Evaluation of the use of Proteins as Moisturising and Skin Tightening Active Ingredients within Skin Care Formulations N I Challoner, S P Chahal and R T Jones Keywords:
protein, skin moisturisation, skin-tightening, Dermal Torque Meter, immediate extensibility
Synopsis Proteins have been used within skin care formulations for over forty years, and during this time have been recognised as offering excellent conditioning benefits to the skin, particularly moisturisation and skin-tightening. Advancements in the fields of cosmetology and dermatology have led to the development of instrumentation capable of allowing scientists to study the effects of cosmetic products on the skin with relative ease and precision. The Dermal Torque Meter is one such instrument, and has allowed the determination of the effect of proteins on the mechanical properties of the skin, which in turn can be related to skin moisturisation and skin-tightening. Proteins have been studied from a variety of cosmetic bases including O/W emulsions, liquid detergents, aqueous gels and simple aqueous systems from both ‘in-house’ and customer formulations. The results have confirmed the functional benefit of the protein, and have illustrated a relationship between functionality and the molecular weight of the protein under evaluation. The data has been used to support marketing claims for moisturisation and skin-tightening when proteins are applied from commercial cosmetic bases. 1 Introduction Proteins were first introduced as cosmetic raw materials over forty years ago, and since this period high molecular weight soluble collagen has probably been the most widely used and recognised protein for high quality skin care products. However, in recent years the range of proteins available for cosmetic use has increased. Proteins from animal, and more recently from marine and vegetable sources, with a range of molecular structures and molecular weights are now commercially available. In general, the functional behaviour of proteins responsible for conditioning may be attributed to the physical characteristics that they possess, such as their ability to retain moisture, their ability to form films and their natural substantivity to the skin through charge-charge interaction. It is in the area of functionality that the molecular weight of the protein has an important part to play. For example, high molecular weight proteins are better at film-formation whilst low molecular weight proteins are better at moisturisation. Croda Colloids Ltd Foundry Lane Ditton Widnes Cheshire WA8 8UB England Tel +44 (0)151 423 3441 Fax +44 (0)151 423 3205 Page1 of 10
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Although 'in-vivo' quantitative evaluation of skin has been carried out, in the field of medicine/dermatology for 25 to 30 years, it is only within the last 10 years that the necessary equipment has been commercially available to allow cosmetic scientists to evaluate the effects of cosmetic products on skin with relative ease and precision. The majority of the devices available operate by measuring the physical and mechanical properties of the skin. This paper presents data on the behavior of a range of proteins on the skin, utilising one such instrument, the Dermal Torque Meter (DTM)†. 2 The Dermal Torque Meter The effect of cosmetic proteins when applied both from aqueous and emulsion bases has been evaluated by our laboratory using the Dermal Torque Meter (DTM). The DTM operates by measuring the mechanical properties of the skin, which may be directly related to the effect of a cosmetic protein on skin moisturisation, skin-firming and skin elasticity. In simple terms, the DTM provides a torsional force to the skin and measures the response of the skin to this force. The force is then removed and the recovery of the skin is measured. The DTM consists of a sensor head, a control unit and an accompanying computer (figure 1). The sensor head is constructed of a torque motor and an angle sensor, with the torque motor being connected to a flat disc at the end of the sensor head. A concentric torque ring around the torque disc defines an annular ring gap, which in turn determines the skin area to be examined. The force is transferred to the skin by securing it to both the torque disc and torque ring using double sided adhesive tape. The torque disc may then be rotated relative to the torque ring, which supports the surrounding skin. Zero pin or switch
Interchangeable guard ring
Fig. 1 - Dermal Torque Meter sensor head
†
Ur Ue 0
15
0
15
Time
Torque (mNm)
Angle, Ø
Central torque disk
Time
Fig. 2 - Schematic representation of time vs. angular displacement for a typical skin sample
Dia-stron Limited, Hampshire United Kingdom
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Torque is applied for a pre-determined time and the resulting angular displacement is recorded. When the torque is removed, restoring forces within the skin attempt to return the torque disc to its original position and this is similarly recorded. These measurements are more clearly illustrated by reference to figure 2 which shows the angular rotation (Ø) of the skin as a function of time on application and removal of the torque. On initial application of torque an immediate deformation (extensibility) of the skin occurs (Ue). This is followed by a slower deformation, which reflects the viscoelasticity of the skin. After a suitable time period (e.g. 15 seconds) the torque is removed and an immediate recovery (Ur) is observed followed by a slower recovery. The data can be expressed in various ways but the most useful parameters to follow as a result of treatment of skin are Ue, Ur and Ur/Ue as defined below. Ue Immediate extensibility (Initial distortion in the elastic region) Ur Immediate recovery (Initial recovery of the skin from an applied distortion) Ur/Ue Elastic recovery (an age related property that illustrates the reduction in the ability of the skin to recover from an applied deformation as the individual becomes older) 2.1 DTM protocol 2.1.1 Test sites The test sites for the DTM sensor head were on the inner forearm between the proximal wrist crease and the flexor aspect. Following the appropriate product application procedure the DTM probe was attached to the forearm before a torque of 10 mNm was applied to the skin for a period of 15 seconds, after which the torque was removed and the relaxation monitored over another 15 seconds. The ring gap on the sensor head was set to 1mm in order to optimise the system to examine the effects of proteins on changes mainly in the stratum corneum1. 2.1.2 Test times The moisture content of the skin is influenced by the atmospheric humidity of the surroundings, and consequently the skin is more hydrated during the summer months. For example, the immediate extensibility of untreated skin averaged over six volunteers was highest in August (1.7) and lowest in February (1.2). In order for the DTM to detect significant changes in skin hydration it is therefore desirable for trials to be performed during periods of low humidity, and consequently all trials were performed throughout the winter months (October – March in the United Kingdom).
2.1.3 Protein types The functionality of cosmetic proteins is intimately linked with the structural composition and molecular weight of the protein itself. Lower-molecular-weight enzyme hydrolysates and amino acids are primarily involved with moisturisation and may exhibit penetration through the stratum corneum and epidermis into the lower dermal layers. Higher-molecular-weight proteins, however, do not penetrate significantly into the epidermis. Instead they remain at the surface of the stratum corneum where they behave as excellent film-formers and modifiers of tensile properties – effects that are perceived as skin tightening or smoothing.
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The immediate extensibility, Ue, of the skin is defined as the initial deformation of the skin (under an applied force) that occurs over the elastic region. De Rigal and Leveque showed that Ue is a direct function of the moisture content of the skin1. Hence, by the utilisation of appropriate experimental protocols, the effect of proteins on the moisture levels of the skin can be determined. In contrast, film-formation lowers the immediate extensibility of the skin since the film resists the torque imparted by the sensor head. Again, by utilising appropriate methods the effect of proteins on the tightness of the skin through film-formation can be determined. A number of proteins, as listed in table 1, have been evaluated to determine their effect on the above parameters. The proteins have been evaluated from aqueous solutions and a variety of cosmetic bases including oil in water emulsions, aqueous gels and liquid detergents. Oil in water emulsions
Molecular weight
Hydrolysed wheat protein (and) hydrolysed wheat starcha Hydrolysed wheat protein polysiloxane copolymerb Hydrolysed whole oatsc Hydrolysed collagen proteind Hydrolysed wheat proteine
1,000 500,000 1,000 50,000 3,000
Liquid detergents Hydrolysed whole oats† Hydrolysed wheat protein (and) hydrolysed wheat starch
1,000 1,000
Aqueous gels Hydrolysed wheat protein polyvinylpyrrolidone copolymerf
40,000
Water Soluble wheat proteing 250,000 Soluble wheat protein (HMW)h 500,000 Hydrolysed wheat protein polyvinylpyrrolidone 40,000 copolymer ____________________________________________________________ The following are trade names registered to Croda: aCropeptide W, b Crodasone W, cCromoist O25, dCrotein C, eHydrotriticum 2000, f Hydrotriticum PVP, gTritisol, hTritisol XM († Formulation unknown)
Table 1 - Proteins evaluated using the Dermal Torque Meter
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2.1.4 Vehicle types The opposing effects on immediate extensibility of moisturisation and skin tightening present a challenge in the design of appropriate scientific protocols. Poleman et al2 have reported the influence of the type of vehicle used on the efficiency of moisturising agents when assessed by conductance and extensibility measurements. The authors found that moisturisation of the skin was most marked from o/w emulsions and was minimal from aqueous bases. Consequently where interest has been focused on moisturisation an o/w emulsion base has been employed, whereas skin-tightening behaviour has been assessed from aqueous solution or gel. 2.1.5 Comparative data The data from the DTM was used to give comparative results rather than absolute values, i.e., differences in angular rotation of the skin between the test and control arms was examined in order to determine the effect of the test protein itself when applied from a cosmetic base. In addition, because the results of DTM experiments are influenced significantly by meteorological conditions such as temperature and humidity, the use of comparative results is scientifically appropriate. 2.1.6 Data analysis The data obtained from the above procedures was subjected to a statistical analysis using the ‘student t-test’. This enabled decisions to be made regarding whether or not the observed differences were a consequence of measurable effects or whether they could be ascribed purely to chance. 3. Skin moisturisation from an O/W emulsion The utilisation of the direct relationship between skin moisturisation and immediate extensibility has allowed the Dermal Torque Meter to be used as a tool for assessing skin moisturisation and hence the functional effect of proteins within skin care formulations. The behaviour of five cosmetic proteins has been evaluated at an inclusion level of 1% ‘active’ within an emulsion base (Formula 1) Cetearyl alcohol and ceteareth-20 (Cosmowax P, Croda Inc.) Cetearyl alcohol (Cetostearyl Alcohol N, Croda Inc.) Mineral oil (paraffinum liquidum), 25 cs @ 25ºC Protein (100% active) Preservatives Deionised water (aqua)
Test
Control
5.0%
5.0%
2.0%
2.0%
5.0%
5.0%
1.0% 0.3% to 100%
0.0% 0.3% to 100%
Formula 1 - O/W emulsion formulation
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3.1 Test protocol The protocol adopted involved the use of six volunteers. The test or control emulsion (0.3g) was applied to the skin between the proximal wrist crease and the flexor aspect of the forearm. Measurements were taken two hours after application utilising the previously described DTM conditions. Studies were performed over a five day period with ‘baseline’ values for untreated skin being obtained on day 1.
3.2 Results and discussion All of the proteins contained within an o/w emulsion resulted in a statistically significant increase in the immediate extensibility (Ue) of the skin when compared to the emulsion alone. This clearly indicates that moisturisation had occurred throughout the four day period of treatment. The fluctuation of Ue from day to day (figure 3) may be attributed to changes in the relative humidity of the environment. The results are expressed as mean difference in the percentage change in Ue between the test and control arms.
Percentage Change in Ue
C
A
E
B
D
10 8
Legend Key
Protein
Average % Change in Ue
P Value
6
A
Hydrolysed wheat protein polysiloxane copolymer
2.8
