In Vitro Antibacterial and Anti-Inflammatory Activity of Arctostaphylos uva-ursi Leaf Extract against Cutibacterium acnes

Cutibacterium acnes (C. acnes) is the main causative agent of acne vulgaris. The study aims to evaluate the antimicrobial activity of a natural product, Arctostaphylos uva-ursi leaf extract, against C. acnes. Preliminary chemical–physical characterization of the extract was carried out by means of FT-IR, TGA and XPS analyses. Skin permeation kinetics of the extract conveyed by a toning lotion was studied in vitro by Franz diffusion cell, monitoring the permeated arbutin (as the target component of the extract) and the total phenols by HPLC and UV-visible spectrophotometry, respectively. Antimicrobial activity and time-killing assays were performed to evaluate the effects of Arctostaphylos uva-ursi leaf extract against planktonic C. acnes. The influence of different Arctostaphylos uva-ursi leaf extract concentrations on the biofilm biomass inhibition and degradation was evaluated by the crystal violet (CV) method. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test was used to determine the viability of immortalized human keratinocytes (HaCaT) after exposure to Arctostaphylos uva-ursi leaf extract for 24 and 48 h. Levels of interleukin (IL)-1β, IL-6, IL-8 and tumour necrosis factor (TNF)-α were quantified after HaCaT cells cotreatment with Arctostaphylos uva-ursi leaf extract and heat-killed C. acnes. The minimum inhibitory concentration (MIC) which exerted a bacteriostatic action on 90% of planktonic C. acnes (MIC90) was 0.6 mg/mL. Furthermore, MIC and sub-MIC concentrations influenced the biofilm formation phases, recording a percentage of inhibition that exceeded 50 and 40% at 0.6 and 0.3 mg/mL. Arctostaphylos uva-ursi leaf extract disrupted biofilm biomass of 57 and 45% at the same concentrations mentioned above. Active Arctostaphylos uva-ursi leaf extract doses did not affect the viability of HaCaT cells. On the other hand, at 1.25 and 0.6 mg/mL, complete inhibition of the secretion of pro-inflammatory cytokines was recorded. Taken together, these results indicate that Arctostaphylos uva-ursi leaf extract could represent a natural product to counter the virulence of C. acnes, representing a new alternative therapeutic option for the treatment of acne vulgaris.


Introduction
Acne vulgaris is a multifactorial chronic inflammatory dermatosis of the pilosebaceous follicles, mainly manifested in adolescents [1]. Although it is not a life-threatening disease, it has a great social and psychological impact on the patient's life [2]. Furthermore, it is estimated to be the eighth most common disease in the world, with 650 million people of antibacterial activity associated with a powerful anti-inflammatory action suggests the use of Arctostaphylos uva-ursi could as a promising source for the treatment of acne vulgaris.

FT-IR/ATR Analysis
FT-IR/ATR analysis was performed through a Spectrum Two PE instrument supplied by PerkinElmer, endowed with a universal ATR accessory (UATR, Single Reflection Diamond/ZnSe). For each sample, FT-IR/ATR spectra were recorded from 400 to 4000 cm −1 with a 4 cm −1 resolution.

Thermal Analysis
Thermogravimetric analysis (TGA) of Arctostaphylos uva-ursi extract was carried out using the PerkinElmer TGA-400 analyzer (PerkinElmer Inc., Waltham, MA, USA). Samples of approximately 5-10 mg were placed in a ceramic crucible and heated under N 2 atmosphere from 30 to 800 • C, with a constant flow rate of 20 • C/min and at a gas flow of 20 mL/min. Thermograms (TG) and their respective derivative curves (DTG) were recorded and analyzed by TGA Pyris series software (version 13.3.1.0014, PerkinElmer Inc., Waltham, MA, USA).

XPS Characterization
XPS characterization was carried out using a scanning microprobe PHI 5000 Ver-saProbe II, purchased from Physical Electronics (Chanhassen, MN, USA), equipped with a micro-focused monochromatized AlKα X-ray source. Specimens were analyzed in HP mode using an X-ray take-off angle of 45 • . The instrument base pressure was set at about 10 −9 mbar. Scanned areas of 1400 × 200 µm were acquired. Pass energy values equal to 117.4 and 29.35 eV were recorded in the FAT mode for survey scans and high-resolution spectra, respectively. For high-resolution spectra curve fitting, MultiPak software (version 9.9.0.8, Physical Electronics, Chanhassen, MN, USA) was used. The carbon C1s correction was performed by setting as reference charge (284.8 eV) the adventitious carbon.

Total Polyphenol Content (TPC) Determination
TPC present in the Arctostaphylos uva-ursi extract was determined in triplicate by the Folin-Ciocalteu spectrophotometric method, described by Fabiano and coworkers, with some modifications [33]. Briefly, for the calibration curve, gallic acid (GA) stock solution at a concentration 5 g/L was prepared by dissolving 0.5 g of GA in 10 mL ethanol and then bringing to 100 mL with distilled water. Successively, dilutions of this stock solution were performed to obtain different concentrations of GA (i.e., 100, 150, 250, 500 mg/L). A 100 µL aliquot of each GA solution was added to 7.9 mL of H 2 O and 500 µL of Folin-Ciocalteu reagent. The solution was left for 8 min before being added with 1.5 mL of sodium carbonate solution (1.9 M). The resulting absorbance was measured spectrophotometrically using a UV-visible Spectrophotometer UV-1900i (Shimadzu, Kyoto, Japan) against the blank at 765 nm. The TPC was calculated by referring to the calibration curve and expressed as mg of gallic acid equivalent per gram of dry extract (GAE/g).

Preparation of a Toning Lotion Loaded with Arctostaphylos uva-ursi Leaf Extract
Toning lotions are water-based formulations usually containing water-soluble active ingredients, used to remove impurities (pollutants, makeup residues, sebum, etc.), refresh the skin and balance the natural pH level. When precious botanical extracts are contained in toning lotions, their action is mainly linked to the extract beneficial properties (astringent, moisturizing, antiaging action, etc.). In this study, an alcohol-free toning lotion formulation was produced, employing a simple recipe based on the solubilization of Arctostaphylos uva-ursi leaf extract in distilled water in a non-cytotoxic and antibacterial effective dose (i.e., 1.2 mg/mL). Finally, an eco-certified preservative (i.e., a mixture of benzyl alcohol and dehydroacetic acid, supplied by Zenstore, Salerno, Italy) was added to the lotion at 0.6% w/w.

HPLC Analysis
HPLC (Prominence Series 20 with SPD-M20A PDA detector, Shimadzu, Milan, Italy) analysis of Arctostaphylos uva-ursi extract was carried out using arbutin as the reference compound, as previously described [34]. Samples were tested in triplicate, and results were reported as mean ± standard deviation. The analytical column used was a Shim-Pack GIST C18-AQ column (150 mm × 4.6 mm, 5 µm Shimadzu) with a mobile phase consisting of solvent A (85%water) and B (15% methanol), with a flow rate of 1 mL/min, using an isocratic mode elution at 30 • C and monitoring the effluent at 282 nm. The samples injection was made through a 20 µL injection loop. The calibration curve was built up by weighing appropriate amounts of the reference compound dissolved in a mobile phase to obtain the four desired concentrations (i.e., 1, 5, 25, 100 µg/mL). Finally, results were elaborated by using the LabSolutions Lite for LC-PDA software (version 5.82, Shimadzu Corporation, Milan, Italy).

In Vitro Skin Permeation Studies
In vitro skin permeation experiments of Arctostaphylos uva-ursi leaf extract were carried out using a jacketed Franz diffusion cell (PermeGear Inc., SES GmbH, Bechenheim, Germany), as reported in a previous study [35]. In detail, 1 mL of toning solution containing the extract (1.2 mg/mL) was placed in the cell donor compartment in contact with the synthetic StratM ® membrane (Merck KGaA, Darmstadt, Germany). This membrane was chosen since it possessed a skin-like porosity, diffusivity and composition. An O-ring joint was put between the donor cell and the membrane, and the whole assembly was fixed with a stainless-steel clamp to guarantee the tight connection between the two compartments. The receptor compartment was filled with 5 mL of PBS (pH 7.4) and kept under continuous agitation by an ATE magnetic stirrer (VELP Scientifica Srl, Usmate, Italy). The temperature was fixed at 32.00 ± 0.03 • C using a CD-B5 heating circulator bath (Julabo GmbH, Seelbach, Germany). PBS aliquots of 500 µL were withdrawn after 2, 6 and 24 h, replaced with fresh PBS, and their arbutin content was assessed by HPLC (Prominence Series 20 with SPD-M20A PDA detector, Shimadzu, Milan, Italy), as described in the HPLC Analysis Section. Furthermore, at the end of the experiments, the Strat-M ® membrane was immersed in the HPLC mobile phase overnight at 25 • C in order to estimate the arbutin engaged in the membrane. At the same time points, total polyphenols permeated and retained by the membrane were also assayed, following the above-reported TPC procedure.

Kirby-Bauer Disk Diffusion Test
An initial screening to assess the Arctostaphylos uva-ursi leaf extract antimicrobial susceptibility against C. acnes ATCC 11827 was performed by the Kirby-Bauer disc diffusion test according to the guidelines of the National Committee on Clinical Laboratory Standards (NCCLS). In detail, a fresh colony was inoculated in a physiological solution up to a turbidity of 1 McFarland. The bacterial suspension was uniformly seeded with a sterile swab on FAA-agar. A blank disc placed on the FAA-agar plate was soaked in 1 mg of extract (UV), while a blank disc soaked in H 2 O was used as solvent control (S). Benzylpenicillin (1 µg) (Thermo Fisher Scientific, Waltham, MA, USA) was used as antibiotic control (PG). The plates were incubated at 37 • C for 48 h in an anaerobic environment. After incubation, the diameters of the areas of inhibition were measured.

Determination of the Minimum Inhibitory Concentration (MIC)
The Arctostaphylos uva-ursi leaf extract antibacterial activity was evaluated through the plate microdilution test according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). Briefly, a colony of C. acnes grown on Schaedler agar plate was inoculated in Schaedler broth and incubated in an anaerobic environment at 37 • C overnight (ON). Then, the inoculum was diluted in 15 mL of fresh medium and incubated until the optical density (OD) at 600 nm of 0.5. The bacterial suspension was diluted to 5 × 10 7 CFU/mL, and 100 µL was added to each well, obtaining a final density of 2.5 × 10 7 CFU/well. Meanwhile, Arctostaphylos uva-ursi leaf extract was serially diluted in PBS 1X in a concentration range from 10 to 0.07 mg/mL. Vancomycin (10 µg/mL) was used as an antibiotic control (CTRL +) and H 2 O as a solvent control (CTRL −) Subsequently, the plates were incubated at 37 • C in anaerobiosis, and the growth rate was evaluated after 48 h using a microplate reader (Tecan, Männedorf, Switzerland).

Time-Killing Assay
To better understand the Arctostaphylos uva-ursi leaf extract kinetics of action in C. acnes planktonic cells, the time killing test was performed in accordance with the American Society for Testing and Materials International (ASTM) standard guidelines. Concentrations of 1.25 (2 × MIC), 0.6 (MIC) and 0.3 ( 1 2 × MIC) mg/mL, and 2.5 × 10 7 CFU/mL of bacteria was added to the Schaedler broth in a final volume of 2 mL/tube. Vancomycin (10 µg/mL) and bacteria treated with the solvent used to dissolve the extract (H 2 O) were used as CTRL + and CTRL −, respectively. The bacterial suspension was incubated at 37 • C under orbital shaking (180 rpm) in an anaerobic environment. After the incubation period (0, 3, 6, 9 and 20 h), a 100 µL aliquot of bacterial suspension was serially diluted and plated on Schaedler-agar plates. The plates were incubated at 37 • C for 48 h in anaerobiosis, and the colonies were counted to define CFU/mL [36].

LIVE/DEAD Staining
Bacteria treated with various concentrations of Arctostaphylos uva-ursi leaf extract as described in Section 2.3 were examined for cell viability using a BacLight LIVE/DEAD staining kit (Invitrogen, Carlsbad, CA, USA). According to the manufacturer's instructions, equal volumes of SYTO ® 9 and propidium iodide (PI) were mixed and added to each well of the 96-well plate. The samples were incubated for 15 min in the dark at room temperature and observed under a fluorescence microscope. Images were acquired through the Nikon ECLIPSE Ti2-U (Nikon Europe B.V., Amsterdam, The Netherlands) inverted fluorescence microscope with beam settings for FITC, TRITC and merged.

Biofilm Inhibition and Degradation
The ability of Arctostaphylos uva-ursi leaf extract to affect biofilm formation and degrade the mature matrix was evaluated using the crystal violet (CV) assay. Briefly, a bacterial inoculum was diluted in a Schaedler broth supplemented with 1% glucose at a final density of 5 × 10 8 CFU/mL. Regarding biofilm inhibition, a volume of 100 µL of bacterial suspension was transferred to each well of a 96-well plate, and 100 µL of fresh medium supplemented with Arctostaphylos uva-ursi leaf extract (10-0.07 mg/mL) was added. The suspension was incubated at 37 • C for 72 h in an anaerobic environment and under static conditions. For the mature biofilm degradation, the bacterial suspension was previously incubated at 37 • C for 72 h in an anaerobic environment and under static conditions. Then, non-adherent cells were removed through two PBS1X washes, and Arctostaphylos uva-ursi leaf extract (10-0.07 mg/mL) was used to treat the mature biofilm for 24 h. Biofilms treated with solvent (H 2 O) and treated with vancomycin (20 µg/mL) constituted the CTRL − and CTRL +, respectively. After treatment, residual planktonic cells were removed, and the biofilm was washed twice with PBS1X. Biofilm biomass was quantified by adding 100 µL of 0.01% CV to each well for 30 min at room temperature under orbital shaking. The excess CV was removed and then solubilized with 98% ethanol for 40 min at room temperature under orbital shaking. The absorbance values at 570 nm were obtained using a microplate reader (Tecan, Männedorf, Swiss) [37].

Cytotoxicity and Hemolysis Profile
Cultured Human Keratinocyte (HaCaT) cells were used to evaluate the cytotoxicity of Arctostaphylos uva-ursi leaf extract by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [38,39]. Cells were cultured in DMEM (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA), at 37 • C with 5% CO 2 in a humid environment. A density of 2 × 10 4 cells/well was seeded into 96-well plates and incubated for 24 h. Arctostaphylos uva-ursi leaf extract was tested in a concentration range between 10 and 0.07 mg/mL. Cells treated with solvent (H 2 O) and cells treated with 100% DMSO represented CTRL − and CTRL +. After 24 and 48 h of treatment, 100 µL of MTT solution was added to each well for 3 h at 37 • C. Then, the formazan crystals were solubilized by adding 100 µL of 100% DMSO, and the viability rate was recorded at OD at 570 nm, using a microplate reader (Tecan, Männedorf, Switzerland). The Arctostaphylos uva-ursi leaf extract haemolytic activity was determined using fresh human erythrocytes from healthy donors [40]. Briefly, 25 mL of blood was centrifuged, and the erythrocytes were washed three times with a NaCl solution (150 mM). Then, the blood was diluted at 1:50 in PBS 1X pH 7.4, and 180 µL of erythrocyte solution was added to the well of a conical bottom 96-well. Arctostaphylos uva-ursi leaf extract was added at the same concentrations mentioned above; solvent (H 2 O) and TritonX 0.1% solution represented CTRL − and CTRL +. The samples were incubated under orbital shaking at 37 • C for 60 min and subsequently centrifuged at 500 rpm for 5 min. Haemoglobin release was monitored by measuring the OD of the supernatant at 540 nm.
The selectivity index (SI) of the Arctostaphylos uva-ursi was calculated through the ratio between CC 50 and MIC 50 . Values greater than 3 render the compound rising for in vivo investigation.

Enzyme-linked Immunosorbent Assay (ELISA)
To evaluate the Arctostaphylos uva-ursi leaf extract anti-inflammatory proprieties, C. acnes fresh colonies were suspended in 5 mL of PBS1X at the final concentration of 5 × 10 8 CFU/mL. The bacteria suspension was incubated at 80 • C for 30 min, and the heat-killed C. acnes was stored at 4 • C until use. Meanwhile, 3 × 10 5 HaCaT cells were seeded in a 12-well and incubated for 24 h. The following day, cells were stimulated by heat-killed C. acnes and (1.2 × 10 7 CFU/mL, MOI = 20) and 1.25 (2 × MIC), 0.6 (1 × MIC) and 0.3 ( 1 2 × MIC) mg/mL of Arctostaphylos uva-ursi leaf extract were added. LPS (10 µg/mL) was used as CTRL+, while untreated cells and cells treated with heat-killed C. acnes, as an anti-inflammatory (cell) and pro-inflammatory (bact) control. After 24 h, the supernatants of the culture medium were collected for the quantification of the pro-inflammatory cytokine. Levels of IL-8, IL-6, IL-1β and TNF-α were analyzed using a sandwich enzyme-linked immunosorbent assay kit (ELISA) according to the manufacturer's instructions (Elabscience, Houston, TX, USA). The absorbance was quantified at 490 nm using a microplate spectrophotometer (Tecan, Männedorf, Switzerland).

Antioxidant Activity
The free radical scavenging activity was evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH ·) assay [41]. Briefly, Arctostaphylos uva-ursi leaf extract was serially diluted in a concentration range of 10-0-0.005 mg/mL and mixed with 50 µM of DPPH in methanol. Ascorbic acid (5 mg/mL) and methanol were used as CTRL + and CTRL −. The antioxidant activity was evaluated by reading the absorbance at 517 nm for 15 min and the percentage of Radical Scavenging Activity (RSA) was calculated according to the formula:

Statistical Analysis
All experiments were performed in triplicate. The data from each experiment represent the mean (SD) of three biological replicates. The ordinary one-way ANOVA, Dunnett's multiple comparison test and cytotoxic concentration 50% (CC 50 ) were performed using GraphPad Prism ver. 8.4.0 for macOS (Software GraphPad, San Diego, CA, USA, www. graphpad.com (accessed on 3 March 2020). Values were considered significant with a p-value < 0.05.

Chemical-Physical Characterization of Arctostaphylos uva-ursi Leaf Extract
In Figure 1A, the FTIR spectra of the Arctostaphylos uva-ursi leaf extract and its main component (arbutin) are reported. The peaks at 1509, 1210, 1127 and 830 cm-1 (see asterisks in Figure 1A) can be correlated to the presence of arbutin, containing an aromatic ring. The other peaks could be ascribable to the carbohydrate fraction present in the extract. In Figure 1B, the thermogram (TG) and the derivative thermogram (DTG) traces of the extract and its target molecule, i.e., arbutin, recorded in a nitrogen atmosphere, were reported. Analyzing the extract, a water/volatiles content equal to 5.6%; successively can be observed. The first thermal event, with a peak temperature centered at 215 • C, within the range 160-260 • C, corresponds to a weight loss of 23.8%. Then, a second broadened thermal event was centered at 298 • C, in the range 260-475 • C (29.5% weight loss) and a third centered at 533 • C in the range 477-600 • C (13.2% weight loss). The residue at 600 • C was equal to 27.8%, denoting a high content of a highly stable and/or inorganic fraction. Pure arbutin was also analyzed, evidencing the following features: (i) no water/volatiles content was detected; (ii) two sharps but partially overlapped thermal events fell at 312 and 331 • C, thus indicating that the arbutin decomposition fell in the thermal event ranging in 260-475 • C of the extract; and (iii) a residue at 600 • C equal to 9.3%. XPS analysis of Arctostaphylos uva-ursi leaf extract revealed a surface chemical composition based on oxygen (36.2%), carbon (58.5%) and silicon (5.3%). The silicon presence could be probably responsible for the inorganic fraction detected in the extract by TGA. Moreover, in Figure 2 the C1s high-resolution spectrum curve-fitting was reported, evidencing a high C-OH content (45.8% of the total carbonaceous species), indicative of a high abundance of phenolic species in the extract, as well as carbohydrates, (hemi-)celluloses, lignin and other important components (flavonoids, tannins, etc.). Moreover, a small but clearly detectable amount of COOR groups could be related to the presence of some important organic acids, such as ursolic acid, tannic acid, gallic acid, etc. [27]. The C1s curve fitting of arbutin also evidenced a 55.6% contribution of C-OH, as previously reported [34]. The presence of   XPS analysis of Arctostaphylos uva-ursi leaf extract revealed a surface chemical composition based on oxygen (36.2%), carbon (58.5%) and silicon (5.3%). The silicon presence could be probably responsible for the inorganic fraction detected in the extract by TGA. Moreover, in Figure 2 the C1s high-resolution spectrum curve-fitting was reported, evidencing a high C-OH content (45.8% of the total carbonaceous species), indicative of a high abundance of phenolic species in the extract, as well as carbohydrates, (hemi-)celluloses, lignin and other important components (flavonoids, tannins, etc.). Moreover, a small but clearly detectable amount of COOR groups could be related to the presence of some important organic acids, such as ursolic acid, tannic acid, gallic acid, etc. [27]. The C1s curve fitting of arbutin also evidenced a 55.6% contribution of C-OH, as previously reported [34]. The presence of saccharides' structures, present both in the glycosylated hydroquinone (i.e., arbutin) and in the cellulosic moieties, were additionally evidenced by the detection of hemiacetal groups (O-C-O) at 288.0 eV.

Total Phenol Content (TPC)
The TPC evaluation for the toning lotion based on Arctostaphylos uva-ursi extract expressed as mg of gallic acid equivalent GAE/g of the dry extract was equal to 315 ± 15 GAEq/g. The obtained result indicates that the extract employed in this work possesses a total phenol content slightly higher than the Arctostaphylos uva-ursi extract TPC values  XPS analysis of Arctostaphylos uva-ursi leaf extract revealed a surface chemical composition based on oxygen (36.2%), carbon (58.5%) and silicon (5.3%). The silicon presence could be probably responsible for the inorganic fraction detected in the extract by TGA. Moreover, in Figure 2 the C1s high-resolution spectrum curve-fitting was reported, evidencing a high C-OH content (45.8% of the total carbonaceous species), indicative of a high abundance of phenolic species in the extract, as well as carbohydrates, (hemi-)celluloses, lignin and other important components (flavonoids, tannins, etc.). Moreover, a small but clearly detectable amount of COOR groups could be related to the presence of some important organic acids, such as ursolic acid, tannic acid, gallic acid, etc. [27]. The C1s curve fitting of arbutin also evidenced a 55.6% contribution of C-OH, as previously reported [34]. The presence of saccharides' structures, present both in the glycosylated hydroquinone (i.e., arbutin) and in the cellulosic moieties, were additionally evidenced by the detection of hemiacetal groups (O-C-O) at 288.0 eV.

Total Phenol Content (TPC)
The TPC evaluation for the toning lotion based on Arctostaphylos uva-ursi extract expressed as mg of gallic acid equivalent GAE/g of the dry extract was equal to 315 ± 15 GAEq/g. The obtained result indicates that the extract employed in this work possesses a total phenol content slightly higher than the Arctostaphylos uva-ursi extract TPC values

Total Phenol Content (TPC)
The TPC evaluation for the toning lotion based on Arctostaphylos uva-ursi extract expressed as mg of gallic acid equivalent GAE/g of the dry extract was equal to 315 ± 15 GAEq/g. The obtained result indicates that the extract employed in this work possesses a total phenol content slightly higher than the Arctostaphylos uva-ursi extract TPC values reported in the literature.

Franz Cell Permeation Tests
As far as the HPLC analysis, a calibration curve of arbutin standards in the concentration range of 1-100 µg/mL was built up and subsequently HPLC analysis of the extract was carried out by monitoring the arbutin peak. The chromatogram obtained for Arctostaphylos uva-ursi extract showed a peak at the retention time of 2.8 min corresponding to arbutin. The toning lotion prepared at a concentration equal to 1.2 mg/mL of the extract, was found to contain 170 ± 20 µg/mL of arbutin. As far as the permeation tests from the toning lotion containing Arctostaphylos uva-ursi leaf extract are concerned, the kinetics of arbutin and total phenols permeated in PBS up to 24 h are reported in Figure 3A,B, respectively. It is Pharmaceutics 2022, 14, 1952 9 of 17 interesting to highlight that the permeated amount at 24 h represents 29 ± 4% of the total arbutin in the toning lotion. No arbutin adsorbed on the membrane was detected. The total phenol content permeated at 24 h represents 19 ± 1% of the total amount present in the toning lotion. On the other hand, the total phenol content detected on the membrane was found to be equal to 49.7 ± 1.5 GAE/g.

Franz Cell Permeation Tests
As far as the HPLC analysis, a calibration curve of arbutin standards in the concentration range of 1-100 µ g/mL was built up and subsequently HPLC analysis of the extract was carried out by monitoring the arbutin peak. The chromatogram obtained for Arctostaphylos uva-ursi extract showed a peak at the retention time of 2.8 min corresponding to arbutin. The toning lotion prepared at a concentration equal to 1.2 mg/mL of the extract, was found to contain 170 ± 20 µ g/mL of arbutin. As far as the permeation tests from the toning lotion containing Arctostaphylos uva-ursi leaf extract are concerned, the kinetics of arbutin and total phenols permeated in PBS up to 24 h are reported in Figure 3A,B, respectively. It is interesting to highlight that the permeated amount at 24 h represents 29 ± 4% of the total arbutin in the toning lotion. No arbutin adsorbed on the membrane was detected. The total phenol content permeated at 24 h represents 19 ± 1% of the total amount present in the toning lotion. On the other hand, the total phenol content detected on the membrane was found to be equal to 49.7 ± 1.5 GAE/g.

Antibacterial Activity on Planktonic C. acnes
The antibacterial screening of Arctostaphylos uva-ursi leaf extract was evaluated by Kirby-Bauer disk diffusion and broth microdilution assays. Both methods indicated an important antibacterial effect exhibited by the Arctostaphylos uva-ursi leaf extract against C. acnes ATCC 11827 planktonic cells. The inhibition area obtained from 1 mg of the compound was 24.0 ± 1.2 mm, while no sensitivity was verified following treatment with the solvent (H2O) used to dissolve the compound (S). On the other hand, the area of inhibition obtained after treatment with 1 μg Piperacillin (PG) was 32.0 ± 0.9 mm ( Figure 4A). To define the MIC value, a dose-response curve was obtained after exposure to Arctostaphylos uva-ursi leaf extract in concentrations ranging from 10 to 0.07 mg/mL. The extract showed a performing bacterial activity, exhibiting MIC50 and MIC90 values of 0.21 and 0.6 mg/mL, respectively. At lower concentrations, a gradual reduction of the inhibitory effect was verified ( Figure 4B). The understanding of the Arctostaphylos uva-ursi leaf extract kinetic action against C. acnes was obtained by time-killing assay. The data obtained showed exponential bacterial growth over time after treatment with the solvent (CTRL −) and with 0.3 mg/mL of Arctostaphylos uva-ursi leaf extract (½ × MIC). Exposure to 1.2 (2 × MIC) and 0.6

Antibacterial Activity on Planktonic C. acnes
The antibacterial screening of Arctostaphylos uva-ursi leaf extract was evaluated by Kirby-Bauer disk diffusion and broth microdilution assays. Both methods indicated an important antibacterial effect exhibited by the Arctostaphylos uva-ursi leaf extract against C. acnes ATCC 11827 planktonic cells. The inhibition area obtained from 1 mg of the compound was 24.0 ± 1.2 mm, while no sensitivity was verified following treatment with the solvent (H 2 O) used to dissolve the compound (S). On the other hand, the area of inhibition obtained after treatment with 1 µg Piperacillin (PG) was 32.0 ± 0.9 mm ( Figure 4A). To define the MIC value, a dose-response curve was obtained after exposure to Arctostaphylos uva-ursi leaf extract in concentrations ranging from 10 to 0.07 mg/mL. The extract showed a performing bacterial activity, exhibiting MIC 50 and MIC 90 values of 0.21 and 0.6 mg/mL, respectively. At lower concentrations, a gradual reduction of the inhibitory effect was verified ( Figure 4B). The understanding of the Arctostaphylos uva-ursi leaf extract kinetic action against C. acnes was obtained by time-killing assay. The data obtained showed exponential bacterial growth over time after treatment with the solvent (CTRL −) and with 0.3 mg/mL of Arctostaphylos uva-ursi leaf extract ( 1 2 × MIC). Exposure to 1.2 (2 × MIC) and 0.6 (1 × MIC) mg/mL maintained the bacterial load constant over time, suggesting a bacteriostatic action. On the other hand, 6 h of exposure to 10 µg/mL of Vancomycin (CTRL +) induced a gradual reduction of the bacterial load, and no living cells were detected after 20 h of treatment ( Figure 4C).

Effect of Arctostaphylos uva-ursi on C. acnes Biofilm
Biofilm formation is one of the main virulence factors of C. acnes; therefore the activity of Arctostaphylos uva-ursi leaf extract on the forming and preformed biofilm was evaluated. Biofilm biomass was quantified using CV in response to treatment in a concentration range between 10 and 0.07 mg/mL. After 72 h of incubation, Arctostaphylos uva-ursi leaf extract inhibited biofilm formation by 73 to 27% at concentrations of 10 to 0.07 mg/mL. In detail, the compound induced inhibition of 50 and 46% compared to the untreated biofilm (CTRL −) at the dose of 0.6 and 0.3 mg/mL, respectively ( Figure 6A). On the other hand, the biofilm eradication involved 72 h of mature matrix formation and subsequently 24 h of treatment with Arctostaphylos uva-ursi leaf extract in the concentration range mentioned above. Mature biofilm was destroyed in a dose-dependent manner from 78 to 9%, recording an eradication rate of 57 and 45% at 0.6 (1 × MIC) and 0.3 ( 1 2 × MIC) mg/mL, respectively. There were no significant changes in the biofilm matrix at concentrations below 0.15 mg/mL ( Figure 6B).

Cytotoxicity Profile
Cell viability was assessed using the HaCaT cell line after 24 and 48 h of Arctostaphylos uva-ursi leaf extract exposure in a concentration range between 10 and 0.07 mg/mL. The cytotoxic effect increased in a dose-dependent manner, showing a mortality rate of 50% at 5 mg/mL, after 24 h of treatment. In a dose range between 1.25 and 0.07 mg/mL, Arctostaphylos uva-ursi leaf extract did not affect cell viability, recording a live cell rate of more than 80%. No alteration was verified in cellular treatment with the solvent (H 2 O) used to dissolve the compound (CTRL −), while 99% cell mortality was detected by treating HaCaT with 100% DMSO (CTRL +) ( Figure 7A). Furthermore, the 50% cytotoxic concentration value (CC 50 ), calculated from the dose-effect curves by non-linear regression analysis, was 4.2 mg/mL and 3.6 mg/mL after 24 and 48 h. The calculated SI-value was 17.2, suggesting Arctostaphylos uva-ursi as a favorable extract for in vivo investigations. The toxicity profile was confirmed by the hemolysis assay, recording 46 and 35% of erythrocyte lysis after treatment with 5 and 2.5 mg/mL of the compound. At lower concentrations, no relevant hemolysis was detected. (Figure 7B).

Induction of Inflammatory Cytokines by C. acnes In Vitro
To investigate the anti-inflammatory effects of Arctostaphylos uva-ursi leaf extract in C. acnes-treated HaCaT cells, ELISA assays were performed to quantify the pro-inflammatory cytokines. The cell line was incubated with increasing doses of Arctostaphylos uva-ursi leaf extract and heat-killed C. acnes for 24 h. Treatment with the extract significantly suppressed cytokine production. In detail, cells exposed to C. acnes secreted 530, 480, 1300 and 600 pg/mL of IL-1β, IL-6, IL-8 and TNF-α, respectively. On the other hand, treatment with 0.6 mg/mL (1 × MIC) resulted in a decrease in proinflammatory cytokines almost comparable to untreated cells, recording a fold-reduction of 1.8, 1.9, 2.27, 5.4 for IL-1β, IL-6, IL-8 and TNF-α, respectively. At lower concentrations, a slight increase in cytokines was recorded (Figure 8).  In detail, the compound induced inhibition of 50 and 46% compared to the untreated biofilm (CTRL −) at the dose of 0.6 and 0.3 mg/mL, respectively ( Figure 6A). On the other hand, the biofilm eradication involved 72 h of mature matrix formation and subsequently 24 h of treatment with Arctostaphylos uva-ursi leaf extract in the concentration range mentioned above. Mature biofilm was destroyed in a dose-dependent manner from 78 to 9%, recording an eradication rate of 57 and 45% at 0.6 (1 × MIC) and 0.3 (½ × MIC) mg/mL, respectively. There were no significant changes in the biofilm matrix at concentrations below 0.15 mg/mL ( Figure 6B).

Cytotoxicity Profile
Cell viability was assessed using the HaCaT cell line after 24 and 48 h of Arctostaphylos uva-ursi leaf extract exposure in a concentration range between 10 and 0.07 mg/mL. The cytotoxic effect increased in a dose-dependent manner, showing a mortality rate of 50% at 5 mg/mL, after 24 h of treatment. In a dose range between 1.25 and 0.07 mg/mL, Arctostaphylos uva-ursi leaf extract did not affect cell viability, recording a live cell rate of more than 80%. No alteration was verified in cellular treatment with the solvent (H2O) used to dissolve the compound (CTRL −), while 99% cell mortality was detected by treating HaCaT with 100% DMSO (CTRL +) ( Figure 7A). Furthermore, the 50% cytotoxic concentration value (CC50), calculated from the dose-effect curves by non-linear regression analysis, was 4.2 mg/mL and 3.6 mg/mL after 24 and 48 h. The calculated SI-value was 17.2, suggesting Arctostaphylos uva-ursi as a favorable extract for in vivo investigations. The toxicity profile was confirmed by the hemolysis assay, recording 46 and 35% of erythrocyte lysis after treatment with 5 and 2.5 mg/mL of the compound. At lower concentrations, no relevant hemolysis was detected. (Figure 7B).

Induction of Inflammatory Cytokines by C. acnes In Vitro
To investigate the anti-inflammatory effects of Arctostaphylos uva-ursi leaf extract in C. acnes-treated HaCaT cells, ELISA assays were performed to quantify the pro-inflammatory cytokines. The cell line was incubated with increasing doses of Arctostaphylos uva-ursi leaf extract and heat-killed C. acnes for 24 h. Treatment with the extract significantly suppressed cytokine production. In detail, cells exposed to C. acnes secreted 530, 480, 1300 and 600 pg/mL of IL-1β, IL-6, IL-8 and TNF-α, respectively. On the other hand, treatment with 0.6 mg/mL (1 × MIC) resulted in a decrease in proinflammatory cytokines almost comparable to untreated cells, recording a fold-reduction of 1.8, 1.9, 2.27, 5.4 for IL-1β, IL-6, IL-8 and TNF-α, respectively. At lower concentrations, a slight increase in cytokines was recorded ( Figure 8).

Induction of Inflammatory Cytokines by C. acnes In Vitro
To investigate the anti-inflammatory effects of Arctostaphylos uva-ursi leaf extract in C. acnes-treated HaCaT cells, ELISA assays were performed to quantify the pro-inflammatory cytokines. The cell line was incubated with increasing doses of Arctostaphylos uva-ursi leaf extract and heat-killed C. acnes for 24 h. Treatment with the extract significantly suppressed cytokine production. In detail, cells exposed to C. acnes secreted 530, 480, 1300 and 600 pg/mL of IL-1β, IL-6, IL-8 and TNF-α, respectively. On the other hand, treatment with 0.6 mg/mL (1 × MIC) resulted in a decrease in proinflammatory cytokines almost comparable to untreated cells, recording a fold-reduction of 1.8, 1.9, 2.27, 5.4 for IL-1β, IL-6, IL-8 and TNF-α, respectively. At lower concentrations, a slight increase in cytokines was recorded ( Figure 8).

Antioxidant Activity
The antioxidant potential of Arctostaphylos uva-ursi leaf extract was investigated by evaluating the discoloration of DPPH·, a free radical that accepts an electron or hydrogen radical to become a stable molecule. Then, the percentage of RSA was calculated in the concentration range from 10 to 0.005 mg/mL. Arctostaphylos uva-ursi leaf extract already showed a performing antioxidant power after 15 min, recording radical scavenging effects of over 80% up to 0.03 mg/mL. On the other hand, considering that arbutin contained in 0.03 mg/mL was about 0.006 mg/mL, at this concentration arbutin alone produced a weak radical scavenging activity (i.e., 13%). However, as shown by Takebayashi et al., arbutin may show its radical-scavenging effect in a long-lasting manner and therefore its antioxidant activity, evaluated after 15 min, is underestimated [44]. A linear reduction was observed at concentrations of 0.01 and 0.005 mg/mL, with an RSA rate of 55 and 43%, respectively ( Figure 9).

Antioxidant Activity
The antioxidant potential of Arctostaphylos uva-ursi leaf extract was investigated by evaluating the discoloration of DPPH•, a free radical that accepts an electron or hydrogen radical to become a stable molecule. Then, the percentage of RSA was calculated in the concentration range from 10 to 0.005 mg/mL. Arctostaphylos uva-ursi leaf extract already showed a performing antioxidant power after 15 min, recording radical scavenging effects of over 80% up to 0.03 mg/mL. On the other hand, considering that arbutin contained in 0.03 mg/mL was about 0.006 mg/mL, at this concentration arbutin alone produced a weak radical scavenging activity (i.e., 13%). However, as shown by Takebayashi et al., arbutin may show its radical-scavenging effect in a long-lasting manner and therefore its antioxidant activity, evaluated after 15 min, is underestimated [44]. A linear reduction was observed at concentrations of 0.01 and 0.005 mg/mL, with an RSA rate of 55 and 43%, respectively ( Figure 9).

Discussion
Currently, multi-drug resistance and the side effects associated with the use of conventional drugs require the need to search for new potential sources to fight C. acnes infections [45]. Traditional herbal medicines could represent promising approaches to overcome the limitations associated with the use of existing drugs for acne vulgaris treatment [46]. Therefore, the present study evaluated the Arctostaphylos uva-ursi leaf-extract ability to fight C. acnes in three different properties: (i) antimicrobial potential; (ii) anti-inflammatory properties; and (iii) antioxidant activity.
A preliminary analytical characterization was performed to gain information about the chemical-physical properties of the investigated extract. Arbutin was chosen as the target molecule in the Arctostaphylos uva-ursi detection due to its high concentration in the vegetal extract. FT-IR/ATR and XPS analyses clearly evidenced the presence of arbutin, as well as the characteristic spectrum relevant to a complex mixture of molecules/macromolecules of vegetal origin (such as carbohydrates, (hemi-) celluloses, lignin, etc.). Moreover, TGA was used to evaluate the thermal stability of the extract; the detected thermal properties revealed a high thermal stability of the extract (Tonset > 150 °C), which makes it suitable for pharmaceutical and/or cosmetic preparations where heating is required.
As far as the Franz cell studies are concerned, the obtained results evidenced that the release and permeation of the different extract compounds followed different pathways. However, it can be concluded that the developed toning lotion carried the Arctostaphylos uva-ursi components across the StratM ® membrane with a noticeable percentage.
Arctostaphylos uva-ursi leaf extract showed a significant inhibitory effect on the growth of C. acnes, exhibiting an inhibition area of 24 mm and a bacteriostatic action of up Figure 9. Free radical scavenging activity of Arctostaphylos uva-ursi leaf extract. Ascorbic acid (5 mg/mL) and Methanol represent the CTRL + and CTRL −, respectively. DPPH assay represents the mean ± standard deviation (SD) of three independent experiments. **** p-value < 0.0001; ANOVA: p-value < 0.0001; R squared = 0.9980.

Discussion
Currently, multi-drug resistance and the side effects associated with the use of conventional drugs require the need to search for new potential sources to fight C. acnes infections [45]. Traditional herbal medicines could represent promising approaches to overcome the limitations associated with the use of existing drugs for acne vulgaris treatment [46]. Therefore, the present study evaluated the Arctostaphylos uva-ursi leaf-extract ability to fight C. acnes in three different properties: (i) antimicrobial potential; (ii) anti-inflammatory properties; and (iii) antioxidant activity.
A preliminary analytical characterization was performed to gain information about the chemical-physical properties of the investigated extract. Arbutin was chosen as the target molecule in the Arctostaphylos uva-ursi detection due to its high concentration in the vegetal extract. FT-IR/ATR and XPS analyses clearly evidenced the presence of arbutin, as well as the characteristic spectrum relevant to a complex mixture of molecules/macromolecules of vegetal origin (such as carbohydrates, (hemi-) celluloses, lignin, etc.). Moreover, TGA was used to evaluate the thermal stability of the extract; the detected thermal properties revealed a high thermal stability of the extract (T onset > 150 • C), which makes it suitable for pharmaceutical and/or cosmetic preparations where heating is required.
As far as the Franz cell studies are concerned, the obtained results evidenced that the release and permeation of the different extract compounds followed different pathways. However, it can be concluded that the developed toning lotion carried the Arctostaphylos uva-ursi components across the StratM ® membrane with a noticeable percentage.
Arctostaphylos uva-ursi leaf extract showed a significant inhibitory effect on the growth of C. acnes, exhibiting an inhibition area of 24 mm and a bacteriostatic action of up to 0.6 mg/mL. Our results were consistent with the literature data, although differences were found due to growth condition variations and/or bacterial strains investigated. In detail, Hassan et al. reported an inhibition diameter between 15 and 30 mm and an MIC-value between 5 and 1.25 mg/mL against Escherichia coli (E. coli), Aeromonas caviae, Bacillus Cereus, Micrococcus luteus, Mycobacterium avium and Paenibacillus alvei [47]. The antimicrobial potential could result from polyphenols, which are secreted by plants as a defense mechanism against pathogens. For Arctostaphylos uva-ursi leaf extract, it could be associated with the arbutin that represents the main extract component (it makes up 7% to 9% of the leaves) and is already known to exhibit antimicrobial activity [48,49]. Moreover, Arctostaphylos uva-ursi was also described to be active against E. coli and possess diuretic properties [50,51]. A study on urinary tract infection prevention reported that 18% (27 individuals) and 0% (30 individuals) of women who were given, respectively, placebo and Arctostaphylos uva-ursi in combination with dandelion root and leaf developed urinary tract infections [50]. In the second set of experiments, we investigated the action of Arctostaphylos uva-ursi leaf extract against the formation and maturation of C. acnes biofilm. The CV assay indicated a strong inhibitory and degradative activity on the biofilm matrix. In detail, biomass degradation exceeded 50% and 40% in response to treatment with 1 × MIC and 1 2 × MIC, thus reaching the deeper layers of a mature biofilm. In acne inflammation pathogenesis, C. acnes plays an important trigger role through IL-6 and IL-8 secretion by follicular keratinocytes and IL-1β, TNF-α, IL-8 and IL-12 by monocytes [52,53]. IL-8, a CXC-type chemokine, is a pro-inflammatory chemotactic factor that primarily affects neutrophils [50]. TNF-α, IL-6 and IL-1β are inflammatory molecules involved in both acute and chronic inflammatory acne [54]. Patients suffering from acne vulgaris presented high levels of IL-8 in peripheral blood mononuclear cells and remarkable expression of IL-6 and IL-8 in keratinocytes [55]. Since these mediators aggravate the initial acne lesion by increasing the inflammatory state, we evaluated whether Arctostaphylos uva-ursi leaf extract could inhibit the secretion of the pro-inflammatory cytokines in HaCaT cells infected with C. acnes. Our evidence showed strong induction of C. acnes to the secretion of IL-1β, TNF-α, IL-8 and IL-6 in keratinocytes [56]. In co-presence with Arctostaphylos uva-ursi leaf extract bacterium, a marked cytokine suppressive effect was recorded, demonstrating a strong anti-inflammatory effect associated with the antibacterial properties mentioned above. Consistent with our findings, a study by Weber et al. screened ninety-nine herbal extracts by evaluating their TLR2-and TLR4-dependent antiinflammatory effects in THP-1 monocytes. Among these, Arctostaphylos uva-ursi leaves, Castanea sativa leaves, Cinchona pubescens bark, Cinnamomum verum bark, Salix alba bark, Rheum palmatum root, Alchemilla vulgaris plant, Humulus lupulus cones, Vaccinium myrtillus berries and Curcuma longa root showed the highest anti-inflammatory properties, suppressing the production of proinflammatory cytokines. In detail, Arctostaphylos uva-ursi inhibited LPS-induced NF-κB translocation, suppressed TNF-α secretion and induced IL-10 release in macrophages, indicating a transition from M1 macrophages (pro-inflammatory) to M2 macrophages (anti-inflammatory) [57]. The predominant molecule responsible for the antioxidant anti-inflammatory activity is arbutin, which reduces the secretion of IL-1β and TNF-α and other related genes, such as the chemotactic protein monocyte-1 and IL-6 [58]. Finally, considering the growing interest in "green production" and in research of natural antioxidants, the radical scavenging activity of Arctostaphylos uva-ursi leaf extract was determined by DPPH assay. The high percentage of RSA obtained up to a concentration of 0.15 mg/mL indicated a performing antioxidant potential, attributable to the high phenol content in Arctostaphylos uva-ursi leaves, as confirmed by the high levels of phenolic compounds detected in the extract (315 GAEq/g).
Considering this information, Arctostaphylos uva-ursi can be considered a valuable plant source for the pharmaceutical and cosmetic fields. Considering that C. acnes in recent years has shown a worrying increase in resistance to erythromycin, clindamycin, doxycycline, tetracycline, trimethoprim/sulfamethoxazole, levofloxacin and minocycline [59], the discovery of green alternative sources as antimicrobial and anti-inflammatory agents for the Acne vulgaris treatment could overcome the limitations associated with C. acnes multidrug resistance and mitigate the side effects of conventional medications. Furthermore, the high phenol content could make Arctostaphylos uva-ursi a promising natural antioxidant additive in herbal preparations. Nevertheless, further investigation to better understand the Arctostaphylos uva-ursi mechanism of action against C. acnes and to designate additional inflammatory targets will be necessary.