo in the present study. Interestingly, however, pre-treatment with Polygonum aviculare extract had a clear protective effect on fibrillin-1 structure, therefore preserving skin elasticity.

UV exposure may not be solely responsible for skin photoaging. An increasing number of studies point toward additional suspects, among which is infrared radiation, especially IRA.4, 18 As mentioned, IR affects the skin in two ways: directly, through ROS generation and ECM degradation, and/or indirectly, through heat generation at the skin surface.19, 20 Exposure to IR at physiologically relevant doses has been shown to activate signaling pathways mediating the up-regulation of MMP-1 and MMP-3.21 Heat has a similar effect on MMP-1 expression, although through a different pathway involving calcium influx.22

In the present study, the authors also found an increased expression of MMP-1 in skin explants upon IR exposure, especially at the DEJ and the upper dermis. Noteworthy, the pattern of IR-induced MMP-1 expression differs from that observed under UV exposure. IR affects MMP-1 within the dermis, while UV strikes essentially at the epidermis level. This is taken as a reflection of the deeper penetration of IRA radiation. As with UV, the IR-induced up-regulation of MMP-1 expression was prevented by pre-treatment with Polygonum aviculare extract.

IR exposure of skin explants also affected the expression of tropoelastin at the DEJ and within the upper dermis. Tropoelastin is a small elastin precursor with high crosslinking potential, known to associate with fibrillin microfibrils to form elastic fibers.23 In the present study, IR caused a strong reduction in tropoelastin expression, an effect that was again prevented by pre-treatment of the skin explants with the extract. This IR-induced disappearance of tropoelastin is somewhat in contradiction with results reported earlier by Chen et al. in 2009; however, different systems were used, i.e., ex vivo vs. in vivo. Importantly, pre-treatment with Polygonum aviculare extract restored the expression and regular distribution pattern of the protein in the upper dermis and at the DEJ.

When tested against placebo in real life conditions, on a panel of human volunteers regularly exposed to strong summer sun, the extract proved to significantly increased skin firmness and elasticity within one month of twice daily application. The product also significantly reduced the appearance of wrinkles in the crow’s feet area. At the end of the study, all volunteers experienced some level of improvement.

Conclusion

Taken together, these results demonstrate that Polygonum aviculare extract is a potent inhibitor of UV and IR-induced elastolysis. The extract is addressing a novel pathway involving cathepsin G inhibition to modulate MMP-1 expression and protect elastic fibers from a broad range of solar effects (see Figure 10). In fact, to the authors’ knowledge, this is the first time that a cathepsin G inhibitor clinically demonstrates anti-wrinkle benefits in humans exposed to photoaging. Its protective effects are seen at all skin levels, opposing UVB at the epidermis as well as UVA and IRA at the dermis (see Figure 10). These experiments also highlight the non-negligible contribution of infrared radiation to solar elastosis. It certainly appears it is time to reconsider photoaging to include this emerging concept of infra’aging.

References
1. GP Pfeifer and A Besaratinia, UV wavelength-dependent DNA damage and human non-melanoma and melanoma skin cancer, Photochem Photobiol Sci 11(1) 90–97 (2012)
2. DL Damian, YJ Matthews, TA Phan and GM Halliday, An action spectrum for ultraviolet radiation-induced immunosuppression in humans, Br J Dermatol 164(3) 657–9 (2011)
3. A Svobodova and J Vostalova, Solar radiation induced skin damage: review of protective and preventive options, Int J Radiat Biol 86(12) 999–1030 (2010)
4. E Dupont, J Gomez and D Bilodeau, Beyond UV radiation: A skin under challenge, Int J Cosmet Sci (2013) Feb 14. doi: 10.1111/ics.12036.
5. J Uitto, The role of elastin and collagen in cutaneous aging: intrinsic aging versus photoexposure, J Drugs Dermatol 7(2 Suppl) s12–6 (2008)
6. ED Son, H Kim, H Choi, SH Lee, JY Lee, S Kim, B Closs, S Lee, JH Chung and JS Hwang, Cathepsin G increases MMP expression in normal human fibroblasts through fibronectin fragmentation, and induces the conversion of proMMP-1 to active MMP-1, J Dermatol Sci 53(2) 150–2 (2009)
7. Y Zheng, W Lai, M Wan and HI Maibach, Expression of cathepsins in human skin photoaging, Skin Pharmacol Physiol 24(1) 10–21 (2011)
8. ED Son, JH Shim, H Choi, H Kim, KM Lim, JH Chung, SY Byun and TR Lee, Cathepsin G inhibitor prevents ultraviolet B-induced photoaging in hairless mice via inhibition of fibronectin fragmentation, Dermatology, 224(4) 352–60 (2012)
9. CE Schmelzer, MC Jung, J Wohlrab, RH Neubert and A Heinz, Does human leukocyte elastase degrade intact skin elastin? FEBS J 279(22) 4191–200 (2012)
10. J Krutmann and P Schroeder, Role of mitochondria in photoaging of human skin: the defective powerhouse model, J Invest Dermatol Symp Proc 14(1) 44–9 (2009)
11. MH Shin, JE Seo, YK Kim, KH Kim and JH Chung, Chronic heat treatment causes skin wrinkle formation and oxidative damage in hairless mice, Mech Ageing Dev 133(2–3) 92–8 (2012)
12. M Costea and FJ Tardif, The biology of Canadian weeds. 131. Polygonum aviculare L, Biology Faculty Publications, Paper 76 (2005), available at scholars.wlu.ca/boil_faculty/76 (Accessed Apr 26, 2013)
13. GJ Fisher, HS Talwar, J Lin and JJ Voorhees, Photochem Photobiol 69(2) 154–7 (1999)
14. JE Wagenseil and RP Mecham, New insights into elastic fiber assembly, Birth Defects Res C Embryo Today 81(4) 229–40 (2007)
15. MJ Rock, SA Cain, LJ Freeman, A Morgan, K Mellody, A Marson, CA Shuttleworth, AS Weiss and CM Kielty, Molecular basis of elastic fiber formation. Critical interactions and a tropoelastin-fibrillin-1 cross-link, J Biol Chem 279(22) 23748–58 (2004)
16. Z Chen, FL Zhuo, SJ Zhang, Y Tian, S Tian and JZ Zhang, Modulation of tropoelastin and fibrillin-1 by infrared radiation in human skin in vivo, Photodermatol Photoimmunol Photomed 25(6) 310–6 (2009)
17. F Rijken, Pathophysiology and prevention of photoaging the role of melanin, reactive oxygen species and infiltrating neutrophils (Thesis), Utrecht University website, available at igitur-archive.library.uu.nl/dissertations/2011-0617-200830/UUindex.html (Accessed Apr 20, 2013)
18. TG Polefka, TA Meyer, PP Agin and RJ Bianchini, Effects of solar radiation on the skin, J Cosmet Dermatol 11(2) 134–43 (2012) Erratum in: J Cosmet Dermatol 11(4) 329 (2012)
19. P Schroeder, C Calles, T Benesova, F Macaluso and Krutmann, Photoprotection beyond ultraviolet radiation–effective sun protection has to include protection against infrared A radiation-induced skin damage, Skin Pharmacol Physiol 23(1) 15–7 (2010)
20. S Cho, MH Shin, YK Kim, JE Seo, YM Lee, CH Park and JH Chung, Effects of infrared radiation and heat on human skin aging in vivo, J Investig Dermatol Symp Proc 14(1) 15–9 (2009)
21. MS Kim, YK Kim, KH Cho and JH Chung, Regulation of type I procollagen and MMP-1 expression after single or repeated exposure to infrared radiation in human skin, Mech Ageing Dev 127(12) 875–882 (2006)
22. MH Shin, JE Seo, YK Kim, KH Kim and JH Chung, Chronic heat treatment causes skin wrinkle formation and oxidative damage in hairless mice, Mech Ageing Dev 133(2–3) 92–98 (2012)
23. MJ Sherratt, Tissue elasticity and the ageing elastic fibre, Age (Dordr), 31(4) 305–25 (2009)

 

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Figure 1. IR can be divided into IRA, IRB and IRC; IRA in particular penetrates deeply into the skin, even reaching the hypodermis.

Figure 1. IR can be divided into IRA, IRB and IRC; IRA in particular penetrates deeply into the skin, even reaching the hypodermis.

IR can be divided into IRA, IRB and IRC, and while all three have low energy and may seem inoffensive, IRA penetrates deeply into the skin, even reaching the hypodermis (see Figure 1).3, 4

Figure 2. Neat inhibition of cathepsin G activity with increasing concentrations of the extract

Figure 2. Neat inhibition of cathepsin G activity with increasing concentrations of the extract

Results presented in Figure 2 show a neat inhibition of cathepsin G activity with increasing concentrations of the extract, with an IC50 value of 0.014%.

Figure 3. Results from MMP-1 immunolabeling; pre-treatment with the extract prevented a UV-induced increase in MMP-1 expression.

Figure 3. Results from MMP-1 immunolabeling; pre-treatment with the extract prevented a UV-induced increase in MMP-1 expression.

Results from MMP-1 immunolabeling are presented in Figure 3.

Figure 4. Immunostaining of fibrillin-1

Figure 4. Immunostaining of fibrillin-1

As can be seen in Figure 4, immunostaining of fibrillin-1 revealed a strong expression of the protein in control samples, with regular distribution at the dermal-epidermal junction (DEJ) and in the upper dermis.

Figure 5. Results from MMP-1 immunolabeling; pre-treatment with the extract prevented an IR-induced increase in MMP-1 expression.

Figure 5. Results from MMP-1 immunolabeling; pre-treatment with the extract prevented an IR-induced increase in MMP-1 expression.

Results from MMP-1 immunolabeling are presented in Figure 5.

Figure 6. Immunostaining of tropoelastin on day nine frozen skin sections; reduced tropoelastin expression was alleviated when pre-treating with Polygonum aviculare extract.

Figure 6. Immunostaining of tropoelastin on day nine frozen skin sections; reduced tropoelastin expression was alleviated when pre-treating with <em>Polygonum aviculare</em> extract.

The same technique was used for immunostaining of tropoelastin on day nine frozen skin sections.

Figure 7. Results from viscoelastic measurements: a) firmness and b) elasticity (D28)

Figure 7. Results from viscoelastic measurements: a) firmness and b) elasticity (D28)

Results from viscoelastic measurements are shown in Figure 7.

Figure 8. Results from skin topography measurements in the crow’s feet area

Figure 8. Results from skin topography measurements in the crow’s feet area

Results from skin topography measurements in the crow’s feet area are shown in Figure 8.

Figure 9. A selection of photographs representative of the clinical results described

Figure 9. A selection of photographs representative of the clinical results described

A selection of photographs, representative of the clinical results described, are presented in Figure 9.

Figure 10. Protective effects are seen at all skin levels, opposing UVB at the epidermis as well as UVA and IRA at the dermis.

Figure 10. Protective effects are seen at all skin levels, opposing UVB at the epidermis as well as UVA and IRA at the dermis.

Its protective effects are seen at all skin levels, opposing UVB at the epidermis as well as UVA and IRA at the dermis (see Figure 10).

Footnotes (CT1309 Loing)

a Elix-IR (INCI: Polygonum Aviculare extract) is a product of Lucas Meyer Cosmetics, www.lucasmeyercosmetics.com.
b The Lourmat RMX 3W UV simulator is manufactured by Vilber, www.vilber.com.
c The IR lamp described is manufactured by Dr. Fisher, www.dr-fischer-group.com.
d The MPA 580 Cutometer is a device from Courage-Khazaka GmbH, www.courage-khazaka.com.
e The Visio3D DermaTOP BLUE system is a device from Eotech SA, www.eotech-sa.com.

Formula 1. Test formulation used in the clinical study

Formula 1. Test formulation used in the clinical study

All subjects were asked to blindly apply a test and control cream (see Formula 1) containing, or not, 2% Polygonum aviculare extract.

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