36 EYE CARE
enzyme (ACE). The ACE is responsible for the extracellular conversion of angiotensin I into angiotensin II, a powerful vasoconstrictor. The test was based on measuring the activity
of ACE in endothelial cells. Human dermal microvascular endothelial cells (HDMEC) were incubated for 24 hours with avocado polyphenols at 0.0005%, 0.001% and 0.005% DM, equivalent to 0.05%, 0.1% and 0.5% active ingredient. After incubation, ACE activity was evaluated in cell extracts, using a fluorimetric test which measures the quantity of His-Leu dipeptide formed by Hippuryl-Histidyl-Leucine (HHL) substrate hydrolysis. Avocado polyphenols significantly stimulated the enzyme activity of the ACE in a dose- dependent manner. This result is in favour of a vasoconstrictive and decongestant effect (Figure 2).
Hyperpigmentation activity The potential depigmenting effect of avocado polyphenols was studied in two separate models, one evaluating melanin production in melanocytes and one evaluating the enzyme activity of tyrosinase, a key enzyme involved in melanin synthesis. In the first test, avocado polyphenols at
different concentrations, or kojic acid at 0.5 mM as a positive reference, were preincubated with tyrosinase extracted from human melanocytes for ten minutes in the cold chamber. The enzyme substrate, L-DOPA at 2mM, was then added. After one hour’s incubation at 37°C, enzyme activity was evaluated by spectrophotometric measurement. In the second, normal human epidermal
melanocytes were grown with NDP-MSH at 10-7
M (α-MSH analogue, an inducer of
melanogenesis) and avocado polyphenols at 0.001% and 0.005% DM, equivalent to 0.1% and 0.5% active ingredient, or kojic acid at 0.25mM (positive reference). After ten days incubation, the melanin was extracted from the cells and quantified by spectrophotometry. The avocado polyphenols significantly
inhibited both the enzyme activity of tyrosinase in a dose-dependent manner (Figure 3) and melanin production by melanocytes stimulated by NDP-MSH (Figure 4). These results confirm their depigmenting potential.
200 180 160 140 120 100 80 60 40 20 0
Control -10%* -19%*** -33%***
3000 2500 2000 1500 1000 500 0
Control
+141%** +112%** +83%**
Av.P. 0.0005% dm Av.P. 0.0001% dm Av.P. 0.005% dm
Figure 2: Effect of the active ingredient on the enzyme activity of ACE in microvascular endothelial cells (**p<0.01 vs. untreated reference – Student t test)
Lipolytic activity Adipocytes, the main cells in human adipose tissue, are specialised in the storage of lipids and are the body’s main energy store. They use their energy reserves by hydrolysing the triglycerides stored under the effect of lipases. The triglycerides stored in the adipose tissue are hydrolysed into fatty acids and glycerol. The fatty acids thus released in the blood can be used by the other tissues for energy purposes. The lipolysis process is seen in a decrease in adipocyte diameter. The effect of avocado polyphenols on adipocyte lipolysis was evaluated directly by assaying the glycerol released during hydrolysis of triglycerides stored in human mature adipocytes and indirectly by measuring adipocyte diameter. Human mature adipocytes isolated from the subcutaneous adipose tissue biopsies of five donors, grown in three sizes, were incubated for four hours with avocado polyphenols at 0.005% DM, equivalent to 0.5% active ingredient, or with Forskolin at 10 µM as a positive reference. After incubation, the glycerol released by adipocyte lipolysis was assayed in the culture media. The avocado polyphenols induced a significant increase in lipolysis in the mature adipocytes (Figure 5).
35 30 25 20 15 10 5 0
Control
Kojic acid 0.5mM Av.P. 0.005% dm
Av.P. 0.05% dm
Figure 3: Effect of the active ingredient on the enzyme activity of tyrosinase (*p<0.05; **p<0.01; ***p<0.001 vs. untreated reference – Student t test)
PERSONAL CARE March 2022 In the second test, human mature adipocytes
from four donors, grown in three sizes, were incubated and treated in the same way as before. After incubation, the mean adipocyte diameters were determined using a cell size counter. The avocado polyphenols induced a significant decrease in adipocyte diameter compared to the reference condition (Figure 6).
Antioxidant effect The antioxidant activity of avocado polyphenols was looked for by evaluating the quantity of reactive oxygen species (ROS) produced by keratinocytes subjected to oxidative stress, which was induced by hydrogen peroxide (H2
). ROS production was evaluated by measuring the fluorescence emitted by the probe in contact with the ROS. Normal human keratinocytes were treated
O2
for 24 hours with the avocado polyphenols at 0.005%, 0.01% and 0.1% DM, equivalent to 0.5%, 1% and 10% active ingredient, or Quercetin at 10 µg/ml as an antioxidant reference before adding the H2DCF-DA probe. The keratinocytes were then stimulated by H2
O2 at 100µM for
20 minutes. It was proved that the avocado polyphenols significantly inhibited the production of ROS by keratinocytes in response to the oxidative stress (Figure 7).
SS -14%* -15%*
-68%***
NDP-MSH 10-7M
Kojic acid 0.25mM
Av.P. 0.001% dm
Av.P. 0.005% dm
Figure 4: Effect of the active ingredient on melanin production by normal
human melanocytes ($$ p<0.01 vs. untreated reference; *p<0.05 ***p<0.001 vs. NDP-MSH - Student t test)
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ACE (nmol His-Leu/mg prot.)
ACE (nmol His-Leu/mg prot.)
Melanin (µg/mL)
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