1. IntroductionIn recent years, scientific research has documented beneficial health effects of polyphenol compounds related with their antioxidant, anti-inflammatory, and antimicrobial activities. The presence of polyphenols in plants is common. Among them, it is possible to mention the presence of this type of compounds in oak leaves (Quercus spp.) Many oak species are endemic to Mexico. Particularly, Durango state is rich in diversity of oak species. Recent studies have shown that some compounds of phenolic nature, present in the oak leaves, can inhibit the growth of Staphylococcus epidermidis [1], a microorganism associated with acne of microbial origin. Acne is a skin condition, which has various causes. One of them is the presence of dysbiosis in the skin microbiota, which results in the appearance of acne. It usually appears in adolescence and youth of human beings, leaving behind scars and severe self-esteem problems [2].Several treatments have been developed to control the bacterial infection caused by the microorganisms associated with the appearance of acne. However, it is complicated because the skin is a barrier that makes difficult the compounds passage through, limiting the topical treatment [3,4]. In this sense, in recent years, the use of new type of materials has been suggested to improve the load and transport of bioactive compounds across the skin; these materials are called bigels, which are semi-solid formulations made up of two gelled immiscible phases [5].The advantages of using bigels have been documented, such as the ability to release hydrophilic and lipophilic compounds, enrichment of the stratum corneum, providing a refreshing and moisturizing effect on the skin, ease of application on the skin, improvement on the permeability of active compounds through the skin, and good physicochemical stability [6]. It is important to mention, that bigels are non-thermoreversible systems. There are various types of bigels, being the most common the oleogel in hydrogels [7]. Once the bigel has been obtained, it is possible to add various elements that prolong its shelf life, such as parabens, as well as bioactive compounds; most of the studies have focused on evaluating the release mechanisms of such bioactives [8], or the mechanical stability of the bigels [9]. The use of bigels includes hydrogels, where several polysaccharides are used as gelators. Hydrogels have been reported showing interactions with polyphenols [10], however, little work has been reported on the influence that bioactive compounds might exert on the mechanical stability of the bigels. Several reports have described the effect of phenolic compounds on hydrogel mechanical stability [11]. In this sense, the development of soft dosage forms as vehicle to transport bioactives for topical use is important in pharmaceutical industry. They are dispersed systems with a distinctive viscoelastic behavior. Their viscous behavior at a given temperature and shear stress is non-linear, depending on the shear rate [12]. This complicates the mechanical analysis because during the developing of soft dosage vehicles (i.e., as bigels), the study of their mechanical properties is useful to determine their stability [13]. Also, it is a useful tool to obtain objective criteria for quality control, production, storage, release of bioactives, and consumer requirements [14]. Several models have been used as the Creep cycles [15], the Palierne model [16], the Krieger-Dougherty model [17] and the semi-empirical model proposed by Lupi et al. [13]. The complexity of the rheological behavior remains unknown.A relatively simple alternative to study rheological behavior is the Carreau-Yasuda model [18]. Some authors [19,20] have used a Carreau-Yasuda model to evaluate the rheological behavior of bigels loaded with vitamin E and organogels loaded with vitamin E, respectively. They found that the use of the Carreau-Yasuda model and their parameters could help to identify changes in the mechanical properties of bigels and organogels. Generally, viscoelastic properties of materials obtained by rheological methods could deliver data about several properties as structure, phase behavior, and physical interactions [21,22]. The most known model for complex viscosity is the Carreau-Yasuda, which correlates complex viscosity to zero complex viscosity, relaxation time, power law index and width of the transition between Newtonian and Power law behavior [23]. The expression is shown next:

η∗(w)=η0∗=η0* [1+(η0*τ*∗w)a] n−1a

(1)

where,

η*(w) is complex viscosity

η0* is zero complex viscosity

λ is the relaxation time

n is the Power law index

τ* is the shear stress at the transition zone

a indicates width of the transition between Newtonian to Power law behavior

And the relaxation time can be defined as:Usually, Equation (1) has been used by researchers in several areas of knowledge, focusing on fitting the model to experimental data and determine the parameters. However, more recently several groups have used this model to obtain more information about material properties [21]. Thus, the objective of this work is to evaluate the effect of the presence of phenolic compounds from oak leaf extracts on the rheological properties of bigels. 2. Materials and Methods

Guar gum and xanthan gum were purchased from Quimica Hércules (Toluca, Edo de México, México), methyl paraben (Sigma, Toluca México), sesame oil, purchased from a local store in Durango, Dgo., México. Sorbitan monostearate was acquired from (Sigma, Toluca, México), procyanidin-B2, (epi)-catechin gallate, (epi)-catechin, procyanidin-B1, catechin, quercetin, rutin, kampferol-3-O-glucoside, taxifolin, naringenin, eriodictyol, acacetin, neohespiridin, phlorizin, and mangiferin were obtained from Sigma-Aldrich (St Louis, MO, USA). Staphylococcus epidermidis (ATCC 14990) and Cutibacterium acnes (ATCC 6919) were obtained from ATCC, (Manassas, VA, USA).

2.1. Oak Leaves Collection and Infusion PreparationQuercus resinosa leaves were collected in Durango, Dgo, México, characterized by botanist Dr. Socorro González-Elizondo and deposited in the CIIDIR-IPN herbarium. The leaves were dried in the shade, ground in an IKA blade mill, and separated in a 100 mesh sieve (0.147 mm). Infusions of oak leaves were prepared, according to Rocha-Guzmán et al. [24]. Briefly, 10 g of powder of Quercus resinosa leaves were placed in a beaker with 1 L of distilled water at 80 °C and stirring for 10 min. The infusion was lyophilized and stored until use. 2.2. Chemical Characterization of the Aqueous Extract of Oak LeavesThe determination of the phenolic profile was carried out according to reported methodology [25]. Briefly, an UPLC coupled to a photodiode array (PDA)-electrospray ionization (ESI)-triple quadrupole tandem (QqQ) (Waters, Milford, MA, USA) was used. An Acquity UPLC BEH C18 column of 2.1 μm × 50 mm × 1.7 μm at 35 °C. The elution gradient consisted of two solvents, phase A was MiliQ water acidified with 7.5 mM formic acid and acetonitrile (B) at a flow rate of 210 µL/min. The gradient applied was as follows, it started with 3% phase B, at 1.88 min; then, 9% B, at 5.66 min, up to 16%, at 16.9 min, up to 50% B, at 19.62 min, then, to 3% B, where it was maintained isocratically for 20 min. The ionization was performed in negative mode with a capillary voltage of 2500 V, the desolvation temperature was 400 °C, the source temperature was 150 °C, the collision gas flow was 800 L/h, the flow of the collision gas was 130 L/min, the collision energy values were 5 for MS and 20 for MS/MS. For the identification and quantification of phenolic compounds, a mixture of the following pure standards was used: Procyanidin-B2, (epi)-catechin gallate, (epi)-catechin, procyanidin-B1, catechin, quercetin, rutin, kampferol-3-O-glucoside, taxifolin, naringenin, eriodictyol, acacetin, neohespiridin, phlorizin, mangiferin (at a concentration of 20 μg/mL), which were used to obtain retention times and MS/MS transitions. The chromatographic and spectrometric systems were controlled using MassLinx software (Waters, Milford, MA, USA). 2.3. Microbiological Assay

The determination of the load of aqueous extracts of oak leaves into the bigels was made by the evaluation of growth inhibition of the two bacterial strains associated with skin diseases, i.e., Staphylococcus epidermidis (ATCC 14990) and Cutibacterium acnes (ATCC 6919). In the case of Staphylococcus epidermidis, nutrient broth was used, while in the case of Cutibacterium acnes, the BHI broth was used. A growth kinetic evaluation was carried out, incubating in a CO2 chamber (5%) at 37 °C, taking readings at 630 nm in a UV/VIS spectrophotometer and seeding in a plate to count the colony-forming units, using the serial dilution method. Once the growth kinetics were determined, the minimum inhibitory concentration of the aqueous extracts of Quercus resinosa leaves on both microorganisms was evaluated according to the following methodology. Samples of 50 μL were taken from each microorganism in the exponential growth phase and their concentration was adjusted to 1 × 104 CFU. They were incubated in a CO2 chamber (5%) at 37 °C for 20 h, when serial dilutions were made and different concentrations of aqueous extract of Quercus resinosa leaves were applied. Benzoyl peroxide and penicillin G were used as positive controls and the concentrations evaluated were penicillin G 100 μg/mL, benzoyl peroxide 500 μg/mL, and concentrations of 500, 1000, 2000, 3000 and 4000 μg/mL of the aqueous extracts of Quercus resinosa leaves.

2.4. Bigels

To obtain the bigels, a hydrogel was prepared with 175 mg of guar gum and 175 mg of xanthan gum, methyl paraben was added as a preservative. Guar gum and xanthan gum were dissolved in water (34.64 mL at 50 °C), while the oleogels were prepared using 12.75 mL of sesame oil and 2.25 mg of sorbitan monostearate. For this purpose, the sesame oil was heated at 80 °C for 10 min, then the sorbitan monostearate was added and kept stirring for 30 min. The oleogel was softly added to the hydrogel at 60 °C under continuous stirring and the bigels were stored at 4 °C for 24 h until use.

2.5. Quercus Leaves Extract Load into the BigelsOnce the oak infusions were obtained [25], they were frozen and lyophilized. Subsequently, in the aqueous phase (hydrogel), different concentrations of the aqueous infusions (i.e., 500, 1000, 2000, 3000 and 4000 μg/mL of sample) were resuspended in the aqueous phase of hydrogel, until their complete dissolution. In the case of the rheological analyses, the control samples were the bigels without load. The higher concentrations 3000 and 4000 μg/mL were tried but presented solubility problems. 2.6. Microscopy Analysis

The samples were observed in a Carl Zeiss microscope (Carl Zeiss de México, México City, México), equipped with a camera. The images were processed using the software Zen Lite (Zeiss, Oberkochen, Germany) and all images were obtained with a resolution of 100× in bright field.

2.7. Rheological Analysis

Steady shear tests were carried out in a DHR-III rheometer (TA-Instrument, New Castle, DE, USA) at 25 °C, in a shear rate range of 0.1 to 100 s−1, with parallel plate geometry. The results were adjusted to the (Ostwald-de-Waale) Power law model, using the ARES software (TA-Instrument, DE, USA). The evaluation of the linear viscoelasticity range was carried out in all samples by means of a strain sweep in a range of 0.001 to 100%. The results were evaluated by means of the Carreau-Yasuda model, using dynamic complex viscosity. A frequency sweep was carried out at 0.1% strain, ranging from 0.01 to 100 rad/s.

2.8. Statistical Analysis

Data analysis was performed using the ANOVA test and mean comparison tests using the Tukey method (p < 0.05). The determination of parameters of the models used was obtained by non-linear estimation, using the Levenberg-Marquadt method, using the Statistica 12 software (StatSoft, Tulsa, OK, USA).

4. DiscussionFrom the results in Table 1, it is possible to observe the high content of flavanols, which are in higher abundance in comparison to other flavonoids compounds. This result agrees with the reported [27] for aqueous extracts of Quercus resinosa. Flavanols have a strong antimicrobial activity against Staphyloccocus spp. [28]. Particularly, it has been reported [29] a strong antimicrobial activity against Staphyloccocus epidermidis from flavonols; also, there are some claims [30] on flavonols with important antimicrobial activity against Cutibacterium acnes. Thus, the flavonoid profile found in aqueous extract of Quercus resinosa leaves could be useful to inhibiting growth of microorganisms related with acne.The doubling time found for Staphyloccocus epidermidis was higher than the reported for this microorganism (81 min) [31]. However, these authors comment that there are notable differences in terms of growth parameters, depending on both the strain used, as well as the culturing conditions. Regarding Cutibacterium acnes, the doubling time observed was lower than the previously found [32] (4.26 h). It is important to mention that changes in the kinetic parameters may be due to many factors such as the strain, culture conditions, and the inoculum used, among others.As can be seen in the table above, there is a concentration-dependent effect on the % inhibition of the growth of both microorganisms, with the greatest inhibitory effect being found at the highest concentration of aqueous extract of Quercus resinosa leaves. However, such concentration turns out to be too high to be kept in solution without precipitation problems, so it was decided to work with just concentrations of 1000 to 3000 μg/mL. The results obtained for inhibition of microorganisms were much higher than those reported by other authors for aqueous extracts of Mugwoort [33], who at a concentration of 20 mg/mL achieved a 37% inhibition of Cutibacterium acnes. These authors did not evaluate the phenolic profile of the aqueous extracts. Besides, according to some reports, it has been found that phloretin, even at low concentrations, has a strong antimicrobial activity against Cutibacterium acnes, but this compound was not identified in the present work, therefore, the presence of flavonols was important (Table 1), This group of phenolic compounds has shown great antimicrobial activity against Cutibacterium acnes [34], likewise good effects against Staphyloccocus epidermidis from flavonol-type compounds have been documented [35].The rheological behavior of bigels is interesting to be discussed. The structuring agents and the gel proportions used were identical, varying only the aqueous extract concentration. Thus, the increase in flow resistance can be attributed to this variable. Some authors have pointed out that the existence of polysaccharides, such as those used in the present experiment in the hydrophilic part (i.e., xanthan gum and guar gum), can establish non-covalent interactions with polyphenols, which are the predominant molecules in leaf extracts of Quercus resinosa. In turn, these interactions can affect the polysaccharide—polysaccharide interactions [36]. However, unlike to these authors, who stated that as the concentration of polyphenols increases, the flow index also increases and the consistency index decreases; in the present experiment, it was observed that there is a decrease of around 10% in the flow index as the concentration of Quercus resinosa leaves extract increases. On the other hand, in the case of the consistency index, the value is lower when no presence of aqueous extract Quercus resinosa leaves (46.95 Pa.s), but atth the lowest amount of extract (500 μg/mL), the value rises to 91.9 Pa.s. Such value remains constant up to a concentration of 2000 μg/mL of Quercus leaves aqueous extract, where the consistency index drops again (83.42 Pa.s). Such behavior has been described as common in bigel systems [37], but the behavior is different when compared to a system with no presence of polyphenol compounds. It is known that the joint presence of polyphenols and polysaccharides can induce aggregation. In the case of bigels, this implies a lower concentration of gum available for the hydrogel formation, causing a greater influence from the oleogel, which is reflected in changes of the consistency index. In the case of the flow index, the observed behavior is typical of an emulsion, that is, a pseudoplastic or thinning behavior was observed when flowing. Regarding the viscoelastic behavior, in unloaded and loaded bigels with aqueous extract of Quercus resinosa leaves, the storage modulus (G’) is greater than the loss modulus (G’’) throughout the observation window, which implies a prevalence of the solid behavior over the viscous one (i.e., elastic modulus range 11 × 102 to 11 × 103 Pa). In all bigel samples, the differences between the G’ and G’’ moduli become more evident at higher frequencies; on the other hand, the value of the moduli increases, both in the viscous and the elastic zones.The behavior modelling using the Carreau-Yasuda model, indicates that the zero-shear viscosity parameter η0 shows the lowest value in the sample with no presence of the Quercus resinosa leaves aqueous extract. This value compares well with other estimates reported for composite gels [38], especially of the polymeric type [39]. With the presence of the aqueous extracts of Quercus resinosa leaves, the above parameter was increased, which is an evidence of the physical interaction between the polyphenol compounds in the extract with the polymer chains (i.e., xanthan gum and guar gum) in the hydrophilic gel. This phenomenon has been documented with various molecules of different nature than the tested in the present experimental work [36].Regarding the relaxation time parameter, this did not show significant differences (pFigure 2, that as frequency increases, the system behavior follows more the Power law, due to the breaking of networks that makes up each of the gels present in the bigel. The above results are reasonable based on whether the high zero shear viscosity at low frequencies or the solid region affects the complex viscosity (i.e., a low zero viscosity at low frequencies would negatively affect the complex viscosity) As the zero complex viscosity is the maximum level during the frequency sweep, this value is directly associated with the complex viscosity of the samples, while a prolonged relaxation time can cause limited molecular mobility. Thus, the complex viscosity depends on two parameters, the viscosity at zero shear and the relaxation time, so it can be said that the Carreau-Yasuda model is appropriate to describe the behavior of bigeles and the effect of polyphenol compounds on its structure. In this sense, it is necessary to mention that similar results to the found in the present experiment were already described [23] to evaluate the effect of carbon nanotubes on composite gels.