108 ANTI-ACNE After 48 hours of colonization, the inoculum
excess was removed before treatment with 5μL of the test item that was applied on epidermal surface for each dedicated series for 24 hours. At the end of the treatment period, media was collected and stored at -20°C for the ELISA assay. The entire assay procedure is shown schematically in Figure 1.
ELISA assay Culture media collected at 72h were analyzed for Caspase-1 and TNF-a by ELISA test using biological triplicate quantified in technical duplicate. Each inflammatory marker in the culture media has been quantified with a commercially available kit and according to internal procedures. â– Human Caspase-1/ICE Immunoassay (Catalog Number DCA100, R&D Systems) â– Human TNF-a (Catalog Number DTA00D, R&D Systems) Optical density (OD) was read at 450 nm using the Tecan Infinite M-200 spectrophotometer and readings at 570 nm were subtracted from the readings at 450 nm. This subtraction corrects for optical imperfections in the plate.
Statistical analysis The OD data was analyzed using the paired student T-Test with statistical significance set at p≤0.05. In all instances examined, the data for the negative control was not reported as all values discussed below in the studies were statistically significant against the negative control.
Results and discussion Solubilized azelaic acid versus suspended azelaic acid An initial investigation comparing solubilized azelaic acid against suspended azelaic acid was undertaken to examine how they performed when examining the release of TNF-a. Results from this study are shown in Figure 2.
0.25 0.2
0.15 0.1
0.05 0
C.Acne Treated Control
5% Solubilized Azelaic Acid
10%
Solubilized Azelaic Acid
1%
Salicylic Acid
2%
Salicylic Acid
1% Salicylic
Acid/5% Solu Azelaic Acid
2% Salicylic
Acid/5% Solu Azelaic Acid
2% Salicylic
Acid/10% Solu Azelaic Acid
Figure 3: Comparison of 5% and 10% solubilized azelaic acid against 1% and 2% concentrations of salicylic acid and combinations of azelaic acid and salicylic acid for expression of TNF-a indicating statistical significance (p≤0.05). All values shown were statistically significantly increased versus the negative control (data not shown)
PERSONAL CARE April 2024
www.personalcaremagazine.com (Optical Density @ 450nm) TNF-a Assay Results
0.12 0.1
0.08 0.06 0.04 0.02 0
p=0.008
5% azelaic acid p=0.0002
10% azelaic acid
C.Acne Treated Control
5% Solubilized Azelaic Acid
5% Suspended Azelaic Acid
10% Solubilized Azelaic Acid
10% Suspended Azelaic Acid
Figure 2: Comparison of 5% and 10% solubilized azelaic acid against similar concentrations of suspended azelaic acid for expression of TNF-a indicating statistical significance (p≤0.05). All values shown were statistically significantly increased versus the negative control (data not shown)
The data indicates that at the lower 5%
concentration of azelaic acid, the suspended azelaic acid appears to elicit a more intense inflammatory response versus the solubilized azelaic acid for TNF-a expression. This difference is not as apparent at the higher concentrations.
Solubilized azelaic acid versus salicylic acid and combinations of both Studies were conducted to examine the influence of solubilized azelaic acid versus salicylic acid and various combinations of salicylic acid and solubilized azelaic acid examining expression of TNF-a. The results of these studies are shown in Figure 3. The data in Figure 3 indicates that both 5% and 10% solubilized azelaic acid had no
(Optical Density @ 450nm) TNF-a Assay Results p=0.008 p=0.0009 p=0.02 p=0.0008 p=0.002 p=0.009 p=0.00009
impact on the upregulation of TNF-a versus the C. acnes treated control. This was not the case for 1% and 2% salicylic acid which showed significant further increases in the expression of TNF-a above those noted for the C. acnes treated control. All the results shown were statistically
increased versus the negative control (data not shown). More importantly, when the solubilized azelaic acid was blended with 1% and 2% salicylic acid, a significant decrease in the expression of TNF-a was seen in all the blends versus their corresponding individual salicylic acid responses. This appears to indicate that adding 5%
or 10% solubilized azelaic acid to 1% or 2% salicylic acid, respectively, can help inhibit the expression of the inflammatory marker TNF-a.
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