Moisturising effect of orange blossom extract

Healthy, glowing skin portrays the image of a healthy person. The majority of cosmetic brands have at least one product or even an entire range dedicated to skin hydration. One of the main issues facing cosmetic companies is to differentiate their product from the competition; one way is through the wording on the label. The scope of the claim can range from a basic term such as ‘hydration’ through to the more colourful and descriptive of, ‘désaltère’, ‘réimbibe’, ‘resource’ ‘strengthens the skin barrier’, ‘boosts the natural mechanisms of hydration’ or ‘combats the dryness of the skin’ Yet, hydration remains a complex and intriguing subject for scientific study. Can we actually increase the amount of water in the skin by using exogenous substances? Can this increase in water reserve be done in a sustainable manner? Is the amount of water in the skin the real marker of hydration? More and more scientific evidence is moving towards an expression of the hydration by the evaluation of the water flux through the skin. Indeed the water/skin condition is the result of balancing supply/disposal, or best diffusion/evaporation. This particular parameter is a variable that can be assessed over time. The concept of the barrier effect here is important to ensure that the state of balance between diffusion and evaporation is met by limiting the evaporation or setting the water in the epidermis. The dermis is the reserve of water in the skin. Some of this water is free and little diffuses through the epidermis and the stratum corneum to evaporate on the surface of the skin. This reserve of water in the dermis is ensured by the presence of the GAG (glycosaminoglycans) and especially hyaluronic acid (HA). GAG are polymers of the unit [uronic acid – ?1?4-N-acetyl hexoamine]. This polyanionic structure gives the GAG a shape stretched by electrostatic repulsion, and also gives them a capacity to hold significant quantities of water, thus occupying a considerable area. These supramolecular structures of considerable size are able to entrap large amounts of water and ions to provide tissues with hydration and turgescence. The compositions and amount of GAG evolve over time, with visible consequences to the skin. The overall content of the GAG decreases with age, with a significant reduction in hyaluronic acid, causing drying and thinning of the dermis and epidermis, and the appearance of wrinkles. Hyaluronic acid, which is part of the GAG family, is distinguished by two points:

•  It is not bound covalently to protein (in contrast to the other GAG).
•  It has no sulphate group.

This is the most effective GAG in the skin tissue water tank constitution. It is able to act like a sponge and absorb the mechanical stress of the skin. HA is present in all connective tissues of the human body and was also characterised some years ago in the epidermis. It is synthesised by the basal keratinocytes. HA plays an important role in the normal epidermis. It also plays a crucial function in the reepithelisation process due to several of its properties. This molecule serves as an integral part of the extracellular matrix of the basal keratinocytes, which are major constituents of the epidermis. In normal skin, HA is found in relatively high concentrations in the basal layer of the epidermis where proliferating keratinocytes are found. The main functions of HA in the epidermis are to maintain the extracellular space and provide an open, as well as hydrated, structure for the passage of nutrients. The content of HA in the skin and in particular in the epidermis, is an important factor in the hydration process of healthy skin. The balance between diffusion/evaporation is directly related to the amount of HA, a substance which can retain and release water. Naturex have been extremely committed to developing a natural solution to this problem. After screening more than 50 plants, Naturex selected a source of orange blossom. The flowers of orange (Citrus aurantium) are obtained mostly on the shores of the Mediterranean basin, particularly in Tunisia and Morocco. These trees, mainly grown for their fruit and flowers, are highly appreciated by perfumers. In addition to the olfactory fraction, the flower of orange contains other interesting phytochemical components that were separated to prepare aurealisTM (Fig 1). Aurealis is an orange flower extract titrated in flavonoids (expressed as synephrine). This extract is clear yellow, fully soluble in water, odourless and contains no cosmetic allergens. Ecocert validated, the phytochemical compounds are obtained using a natural process without chemical solvent. The INCI name is different for US [Citrus Aurantium Amara (Bitter Orange) Flower Extract] and for EU [Citrus Aurantium Amara Flower Extract]. To demonstrate the effectiveness of aurealis (now referred to as ‘the orange flower extract’) on skin hydration, studies have been conducted on skin and cell tissues.

Hyaluronic acid expression and secretion – in vitro keratinocytes culture

Objectives of the study

The effects of the orange flower extract on the expression/secretion of hyaluronic acid by normal human epidermal keratinocytes (NHEK) were tested. This compound was selected following a primary screening performed as part of a previous study. The effects of this compound were evaluated by measuring:

•  The quantity of hyaluronic acid (HA) secreted in the keratinocytes cultures supernatants.
•  The expression of hyaluronic acid in keratinocytes cultures using in situ immunolabelling and image analysis.

The biological model used is of a normal human epidermal keratinocytes (NHEK), control is without treatment, reference is retinoic acid (10–7 M).

Culture and treatments

The keratinocytes were inoculated and cultured in the culture medium for 24 hours. After incubation, the culture medium was replaced with a culture medium either containing or not containing (control) the test or reference compounds, and the cells were then incubated for 72 hours. All experiments were processed in triplicate. After incubation, the culture supernatants were sampled in order to quantify the hyaluronic acid present by ELISA. And immunolabelling of hyaluronic acid was performed on the cells layer.

Hyaluronic acid secretion – ELISA assay

At the end of incubation time, the hyaluronic acid content was assayed in culture supernatants by using a modified ELISA assay based on the use of the ‘hyaluronan binding protein’. The reference, retinoic acid tested at 10–7 M, stimulated the synthesis/secretion of hyaluronic acid by the keratinocytes (176% of the control). This result was expected and confirmed the literature and validated the trial. The orange flower extract compound, tested at two dosages (0.008 mg/mL and 0.04 mg/mL), stimulated the synthesis of hyaluronic acid by the NHEK (131% and 144% of the control, respectively) (Table 2). The action of the orange flower extract regarding the stimulation of the HA synthesis by the basal keratinocytes has not yet been clarified. The present study demonstrates that the orange flower extract acts in the same way as retinoic acid, leading to an accumulation of HA in the extracellular compartment.

Hyaluronic acid expression – in situ immunolabelling

The culture medium was sampled and the cells were rinsed, fixed, and permeabilised. The cells were then marked with the primary antibody directed against the protein of interest (AH). This antibody was revealed by a secondary antibody coupled with a fluorochrome. Tests without primary antibodies (secondary antibodies only) were also performed. For each well, five digital images were captured. Labellings were quantified by measuring the fluorescent intensity of the cells in relation to the number of nuclei identified by the Hoechst stain. Intergroup comparisons were made using the Student’s t-test. In the controls, labelling was localised mainly to the intracellular level. The retinoic acid treatment, tested at 10–7 M, increased the intracellular content of hyaluronic acid in the keratinocytes, slightly but significantly (107% of the control). This result was expected and validated the trial. In the assay the orange flower extract compound was tested between 0.008 mg/mL and 0.2 mg/mL, and the observations have shown a modification of the intracellular expression of hyaluronic acid by the NHEK (Fig. 3). Most of the cells contained more hyaluronic acid in their cytoplasm. But this was not a reproducible modification of the intracellular expression of hyaluronic acid. This increase in the expression of intracellular hyaluronic acid was not observed in all parts of the cultures. Cell cultures used to perform this experiment were the same as the one used to measure the release of HA in the extracellular matrix. On the one hand, we observed that keratinocytes release the HA, on the other hand the observed levels of intracellular HA were not higher in the basal level. In conclusion, the orange flower extract stimulates intracellular hyaluronic acid biosynthesis pathways and at the same time promotes the release of the new molecule formed into the extracellular medium. Indeed, if this compound only stimulated the cellular synthesis of hyaluronic acid, an increase in the expression of intracellular hyaluronic acid would have been observed. Likewise, if this compound only stimulated the secretion of hyaluronic acid without modifying its cellular synthesis, a decrease in the expression of intracellular hyaluronic acid would have been observed in relation to the control. Hyaluronic acid is continuously synthesised and secreted, which explains why there is no notable accumulation in the cell.

Conclusion

The orange flower extract simultaneously stimulates both the intracellular synthesis of hyaluronic acid and the secretion outside the cells. This result, obtained on a cells culture, was confirmed by a more accurate model as ex vivo human explant.

GAG and hyaluronic acid expression – ex vivo skin explant

A second demonstration has been done ex vivo on human skin explant. This study aims to highlight the activity of a product on the synthesis of hyaluronic acid on human skin explants. The activity was evaluated by:

• The observation of general morphology after staining with Masson-Goldner trichrome.
• Staining of acid glycoaminoglycans (GAG).
• Labelling of the hyaluronic acid, using HABP (Hyaluronic Acid Binding Protein).

The quantification of the results is made by image analysis, and results are expressed through statistical analysis. From a normal human plasty, nine explants (average diameter 10 mm) were prepared and kept in a specific explant medium. Explants have been organised as shown in Table 2. The mother solution was prepared at 1 mg/mL in PBS solution, and then stored in a sterile tube at +4°C. At D0, D2, D6 and D8, the explants were exposed for four hours with 30 ?L of the orange flower extract by using a soaked paper filter. References are explants without the orange flower extract. Glycoaminoglycans were visualised by blue alcian staining, in the epidermis and papillary dermis. GAG appear in blue, and are mainly acid GAG. HABP is useful to detect HA in a section of tissue. HABP labelling is realised on paraffin slices treated with Boin’s fixative, with a biotinylated protein anti hyaluronic acid, at 1/125°, for two hours at room temperature, with biotin/streptavidin amplification system. Nucleuses are coloured with nuclear Fast Red.

 Results

After 10 days’ culture, the explants without treatment showed the same general morphology as the one observed at D0. It means that the culture conditions are safe for the tissues. The general morphology of treated explants remained the same compared to explants without treatment at D10. The number of cell layers was quite equal and a thickening effect on the epidermis was observed. Glycoaminoglycans On the reference without treatment, GAG were slightly expressed in the epidermis basal layer. It was seen to demonstrate a good viability and metabolism of the explants in the cultures. With the orange flower extract, the acid GAG were very strongly expressed on the epidermis, on 2-3 cell layers (Fig. 5). The increase of expression of GAG with the application of the orange flower extract is quantified to +183% (Fig. 4). Following Student’s t-test, the result is very highly statistically significant (control D10 vs. the orange flower extract D10 : P<0.0001).

Hyaluronic acid

At D0, visualisation of HABP shows a moderate but regular quantity of hyaluronic acid in the suprabasal layer of the epidermis. This quantity is lower and irregular on the basal layer. In the papillary dermis, quantity of HABP is low along the DEJ, and moderate in the underlying reticular dermis. At D10, in the reference, the quantity of HABP was moderately higher in the epidermis basal layer, compared to D0. It can be concluded that treatment with the orange flower extract leads to a significant increase in the expression of HA in the basal and suprabasal layers of the epidermis (Fig. 7). The increase of expression of HA with the application of the orange flower extract is quantified to +33%. Following Student’s t-test, the result is very highly statistically significant (control D10 vs. the orange flower extract D10, P=0.0006) (Fig. 6).

Thickness of the epidermis

As a consequence of the increase in synthesis of GAG and HA, the thickness of the epidermis is increased, and quantified to +82%, as a statistically significant result (P<0.0001) (Fig. 8). The increased quantity of binding-water macromolecules GAG and HA occurs only in the basal and suprabasal layers of the epidermis, without affecting the stratum corneum. The increase of the thickness of the epidermis through an increase of the quantity of GAGs and HA in the basal layer demonstrates a visible re-plumping effect in the skin (Fig. 8).

Conclusion

On an ex vivo application (human explants in culture, during 10 days’ treatment), the compound aurealis significantly induces:

•  A strong expression of acid GAG in the epidermis : +183% (P<0.0001).
•  A strong expression of hyaluronic acid in the epidermis : +33% (P=0.0006).

This increase of big macromolecules in the extra cellular matrix in the basal layers results in the thickening of the epidermis: +33% (P<0.0001). The compounds of acid family (GAG) including HA are directly responsible for the retention of water in the epidermis. This means an increase in the quantity of these compounds in the skin, creates a high level of skin hydration. This new water reserve is very useful for the balance of the diffusion/evaporation, which is the most important parameter of the skin hydration process. The increase of GAG in the epidermis thanks to aurealis allows the skin to fight against ageing, drying and thinness of the skin, and leads to an increase of the thickness of the skin for a re-plumping effect.

References

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