The study was conducted to assess the immediate efficacy and delayed of an active in attenuating the signs of skin aging of the face. In the present research, subjective and objective techniques were used to evaluate the elasticizing, moisturizing and anti-wrinkle effects.

MATERIALS AND METHOD

PRODUCT

The tested product is an antiwrinkle active, called ANTIWRINKLES TOUCH ® , produced by the company SOCRI – GREENGREDIENTS ®. The raw material has the following INCI denomination: Polyglyceryl-4 Betaine, Agrimonia Eupatoria Extract, Potentilla Erecta Extract, Ascophyllum Nodosum Extract, Saccharomyces Cerevisiae Lysate, Vaccinium Angustifolium (Blueberry) Fruit Extract.

It has been tested at 4% in a cream with the following INCI formula: Aqua, Caprylic / Capric Triglycerides, Ethyl Hexyl Stearate, Glycerylstearate, Cetearyl Alcohol, Glycerin, Cetyl Alcohol, Ceteareth-20, Phenoxyethanol, Dimethicone, Citric Acid, Sodium Dehydroacetate, Benzoic Acid, Lecithin, Ethylhexylglycerin, Tocopherol, Ascorbyl Palmitate.

SELECTION OF VOLUNTEERS

For this study a standard female sample was selected having the following inclusion and exclusion criteria: age between 35-55 years, non-smokers, Caucasians, phototype 3 , black eyes and black hair, a negative history of sunburns on the face, slight signs of photo aging (some spots and solar freckles, absence of keratoses and basaliomas), presence of linear wrinkles of mild-moderate degree as described by Griffiths on the cheeks and around the eyes (Griffiths 1992), presence of dry skin, a negative history of skin diseases of the face, atopy and DAC and anti-wrinkle skin treatments in the last 6 months.
During home experimentation, exposure to the sun or artificial UV rays and the use of topical pharmaceutical products or other anti-wrinkle cosmetic products have been banned. All subjects were informed about the study procedures and signed an informed consent.

METHODOLOGY

Effectiveness in improving the skin was assessed:

A) instrumentally measuring the degree of hydration, elasticity through the sensors of a Multiprobe Adapter System MPA5 of the Courage & Khazaka Electronic GmbH (Cologne, Germany); the degree of roughness by means of VISIOSCAN® VC 98 from Courage & Khazaka Electronic GmbH (Cologne, Germany).

B) With subjective clinical evaluation and / or with the help of macro photography obtained through the Visioface 1000D (Courage + Khazaka Electronic GmbH), by the dermatologist, the facial roughness and safety parameters after the application of the two product.

Evaluation of the cutaneous hydration index

The measurement of cutaneous hydration was evaluated using the internationally recognized Corneometer method. The measurement is based on the evaluation of the dielectric constant of the water. The measuring probe shows changes in the water content of the sample to be measured. A diffuse electric field, generated by the probe head, penetrates the first layers of the skin and determines the dielectricity.
The CORNEOMETER® CM825 (Courage + Khazaka, electronic GmbH) has been used for the instrumental evaluation of the hydrating capacity of the products it allows to measure skin hydration through capacitance measurements between the stratum corneum and the sensor probe of the device. By pressing the front surface of the sensor on the skin, a number appears on the computer monitor: this number is related to the level of hydration on the skin surface.
The measurement was performed on a cutaneous area, as flat as possible, taking care to exert a constant pressure for a time set by the instrument itself. The sensor has been kept clean from time to time. A healthy skin in conditions of about 20 ° C of temperature and about 40-60% of environmental humidity should, in the monitored region, have a hydration index> 45.

Evaluation of cutaneous elasticity

The skin elasticity was evaluated using the suction / elongation method and the subsequent release of the skin within a measurement probe. During the suction / elongation phase the instrument generates a constant negative pressure able to suck the skin inside the probe.
This is followed by a release phase, in which the pressure inside the probe is brought to zero millibar thus allowing the skin to return to a state of rest. An optical detection system evaluates the depth reached by the skin inside the probe during the two measurement phases.
The computerized suction instrument, equipped with a 2 mm probe, used by us was the CUTOMETER MPA580® (Courage + Khazaka Electronic GmbH, Köln, Germany). Before the test, it was necessary to proceed to the setting of the measurement technique, of the depression level and time, of the subsequent release time and of the number of successive measurements on the same test area.
In this case it was considered appropriate to choose the measurement technique that provided, at a standard pressure of 450 mBar, a depression that increased steadily in a time of 10 seconds and an equally constant release in the same period of time, for 5 measurements later.
This choice is motivated by the fact that, by applying weakly weak suction forces, it is possible to investigate the elasticity of the epidermal layers and the superficial dermis, ie the cutaneous areas that can be most affected by a cosmetic treatment.

The figure above shows graphically the behavior of the skin in the suction / elongation phase (from 0 to 5 seconds) and then in the release phase (from 5 to 10 seconds).
As you can see from the figure the skin reacts to the negative pressure stimulation with a biphasic type response characterized by a first phase of rapid relaxation (Ue) followed by a second phase in which the skin tends to oppose the stimulus (Uv) reaching its maximum degree of deformation (Uf).
In literature the first part of the curve is known as the elastic component of the skin while the second part characterizes the viscoelastic component, represented for the most part by the plastic component.
Analogous is the behavior of the skin at the end of the negative pressure stimulus in the release phase.
In fact, if in a first phase (Ur) the skin tends to return to its original state of rest – give its elastic qualities – in a second phase the plastic component exceeds the elastic one by slowing down the speed of redefining the skin (Ua-Ur) up to keep the skin in a deformed state (Uf-Ua); this phenomenon is known as hysteresis. The deformation curves of the skin were analyzed using the Win-Cutometer MPA software which provided the parameters listed in the table below.

These skin parameters have been divided into two main categories: elastic and viscoelastic and further subdivided into absolute and relative.
Absolute parameters are: Ue, Uv, Uf, Ur, R8 (Ua) and R0 (Ue + Uv), R1 (Uf-Ua). Relative parameters are: R2 (Ua / Uf), R5 (Ur / Ue), R6 (Uv / Ue) and R7 (Ur / Uf).

The absolute parameters are measured in millimeters, while the relative parameters are presented with a number, which represents a ratio between the values of two absolute parameters – whose maximum value is 1 ( 100%). Absolute parameters are believed to depend on the thickness of the skin, which varies with age, gender and body region.

This is the reason why in comparative studies, the thickness of the skin must first be standardized with ultrasound.
Since this is not always possible, it is preferable to use, in comparisons, the relationships between absolute parameters, ie the relative parameters.

It is admitted that these do not depend on the thickness of the skin and can be compared between subjects and anatomical regions (Berndt 2002).
The reason is that the relative parameters are composed of two parts: the numerator and the denominator, and the same value can be the result of an increase of the numerator or a decrease of a denominator and vice versa (Dobrev 2000, Dobrev 2002).

Their biological information the R parameters are divided into three groups (Barel 1998, Dobrev 2000, Dobrev 2002): Absolute elastic parameters [Ue, Uf and Ur] and related Elastic Parameters [R2 (Ua / Uf), R5 (Ur / Ue) and R7 (Ur / Uf)].

Absolute viscoelastic parameters [Uv and H] and related Viscoelastic Parameters – Uv / Ue. Mixed parameters [R1 and R4]. In the present study the mechanical parameters used are some of the most recommended to characterize the main mechanical properties of the skin: Ue and Uf (distensibility), Ue / Uf and Ur / Uf (elasticity) and Uv and Uv / Ue (viscoelasticity) (Dobrev 2000, Dobrev 2002).

The distensibility of the skin is the ability of the skin to undergo stress or it can be understood as the resistance of the skin to change shape under the influence of stress.

The elasticity of the skin is the ability of the skin to recover the original shape after a deformation. The viscoelasticity of the skin is the timedependent deformation with a phenomenon non-linear “plastic sliding” and “hysteresis”. Description of the parameters used: R0 (absolute parameter) is called final distension or skin distensibility and is the highest point of the first curve (Uf). This parameter evaluates the solidity (firmness, rigidity) of the skin.

It represents the passive behavior of the skin to external forces and is an absolute parameter that depends on the thickness of the skin: if it increases, in its components, its flexibility is reduced (Jachowicz 2008).

The more the value tends to the more elastic the skin will be. In fact, the decrease of R0 represents an increase in the thickness of the skin and the corneous state and / or an increase in thickness or a decrease in the length of the collagen fibers that cause a skin stiffness.

The skin stiffness can be reduced with the help of emollients: in this way the tension between the stratum corneum and the epidermis and the underlying dermis is diminished as the lines and wrinkles of the skin are smoothed.
A decrease in R0 may also be due to a firming effect induced by a cosmetic product capable of producing changes in the skin’s properties inducing a greater skin tension (Dobrev 1998).

R6 (relative parameter) It is a relative parameter and represents the relationship between viscoelasticity and elastic distension ie Uv / Ue. It is the portion of visco-elasticity on the elastic part of the first curve. Indicates the relative contribution of viscoelasticity to elastic deformation of the skin and is attributed to the movement of interstitial fluid through the fibrous network (Hashmi 2007, Jachowicz 2008).

The lower the value, the greater the elasticity. This parameter is the most indicative of the epidermal and dermal water content (Dobrev 2000). This is because the mechanical and electrical properties of the skin are influenced by the level of skin hydration. The increase in Uv / Ue indicates a decrease in interstitial viscosity, as a result of an increase in water content and changes in the composition of proteoglycans.

The accumulation of water in the dermis decreases the friction between the fibers and facilitates the movement of the interstitial fluid. At the epidermal level the moisturizers cause a softening of the outer layers of the epidermis, mainly of the stratum corneum, increasing its plasticity (Ryu 2008).

In one study, the skin slightly dehydrated by the use of an alkaline soap showed a decrease in the parameters R6 and R5 (Maia Campos 2008). In another study, Dobrev demonstrated a positive correlation between epidermal hydration and parameters R5 and R6 (Dobrev 2000). R7 (relative parameter) or biological elasticity (Paye 2007).

It is the portion of elasticity compared to the final distension. It represents the relationship between the immediate retraction and the final distension ie Ur / Uf (Dobrev 2000). The more the value is close to 1 (100%) the more the skin is elastic.

Evaluation of the cutaneous texture

The VISIOSCAN® VC 98 with SELS 2000 software (Courage + Khazaka, electronic GmbH) was used to evaluate the changes induced by treatment on the skin design.

The Visioscan VC98 is a special high-resolution UVA video camera. In fact, this device uses a special video sensor with a very high resolution, and a UVA light source placed inside in a small, easily manageable, ergonomically shaped plastic probe. Two special halogen lights, arranged on opposite sides, uniformly illuminate the skin.

The spectrum of light, its intensity and the path are pre-established so that only the stratum corneum, without reflections of the deeper layers, is analyzed. This special light excludes the majority of unwanted light reflections of the skin, allowing very clear and non-glossy skin images to be obtained.

The size of the measured area is 6 x 8 mm. The skin image is taken from a CCD camera that is inside the probe and transmitted to a PC in 256 levels of gray, where 0 is black and 255 is white.
The image obtained will then be analyzed through the SELS software (Surface Evaluation of the Living Skin) which was developed by Prof. Tronnier of the University of Witten in Germany and offers countless possibilities for analysis of the skin surface providing four clinical parameters.

The parameters correspond quantitatively and qualitatively to the physiological condition of the skin surface. They have been purposely derived from conventional parameters for the roughness of the materials.
The SELS parameters are: SEsc (Scaliness) which indicates the degree of desquamation or the level of dryness of the stratum corneum. The smaller the value of SEsc, the greater the moisture in the skin as a result of treatment with moisturizing or anti-aging formulations.

SEr (Skin Roughness) understood as a parameter of skin roughness, which seems to be a parameter opposite to SEsm. It calculates the percentage of dark pixels present in the entire image. A decrease in values indicates less skin roughness. SEw (wrinkless) identifies the degree of aging and wrinkles by calculating the proportion of horizontal and vertical wrinkles.

Lower values for the SEW parameter indicate that there are fewer wrinkles on the skin. SEsm (Skin smoothness) is the parameter of skin softness or smoothness. It is calculated as an average value of the width and depth of wrinkles.

The tighter the skin, the better its state (eg in youth) and the higher its values. Scaling Severity Score (SSS) During the study a subjective clinical evaluation was carried out regarding the entity of firmness, tone and of skin roughness by a Dermatologist, using the Scaling Severity Score (SSS) (0 = absence, 1 = slight presence, 3 = moderate presence, 3 = evident presence).

EXECUTION METHOD

The product has been applied according to its characteristics of use: used on the face in the evening. Its effectiveness was assessed in a 24-hour short-term test and a 28-day Long Term Test.
The area used for the test was one of the two sides of the face and in particular the crow’s leg wrinkles. sides of the eyes (for roughness and SSS), the zygomatic region (for hydration, elasticity and SSS).
Clinical-instrumental evaluations were performed in the short term test at time t0 (baseline), after 30 minutes (t30m) and 2 hours (t2h); in the long term test after 7 days (t7d) and 28 days (t28d). Before the measurements the subjects remained for 30 minutes in the medical clinic, to acclimatize the skin to the temperature and humidity of the air-conditioned room in which the tests were carried out.
After the basal values were detected, the product was delivered with the instructions for use. At each study time a dermatological examination was carried out to evaluate any collateral effects.
The hydration index was obtained through 4 measurements carried out in the middle-lateral direction under the zygomatic arch. In each study period, if, after the acclimatization phase, at least 2 very different hydration values were observed (greater than ± 5 corneometric units), more time was expected before proceeding with the evaluation. The elasticity values were immediately underneath the zygomatic arch at the level of a vertical line intersecting the lateral ocular commissure.

TOLERABILITY EVALUATION

For the evaluation of tolerability were recorded all the undesirable local events, immediate or delayed, occurred during the study, evaluating the severity, the time of onset, the duration and possible relationship with the treatment.

STATISTICAL ANALYSIS

All results were compared, with the baseline measurement at each time, using the Student’s t test for paired data or with a test not parametric (Wilcoxon signed rank test) when the difference in averages did not show the characters of normalcy verified using the Shapiro-Wilk test.
The results were considered significant if p <0.05 (95% Confidence Level) and were calculated using a Microsoft® Excel spreadsheet.
All data were presented as mean ± standard deviation and in addition to the measured value they were also reported as differences vs basal (difference and percentage value).