453 DELIVERING SUSTAINABLE SOLUTIONS TO IMPROVE WELLBEING
fluorescence intensity is proportional to the amount of the product in the PCR reaction.
Cycling conditions in the Bio-Rad CFX96 instrument are as follows: 95°C for 3 minutes,
followed by 40 cycles of denaturing at 95°C for 5 seconds, and annealing and elongation at
60°C for 30 seconds. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) and HRPT1
(hypoxanthine phosphoribosyltransferase 1) were used as endogenous controls. Fold change
relative to the expression of the sample genes and reference genes was calculated using a
normalized expression (ΔΔ(Ct)) method using CFX manager software (Bio-Rad). Results
were expressed as a percentage of gene expression normalized by the corresponding control
(cells treated with the medium alone). Evaluated genes for retinoic acid pathway were as
follows: RBP (retinol binding protein), RAR (retinoic acid receptor), and RXR (retinoid
X receptor). Evaluated genes for skin regeneration genes were: HB-EGF (heparin-binding
EGF-like growth factor) and HAS2 (hyaluronan synthase).
EVALUATION ON DERMIS REMODELLING: TYPE I COLLAGEN AND ELASTIN INDUCTION, AND
MATRIX METALLOPROTEASES 1 AND 3 (MMP-1 AND MMP-3) INHIBITION IN HUMAN DERMAL
FIBROBLASTS COCULTURED WITH HUMAN EPIDERMAL KERATINOCYTES
HEKa and HDFa were independently trypsinized and seeded in 12-well plates. The
cells in the coculture were incubated at 37°C in 5% CO
2 for 24 hours. After this
incubation period, the medium was removed, and fresh coculture medium was added
with testing products (S rebaudiana extract at 0.01% and retinoic acid at 0.05 µg/mL)
prepared in the same medium. Cells treated with coculture medium alone were used
as the coculture control. Cells were incubated for an additional 24 hours (for MMPs),
48 hours (for type I collagen), and 72 hours (for elastin), respectively, at 37°C in 5%
CO
2 humidified air. After the indicated times, supernatants were collected. A protease
inhibitor cocktail was added to the supernatants, and these were kept at -80°C until
they were analyzed by AlphaLISA (PerkinElmer, Inc., Waltham, MA, USA for type I
collagen), or ELISA (Cusabio, Houston, TX, USA, for elastin and ABCAM for MMP-1
and MMP-3) according to the manufacturer’s protocol. These results were normalized
with the total viable cell number for each condition calculated by the PrestoBlueTM
staining assay (Thermo Fisher Scientific, Waltham, MA, USA). The obtained values
were normalized compared to the coculture control and represent the means of three
independent experiments performed in duplicates. The statistical analysis used was the
unpaired student’s t test.
GENE EXPRESSION MODULATION ON HUMAN DERMAL FIBROBLASTS AND KERATINOCYTES
Keratinocytes or fibroblasts were seeded in a 24-well plate (NHEK) or 12-well plate
(NHDF) and then cultured in culture medium for 48 hours with the medium renewed
after 24 hours of incubation.
After this incubation time, cell culture medium was removed and replaced with the
medium containing or not containing (control) S rebaudiana extract, and cells were
incubated for 24 hours. All the experimental conditions were realized in n =3. At the end
of the incubation, culture supernatants were removed, and the cells were rinsed with a PBS
solution. The plates were immediately frozen dry at -80°C. Prior to RNA extraction, the
plates were thawed, and technical replicates were pooled. The total RNA of each sample

454 JOURNAL OF COSMETIC SCIENCE
was extracted using a TriPure Isolation Reagent® (Sigma-Aldrich, St. Louis, MO, USA)
according to the protocol recommended by the supplier. The quantity and quality of the
RNAs were evaluated by capillary electrophoresis (Bioanalyzer 2100, Agilent, Santa Clara,
CA, USA). Gene expression was measured using a DNA microarray technique. A full
transcriptomic analysis (Affymetrix, Santa Clara, CA, USA) was developed by the external
company Bioalternatives SAS. The “fold change” thresholds (value corresponding to the
ratio: signal intensity value of a probe corresponding to the processed sample divided
by the signal intensity value of a probe corresponding to the control) have been defined
and applied to standardized data to filter more relevant upregulated and downregulated
genes. The percentage of fold change values are represented for selected genes. The studied
genes for antioxidant response were as follows: HMOX1 (heme oxygenase-1), TXNRD1
(thioredoxin reductase) and OSGIN-1 (oxidative stress induced growth inhibitor 1). These
genes were found in fibroblasts treated with 1.1% S rebaudiana extract. Studied genes for
evaluating inflammatory response were PTGS2 (Prostaglandin-endoperoxide synthase 2,
also known as COX-2) in keratinocytes treated with 0.37% S rebaudiana extract.
ORAC METHOD: TEAC ASSAY
The oxygen radical absorbance capacity (ORAC) method evaluates the ability of antioxidants
in a sample to limit the oxidation of fluorescein, a fluorescent probe that is sensitive to
oxidation, by generated peroxyl radicals by a thermolabile oxidizing compound (AAPH).
The oxidation of fluorescein is accompanied by a decrease in fluorescence measured over
time (excitation is 485 nm, emission is 520 nm). The antioxidant compounds involved in
this reaction thus limit, at least for a time, the loss of fluorescence. The sample’s antioxidant
potential is determined by the difference in area under the curve with the area under
the curve of a control without antioxidants. The antioxidant potential is expressed in a
concentration that is equivalent to an antioxidant of reference (Trolox), where 1 ORAC unit
is equivalent to 1 µmol of Trolox per g or per 100 g (extracts and fruits) or 1 µmol of Trolox
per µmol of pure compound.
INHIBITION OF INTERLEUKIN 6 (IL-6) RELEASE IN HUMAN EPIDERMAL KERATINOCYTES
HEKa were seeded in a 96-well black plate (PerkinElmer, Inc., Waltham, MA, USA) in
culture medium. After 24 hours of incubation at 37°C in 5% CO
2 humidified air, the
medium was removed and keratinocytes were treated with the proinflammatory TNF-α
at 0.25 ng/mL, which shows an inflammatory effect with an increase in IL-6 levels this
medium was then used as a positive control. Keratinocytes were also treated with the
combination of TNF-α and either 0.05 µg/mL retinoic acid, 0.005% S rebaudiana extract,
or 0.005% bakuchiol. Cells treated with the medium alone were used as a control. After
48 hours of incubation, all supernatants were collected and kept at -20°C until they were
analyzed by ELISA for interleukin-6 protein (Thermo Fisher Scientific, Waltham, MA,
USA) following the provider’s instructions. These results were normalized with the total
viable cell number for each condition calculated by the PrestoBlueTM staining assay (Thermo
Fisher Scientific, Waltham, MA, USA). The obtained values were normalized compared
to the control and represent the mean of three independent experiments performed in
triplicates. The statistical analysis used was the unpaired student’s t test.