468 JOURNAL OF COSMETIC SCIENCE
To evaluate the antioxidant capacity of stevia extract obtained through different extraction
methods, the samples were analyzed using the ORAC test (Figure 16). The stevia extract
obtained through subcritical water technology resulted in a much higher antioxidant
efficacy compared to the extract derived from common extraction processes with glycerin
or water, which suggests a higher recovery of antioxidant compounds with the subcritical
water technology.
Moreover, stevia extract was found to downregulate the expression of the cyclooxygenase-2
(COX-2) enzyme gene by 65% (data not shown) in HEKa treated with 0.37% S rebaudiana
extract for 24 hours. COX-2 encodes an enzyme responsible for producing inflammatory
prostaglandins and is a key player in the induction of inflammatory responses. The
Figure 15. Relative gene expression (fold change) in HDFa cultures treated with S rebaudiana extract at 1.1%
for 24 hours respect to control that is evaluated by microarray analysis.
Figure 16. Antioxidant capacity for stevia extract versus common extraction methodologies analyzed by
ORAC test.

469 DELIVERING SUSTAINABLE SOLUTIONS TO IMPROVE WELLBEING
attenuation of COX-2 production provides anti-inflammatory properties that can help
prevent irritation, thus making the active ingredient a safer natural alternative to retinoids.
To further confirm the anti-inflammatory properties of the extract (Figure 17), HEKa were
treated with the proinflammatory TNF-α (0.25 ng/mL) and showed a pro-inflammatory
effect with an increase in IL-6 levels. Keratinocytes treated with TNF-α were also treated
with 0.05 µg/mL retinoic acid, 0.005% S rebaudiana extract, or with 0.005% bakuchiol for
48 hours. Retinoic acid did not reduce the inflammation caused by TNFα and even slightly
increased it. On the other hand, both bakuchiol and S rebaudiana extract significantly
decreased the level of the pro-inflammatory cytokine IL-6.
Bakuchiol is known in the industry as a gentle alternative to retinoids, and S rebaudiana
extract has shown the same anti-inflammatory effects. Additionally, the previously
mentioned comparative results with retinoic acid indicates that S rebaudiana extract
operates on the same pathway as retinoids, thereby exhibiting the same antiaging effects as
retinoids but without the side effects.
A clinical test was carried out on 21 female volunteers of Caucasian descent between 41
and 60 years old. These volunteers applied a cream containing S rebaudiana extract at 2%
to half of the face, and a placebo cream to the other half twice a day for 28 days. The effect
of the products on the crow’s feet wrinkles, skin tone homogeneity, and the number of
pigmented dark spots were evaluated after 28 days of treatment.
The wrinkle area and wrinkle length parameters were evaluated before and after 28 days of
product treatment on the crow’s feet area. The active cream showed a reduction of the area
of wrinkles by 16.5% and of the length of wrinkles by 17.1% with respect to initial time
(Figure 18A). The placebo treatment did not induce any improvement either in wrinkle area
or wrinkle length. Moreover, a visual reduction in the appearance of wrinkles is observed
from the facial images (Figure 18B).
In addition, to determine the skin tone evening effect, variations in the skin tone homogeneity
and number of pigmented spots on the cheek area were evaluated. The results showed
an improvement in the skin tone homogeneity with an increase of 4.8% after 28 days of
using the product with respect to initial time (Figure 19A, left panel). In the images of the
volunteer’s cheek area (Figure 19A, right panel), a more homogeneous skin tone can be seen.
Figure 17. Percentage of IL-6 release in HEKa cell culture after TNF-a treatment during 48 hours in
presence of different treatments respect to inflammation control (TNF-a). Data shown as MEAN ± SEM of
the mean of three independent experiments. Statistical significance: **p 0.01, calculated using an unpaired
student’s t test.