54 NATURALS
CosPET method Cosmetic Preservative Efficacy Test, semiquantative
Criteria
S. aureus S. epidermidis
P.aeruginosa
P.gergoviae P.putida B.cepacia
E.coli
K. pneumoniae 105 -106 fcu/a Figure 1: Germs and assessment of CosPET
are separately inoculated with a bacterial and a fungal suspension (Figure 1). Simultaneously, streak cultures of each batch are made before inoculation. The microbial growth is evaluated semi-quantitatively. After inoculation the germ count is expected to decrease over time. The final assessment is performed in accordance with European Pharmacopoeia.
Antioxidation To determine the radical scavenging efficacy of raspberry ketone, a DPPH assay was performed – a free radical method based on electron- transfer that produces a violet solution in ethanol. When DPPH reacts with an antioxidant compound, which can donate hydrogen, it is reduced subsequently. The changes in colour (from deep violet to light yellow) were read at 517 nm after 30 min of reaction using a UV/VIS spectrophotometer.1
The resulting
decolourization is stoichiometric with respect to the number of electrons taken up. Another popular tool to measure the
antioxidative activity is the FRAP assay. The ferric reducing antioxidant power (FRAP) assay is a method that measures the reduction of a ferric ion (Fe3+
coloured ferrous (Fe2+
)-ligand complex to the intensely blue- ) complex by antioxidants
in an acidic medium. Antioxidant activity is determined via optical density evaluated with a colorimetric probe at 700 nm using a UV/VIS spectrophotometer.2
Peroxidation protection Lipids peroxidation is a process in which free radicals initiate a cascade of reactions attacking the lipid membranes, resulting in cell damage and eventual cell death. Lipids peroxidation is involved in premature aging and in the progression of comedogenesis. The percentage of tested and preserved lipid is measured via an in tubo oxygen peroxide stress assay and evaluated with a HPLC-MS quantification of peroxidized linoleic acid.3
Enzymes inhibition Inhibiting collagenase and elastase skin enzymes helps to protect the skin elasticity which may be stressed by UV, IR or blue light. In tubo collagenase inhibition activity is used to understand the characteristics of a substrate to protect the ECM (extracellular matrix) from the digestion by MMPs (matrix metalloproteinases) such as collagenase or elastase.
PERSONAL CARE March 2023
Figure 2: o/w cream CosPET results Light O/W cream
Unpreserved
0.5% optiphen od (Caprylyl Glycol) 1.0% optiphen hd (1,2-Hexanediol) 0.5% optiphen po (Phenoxyethanol)
0.5% phyteq raspberry 0.5% optiphen od
0.5% phyteq raspberry 1.0% optiphen hd
0.5% phyteq raspberry 1.0% optiphen po
Assessment F= failed A= pass criteria A B= pass criteria B
– = Free of microbial growth + = Slight growth ++ = Moderate growth
Figure 2: o/w cream CosPET results
Gelatinase/Collagenase Assay Kit, 250-2 with some modifications,4
EnzChek Elastase Assay Kit.5
Anti-inflammation Intracellular cytokine staining is a flow cytometric technique consisting of culturing stimulated cytokine-producing cells in the presence of a protein secretion inhibitor. To understand anti-inflammation capabilities a decrease of IL-1R1 expression is evaluated with the help of a Volocity Acquisition 3D image capture software.
Results and discussion Antimicrobial activity The raspberry multifunctional was tested in various formulations at concentrations from 0.5% to 1.5%. Hereby, boosting and broad-spectrum efficacy without pH limitations at a low dosage (0.5 – 1%) could be demonstrated. Challenging applications in cosmetics are
water containing systems such as micellar waters, o/w emulsions or rinse-off products. Here, combinations of 0.5% raspberry multifunctional ingredient with additional antimicrobials such as optiphenTM
hd multifunctional (1,2-Hexanediol)
or optiphen od multifunctional (Caprylyl Glycol) provide a reliable protective strategy (Figure 2).
Antioxidation - DPPH assay (α, α-diphenyl-β- picrylhydrazyl) In this assay, the test substrate DPPH has been
The test was performed using the EnzChek and the assay kit E12056,
mixed individually with the following test substances: 0.5% raspberry multifunctional ingredient, 0.5% hydroxy acetophenone, and 0.5% ascorbic acid. Ascorbic acid hereby represents a well-known antioxidative benchmark in this test scenario.1 The scavenging test was performed for
30 minutes in dark measuring the reaction potential of a test substrate (prepared in DMSO) and the stable free DPPH-radical (DPPH prepared in ethanol) being reduced to DPPH-H. This reduction causes a colour shift from deep purple to colourless or pale yellow.2 The raspberry multifunctional ingredient
can scavenge 70% of the free radicals after 30 minutes of reaction time (Figure 3). Hydroxy acetophenone only provides limited antioxidant properties (10%). Ascorbic acid achieves 95%.
Ferric reducing antioxidant power (FRAP) The FRAP assay is a widely used method wherein Fe3+
is reduced to Fe2+ ; which then reacts with ferric
chloride to form a ferric-ferrous complex that has an absorption maximum at 700 nm. The assay was done using 0.5% raspberry multifunctional ingredient, 0.5% hydroxy acetophenone, and 0.5% ascorbic acid and was carried out under acidic pH conditions (pH 3.6) in order to maintain iron solubility and, more importantly, drive electron transfer.2 Results indicate that 0.5% raspberry
multifunctional ingredient provides significant reducing power, while hydroxy acetophenone
www.personalcaremagazine.com Evaluation
pH Sterility control
2d
5.3 – 5.3 – 5.3 – 5.3 –
5.3 – 5.3 –
7d Bacteria
14d 28d Assess- ment
C C C C – – – – – – – – – – – –
– – – – – – – –
5.3 – ++++ ++++ C C
+++ = heavy growth ++++ = Massive growth C = completely overgrown
F A A A
A A F
Fungi
14d 28d Assess- ment
C C
+++ +++ +++ +++ +++ +++
– – – – ++ –
F A A A
A A A
Assess- ment
F F F F
A A F
C.albicans
A.brasiliensis
Bracteria Ffungi
evaluation time points bacteria 2, 7, 14, 28 days fungi: 14, 28 days
A B A
B
2d 2
–
n.d. n.d.
n.i. - no increrase n.d. - not determined
7d 3
–
n.d. n.d.
14d 3
3 3
2
28d n.i.
n.i. n.i.
n.i.
Germ reduction
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