28 ANTI-AGEING Untreated control Propanediol 0.1% SEBOCLEARTM MP 0.1% Fibroblast control
Figure 3: Bioflavonoids from Maclura cochinchinensis change the cell morphology of sebocytes. Untreated cells show the typical roundish form of lipid- producing sebocytes. The addition of propanediol has no effect. Treatment with 0.1% active ingredient transforms the cell morphology into a fibroblast-like appearance typical for motile precursor cells.
legumes like soya beans have elevated levels of isoflavones (tofu or fermented miso, for example). Although Maclura is a member of the Moraceae family of plants (mulberry), the leaves contain high amounts of isoflavones. Bioflavonoids are known to bind nuclear receptors regulating gene expression. Here we investigated their function as retinoic acid like effector molecules in ageing skin.
Methods
Binding of bioflavonoids to the RAR and RXR receptors To investigate the potential of Maclura cochinchinensis bioflavonoids for activating the retinoic acid receptor / retinoid X receptor complex (RAR/RXR), a ligand replacement assay (agonist) with radioactively labelled [3
H] 9-cis retinoic acid
bound to human recombinant RAR and RXR receptors was performed. For RAR, 1 nM ligand was used, for RXR, 5 nM were used. Put more simply, the addition of agonist (0.01 % or 0.1 % bioflavonoids) will displace the radioactively labelled [3
H] 9-cis retinoic
acid from the ligand binding site of the receptors. Liberated [3
is detected by scintillation counting6
180 minutes for RAR and after 60 min for RXR.
0.3 0.25 0.2 0.15 0.1 0.05 0
Figure 4: Treatment with bioflavonoids from Maclura cochinchinensis has no adverse effect on the viability of sebocytes. This data confirms the good compatibility of the active ingredient and supports the hypothesis of a dedifferentiation of sebocytes as explained in Figure 3.
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H] 9-cis retinoic acid after
Re-programming sebocytes To visualise the effects of Maclura cochinchinensis bioflavonoids on cultured primary sebocytes a bright-field microscopic study of the cell morphology was performed. Primary sebocytes from facial location were cultivated in differentiation medium. Terminal differentiation was checked by labelling with keratin-7 antibody and staining with a CY3 labelled secondary antibody. The differentiation was confirmed by FACS analysis. For the viability assay, cultured primary sebocytes were incubated with the tetrazolium salt WST-1, which is converted by the mitochondrial succinate-tetrazolium reductase system into formazan. Formazan can be detected at 450 nm in a spectrophotometer. The colour conversion can only take place in living and metabolically active cells. The assay is comparable to the MTT assay.
Rejuvenation effect on ageing skin The in vivo study has been performed in accordance with the principles of good laboratory practice (GLP), good clinical practice (GCP), and in compliance with the quality assurance system requirements. The study was conducted in accordance with the World Medical Association’s Declaration
n Untreated control n Active 0.1%
80 70 60 50 40 30 20 10 0
of Helsinki. All study participants signed a written informed consent at the beginning of the study.
A double-blind, placebo controlled, randomised study on 21 female subjects, aged 45 – 64 years (average 56.9) and with healthy, Caucasian skin was performed. The study subjects with signs of ageing skin applied the placebo or verum formulation with 3% bioflavonoids from Maclura cochinchinensis in a hemi-face approach twice daily. Each study participant was her own control. The study duration was 3 months with measurements at day 0, day 28 and day 84. Firmness and elasticity of the periocular region were assessed with a standard cutometer (MPA 580, dual, 2 mm probe). Wrinkles in the nasolabial and crow’s feet area were determined using a PRIMOS 3D imaging system. The visible appearance of wrinkles and skin texture were assessed by objective evaluation by a dermatologist. The wrinkle appearance was assessed by using the crow’s feet wrinkle grading score (WGS), a photo grading scale of 8 degrees,7
whereas the wrinkle
appearance in the nasolabial area was assessed by using the nasolabial wrinkle grading system (scale of 5 degrees).8
The
skin texture parameters of evenness, roughness and suppleness were assessed
n Placebo n Active 3% **
*** *** * * *** *** = p<0.001 ** = p<0.01 * = p<0.05
Firmness
Elasticity
Figure 5: Bioflavonoids from Maclura cochinchinensis rejuvenate the skin. Firmness and elasticity are significantly improved above the placebo by up to 43 % over initial condition. Wilcoxon signed-rank test. Statistical values in black refer to comparison with baseline while values in blue refer to placebo.
June 2018
Mean + SEM; n=21;
Mean + SEM; n=6
Optical density at 450 nm (au)
Relative to initial condition %
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