A highly soluble form of rutin for instant resolution of mask‐wearing related disorders

The COVID‐19 pandemic brought about a new normal, necessitating the use of personal protective equipment (PPE) like face shields, surgical masks, gloves, and goggles. However, prolonged mask‐wearing introduced skin‐related issues due to changes in the skin's microenvironment, including increased humidity and temperature, as well as pressure on the skin. These factors led to skin deformation, vascular issues, edema, and inflammation, resulting in discomfort and cosmetic concerns. Clinical reports quickly highlighted the consequences of long‐term mask use, including increased cases of “maskne” (mask‐related acne) or mask‐wearing related disorders such as rosacea flare‐ups, skin‐barrier defects, itching, erythema, redness, hyperpigmentation, and lichenification. Some of these issues, like inflammation, oxidative stress, and poor wound healing, could be directly linked to acne‐related disorders or skin hypoxia.


| INTRODUC TI ON
The worldwide COVID-19 pandemic introduced new habits and an obligation to wear personal protection equipment (PPE) such as face shields, surgical masks, gloves, or goggles, leading to the appearance of skin-related dermatoses.Indeed, mask-wearing triggers a modification of skin microenvironment (higher relative humidity and temperature) and prolonged pressure, generating tissue deformation, vascular occlusion, edema, and inflammation 1 leading to major cosmetic concerns and discomfort, 2,3 even though, the mode of action is not clearly established.Moreover, it took only a very short time before several clinical reports highlighted the many consequences of long-term mask-wearing.
][8][9][10] Some consequences, such as high inflammatory and oxidative states, and poor wound healing (scarring), can be linked directly to acne-related disorders or can be triggered by skin hypoxia. 11,12Since these are widely described disorders, they can be a starting point for the resolution of mask-wearing related disorders.Indeed, since dermatologists raised the importance of finding cosmetic solutions to the skin damage related to mask-wearing, we identified rutin (quercetin-3-Orutinoside) among the natural and bioactive compounds with pharmacological properties that can fill this role. 131][22] In the present study, we designed a poly-glucosylated rutin through biocatalysis by adding to its backbone a set of natural sugars in order to improve its water solubility by more than 14 000 times, offering the possibility of use at higher concentrations.The biological activity of poly-glucosylated rutin was then assessed with in vitro and ex vivo tests and a clinical trial of its effect on the main disorders identified in mask-wearing related disorders, offering a cosmetic solution for their resolution.

| Active ingredient preparation
The soluble form of rutin was obtained via alpha glucosylation by cyclodextrin glucanotransferase.The reaction was conducted in water with 3.0 L Toruzyme® (1.3 mL.L −1 , from Novozymes), rutin (26 g.L −1 , from Naturex) and alpha-cyclodextrin (132 g.L −1 , from Sigma-Aldrich), q.s. 5 L sodium acetate buffer (pH 5.7), for 20 h.The reaction medium was purified on adsorbent resin to retrieve rutin alpha glucosides.The product was concentrated in water and its composition was determined by HPLC on column Kinetex C8 2.6 μm 100 × 4.6 mm (Phenomenex).Solvent A was water at 0.1% acetic acid, and solvent B was methanol at 0.1% acetic acid.Flow rate was 1 mL.min −1 .Gradient was as follows: 0 min-20% B; 2 min-20% B; HPLC quantification of rutin glucosides was conducted during a stability study of 1 year in water at room temperature (20°C).Color stability was evaluated by measurement of color on Gardner scale with Lovibond comparator 2000+ (Tintometer GmbH).Solubility of the polyglucosyl rutin was also evaluated in water.

| DPPH assay for measurement of direct antioxidant activity
DPPH solution was prepared in absolute ethanol at 0.0545 mg/mL.Gallic acid was used as positive control with concentration ranging from 1 to 7.52 μg/mL prepared in absolute ethanol.Samples of poly-glucosylated rutin and rutin were prepared in water from 10 to 80 μM.Four dilutions of the samples were tested in order to deduce the IC50.Each dilution of the compounds, each blank and each positive control were deposited in triplicate in a 96 wellsmicroplate (VWR®, sterilized and non-treated) with DPPH solution (diluted in 1:2).The plate was incubated with a lid for 30 min at room ingredient application, indicating inflammation and erythema reduction.Volunteers reported improved skin comfort.

Conclusion:
In summary, the COVID-19 pandemic led to various skin issues associated with mask-wearing.A highly soluble form of rutin was developed, which effectively addressed these concerns by reducing inflammation, oxidative stress, and hyperpigmentation while promoting wound healing.This soluble rutin offers a promising solution for the rapid treatment of maskne-related disorders and other skin problems caused by prolonged mask use.

| Measurement of intracellular ROS scavenging activity in primary keratinocytes
Normal human primary epidermal keratinocytes (NHEKs) were seeded in a black plate with a glass bottom at 20000 cells/well in 96wells plates with a type I collagen pre-coating (54 907 100, Roche), in quadruplicate.The cells were incubated for 24 h in Epilife medium (MEPI500CA, Gibco™, Thermo Fisher Scientific, Carlsbad, CA, USA,) supplemented with HKGS factors (S0015, Gibco™, Thermo Fisher Scientific) and 1% antibiotics (Sigma-Aldrich), at 37°C with 5% CO 2 for 24 h.After culturing, the cells were treated in complete medium with the positive reference, with either resveratrol (D6883, Sigma-Aldrich, St. Louis, MO, USA) at 200 μM, or the active ingredient at 0.1% (v/v) or 0.5% (v/v).Skin cells, untreated and cultivated with complete medium, were used as a negative control.Cells were then incubated for 24 h at 37°C, 5% CO 2 .After incubation, the 2′,7′-dichlorofluorescin diacetate (DCFH-DA, Sigma-Aldrich, ENA490047502) (50 μM) probe was added to the wells for at least 30-45 min at 37°C.Cells were then washed twice with PBS buffer (14190-0144, Gibco™, Thermo Fisher Scientific) and an oxidative stress was induced with tert-butyl hydroperoxide solution (TBP) at 5 mM (458 139, Sigma-Aldrich) in PBS buffer.The untreated cells remained in PBS buffer.Finally, after 1 h of incubation, the emitted fluorescence was measured in darkness by an excitation wavelength of 488 nm and an emission wavelength of 525 nm with the microplate reader (Spark®, TECAN, Switzerland).

| Wound healing activity assessment in primary keratinocytes
NHEKs were seeded in a type I collagen pre-coated (Roche) 12-well-plate at 200000 cells/well in triplicate.
After 48 h of incubation in Epilife medium supplemented with HKGS medium (Gibco™) and 1% of antibiotics (Sigma-Aldrich) at 37°C with 5% CO 2 , cells were rinsed twice with PBS buffer (Gibco™) and incubated for 8 h in basal medium (Gibco™).After incubation, the cells were treated overnight at 37°C with 5% CO 2 either with the active ingredient at 0.5% (v/v) or the positive reference, HB-EGF (E4643, Sigma-Aldrich), at 1 ng.mL −1 .Untreated cells that were cultivated with medium were used as a negative control.The next day, a scratch was inflicted on the cell layer, and cells were rinsed twice with PBS buffer in order to eliminate floating cells.The cells were again treated with the previous conditions.Photographs were taken immediately (×10 magnification, Axiovert microscope, Zeiss) and after 24 h of incubation at 37°C with 5% CO 2 .Wound closure was then assessed with ImageJ software (NIH, USA) to calculate wound-healing activity.

| Anti-inflammatory-activity assay in human skin tissue
Human skin explants were topically pre-treated with the active ingredient diluted in distilled water at 1% (v/v) for 24 h.The untreated explants were treated only with vehicle (distilled water).After pretreatment, an inflammatory stress was applied by topical application of Phorbol 12-myristate 13-acetate (PMA) solution at 75 mg.mL−1 .
The active ingredient was then re-applied for 48 h.The culture medium was renewed every other day.After culturing, the skin explants were sampled and frozen at −80°C.The skin samples were then lysed and homogenized with a Precellys apparatus and CKMIX tubes containing specific beads and MeOH-BHT solution.Samples were extracted using an Oasis HLB 96-well solid phase extraction system (Waters Corporation, Milford, MA, USA).The LC-MS/MS analysis was performed on a UHPLC system (EXION LC AD, SCIEX) coupled to a QTrap 6500+ MS (SCIEX) equipped with electrospray ionization operating in negative mode.The experiment was conducted in triplicate.

| Evaluation of lightening activity in human skin tissue
Human skin explants with a dark skin phototype were topically treated for 7 days either with the active ingredient diluted in distilled water at 1% (v/v), or with the positive reference, kojic acid (K3125, Sigma-Aldrich) at 2% (v/v) diluted in distilled water.The untreated explants were treated only with the vehicle (distilled water).The medium was renewed every other day.After culturing, the skin explants were sampled and put in formalin before being dehydrated and embedded in paraffin.Sections (4μm thick) were dewaxed and then stained using the Fontana-Masson silver standard method for melanin detection.Twice daily, volunteers applied a cream containing 1% active ingredient, or placebo, on hemi face for 4 days.
Skin reactivity was analyzed by measured red spots after 1 day of application; hyperpigmentation was evaluated by analyzing the brown spots after 4 days.Volunteers were asked their own perception of the efficacy via a self-assessment questionnaire.

| Red-spot analysis
Digital photographs of the face were obtained at different times with a Visia® CR 2.3 skin analysis visualizing system (Canfield Scientific, Parsippany, NJ, USA).The control of repositioning took place directly on the data-processing screen using an overlay visualization of the images at each time of acquisition.The Visia® CR 2.3 allows for taking photographs under different types of illumination and at a very rapid capture of images.A series of photographs was taken under multi-spectral imaging and analysis allowing for the capture of visual information affecting the appearance of the skin.The photographs were taken of each hemi face.
The analyses were performed using Maestro® software on RBX photographs for red-spot measurements.

| Brown-spot analysis
Digital photographs of the face were done at different times with the same Visia® CR 2.3 system mentioned above.The control of repositioning takes place directly on the data-processing screen using an overlay visualization of the images at each acquisition.A series of photographs was taken under multi-spectral imaging and analysis to allow capturing visual information affecting appearance of the skin.
The photographs were taken of each hemi face.
The analyses were performed using Maestro® software on standardized photographs for brown-spot measurement.

| Statistical analysis method
For in vitro and ex vivo studies, the results are presented as mean ± standard error of mean (SEM) of three independent triplicates.
In vivo results are expressed in percent of variation relative to D0.
A Shapiro-Wilk test was used to determine Normality following Gaussian Law.In case of Normally-distributed data, the mean values were compared using either an unpaired t test (≤2 groups) or One-way ANOVA followed by post-hoc test (≥2 groups).In case of non-Normally-distributed data, a Kruskal-Wallis test followed by a Mann-Whitney U test was used for unpaired data.

| DPPH assay comparing poly-glucosylated rutin and rutin
The IC50 of rutin and the poly-glucosylated rutin obtained were similar (Table 2), evidencing that the enzymatic alpha-1,4-glucosylation of rutin preserved its antioxidant activity in tubo.

| Antioxidant property
Acne is characterized by a high level of oxidative stress in cells, so we first evaluated the antioxidant property of the active ingredient to demonstrate its ability to fight this first mask-wearing related disorder.NHEKs were challenged with Tert-butyl peroxide to induce an oxidative stress by production of reactive oxygen species (ROS).The model was validated with a positive control, resveratrol at 200 nM, which showed a significant reduction of ROS production of 77%.
In the presence of the active ingredient, at the highest concentration (0.3%), production of ROS was significantly lower than in the stressed control.The ROS production was reduced by −23% and −29% at 0.1% and 0.5% active ingredient, respectively (Figure 2).

| Wound healing activity
Poor wound healing is described in acne and mask-wearing related disorders, so the active ingredient was tested for its effect on this particular biological process.A physical scratch was induced on the cell layer of the NHEKs in order to trigger the wound-healing properties of keratinocytes.After 24 h of incubation, the positive reference demonstrated a significant stimulation of wound closure.In the same condition, the active ingredient showed a similar effect, stimulating the closure significantly by 113% over the untreated condition (Figure 3).Therefore, we concluded that the active ingredient stimulated wound healing.

| Analysis of skin redness reduction
The 3 biological functions analyzed above can be evaluated clinically by the measurement of red spot, characterizing the skin redness.A panel of volunteers wearing face masks topically applied a formula containing or not the active ingredient at 1%.After 1 day of application, skin redness was measured by analyzing the red spot from images taken by Visia® CR 2.3.As observed by the counting of red spots, we found a significant reduction with the active ingredient by 23.1% (relative to Day 0).This variation was significantly different from the placebo-treated area (Figure 5).These results are accompanied by representative images showing the red-spot-counting mask (Figure 6), and images taken with cross-polarized light showing vascularization of the skin of the volunteers (Figure 7).The results show a clear reduction of the inflammation that had been triggered by face mask-wearing, with the active ingredient, thereby validating the results observed in the pre-clinical studies.
Efficacy perceived by the volunteers were assessed by selfassessment questionnaire.The volunteers perceived in a higher manner that their active-treated skin area was less tight and softer, giving a more hydrated feeling but more importantly, the skin was significantly more comfortable in the presence of the active ingredient (p < 0.05).

| Lightening activity ex vivo
In this study, we wanted to evaluate the lightening activity of our active since hyperpigmentation has been pointed as a consequence of mask-wearing.For this purpose, dark skin tissues were topically treated with the active at 1% for 7 days and its activity was compared to a well-known positive reference, the kojic acid.The melanin index was obtained from the analysis of Fontana-Masson staining that was performed on the sectioned dark skin tissues.The analysis showed that the positive reference, at 2%, significantly reduced the melanin index by 14%, validating the responsiveness of the skin.The active ingredient produced a significant reduction, by 37% of the melanin index, indicating lightening activity (Figure 9 and Figure 10).The effect was even higher than the one observed with the positive reference treatment.

| Lightening activity in vivo
In order to confirm the observation made in the ex vivo test, the brown spots were measured in the same panel of volunteers wearing face masks before and after having topically applied, for 4 days, a formula either containing or not containing the active ingredient at 1%.After this period, the brown spots were counted using image analysis.The results revealed a significant reduction of brown spots on the masked area, with the active ingredient, by −2.4% (Day 4 vs Day 0), which was significantly more than the placebo-treated area (Figure 11).Representative images, with the brown-spot counting mask, are presented in Figure 11.The topical application of the active ingredient reduced the presence and limited the appearance of brown spots that had been triggered by face mask-wearing.

| DISCUSS ION
Wearing face masks is the new routine when hygienic and safety measures are required.Nevertheless, since the pandemic began, various skin concerns have been raised and have been correlated 6][7][8][9][10] It was important to find a cosmetic solution rapidly; we choose a powerful and well-known molecule, having a wide range of biological activities: rutin. 14,17Nevertheless, rutin is poorly water-soluble so we designed a highly water-soluble and stable form of rutin to allow an easier integration into cosmetic formulas [20][21][22] (Table 1).
The first step was to confirm that the alpha-1,4-glucosylation of rutin had no impact on the antioxidant activity of the new version of rutin.The DPPH assay demonstrated that rutin and the polyglucosylated rutin had a similar IC50, proving that the activity was preserved (Table 2).
We assessed the biological activity of this highly water-soluble form of rutin through in vitro, ex vivo tests, targeting the main skin concerns triggered by face-mask wearing.Maskne, or mask-related acne, was the major skin concern reported among various countries and populations, so we designed three in vitro tests to examine a beneficial effect for acne management.4][25] The antioxidant activity of the active ingredient was evaluated in primary keratinocytes that were challenged with a pro-oxidative molecule triggering ROS production.In these conditions, the data demonstrated that the active ingredient significantly reduced the production of ROS to 29% (Figure 2).
Since acne can lead to scarring, 25 we evaluated the woundhealing activity of the ingredient on primary keratinocytes using a scratch assay.Wound closure was then quantified using image analysis.The application of the active ingredient led to a significant stimulation of wound-healing activity relative to the untreated Representative pictures of volunteer skin vascularization using cross-polarized light (RBX quality), obtained with the Visia® CR 2.3 skin analysis visualizing system.

F I G U R E 8
Answers of self-assessment questionnaire, expressed in % of total volunteers.A chi-square contingency test was used to determine the difference between the two groups.condition, with a similar benefit to that of the positive reference, hB-EGF (Figure 3).Finally, the anti-inflammatory activity was assessed in a skin explant model.The quantification of eicosanoids (lipid-mediator molecules) was conducted because the biological activities of rutin were already known regarding the arachidonic acid cascade. 17,19,26,27Skin explants were challenged with PMA in order to induce inflammation, and then they were topically treated with the active ingredient.LC-MS/MS quantification revealed a significant reduction of this lipid mediator, indicating a reduction of skin inflammation (Figure 4).
The active ingredient demonstrated its ability to manage various consequences of mask-wearing: oxidative stress, inflammation, and defective wound healing.These biological effects were then confirmed in the clinical trial by analyzing skin redness and skin vascularization (by using cross-polarized light).In the clinical study, the volunteers were required to wear surgical mask for at least 2 h.The active at 1% was topically applied for 1 day and then measurements were made using Visia CR 2.3.The red-spot number was increased in the masked area of placebo-treated subjects, whereas it was significantly decreased in the active ingredient-treated area (comparison of Day 1 to Day 0).This effect was significantly different in comparison with placebo (Figure 5).Moreover, as observed in Figure 6, the skin vascularization was drastically reduced on Day 1 (in comparison with Day 0), indicating a clear reduction of inflammation and erythema by the highly water-soluble form rutin. 28 The volunteers perceived a fast improvement with a significant increase of skin comfort (Figure 8).
Hyperpigmentation brought on by mask-wearing is another major skin concern. 1,2,6,7,9We wanted to find a solution for this problem so we evaluated the lightening activity of the active ingredient in dark skin explants.Image analysis revealed that the active ingredient significantly reduced the melanin index (melanin content) by 37% compared to the untreated condition, indicating a lightening activity of the active ingredient (Figure 9 and Figure 10).This activity was also evaluated in the clinical study by quantifying brown spots.A significant reduction was observed after 4 days of application in the activetreated area (compared to Day 0) and it was significantly lower than in the placebo-treated area (Figure 11).This rapid effect could be explained by the other biological activities of the ingredient (antioxidant and anti-inflammatory), since melanogenesis is intrinsically related to ROS production and to a post-inflammation state. 1,29,30

| CON CLUS ION
The COVID-19 pandemic established a turning point in the dermocosmetic industry with the rise of new skin disorders.Indeed, wearing personal protection equipment such as surgical masks triggered the appearance of maskne (mask-related acne), rosacea flare, skin-barrier defect, itching, erythema, redness, hyperpigmentation, poor wound healing and lichenification (Figure 12).

K
E Y W O R D S inflammation, mask-wearing disorders, pigmentation, rutin, skin repair temperature in the dark.The absorbance at 540 nm was read after removing the lid.Percentage of DPPH inhibition was evaluated by calculation using the formula: [1 − (DO540 of sample − DO540 of blank sample)]/ DO540 blank DPPH.The graph representing the inhibition percentage was drawn as a function of the different concentrations of the sample tested, allowing the determination of a linear equation and the calculation of 50% DPPH inhibition called IC50.At least three points around the IC50 are required to trace the trend curve and calculate the linear equation.

Four
Photomicrographs (jpeg format) of Fontana-Masson tissue sections were opened in the GIMP-GNU open-source software program.The brown-to-black color signals corresponding to melanin pigment amount were selected, copied, and pasted into a new image on a white background.Images were saved in jpeg format.These images were subsequently opened in the ImageJ open-source software program.The image was then inverted and the mean intensity was measured.The experiment was conducted in triplicate.

2. 7 |
Subjects for clinical evaluationThe clinical study was carried out in a double-blind and placebocontrolled procedure on one group of 19 volunteers aged over 18 years old, mean age = 39.5 years, SD = 9 years.Volunteers were recruited according to inclusion criteria, which were: (a) having sensitive and reactive skin to the use of surgical masks; and (b) wearing masks every day in their daily life.The subjects were requested to wear the surgical masks for at least 2 h before coming to the clinical laboratory at each visit.All the subjects participating in the study gave informed consent signed at the beginning of the study.The study followed, and was in compliance with, the ethics tenets of the Declaration of Helsinki.
of the polyglucosyl rutin was also stable along the stability study at 7.5 on the Gardner scale.Water solubility of polyglucosyl rutin was evaluated at 1830 g.L −1 of water; meaning it is 14 000

F I G U R E 5 6
Analysis of red-spot count on masked area after 1 day of cream application containing the active ingredient at 1%, or no active ingredient.Results are expressed in percentage of variation between Day 0 and Day 1 measurements.Statistical analyses used were the Wilcoxon test for paired conditions and the Mann-Whitney U test for unpaired conditions: # p < 0.1; *p < 0.05.Representative photographs of volunteers and the area measured for red spots, obtained with the Visia® CR 2.3 skin analysis visualizing system.

F I G U R E 9
As the clinical reports were published in number around the world, it became crucial to find a quick and efficient solution for consumers.Rutin is a well-known flavonoid widely recognized for its numerous benefits but its poor water solubility made its formulation a difficult task at high concentration.In order to facilitate its formulation for most different galenics, we designed a highly soluble form of rutin.This active ingredient demonstrated the capacity to reduce Quantification of melanin index based on analysis of Fontana-Masson staining of skin explants treated with or without the active ingredient.Results are expressed in percentage of untreated condition ±SEM.Statistical analysis with Mann-Whitney test: # p < 0.1; **p < 0.01; ***p < 0.001.Representative images of skin sections with Fontana-Masson staining.F I G U R E 11 Analysis of brown-spot count by Visia® CR 2.3 measurement on the face-masked area after 4 days of cream application that either contained the active ingredient at 1%, or not.Results are expressed in percentage of variation between Day 0 and Day 4 measurements.Statistical analysis with Student-t test for paired conditions and unpaired conditions: *p < 0.05.