3.1. Organoleptic Characteristics and Determination of pH
The overall appearance of all formulations, labeled S, M and MS (for both A and B cream bases), remained unchanged throughout the 90 days of testing, there being no phase separation or any sort of precipitation.
Regarding stability of color and smell, it was found that after 60 days of testing, sample M, for both cosmetic emulsions, suffered only minor changes due to temperature variations or exposure to sunlight.
The pH range of all tested formulations remained between 5.9 and 6.3; thus, the pH of the formulations was considered not detrimental and suitable to applying to epidermal tissues since human skin has a physiological pH that ranges from 4.6 to 5.8 [
11].
Formulations prepared with both cream bases (
A and
B) had pH variations of no more than 0.2 units at the three temperature study conditions of 5.0 ± 0.5, 45.0 ± 0.5 and 25.0 ± 2.0 °C throughout all 90 days of testing, the only exception being the
S formulation incorporated into cream base B. This formulation, when incubated at 45.0 ± 0.5 °C, had its pH increased from 6.2 (t0) to 6.6 (t90), a total of 0.4 pH units. Such pH variation is understandable, as it only demonstrates the impactful effects of higher temperatures and humidity on the formulation; considering current legislation and comparing the effects on pH variation with those observed on the formulations tested under normal temperature conditions (which did not vary significantly at all), such pH variation can be considered acceptable [
8].
The MS formulation suffered less impactful variations on organoleptic parameters and pH values under the analyzed conditions, demonstrating that rutin succinate associated with sunscreens (MS) improves stability of formulations prepared with both cream bases (A and B).
3.2. Analysis of Functional Features: Antiradical Activity and Photoprotective Effectiveness
Regarding physicochemical functional parameters (antiradical activity), samples
S and
MS showed a DPPH· radical inhibition interval that ranged from 32.0 to 36.0% (t0). Analysis of variance homogeneity (ANOVA F-test) indicated that
MS formulation samples suffered no variations throughout the 90-day testing period, especially when considering only formulations made with cream base (
A). However, cream base (
B) formulations allowed for significant changes of antiradical activity when kept under refrigeration (5.0 ± 0.5 °C), starting from day 15 of storage, and also when kept in incubators (45.0 ± 0.5 °C), starting from day 60 (
Table 2). The DPPH method was used to assess antiradical activity of photoprotective formulations because of the hydrophilic properties of rutin succinate and due to the solubility of chemical filters in alcohol and interactions by hydrogen donation.
Significant variations of antiradical activity were found for S formulations under different conditions of analysis throughout storage time, regardless of the cosmetic base used. Stability of the antiradical potential of these formulations improved due to association of rutin succinate with sunscreens (MS).
Rutin succinate is a phenolic compound derivative of rutin, capable of scavenging oxidized species and thus decreasing damage caused by UV radiation. In comparison with rutin alone (log P = 0.85 ± 0 .05), the insertion of carboxylate groups on hydroxyls of the rutin disaccharide increases its water solubility by 80 times (log P = −1.13 ± 0.02). However, its antioxidant activity and ability to prevent lipid peroxidation (as determined by assessing formation of malondialdehyde, with IC50 = 13.46 μM) [
17] remains intact.
The association of rutin succinate with sunscreens in formulations (
MS) increased by nearly 70% their antiradical activity when compared to formulations which contained sunscreens only (
M), as shown in
Table 2. Evaluation of this parameter is vital for proper assessment of photostability of the sunscreens incorporated in the formulations, as antiradical compounds can scavenge reactive species and prevent photodegradation.
Thus, the chemical filters 2-hydroxy-4-methoxybenzophenone and 2-ethylhexyl 4-methoxycinnamate, which are capable of filtering UVA and UVB radiation, respectively, were chosen for association with rutin succinate in this study. These filters are widely used in commercial sunscreens. However, they do tend to suffer photodegradation, limiting their use in broad-spectrum products. When chemical filters lose photostability, structural changes take place and the filters may interact with other molecules of the formulation, decreasing their ability to absorb UV radiation, leading to a reduced photoprotective efficacy.
The 2-ethylhexyl 4-methoxycinnamate filter can undergo photodegradation, in which isomerization occurs decreasing molar extinction coefficient from 23,300 mol
−1 cm
−1 (λ = 311 nm) to 12,600 mol
−1 cm
−1 and also decreasing absorption wavelength to a maximum of 301 nm [
18]. The 2-hydroxy-4-methoxybenzophenone filter, with maximum absorption in two spectral regions, UVB (λ = 288 nm,
= 14,000 mol
−1 cm
−1) and UVA II (λ = 325 nm,
= 9400 mol
−1 cm
−1), can undergo oxidation when in its triplet state, which can easily react with phospholipids found in cosmetic formulations, resulting in the formation of peroxyl radicals [
19].
Evaluation of the photoprotective efficacy of the studied formulations (
Table 3) showed there was no significant variation of SPF, UVA/UVB ratio and critical wavelength values (>370 nm) for the
MS formulation throughout the 90-day testing period under the three studied conditions. Such results are due to the association of chemical filters with rutin succinate in the formulation, as it caused the chemical filters to resist UV radiation-mediated degradation. As a flavonol, rutin succinate prevents lipid peroxidation by scavenging initiator radicals such as singlet excited oxygen, hydroxyl radicals and superoxide ions [
6,
7]. Formulation
M, on the other hand, proved to be unstable, as SPF values for formulations prepared with cosmetic base
A when studied at 25.0 ± 0.5 °C decreased 48.5% throughout the 90-day period; this is a case for concern, as RT conditions are precisely the conditions of use of the average consumer. Still, from day 15 to day 30, there was an increase of FPS values determined for formulation
M when incorporated into cream base
A, which further reinforces the fact that such chemical filters (2-hydroxy-4-methoxybenzophenone and 2-ethylhexyl 4-methoxycinnamate) are relatively unstable and susceptible to photodegration, as already mentioned above.
Formulations prepared with incorporation of sunscreens in cream base
A (
Table 3) showed SPF values approximately 50% higher when compared to those formulations incorporated in cream base
B (
Table 4), which were the formulations
M (19.19; 10.31) and
MS (20.25; 9.73), respectively. Rheological behavior and pH values also did not change significantly throughout the study, and these data can be correlated with the data for SPF values, especially when formulations were prepared with cream base
A, which was found to allow preparation of a more stable formulation. Despite the fact that both cream bases used share structural similarities, the formation of micelle-based structures in cream base
A seems to be more robust than in cream base
B.
3.3. Analysis of Rheological Behavior of Formulations
Current research shows great interest on the study of the rheological behavior of emulsions, as it is closely related to properties of formulations that define their stability. This study was thus carried out in order to investigate whether rutin succinate could be used in sunscreen formulations as an auxiliary component which could improve the efficacy of photodegradable sunscreens. The ability of these formulations to be applied to skin and variations on FPS and UVA/UVA ratio values, consistency and spreadability were also assessed.
The rheological behavior of the formulations was assessed by analysis of apparent viscosity, flow curve and hysteresis area. After formulation samples have undergone heat and light stress at pre-established periods of time, their resulting rheological behaviors allowed for detection of changes that destabilize the sunscreen formulation, and such changes are not always perceived by organoleptic analyses [
15,
16].
The tests mentioned above were carried out only with the formulations incorporated in cream base A, as the formulations prepared with this cream base showed more favorable results compated with formulations incorporated in cream base B for all other tests previously described.
Both self-emulsifying bases (
A and
B) are systems with phosphate anionic O/W characteristics; they were chosen for this study as they are good alternative bases for the incorporation of many cosmetic actives, including sunscreens, while also having biomimetic properties, as they have characteristics similar to those of skin phospholipids [
20].
Only cream base
A possesses two phosphoric esters linked to chemical structures of both high and low degree of ethoxylation (dicetyl phosphate and ceteth-10 phosphate), which allowed association with sorbitan monostearate 20 EO (nonionic emulsifier). This combination induces an electrosteric stabilization mechanism, which decreases repulsion of negative charges and consequently reduces the size of micelles [
20,
21].
Emollients used in the studied formulations were chosen according to their ability to properly spread when applied on skin, covering more area and protecting more skin tissue, and also according to their sensory properties, which should be pleasant to the user, such as the capability of the formulation to cause a dry and velvety feeling when applied. All of these characteristics can be found in volatile silicones (cyclomethicones) associated with other thickener/stabilizer silicones that stabilize micelles (PEG-PPG 18/18 dimethicone and vinyl dimethicone crosspolymer) and with polysiloxanes, responsible for improving the formulation resistance to water.
Formulations containing rutin succinate when incorporated into cream base (
A), associated or not with chemical and physical filters, were found to be stable regarding their apparent viscosity throughout the 90 days of study (
Table 5). However, there were increases of apparent viscosity in
M formulations of 43.4% (0.972 to 1.394 Pa.s) and 90.0% (0.972 to 1.852 Pa.s), when under storage conditions of 5.0 ± 0.5 °C and 45.0 ± 0.5 °C, respectively. Variations of apparent viscosity observed for
S formulations were not greater than 20%, but significant variations when the formulations were under storage conditions of 45.0 ± 0.5 °C and 25.0 ± 2.0 °C started to occur as soon as after day 15 of study.
Analysis of hysteresis areas from
MS formulations prepared in cream base
A resulted in no significant variations throughout the 90-day study period for all storage conditions tested: 5.0 ± 0.5 °C, 45.0 ± 0.5 °C and 25.0 ± 2.0 °C (
Table 5). However,
M formulations showed an increase of hysteresis area of 32.6% (from 34,693 to 45,817 mPa/s) when stored at 5.0 ± 0.5 °C, considering the whole t0 to t90 period.
S formulations showed an increase of hysteresis area of 50% at the end of the 90-days study period when kept under all three study conditions: 5.0 ± 0.5 °C, 45.0 ± 0.5 °C and 25.0 ± 2.0 °C, and significant changes could be seen as soon as after 15 days of storage. Both apparent viscosity and hysteresis area suffered significant variations when assessed on
M and
S formulations after 15 days of storage, which can be taken as indicative signs of instability of their structures.
Given the results observed in the rheograms (
Figure 1A), we chose to evaluate the different rheological profiles of formulations
S,
M and
MS by adjusting the flow curves according to the power-law mathematical model, also called Ostwald-Waele model (t = K·γ
n). After proper adjustments, analysis of the new values in correlation with the non-adjusted values resulted in correlation coefficients (R
2) between 0.9900 and 0.9970. Yield stress was considered as being nearly zero, and therefore negligible for further calculations (τ
0 = 0). This model was used in order to determine flow behavior index (n) and consistency index (K), making the above mentioned equation linear (log t = log K + n log γ), where
n represents the angular coefficient (slope) and
log K represents the linear coefficient. All calculations were carried out with the aid of Excel 2003
® software (
Table 6).
Pseudoplastic materials are characterized by flow curves in which shear stress decreases while shear rate increases [
16], a behavior shared by a large number of (bio) technologically relevant systems. Such behavior is a consequence of orientational rearrangements of the internal structure within the flow area, which diminish the material resistance to the applied shear stress [
22]. This is found to be the case for the systems studied in this work.
The intensity of this property is reflected on factor
n of the Ostwald de Waele equation (the lower
n is from 1.0, the more intense is pseudoplasticity).
Table 6 depicts the significant pseudoplasticity of the studied formulations. This is a desirable aspect in formulations meant to be spread on skin, as is the case in this study.
Table 6 also displays the consistency indexes K of the assessed formulation samples. The higher the K index, the more consistent, or “viscous”, is the formulation. Results show that K values are situated around 2000 mPa.s.
Since viscosity is defined as the ratio between shear stress and shear rate, η = dτ/(dγ/dt), in the case of pseudoplastic fluids, it necessarily diminishes as flow curves are drawn. That is the why viscosity of non-ideal fluids (pseudoplastic included) is called “apparent viscosity”; for such systems, viscosity does not vary independently, as it essentially depends on either shear rate or shear stress applied [
16,
22].
Another characteristic observed in the studied emulsions was that apparent viscosities varied with time. In the formulation samples assessed, apparent viscosities at a constant shear rate (200 s
−1) diminished with time (
Figure 1B), which characterizes the so-called thixotropic behavior (this shear rate was chosen due to the fact that it closely resembles the shear rate of a formulation being spread on skin). Such behavior is desirable, considering that a decrease of viscosity with time might speed up the absorption process at the skin when rubbing is required during application. Thixotropy is a property of a number of different systems, such as dispersions and emulsions.
Viscosity variations during storage reflect internal microstructural changes in the formulations. In some cases, decrease of viscosity may lead to unavoidable effects such as flocculation, coalescence or sedimentation, which, however, have not been observed for the formulations studied in this work.
Some authors, such as Brummer (2006) [
23] and Tadros (2004) [
16], discuss the relevance of thixotropy (phenomenon where apparent viscosity decreases with time) (
Figure 1B) and thinning (pseudoplastic behavior, phenomenon where apparent viscosity decreases while shear rate increases). They correlate both phenomena to the gradual breakdown of the structure of formulations and flattening of emulsion droplets leading to possible breakage of aggregates, thus aligning molecules and droplets within a flow. For consumers, this would mean the formulation becomes more fluid, spreading more easily and without sagging when applied on skin, forming a homogeneous film that comes in intimate contact with the skin microrelief. As a result, an increase in product effectiveness would occur due to both the release of filters during the process where structural changes of the emulsions take place and to the longer time available for a better accommodation of the product on the skin, as indicated by the higher hysteresis area observed (anionic phosphate cosmetic base employed) [
16,
22].
Larger hysteresis areas indicate that the return to original structural conditions while shear application decreases takes longer for some samples (e.g., greater hysteresis area for MS formulations = 36,581 mPa/s compared to S formulations = 13,423 mPa/s). The differences of behavior between these formulations could be associated with the presence of a dispersed physical filter in the MS formulation that impairs recovery of the structural system.
Structural recovery of the
MS formulation was constant throughout the whole study period of 90 days. The variations on hysteresis area values determined for the
M formulation, however, were greater for most conditions tested and the variations in its hysteresis areas were also found to be greater than those observed for formulations
MS and
S. This suggests that rutin succinate, which is present in formulations
MS and
S, might have improved the alignment of the droplet stream. This could have been partly due to the nature of rutin succinate hydrophilic (log P = −1.13 ± 0.02) [
17], and to its role in the inhibition of peroxyl radicals formation, which could react with components of the oil phase, destabilizing the system and ultimately affecting its rheological behavior.