Ternary mixture provides significant potency

GAGs are known for their extremely high water binding capacity, thus making them ideal moisturisers. Collagen and GAGs play a significant role in wound healing as well. During ageing, intrinsic and extrinsic factors such as alteration in the genomic structure, exposure to UV light or noxious environmental substances lead to collagen and consequently to dry skin and wrinkle formation via an increased synthesis rate of matrix degrading collagenases and/or a decrease in de novo collagen synthesis. GAG reduction during chronologic ageing is also described by several authors.1-4 There are two ways to counteract the undesired effects of collagen and GAG decrease: either via supplementation through topical application of natural and synthetic collagen and GAG derivatives or via stimulation of skin endogenous collagen and GAG biosynthesis. The objective of our research was to develop an active ingredient that counteracts age related collagen and GAG degradation via stimulation of de novo biosynthesis. In the dermal layer of human skin, fibroblasts produce primarily collagen type I (80%-95%) and type III (5%-15%). Initial in vitro substance and substance mixture screenings showed that a ternary mixture of Aloe leaf concentrate, magnesium ascorbylphosphate and raspberry leaf extract synergistically enhances total collagen production in normal human dermal fibroblast culture by approximately 150%. In further studies, we investigated the influence of the ternary mixture both on collagen I and on GAG synthesis in human ex vivo skin models. For that purpose we cultivated human skin explants over a 12-day period in the presence of either placebo or actives formulations comprising 0.5% and 1% of the ternary mixture. Quantitative immunostaining showed that dermal collagen I content could be increased in human skin explants up to 80%. GAG content could be increased even more significantly up to approximately 250%. In conclusion, we consider the ternary mixture as a potent new multifunctional active for use in antiageing, sun care, skin soothing, skin cleansing, mouthwash, shaving, aftershave, and wound healing products.

Materials and methods

Test products: approximately 90 samples comprising synthetic and natural products were prepared. Aloe extract (200-fold Aloe vera powder concentrate), powder concentrates obtained from aqueous extracts of raspberry leaf (Rubus idaeus L.), strawberry leaf (Fragaria vesca L.) and Queen of the Meadow herb (Filipendula ulmaria L.), Mg ascorbyl phosphate and combinations thereof were investigated in detail.
In vitro collagen assay with NHDF cell culture: the collagen synthesis enhancing effect of the samples was tested on Normal Human Dermal Fibroblasts (NHDF, in vitro aged; >8th passage) seeded in 96-well microplates (2 x 104 cells per well). The maximum sample concentration used in the collagen assay depended on the IC20 cytotoxicity value, determined with the Resazurin test on 3T3 mouse fibroblasts. To avoid influence of any cytotoxic effect on test results, 1/10 of the IC20 value was used as maximum sample concentration in the collagen assay. After incubation of the cells with samples for 48 h, total collagen content was determined with the Sirius Red staining method,5 a standard histological staining procedure, which is specific for collagen molecules. The collagen-bound dye can be measured by absorbance at 540 nm. An increase in collagen synthesis is proportionally linked to an increase of the absorbance (OD). To evaluate potential synergistic effects of ternary mixtures, the single products were mixed based on corresponding IC20 values in a ratio of 0.33 x conc 1/10 IC20 sample A + 0.33 x conc 1/10 IC20 sample B + 0.33 x conc 1/10 IC20 sample C and tested under identical conditions in the collagen assay. Synergy indices (SI) were calculated with Kull’s equation according to published procedure.6,7
Ex vivo collagen assay on human skin explants: ex vivo collagen stimulation studies were performed with a highly active synergistic mixture comprising Mg ascorbyl phosphate and Aloe extract and raspberry leaf extract at a concentration ratio of 9:90:1 (w/w/w). Four samples were compared in the ex vivo experiment, which included a placebo o/w formulation and two actives o/w formulations comprising 0.5% and 1.0%, respectively of the ternary combination. An o/w formulation comprising 0.1% retinol was used as positive control.
Explants: ex vivo studies were performed on an abdominal plasty coming from a 52-year-old Caucasian woman. Explants of an average diameter of 10 mm were prepared. The explants were kept in survival in culture medium at 37°C in a humid, 5%-CO2 atmosphere. These explants were distributed into different batches as following: batch T (untreated control explants), batch R (explants treated with the positive control – cosmetic formulation comprising 0.1% retinol); batch P1/P2 (explants treated with actives formulation at 0.5% and 1.0%); batch P3 (explants treated with placebo formulation).
 Product application: the products tested (P1, P2 and P3) were applied topically on the basis of 2 mg per explant, on day 0, 1, 2, 5, 7 and 9. Because of irritant side effects at higher concentration, the retinol positive control (R) was applied topically on the basis of only 1 mg per explant, on day 0, 2, 5 and 8. S
ampling for histology/ immunostaining: on day 0, three explants from the untreated control batch were collected. On day 6 and 12, three explants from each batch were collected. They were instantly cut in two parts: one half was fixated in ordinary Bouin solution, the other half was frozen at –80°C.
 Immunostaining of type 1 collagen: immunostaining of type-I collagen, making approx 80%-95% of total dermal collagen was realised on frozen sections with a polyclonal, anti-collagen I antibody (Monosan ref PS 047) with a biotin/streptavidin enhancement system and revealed by FITC. Nuclei were counterstained with propidium iodide.
Microscopy: the microscopic observation was made using a Leica DMLB microscope, with a x40 objective. The pictures were taken with a tri CDD DXC 390P Sony camera and stored using the Leica IM 1000 data storing software.
Quantitative image analysis of collagen I: for each batch, 9 microscopic fields were analysed with the Leica QWIN software using the following process: “07E1473 Surf et Int Coll I.Q5R”.
Ex vivo glycosaminoglycan assay on human skin explants: ex vivo glycosaminoglycan stimulation studies were also performed with the highly active synergistic mixture comprising Mg ascorbyl phosphate and Aloe extract and raspberry leaf extract at a concentration ratio of 9:90:1 (w/w/w). Three samples were compared in the ex vivo experiment, which included a placebo o/w formulation and two actives o/w formulations comprising 0.5% and 1.0%, respectively of the ternary combination.
Explants: ex vivo studies were performed on an abdominal plasty coming from a 52-year-old Caucasian woman. Explants of an average diameter of 10 mm were prepared. The explants were kept in survival in culture medium at 37°C in a hum id, 5%-CO2 atmosphere. These explants were distributed into different batches as following: batch T (untreated control explants); batch P1/P2 (explants treated with actives formulation at 0.5% and 1.0%); batch P3 (explants treated with placebo formulation).
Product application: the products tested (P1, P2 and P3) were applied topically on the basis of 2 mg per explant, on day 0,1,2,5,7 and 9.
Sampling for histology/ immunostaining: on day 0, three explants from the untreated control batch were collected. On day 12, three explants from each batch were collected. They were instantly cut in two parts: one half was fixated in ordinary Bouin solution, and the other half was frozen at -80°C.
Detection and quantification of glycosaminoglycans were realised on frozen sections by Mowry staining.
Microscopy: the microscopic observation was made using a Leica DMLB microscope. The pictures were taken with a tri CDD DXC 390P Sony camera and stored using the Leica IM 1000 data storing software.
Quantitative Image analysis of GAGs: for each batch, 9 microscopic fields were analysed with the Leica QWIN software using the following process: “08E1545 GAGs Surf et Int en absal.Q5R”.