54 UPCYCLED INGREDIENTS
Control ■ Reference (dexamethasone) ■ Chios mandarin extract ■
400 300 200 100 0 10 µM Figure 2: Chios mandarins
for an additional six hours. After the treatment, cells were washed and collected. Nuclear proteins were isolated using an extraction protocol. Following this, active NF-κB was quantified.
Gene expression analysis under chronic inflammation in keratinocytes Human keratinocytes were incubated for 24 hours with Chios mandarin extract or left untreated. Chronic inflammation was then induced in human keratinocytes by the addition of an inflammatory cocktail containing LL-37, calcitriol, and IL-17 for 48 hours. Following this, the expression of candidate genes that are important for the inflammatory response was analyzed.
Fibroblasts treatment with inflammatory keratinocyte secretion To induce inflammatory keratinocyte secretion, cells were exposed to UV radiation (275 mJ/ cm2
UVB and 2.1 J/cm2 UVA) and subsequently
incubated for 48 hours. Dermal fibroblasts were treated for 24 hours with or without 0.33% and 1% Chios mandarin extract. After the incubation time, the treatments
were refreshed, and the supernatant of the irradiated keratinocytes was added for 72 hours. At the end of the study, the expression of candidate genes that are important for the inflammatory response was analyzed.
Procollagen I secretion In this assay, skin fibroblasts were treated with
Figure 3: Reduced activation of NF-κB
or without 0.33% Chios mandarin extract for 24 hours. To induce oxidative stress, 800 µM H2
O2
was added for two hours. Following this, the cell culture medium
was replaced with fresh medium containing Chios mandarin extract at the concentration used before. After 72 hours of incubation, the secretion of procollagen I was analyzed.
Production of newly formed collagen Human skin explants were treated topically with a cream containing 0.5% and 1% Chios mandarin extract or a placebo cream. Following seven days of treatment, punches of the skin explants were fixed and prepared for staining. After Herovici staining, images of the skin
explants were captured using brightfield microscopy (Nikon microscope from Nikon Corporation). In the images taken, the collagen fibers were localized in the dermis of the human skin explants. Young collagen is represented in blue, while mature collagen is represented in red magenta.
Clinical studies We aimed to investigate the impact of our Chios mandarins active in vivo on a panel of volunteers with signs of skin redness as well as photo-aging. Twenty women aged between 51 and 68 years (mean age: 60.9 years) were included in this randomized, placebo- controlled clinical study. The volunteers applied a cream containing
2% Chios mandarins active or a corresponding placebo cream on each side of their face
and on two separate areas on the forearms twice daily for 28 days. Facial skin redness was determined at days 0, 7, and 28 using the Spectrophotometer® CM-700d from Konica Minolta, and images were captured using the ColorFace® from Newtone Technologies. In addition, skin elasticity and skin firmness
of the face were analyzed at days 0, 7 and 28 using the Cutometer® dual MPA 580 from Courage and Khazaka, while skin density was measured on the forearms of the volunteers using the Dermalab® ultrasound device from Cortex Technology.
Results and discussion To investigate the effect of Chios mandarin extract on inflammation, in vitro studies were performed using keratinocytes and dermal fibroblasts. Pretreatment of human dermal fibroblasts
with the Chios mandarin extract significantly reduced the activation of NF-κB to the same extent as the anti-inflammatory reference compound dexamethasone, showing that our Chios mandarins active suppresses the NF-κB signaling pathway (Figure 3). For the keratinocyte assay, cells were
treated with an inflammatory cocktail to stimulate chronic inflammation in the presence and absence of Chios mandarin extract. Analysis of genes important for the inflammatory response revealed that co-treatment with Chios mandarin extract decreased the expression of inflammatory markers in keratinocytes.
Control ■ 0.33% Chios mandarin extract ■ 1 % Chios mandarin extract ■
250 200 150 100 50 0
Control Figure 4: Reduced inflammatory response PERSONAL CARE September 2024
www.personalcaremagazine.com CXCL1 CCL2 IL-6 0.01% *p<0.001 versus untreated control **p<0.001 versus stressed control
Gene expression changes compared to untreated control in %
Keratinocyte secretion after UV radiation
NF-kB activation compared to untreated control in %
Inflammatory stress
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