In Vivo Evaluations of Emulsion O/W for a New Topical Anti-Aging Formulation: Short-Term and Long-Term Efficacy

This study aims to evaluate the in vivo efficacy in terms of elasticity, hydration and anti-wrinkle enhancement of a new cosmetic cream formulation: Apollinea anti-ageing facial cream. The latter is O/W (oil/water) emulsion with high volume percentages of snail secretion filtrate and donkey milk serum in its composition. The investigation has been performed on (twelve) healthy subjects: 8 (eight) females, and 4 (four) males, all presenting evident skin wrinkles in typical facial areas (e.g. forehead and eye contour). The subjects had to apply about 2 grams of Apollinea anti-ageing facial cream every day. The efficacy of the new cosmetic formulation was assessed before and after the cosmetic treatment, precisely 2 hours, to assess the short-term efficacy, and 40 days after, this to evaluate the product long-term efficacy. Elasticity enhancements have been tested through measurements of the viscoelastic properties of the skin, whereas the hydration parameter through measurements of the skin and subcutaneous tissues dielectric constant. All the latter measurements have been performed with specific instrumentation, as discussed more in detail in the following. To evaluate the anti-wrinkles efficacy instead, we proceeded with digital camera imaging and image analysis processed through ImageJ software. The new Apollinea facial cream cosmetic formulation, based on donkey milk whey and snail secretion filtrate from Helix Aspersa Müller, showed significant enhancement effects on skin elasticity and hydration as well as an important skin-wrinkles-reduction.


Introduction
Skin ageing is influenced by a number of intrinsic and extrinsic factors, promoting the accumulation, in the cutaneous structure, of physiochemical alterations [1] [2] leading to a progressive loss of structural integrity and physiological functions [3]. Such aforementioned alterations result in visible changes to the cutaneous tissue, of which few examples are wrinkling, dyschromia and elastosis [4]. Thus, a modern anti-aging cosmetic product must attain a variety of effects like moisturizing the skin, making it more elastic and reducing the visibility of fine and deep wrinkles. The aim of this study is to evaluate the short term (2 hours) and long term (40 days) efficacy of a new cosmetic formulation, Apollinea cosmetic cream (O/W emulsion), in skin elasticizing function, hydration and wrinkles reduction. The main ingredients in this new cosmetic formulation "Apollinea facial cream'' are donkey milk serum, snail secretion filtrate and vegetable lipids, such as sweet almonds oil and Karite butter, all ingredients that strongly contribute to the anti-age efficacy of the final product. Among the latter donkey milk is the most suited for a dermo-cosmetic application given its high percentage of milk serum proteins and other milk compounds, well known for their high degree of uptake and water retention that promotes skin hydration and prevention of epidermis cells degeneration [5] [6] [7]. Prominent donkey milk serum-proteins are β-lactoglobulin (β-LG), α-lactalbumin (α-La), and lysozyme, with an average percentage respect to the total serum-proteins of 29.8%, 22.6% and 21.0%, respectively. The remaining of serum-proteins is the sum of minor compounds average percentages as lactoferrin, serum-albumin and immunoglobulin [8]. The latter has a high iron-chelating property that prevents free radical production and the degradation of the epidermal cells [5] [6] as well as anti-microbial and anti-viral properties [9]. Donkey milk serum contains 3.75 g/L of β-LG which is a protein that binds and carries various small hydrophobic molecules, and 1.8 g/L of α-La, which concentration is very close to the one found in human milk. The lysozyme is an anti-bacterial agent belonging to the hydrolase class and specifically is a glucosidase [9]. Its concentration in donkey milk serum spans from 0.76 -3.75 g/L but this strongly depends on the analytic method applied for the measure [10] [11] [12]. As already reported from J. Cotte back in 1991, α-La and β-LG have been widely applied in cosmetics as hydrating agents that makes them effective in temporarily masking cutaneous wrinkles [13]. The capability in calming irritation symptoms [14] [15] and in restructuring skin-ageing processes [16] [17] of Serum-proteins as β-lactoglobulin and lysozyme and essential amino acids beautifying properties are also well known [18].
The second key ingredient of the cosmetic product under investigation is snail secretion filtrate, precisely from Helix Aspersa Muller, snail belonging to the gasteropods class and native of the Mediterranean area [19]. Snails are induced into mucus secretion through a harmless process. The clammy secretion covering the snail outer surface may serve in preventing the moisture evaporation, in helping smooth movements and in protecting the body from mechanical injuries [20]. In 1961 Campion [21], carried out a histological and histochemical study of the Helix Aspersa mantle collar glands that secrete drool. He spotted at least eight different kinds of glands, four extruding different kinds of mucus, one rich in proteins, one in calcium carbonate granules, one is a pigmented secretion with a flavone, and one containing fat-globules. Campion observed that the mantel collar glands were producing mucus combined with proteins [21] [22]. In recent years, scientific studies of the secretions of the snail Helix Aspersa, have shown that the mucus contains a complex combination of natural substances, including allantoin and glycolic acid [23] [24] [25] [26]. According to USA Food and Drug Administration (FDA), allantoin is a safe and effective active compound for skin protection [27], is a well-known anti-irritating and hydrating [28] agent as well as a promoter of epithelial stimulation [29]. In particular allantolin promotes keratolytic activity [30] [31] [32] [33] as well as glycolic acid, an alpha-hydroxy-acid, effective as a peeling agent. It eliminates superficial dead skin cells and promotes their replacement with new corpuscles [32] [34].
Skin ageing is indeed an inevitable biological phenomenon affecting both dermis and epidermis through changes in the integumental system, which determines its most common manifestations [35]. The most reproducible and recognizable feature of skin ageing is the flattening of the dermo-epidermic junction [36] [37]. There happens a general atrophy of the extracellular matrix, reflecting in a decrease in the number of fibroblasts and in low levels of collagen [38] and elastin thus resulting in a compromised arrangement of the latter [37]. Such changes are partially the result of cumulative endogen damage due to the increasing formation of oxygen-reactive species (ROS) generated during the cellular oxidative metabolic process [39]. Moreover, ageing is speed up on the skin areas often exposed to sun light, also called photo-ageing [40]. To clarify photo-ageing dynamics, Brieva et al., via in vivo studies, have shown that snail secretion has anti-oxidant efficacy through the superoxide dismutase (SOD), as well as Glutathione-S-transferase (GST) [41] [42] [43] [44]. Other studies carried out on snail drool have shed light on the possible mechanisms behind its regenerative properties [42] [43]. SOD is a key enzyme in the radical superoxide anion (O2 -) as well as hydrogen peroxide (H 2 O 2 ) deactivation process, while GST is one of the enzymes responsible of the detoxification from xenobiotic substances and free radicals. Snail drool promotes fibroblasts proliferation, the rearrangement of the actin-cytoskeleton and favors the extra-cellular matrix (ECM) assembly [42] [43] thus reducing the progressive degeneration of the cutaneous mantel [41] [42] [43] [44] [45].
In the formulation of the O/W "Apollinea'' emulsion an abundant portion of lipid compounds has been added, mainly consisting in butters and vegetable oils with noticeable protective and emollient effects.
The main lipid compound of the product on test is Karite butter. The latter contains fat acids such as: stearic, oleic, linoleic, arachidic, eicosenoic, docosanoic The current study aims to evaluate the skin-care effects of the product under investigation taking carefully into account the specific combination of ingredients.

Snail Secretion Filtrate: How to Collect It
The snail secretion filtrate used as a dispersing medium in the formulation of the process snail secretion appears like a clear ambered fluid. The latter is completely miscible in water, displays a limited compatibility with Ethanol but it is insoluble in vegetable or mineral oils and its pH may vary between 6.5 and 7.5.

Donkey Milk and Its Serum Fraction
To obtain the donkey milk serum, used in the formulation of the cosmetic product Apollinea O/W emulsion, the donkey milk is centrifuged at first to eliminate the lipid fraction. The second step of the process consists in precipitating the casein fraction by reducing the pH to the exact value of 4.2, value at which the serum proteins, instead, are still stable and well dispersed.

Anti-Age Facial Cream Formulation
The dermo-cosmetic product under evaluation has been prepared in agreement with the admitted substances listed for cosmetic and personal hygiene formula-

Experimental Study
The aim of the study was to evaluate the anti-wrinkle efficacy (reduction of facial skin roughness), hydration and elasticity of the skin of the face, of a dermo-cosmetic treatment, which involves the distribution until complete absorption on the anatomical districts of the face skin tested for efficacy, about 2 (two grams) of the O/W Apollinea-anti-aging facial facial cream over specific anatomic districts, subject to efficacy evaluation.
To this end, a study conducted in 12 healty subjects: 8 females and 4 males, with facial skin characterized by skin wrinkles. Thus, the study aims to investigate the "in vivo'' efficacy of the Apollinea-anti-aging facial cream formulation on the short-term (2 hours) and long-term (40 days), after the treatment through non-invasive techniques able to quantify skin elasticity and hydration enhancement as compared to a reduction of the skin wrinkles. The current experimental investigation has been held in agreement with the Helsinki declaration (Ethical principles for medical research involving human subjects). The efficacy of the cosmetic product has been evaluated 2 hours and 40 days after the treatment through non-invasive techniques able to quantify skin elasticity and hydration enhancement as opposed to a reduction of the skin wrinkles.
Twelve healthy volunteers between 35 and 67 in age, have been selected with the following inclusion criteria: • To be generally in good health; • Subjects willing to use solely the cosmetic product being tested through all the test time in the skin areas devoted to the cosmetic evaluation; • Subjects willing to sign the informed agreement.
And the following exclusion criteria: • Subjects not belonging in the abovementioned criteria; • Subjects under a specific pharmaceutical treatment which may interfere with the experimental study in question; • Participation to a similar test study within the last 60 days; • Subjects allergic to compounds in the proposed cosmetic formulation.

Topical Application
The product is applied by the experimenter massaging till complete absorption over the anatomic zones subject to evaluation. The levels of skin elasticity, hydration and rugosity were measured before the treatment (thus at time t = 0), t = 2 hours after and t = 40 days after in order to check on the efficacy of the aforementioned cosmetic formulation. On the first day, the experimenter provides to instruct the participants to the study about how to correctly apply the emulsion and in particular: 1) The facial skin must be thoroughly cleansed so to remove any other cosmetic eventually used before; 2) The emulsion is applied till complete absorption (t = 40 days) in wrinkles areas, through a smooth massage; 3) The experimenter the first day makes an assessment of skin elasticity, hydration and rugosity in order to evaluate the efficacy of the applied cosmetic product on the short-term (t = 2 hours).

Evaluation of the Skin Elasticity
The measurement of the skin elasticity level has been carried out through "soft-plus'' instrumentation from Callegari S.p.A. Parma (Italy), provided with single measurement probes.
Measurement Principle: the evaluation of the cutaneous elasticity allows the monitoring of the skin viscous-elastic properties respect to immediate deformation and its recovery capability.
Through the measurement of the skin deformation under exerting negative pressure from probe, the index of skin elasticity can be determined.

Hydration Parameter Evaluation
The measurement of skin hydration has been carried out using a specific probe for single measurement, provided from the "soft plus'' instrumentation form

Anti-Wrinkles Efficacy Evaluation via Imaging Data Analysis
The anti-wrinkles efficacy evaluation has been carried out using a micro-camera, provided from the "soft plus'' instrumentation form Callegari company s.p.A.
(Italy), to obtain digital images of the skin areas of interest for such study, that can be used for further imaging-data analysis. The latter micro-camera is pro-

Statistical Analysis
Skin elasticity, hydration and wrinkles-profile have been analyzed through an ANOVA 1-way test, given the three different sampling times: t = 0, thus before treatment, and t = 2 hours, t = 40 days, after treatment.

Skin Elasticity Parameter
The skin elasticity parameter evaluates the skin viscous-elastic properties as a response to an immediate deformation of the skin surface. Figure 1 and Figure   2, show the difference in elasticity for direct instantaneous evaluation after applying a negative pressure to t = 0, t = 2 hours and t = 40 days (Table 1 and Table 2).
Measurements show an increase in skin elasticity at short term (t = 2 hr) of 30.80% and 49.60% at long-term (t = 40 days). The p-value corresponding to the F-statistic of one-way ANOVA is lower than 0.05, suggesting that the treatments are significantly different.

Skin Hydration Parameter
The measurements about skin hydration have been carried out through a capacitor sensor able to monitor the dielectric constant of the skin and underlying skin layers. The latter measurements are directly proportional to the water content in the tissues under examination (Figure 3 and Figure 4, Table 3 and Table 4).
The measurement of the skin hydration parameter via capacitor sensor (skin and underlying tissues dielectric constant) shows an increase in facial skin hydration at short-term (t = 2 hr) of 30.67% and 68.70% at long-term (t = 40 day).
The p-value corresponding to the F-statistic of one-way ANOVA is lower than 0.05, suggesting that the treatments are significantly different.

Anti-Wrinkles Efficacy Evaluation
For the objective evaluation of the images analyzed, Image J allows you to select the same skin portions, peculiarly, pixel measurements on the wrinkled profile in question: area, height, and integral density, which is equivalent to the average product of the mean for the areas (Figures 5-7, Table 5 and Table 6).
Measurements show a decrease the wrinkle height at short-term (t = 2 hr) of 35.44% and 62.44% at long-term (40 day). The p-value corresponding to the F-statistic of one-way ANOVA is lower than 0.05, suggesting that the treatments are significantly different.           [50]. Almonds oil showed high efficacy in the treatment of dermatological pathologies such as psoriasis and eczema. Morover, thanks to its moisturizing properties, smoothes the skin and promotes cicatrization [51].

Conclusion
The tested product has proven to be effective and safe as each ingredient used in its formulation. Notably, the donkey milk serum promotes skin hydration and smoothening visibly contrasting skin wrinkles formation. The karité butter, thanks to its unsaponifiable components (tocopherols and catechin, triterpenes and sterols, Vanillin, Karitene and Allantolin) also displays hydrating, protecting, anti-inflammatory and anti-wrinkles properties. At last, the snail secretion promotes fibroblasts longevity and the assembly of the extracellular matrix thus minimizing the manifestations of skin ageing. Moreover, thanks to its unique and complex composition, the cosmetic product under investigation, favors the natural skin equilibrium as showed out from the testing over 12 volunteers that revealed a substantial increase in skin elasticity and hydration together with a sensible reduction of the skin-wrinkles.