Efficacy of Two Moroccan Cistus Species Extracts against Acne Vulgaris: Phytochemical Profile, Antioxidant, Anti-Inflammatory and Antimicrobial Activities

Background: The genus Cistus L. (Cistaceae) includes several medicinal plants growing wild in the Moroccan area. Acne vulgaris (AV) is a chronic skin disorder treated with topical and systemic therapies that often lead to several side effects in addition to the development of antimicrobial resistance. Our study aimed to investigate the bioactivity of extracts of two Moroccan Cistus species, Cistus laurifolius L. and Cistus salviifolius L., in view of their use as potential coadjuvants in the treatment of mild acne vulgaris. Methods: Targeted phytochemical profiles obtained by HPLC-DAD and HPLC-ESI/MS analyses and biological activities ascertained by several antioxidants in vitro chemical and cell-based assays of the leaf extracts. Moreover, antimicrobial activity against Gram-positive and Gram-negative bacteria, and Candida albicans was evaluated. Results: Analyses revealed the presence of several polyphenols in the studied extracts, mainly flavonoids and tannins. Cistus laurifolius L. and Cistus salviifolius L. possessed good biological properties and all extracts showed antibacterial activity, particularly against Staphylococcus aureus, S. epidermidis, and Propionibacterium acnes, identified as the main acne-causing bacteria. Conclusion: The results suggest that examined extracts are promising agents worthy of further studies to develop coadjuvants/natural remedies for mild acne treatment.


Introduction
Skin is the largest organ and the main interface of the human body with the external environment. The skin condition largely depends on the overall state of health of individuals and acne vulgaris (AV) is one of the most common and severe skin disorders. Acne lesions are typically classified as non-inflammatory (open and closed comedones) or inflammatory, including papules and pustules or nodules [1,2]. It is mainly seen on the subjects' face, the upper part of their chest, and on their back [3]; however, these disorders have a negative impact on the mental health, self-esteem, and quality of life of individuals [4,5].
The clinical and histological features define acne as a chronic inflammatory disease of the pilosebaceous unit [6]. Therefore, AV is pathophysiologically triggered by an increased sebum's production, and follicular hyperkeratinization leading to clogging of the sebaceous/oil duct and colonization of bacteria, especially the Propionibacterium acnes (recently Table 1. Phytochemical analysis and yield extraction of C. laurifolius L. and C. salviifolius L. crude extracts and their polyphenol fractions.

HPLC-DAD and HPLC-ESI-/MS Based Secondary Metabolic Profiles
The detailed composition of CLLE and CSLE together with the corresponding phenolic fractions CLLE PF and CSLE PF are reported in Table 2. The crude extracts are characterized by the presence of many polyphenols belonging to three main biochemical classes: flavonoids, hydroxycinnamic acids, and tannins. Among the 36 peaks detected and tentatively identified, numerous methylated flavonoids stand out, such as methyl and di-methyl derivatives of flavonols quercetin and kaempferol as well as those of flavones apigenin and luteolin (Tables 2 and S1 and Figure S1).

Antioxidant Activity
In most electron transfer (ET)-based assays, the antioxidant reaction is simulated with a suitable redox-potential agent. The antioxidants react with a fluorescent or colored oxidizing agent instead of peroxyl radicals. ET-based assays include ferric reducing/antioxidant power (FRAP) assay, ABTS assay, DPPH assay and others assays [18]. Considering the hypothesis that suggests the oxygen free radicals generated by neutrophils in the follicular wall could be involved in the pathogenesis of acne [19], the evaluation of the antioxidant potential of our extracts seemed necessary.
The measurement of free radical scavenging indicated that all extracts possessed a noteworthy activity ( Table 3). The higher DPPH scavenging activity was demonstrated for the polyphenolic fraction of CSLE (mmol TE/mg 2.593 ± 0.572) followed by the polyphenolic fraction of CLLE (mmol TE/mg 1.284 ± 0.327) ( Table 3). The ABTS assay reveals that all extracts were equally efficient in reducing the ABTS cations. Furthermore, all extracts possess good ferric trichloride reduction capacity, particularly the CLSE PF extract.

Anti-Inflammatory Activity
The HClO-induced BSA denaturation was carried out to investigate the antiinflammatory capability of the extracts under study. It is well-known that HClO reacts with a wide variety of biological molecules producing tissue damage that impacts the function of biological systems, and that protein degradation is a well-documented cause of inflammatory diseases [20]. Among the tested samples, the extracts of C. salviifolius L. possessed the higher capacity to protect albumin against HClO-induced degradation comparable to Trolox used as standard ( Figure 1 and Table 4).

Anti-Inflammatory Activity
The HClO-induced BSA denaturation was carried out to investigate the anti-inflam matory capability of the extracts under study. It is well-known that HClO reacts with wide variety of biological molecules producing tissue damage that impacts the functio of biological systems, and that protein degradation is a well-documented cause of inflam matory diseases [20]. Among the tested samples, the extracts of C. salviifolius L. possessed the higher capacity to protect albumin against HClO-induced degradation comparable t Trolox used as standard ( Figure 1 and Table 4).

Antimicrobial Activity
The MIC values analysis (Table 5) showed that the plant extracts possessed an inhib itory effect against tested strains. However, they demonstrated more efficacy agains Gram-positive bacteria than against Gram-negative bacteria and C. albicans ATCC 10231 All Cistus extracts showed antibacterial activity against S. aureus ATCC 6538, methicillin resistant S. aureus (MRSA) ATCC 43300, S. epidermidis ATCC 35984 and P. acnes ATCC

Antimicrobial Activity
The MIC values analysis (Table 5) showed that the plant extracts possessed an inhibitory effect against tested strains. However, they demonstrated more efficacy against Gram-positive bacteria than against Gram-negative bacteria and C. albicans ATCC 10231. All Cistus extracts showed antibacterial activity against S. aureus ATCC 6538, methicillinresistant S. aureus (MRSA) ATCC 43300, S. epidermidis ATCC 35984 and P. acnes ATCC 11827.
In particular, C. salviifolius L. extracts revealed the highest antibacterial activity with MIC Molecules 2023, 28, 2797 6 of 13 values of 62.5-125 µg/mL. None of the tested strains was inhibited by DMSO (maximum 1% v/v) used as negative control. The determination of the killing activity revealed that the extracts possessed a bacteriostatic rather than bactericidal effect. In this context, the MBC values ranged from 1000 to >2000 µg/mL, except for P. acnes, which showed MBC values of 500-1000 µg/mL. These findings were confirmed by the MBC/MIC ratio of 4 for CSLEand CSLE PF against P. acnes (confirming a bactericidal effect) [21], and MBC/MIC ratio values higher than 4 for the other strains.

Cytotoxicity Evaluation
To characterize the cytotoxic effect of Cistus extracts, the viability of NIH/3T3 exposed for 48 h to different concentrations of the extracts was investigated. The findings indicated that the cytotoxic activity of the crude hydroalcoholic extracts was weaker with respect to the polyphenol fractions, suggesting that crude extracts are probably safer (Table 5).

Discussion
The management and the therapy of acne vulgaris aim to reduce sebum production, and follicular hyper keratinization. The drugs used should primarily exhibit antioxidant, anti-inflammatory, and antimicrobial activities, without having cytotoxic effects. Some synthetic remedies have limitations because of their highly toxic activity, and their ability to induce adverse side effects. Thus, a growing interest in the use of herbal medicines as coadjuvants for the treatment of acne is increasingly recognized.
In our research, we assessed the composition and evaluated the biological properties of various extracts from the leaves of Cistus laurifolius L. and C. salviifolius L., grown in Morocco. Thus, we identified the main compounds present in both Cistus spp. and determined the antioxidant, anti-inflammatory, antimicrobial, and cytotoxic properties of such extracts (crude and polyphenolic fraction).
The Cistus genus was recently reviewed for its composition by Papaefthimiou et al. [22]. The authors reported C. laurifolius L. and C. salviifolius L. as a source of flavonoids, hydroxycinnamic acids, and tannins. In our study, 36 chromatographic signals were detected, tentatively identified, and quantified, as shown in Table 2. When considering the polyphenolic profile from the crude hydroalcoholic extracts of these two species (CLLE and CSLE in Table 2), at least two significant differences can be evidenced: the presence in CLLE of several hydroxycinnamic acids (mainly para-coumaric) derivatives (peaks 1, 3, 6, 8, and 12) as well as that of a bulky group of methyl-and polymethyl-flavonoids (peaks 22-26 and 28-36), that CSLE shares only to a minimum extent (Table 2). This particular richness in methyl-flavanoids of CLLE aligns with what reported in the literature [23]. On the other side, in CSLE, a series of ellagitannins were troublesomely identified, of which two couples of anomers (peaks 2 + 7 and 4 + 9) corresponding to terflavin A and cistusin, respectively, reported to belong to the secondary metabolism of C. incanus [24]. A gallic acid derivative (peak 5) and an ellagic acid derivative (peak 14) complete the ellagitannin series in CSLE. No punicalagin were detected, this time in disagreement with what was reported in the literature [15][16][17][18][19][20][21][22][23][24][25]. Regarding the quantitative analyses, small differences were detected between CLLE (3.74 mg polyphenols/100 mg extract) and CSLE (2.98 mg polyphenols/100 mg extract) ( Table 2).
As well documented in the literature, the extracts of C. laurifolius L. and C. salviifolius L. showed important biological effects. In particular, a recent review by Zalegh et al. [26] described the antioxidant and anti-inflammatory effects of Cistus spp. extracts and their correlation with the polyphenolic content. Semwal et al. [27] reported that myricetin and its derivates are known for radical scavenging, immunomodulating and anti-inflammatory activities by interfering with the NF-kB signaling pathway and, as described by Imra et al. [28], the antioxidant activity of myricetin is attributed to the presence of three hydroxyl groups on ring B, as compared to other flavonoids. Furthermore, Lee and Lee [29] demonstrated that myricetin was able to exhibit differential anti-inflammatory effects on the production of inflammatory mediators in various cells and with distinctive stimulants.
Actually, quercetin is the one of the most powerful antioxidant flavonoids in nature. Recently, its biological activity was evaluated by Srimathi Priyanga and Vijayalakshmi [30] and Azeem et al. [31]. The authors highlighted the potential role in supporting the immune system against oxidative stress and its related disorder, the antimicrobial and the antiinflammatory effects of this flavonoid.
Concerning tannins, de Melo et al. [32] described their ROS reduction characteristics. The authors reported that their activities are associated with the number and degree of polymerization of reactive hydroxyl groups present in the phenolic rings and that those characteristics are associated with anti-inflammatory, anti-aging, and health benefits.
Therefore, the presence of myricetin and quercetin derivatives and hydrolysable tannins may partly explain the antioxidant and anti-inflammatory activities of the extracts under examination.
Regarding antimicrobial activity, the best efficacy of our extracts was detected against Gram-positive bacteria such as S. aureus, including MRSA strain, S. epidermidis, and P. acnes. In contrast, the extracts were found to have a low activity on Gram-negative bacteria. This different effect can be partly due to the differences in cell wall structure among these microorganisms; the Gram-negative bacteria have an outer membrane that pro-vides an additional selective permeation barrier.
The antimicrobial activity of some Cistus spp. has been explored. However, it is difficult to compare the data with the literature because different variables, such as the different collecting site, extraction solvent and antimicrobial method, can influence the results. C. laurifolius and C. salviifolius methanol extracts were reported to show activity against Bacillus subtilis, B. cereus and S. aureus, but no efficacy against P. aeruginosa and C. albicans. [33]. Tomàs-Menor et al. [25] investigated the relationship between the antimicrobial activity of C. salviifolius extracts and phytochemical composition. In this context, Zeouk et al. [34] reported that C. salviifolius methanol extracts with high amounts of total phenols and flavonoids showed antibacterial activity against MRSA and S. epidermidis strains. Moreover, Álvarez-Martínez et al. [35] investigated the activity of C. salviifolius against 100 S. aureus clinical isolates and referred MIC 50 values ranging between 50-80 µg/mL. The authors also documented that the Cistus extracts containing hydrolysable tannins and flavonoids such as myricetin and quercetin derivatives showed the higher activity against MRSA isolates.Among the Cistus extracts, C. laurifolius exhibited antibacterial efficacy against S. aureus and E. coli [36] and inhibitory activity against microbial biofilm production [37].
Considering the literature and based on our results on antioxidant, anti-inflammatory, antimicrobial and citotoxic activities, we can say that C. laurifolius L. and C. salvifolius L. extracts possess a potential to treat mild acne.

Preparation of the Extracts
The powder of dried leaves of both plants was subjected to extraction with Ethanol/ Water 70% (v/v) under permanent agitation at room temperature for 3 days. The obtained extracts were filtered and concentrated under reduced pressure to obtain dried residues of the ethanolic extracts (CLLE, CSLE) ( Figure 2). The extraction yields are expressed in the percentage of dried plant material ( Table 1).
As shown in Figure 2, the obtained hydroalcoholic crude extracts were dissolved in hot distilled water. They were processed with successive solvent extraction using three solvents with different polarities (n-hexane, dichloromethane, ethyl acetate). The ethyl acetate extract of each plant was filtered and evaporated under reduced pressure to obtain the respective polyphenolic-rich fractions (PF). against 100 S. aureus clinical isolates and referred MIC50 values ranging betwee µg/mL. The authors also documented that the Cistus extracts containing hydrolys nins and flavonoids such as myricetin and quercetin derivatives showed the high ity against MRSA isolates.Among the Cistus extracts, C. laurifolius exhibited anti efficacy against S. aureus and E. coli [36] and inhibitory activity against microbia production [37].
Considering the literature and based on our results on antioxidant, anti-in tory, antimicrobial and citotoxic activities, we can say that C. laurifolius L. and C. s L. extracts possess a potential to treat mild acne.

Preparation of the Extracts
The powder of dried leaves of both plants was subjected to extraction wi nol/Water 70% (v/v) under permanent agitation at room temperature for 3 days. tained extracts were filtered and concentrated under reduced pressure to obtain d idues of the ethanolic extracts (CLLE, CSLE) ( Figure 2). The extraction yields are ex in the percentage of dried plant material ( Table 1).
As shown in Figure 2, the obtained hydroalcoholic crude extracts were diss hot distilled water. They were processed with successive solvent extraction usi solvents with different polarities (n-hexane, dichloromethane, ethyl acetate). The etate extract of each plant was filtered and evaporated under reduced pressure t the respective polyphenolic-rich fractions (PF).  Phytochemical analysis of the extracts was performed to determine the content of secondary metabolites such as total phenols, flavonoids, and flavonols. In all the tests, results were expressed as the mean ± SD of the three different experiments.

Total Polyphenol Content
The total polyphenol content of each extract was determined using the Folin-Ciocalteu reagent according to the method described by Tomaino et al. [38]. A volume of 0.5 mL of Folin-Ciocalteu reagent was mixed with 0.1 mL of each extract concentration previously diluted with distilled water (1:10). The solution was allowed to stand for 3 min at 25 • C before adding 1.7 mL of a 10% sodium carbonate solution (NA 2 CO 3 ). The absorbance was measured at 786 nm after 1 h of incubation with agitation at room temperature. Total polyphenol content was determined using a standard curve prepared with gallic acid. The results are expressed as mg of gallic acid equivalents (GAE) per gram of dry extract (DE).

Flavonoids Content
The flavonoid content was measured using the method described by Bouaziz et al. [39]. Briefly, 1 mL of each sample at various concentrations ranging from 0.125 to 1 mg/mL, was mixed with 4 mL of distilled water, and 300 µL of a 5% sodium nitrate solution (NaNO 2 ) was added. After 5 min, 300 µL of aluminum trichloride solution (AlCl 3 , 10%) was added to the mixture, followed 1 min later by the addition of 2 mL of sodium hydroxide (NaOH, 1 M). The absorbance was measured at 510 nm using a UV-VIS spectrophotometer. Total flavonoid content was calculated from a calibration curve using catechin as a standard and expressed as mg catechin equivalents (CatE) per g of DE.

Flavonols Content
The flavonols content of each extract was determined using the method described by Russo et al. [40]. Each extract (1 mL) at various concentration (0.125 to 1 mg/mL) was mixed with 1 mL of a 20% aluminum trichloride solution (AlCl 3 ). Then, a volume of 3 mL of sodium acetate solution (C 2 H 3 NaO 2 , 50 mg/mL; w/v) was added. The mixed solutions were incubated in the dark for 2.5 h at room temperature. The absorbance was measured at 440 nm using a UV-Vis spectrophotometer. The content of flavonols was determined from a calibration curve, using quercetin as a standard. The results are expressed as mg of quercetin equivalents (QE) per g of dry extract.

HPLC-DAD and HPLC-ESI-/MS Based Secondary Metabolic Profiles
Chromatographic analyses were carried out on an Ultimate3000 UHPLC focused instrument equipped with a binary high-pressure pump, a Photodiode Array detector, a Thermostated Column Compartment, and an Automated Sample Injector (Thermo Fisher Scientific, Inc., Milan, Italy). Collected data were processed through a Chromeleon Chromatography Information Management System v. 6.80. Chromatographic runs were performed using a reverse-phase column (Gemini C18, 250 × 4.6 mm, 5 µm particle size, Phenomenex Italia s.r.l., Bologna, Italy) equipped with a guard column (Gemini C18 4 × 3.0 mm, 5 µm particle size, Phenomenex Italia s.r.l., Bologna, Italy). Components of the hydroalcoholic extracts of Cistus spp. were eluted using a gradient of B (2.5% formic acid in acetonitrile) in A (2.5% formic acid in water): 0 min: 5% B; 10 min: 15% B; 30 min: 25% B; 35 min: 30% B; 50 min: 90% B; 57 min then kept for other 7 min, 100% B. The solvent flow rate was 1 mL/min, the temperature was kept at 25 • C, and the injector volume selected was 10 µL. Quantification was carried out at 330 nm using p-coumaric acid, ferulic acid, and apigenin commercial standards to build the calibration curves; for kaempferol, quercetin, and myricetin derivatives, the following standards were used at 350 nm: kaempferol 3-O-rutinoside, quercetin 3-O-glucoside, and myricetin; finally, punicalagin was used to calibrate instrument's response for ellagitannins at 360 nm. In order to confirm peak assignments, HPLC/ESI-MS analyses were also performed. The HPLC apparatus was the same as described above, while ESI mass spectra were acquired by a Thermo Scientific Exactive Plu Orbitra MS (Thermo Fisher Scientific, Inc., Milan, Italy) using a heated electrospray ionization (HESI II) interface. Mass spectra were recorded operating in negative ion mode. Analyses were always carried out in triplicate.

Anti-Inflammatory Activity
Anti-inflammatory activity was evaluated through the study of the ability of extracts to protect albumin (BSA) against HClO-induced denaturation. BSA was incubated in the absence or presence of 177 mM HClO and with increasing amounts of the extracts (0.05, 0.1, 0.25 mg/mL), then subjected to SDS-PAGE and developed with Coomassie Blue. Trolox at different concentrations (0.25, 0.088, and 0.062 mg/mL) was used as a positive/standard control [41]. Experiments were carried out in triplicate, and the results were expressed as IC50 values (mg extract/mL) and 90% C.L.

MIC and MBC/MFC Determination
The minimum inhibitory concentration (MIC), the minimum bactericidal concentration (MBC), and the minimum fungicidal concentration (MFC) of plant extracts were determined using a broth dilution micro method in 96-well microtiter plates according to Clinical and Laboratory Standards Institute (CLSI) guidelines with some modification [43,44]. Briefly, Cistus laurifolius L. and Cistus salviifolius L. extracts and their polyphenolic fractions were dissolved in pure dimethylsulfoxide (DMSO) to prepare respective stock solutions (200 mg/mL) from which serial twofold dilutions were made in MHB, BHI or SBD at final concentrations ranging from 2000 to 1.95 µg/mL. Microbial cultures were inoculated to yield a final concentration of 5 × 10 5 CFU (bacteria) and 2.5 × 10 3 CFU (yeast). All determinations were performed in duplicate and growth control consisting of MHB and MHB with DMSO 1% were included as controls. Ofloxacin and amphotericin B were used as positive reference standard drugs. The MIC was considered as the lowest concentration of each extract, giving a complete inhibition of visible bacterial growth after incubation for 24 h (bacteria), 48 h (yeast), and in anaerobic conditions for 4 to 5 days (P. acnes). The MBC was determined by seeding 20 µL from all clear MIC wells onto agar plates and was defined as the lowest concentration of plant extracts that killed 99.9% of the inoculum. Each determination was performed in three independent experiments, and modal results were calculated.

Cytotoxicity Evaluation
The biocompatibility of both plant extracts was assessed on NIH/3T3 fibroblasts cell line (ATCC CRL-1658; American Type Culture Collection, Rockville, MD, USA) using the Sulforhodamine B (SRB) assay. The NIH/3T3 cells were exposed for 48 h to different concentrations (15.5, 31, 62.5, and 125 µg/mL) of tested extracts added to the cell culture medium, or to the vehicle alone (DMSO) used as the control [45].

Statistical Analysis
Results are statistically analyzed by a one-way or a two-way analysis of variance (ANOVA) test, followed by Tukey's honest significant difference, using the statistical software ezANOVA (https://people.cas.sc.edu/rorden/ezanova/index.html, accessed on 28 February 2023)

Conclusions
Overall, the results suggest that the crude extracts of Cistus laurifolius L. and C. salviifolius L. from the High Atlas Mountain of Morocco, despite the slightly different activity, can be regarded as promising agents to develop coadjuvants/natural remedies for the treatment of mild acne, and it is worth noting that extracts could be subjected to in vivo study.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/molecules28062797/s1, Figure S1: HPLC/DAD chromatograms, visualized at 330 nm, of the Cistus spp. crude extracts object of this study. Peak numbers correspond to Table 2.; Figure S2: Composition (%) in polyphenols, divided into biochemical subclasses, of the Cistus spp. extracts. See text for details.; Table S1: Peak list and diagnostics of selected metabolites from the Cistus spp. extracts object of this study. See text for details.